Caloric Restriction Reprograms the Single-Cell Transcriptional Landscape of Rattus Norvegicus Aging.

Aging causes a functional decline in tissues throughout the body that may be delayed by caloric restriction (CR). However, the cellular profiles and signatures of aging, as well as those ameliorated by CR, remain unclear. Here, we built comprehensive single-cell and single-nucleus transcriptomic atlases across various rat tissues undergoing aging and CR. CR attenuated aging-related changes in cell type composition, gene expression, and core transcriptional regulatory networks. Immune cells were increased during aging, and CR favorably reversed the aging-disturbed immune ecosystem. Computational prediction revealed that the abnormal cell-cell communication patterns observed during aging, including the excessive proinflammatory ligand-receptor interplay, were reversed by CR. Our work provides multi-tissue single-cell transcriptional landscapes associated with aging and CR in a mammal, enhances our understanding of the robustness of CR as a geroprotective intervention, and uncovers how metabolic intervention can act upon the immune system to modify the process of aging.

Meningoencephalitis, coronary artery and keratitis as an onset of brucellosis: a case report.

BACKGROUND: Brucellosis is a zoonotic disease caused by brucella. It has been an increasing trend in recent years (Wang H, Xu WM, Zhu KJ, Zhu SJ, Zhang HF, Wang J, Yang Y, Shao FY, Jiang NM, Tao ZY, Jin HY, Tang Y, Huo LL, Dong F, Li ZJ, Ding H, Liu ZG, Emerg Microbes Infect 9:889-99, 2020). Brucellosis is capable to invade multiple systems throughout the body, lacking in typical clinical manifestations, and easily misdiagnosed and mistreated. CASE PRESENTATION: We report a case of a male, 5-year-and-11-month old child without relevant medical history, who was admitted to hospital for 20 days of fever. When admitted to the hospital, we found that he was enervated, irritable and sleepy, accompanied with red eyes phenomenon. After anti-infection treatment with meropenem, no improvement observed. Lumbar puncture revealed normal CSF protein, normal cells, and negative culture. Later, doppler echocardiography suggested coronary aneurysms, and incomplete Kawasaki Disease with coronary aneurysms was proposed. The next day, brucellosis agglutination test was positive. Metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid suggested B.melitensis, which was confirmed again by blood culture. The child was finally diagnosed as brucellosis with meningocephalitis, coronary aneurysm and keratitis. According to our preliminary research and review, such case has never been reported in detail before. After diagnosis confirmation, the child was treated with rifampicin, compound sulfamethoxazole, and ceftriaxone for cocktail anti-infection therapy. Aspirin and dipyridamole were also applied for anticoagulant therapy. After medical treatment, body temperature of the child has reached normal level, eye symptoms alleviated, and mental condition gradually turned normal. Re-examination of the doppler echocardiographic indicated that the coronary aneurysm was aggravated, so warfarin was added for amplification of anticoagulation treatment. At present, 3 months of follow-up, the coronary artery dilatation gradually assuaged, and the condition is continued to alleviate. CONCLUSION: Brucellosis can invade nervous system, coronary artery, and cornea. Brucellosis lacks specific signs for clinical diagnosis. The traditional agglutination test and the new mNGS are convenient and effective, which can provide the reference for clinical diagnosis.

Peptide probes derived from pertuzumab by molecular dynamics modeling for HER2 positive tumor imaging.

A high level of HER2 expression in breast cancer correlates with a higher tumor growth rate, high metastatic potential, and a poor long-term patient survival rate. Pertuzumab, a human monoclonal antibody, can reduce the effect of HER2 overexpression by preventing HER2 dimerization. In this study, a combination protocol of molecular dynamics modeling and MM/GBSA binding free energy calculations was applied to design peptides that interact with HER2 based on the HER2/pertuzumab crystal structure. Based on a ß hairpin in pertuzumab from Glu46 to Lys65-which plays a key role in interacting with HER2-mutations were carried out in silico to improve the binding free energy of the hairpin that interacts with the Phe256-Lys314 of the HER2 protein. Combined the use of one-bead-one-compound library screening, among all the mutations, a peptide (58F63Y) with the lowest binding free energy was confirmed experimentally to have the highest affinity, and it may be used as a new probe in diagnosing and treating HER2-positive breast cancer.

Microarray based screening of peptide nano probes for HER2 positive tumor.

Peptides are excellent biointerface molecules and diagnostic probes with many advantages such as good penetration, short turnover time, and low cost. We report here an efficient peptide screening strategy based on in situ single bead sequencing on a microarray. Two novel peptides YLFFVFER (H6) and KLRLEWNR (H10) specifically binding to the tumor biomarker human epidermal growth factor receptor 2 (HER2) with aKD of 10(-8) M were obtained from a 10(5) library. Conjugated to nanoparticles, both the H6 and H10 probes showed specific accumulation in HER2-positive tumor tissues in xenografted mice by in vivo imaging.

Computational study on the molecular mechanisms of drug resistance of Narlaprevir due to V36M, R155K, V36M+R155K, T54A, and A156T mutations of HCV NS3/4A protease.

Narlaprevir is a novel NS3/4A protease inhibitor of hepatitis C virus (HCV), and it has been tested in a phase II clinical trial recently. However, distinct drug-resistance of Narlaprevir has been discovered. In our study, the molecular mechanisms of drug-resistance of Narlaprevir due to the mutations V36M, R155K, V36M+R155K, T54A, and A156T of NS3/4A protease have been investigated by molecular dynamics (MD) simulations, free energy calculations, and free energy decomposition analysis. The predicted binding free energies of Narlaprevir towards the wild-type and five mutants show that the mutations V36M, R155K, and T54A lead to low-level drug resistance and the mutations V36M+R155K and A156T lead to high-level drug resistance, which is consistent with the experimental data. The analysis of the individual energy terms indicates that the van der Waals contribution is important for distinguishing the binding affinities of these six complexes. These findings again show that the combination of different molecular modeling techniques is an efficient way to interpret the molecular mechanism of drug-resistance. Our work mainly elaborates the molecular mechanism of drug-resistance of Narlaprevir and further provides valuable information for developing novel, safer, and more potent HCV antiviral drugs in the near future.

Rapid screening of peptide probes through in situ single-bead sequencing microarray.

Peptide ligands as targeting probes for in vivo imaging and drug delivery have attracted great interest in the biomedical community. However, high affinity and specificity screening of large peptide libraries remains a tedious process. Here, we report a continuous-flow microfluidic method for one-bead-one-compound (OBOC) combinatorial peptide library screening. We screened a library with 2 × 10(5) peptide beads within 4 h and discovered 140 noncanonical peptide hits targeting the tumor marker, aminopeptidase N (APN). Using the Clustal algorithm, we identified the conserved sequence Tyr-XX-Tyr in the N terminal. We demonstrated that the novel sequence YVEYHLC peptides have both nanomolar affinity and high specificity for APN in ex vivo and in vivo models. We envision that the successful demonstration of this integrated novel nanotechnology for peptide screening and identification open a new avenue for rapid discovery of new peptide-based reagents for disease diagnostics and therapeutics.

A single-nucleus transcriptomic atlas of primate liver aging uncovers the pro-senescence role of SREBP2 in hepatocytes.

Aging increases the risk of liver diseases and systemic susceptibility to aging-related diseases. However, cell type-specific changes and the underlying mechanism of liver aging in higher vertebrates remain incompletely characterized. Here, we constructed the first single-nucleus transcriptomic landscape of primate liver aging, in which we resolved cell type-specific gene expression fluctuation in hepatocytes across three liver zonations and detected aberrant cell-cell interactions between hepatocytes and niche cells. Upon in-depth dissection of this rich dataset, we identified impaired lipid metabolism and upregulation of chronic inflammation-related genes prominently associated with declined liver functions during aging. In particular, hyperactivated sterol regulatory element-binding protein (SREBP) signaling was a hallmark of the aged liver, and consequently, forced activation of SREBP2 in human primary hepatocytes recapitulated in vivo aging phenotypes, manifesting as impaired detoxification and accelerated cellular senescence. This study expands our knowledge of primate liver aging and informs the development of diagnostics and therapeutic interventions for liver aging and associated diseases.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner.

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Unraveling the allosteric inhibition mechanism of PTP1B by free energy calculation based on umbrella sampling.

Protein tyrosine phosphatase 1B (PTP1B) is a promising target for the treatment of obesity and type II diabetes. Allosteric inhibitors can stabilize an active conformation of PTP1B by hindering the conformational transition of the WPD loop of PTP1B from the open to the closed state. Here, the umbrella sampling molecular dynamics (MD) simulations were employed to compute the reaction path of the conformational transition of PTP1B, and the snapshots extracted from the MD trajectory were clustered into 58 conformational groups based on the key conformational parameter. Then, the impact of the conformational change of the WPD loop on the interactions between the allosteric site of PTP1B and an allosteric inhibitor BB3 was explored by using the MM/GBSA binding free energy calculations and free energy decomposition analysis. The simulation results show that the binding free energy of BB3 increases gradually from the open to the closed conformation of the WPD loop, providing the molecular mechanism of allosteric inhibition. Correlation analysis of the different energy terms indicates that the allosteric inhibitor with more negative van der Waals contribution cannot only exhibit stronger binding affinity but also hinder the swing of the WPD loop more effectively. Besides, it is found that the energy contribution of Lys292 in the α7 helix undergoes significant change, which reveals that Lys292 is not only the key residue for ligand binding but also plays an important role in hindering the conformational change of the WPD loop.

Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration.

Hair loss affects millions of people at some time in their life, and safe and efficient treatments for hair loss are a significant unmet medical need. We report that topical delivery of quercetin (Que) stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice. We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1α in endothelial cells. Skin administration of a HIF-1α agonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que. Together, these findings provide a molecular understanding for the efficacy of Que in hair regrowth, which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine, and suggest a route of pharmacological intervention that may promote hair regrowth.

Genome-wide CRISPR activation screening in senescent cells reveals SOX5 as a driver and therapeutic target of rejuvenation.

Our understanding of the molecular basis for cellular senescence remains incomplete, limiting the development of strategies to ameliorate age-related pathologies by preventing stem cell senescence. Here, we performed a genome-wide CRISPR activation (CRISPRa) screening using a human mesenchymal precursor cell (hMPC) model of the progeroid syndrome. We evaluated targets whose activation antagonizes cellular senescence, among which SOX5 outperformed as a top hit. Through decoding the epigenomic landscapes remodeled by overexpressing SOX5, we uncovered its role in resetting the transcription network for geroprotective genes, including HMGB2. Mechanistically, SOX5 binding elevated the enhancer activity of HMGB2 with increased levels of H3K27ac and H3K4me1, raising HMGB2 expression so as to promote rejuvenation. Furthermore, gene therapy with lentiviruses carrying SOX5 or HMGB2 rejuvenated cartilage and alleviated osteoarthritis in aged mice. Our study generated a comprehensive list of rejuvenators, pinpointing SOX5 as a potent driver for rejuvenation both in vitro and in vivo.

Computational insights into the selectivity mechanism of APP-IP over matrix metalloproteinases.

In this work, selectivity mechanism of APP-IP inhibitor (ß-amyloid precursor protein-derived inhibitory peptide) over matrix metalloproteinases (MMPs including MMP-2, MMP-7, MMP-9 and MMP-14) was investigated by molecular modeling methods. Among MMPs, MMP-2 is the most favorable one for APP-IP interacting based on our calculations. The predicted binding affinities can give a good explanation of the activity difference of inhibitor APP-IP. In Comparison with MMP-2/APP-IP complex, the side chain of Tyr214(MMP-7) makes the binding pocket so shallow that the whole side chain of Tyr3(APP-IP) can not be fully embraced, thus unfavorable for the N-terminal of APP-IP binding to MMP-7. The poor selectivity of APP-IP toward MMP-9 is mainly related with the decrease of interaction between the APP-IP C-terminal and MMP-9 due to the bulky side chains of Pro193 and Gln199, which is in agreement with experiment. The mutations at residues P193A and Q199G of MMP-9 alternate the binding pattern of the C-terminal of APP-IP by forming two new hydrogen bonds and hydrophobic interactions with MMP-9. The mutants favor the binding affinity of MMP-9 largely. For MMP-14/APP-IP, the large steric effect of Phe204(MMP-14) and the weak contributions of the polar residues Asn231(MMP-14) and Thr190(MMP-14) could explain why MMP-14 is non-selective for APP-IP interacting. Here, the molecular modeling methods were successfully employed to explore the selective inhibitor of MMPs, and our work gives valuable information for future rational design of selective peptide inhibitors toward individual MMP.

Effectiveness of Selenium on Chondrocyte Glycoprotein Glycosylation Which Play Important Roles in the Pathogenesis of an Endemic Osteoarthritis, Kashin-Beck Disease.

In this study, we aimed to explore the effectiveness of selenium on the chondrocyte glycoprotein glycosylation which plays important roles in the pathogenesis of Kashin-Beck disease (KBD). Cartilage samples were collected from KBD patients after total knee replacement surgery. Chondrocytes were cultured with sodium selenium. The group of chondrocytes which were cultured without adding sodium selenium was considered as control group. Lectin microarray was used to screen the differences in lectin levels between KBD and KBD with selenium groups. Stronger signals for Bandeiraea simplicifolia (BS-I), Hippeastrum hybrid lectin (HHL), Pisum sativum agglutinin (PSA), Psophocarpus tetragonolobus lectin I (PTL-I), Psophocarpus tetragonolobus lectin II (PTL-II), Sophora japonica agglutinin (SJA), Lotus tetragonolobus lectin (LTL), and Triticum vulgaris (WGA) were observed in the KBD group. Meanwhile, Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), Lycopersicon esculentum (tomato) lectin (LEL), Peanut agglutinin (PNA), and Sambucus nigra lectin (SNA) signals were lower in the KBD group. Selenium may have the function of influence the expression levels of carbohydrate chains Galα1,3-Gal, high mannose, and GlcNAc.

Cross-species metabolomic analysis identifies uridine as a potent regeneration promoting factor.

Regenerative capacity declines throughout evolution and with age. In this study, we asked whether metabolic programs underlying regenerative capability might be conserved across species, and if so, whether such metabolic drivers might be harnessed to promote tissue repair. To this end, we conducted metabolomic analyses in two vertebrate organ regeneration models: the axolotl limb blastema and antler stem cells. To further reveal why young individuals have higher regenerative capacity than the elderly, we also constructed metabolic profiles for primate juvenile and aged tissues, as well as young and aged human stem cells. In joint analyses, we uncovered that active pyrimidine metabolism and fatty acid metabolism correlated with higher regenerative capacity. Furthermore, we identified a set of regeneration-related metabolite effectors conserved across species. One such metabolite is uridine, a pyrimidine nucleoside, which can rejuvenate aged human stem cells and promote regeneration of various tissues in vivo. These observations will open new avenues for metabolic intervention in tissue repair and regeneration.

The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis.

Arabinogalactan proteins are widely distributed in plant tissues and cells, and may function in the growth and development of higher plants. To our knowledge, there is currently no direct evidence concerning the involvement of fasciclin-like arabinogalactan proteins (FLA) in sexual reproduction in Arabidopsis. In this study, Arabidopsis FLA3 was found to be specifically expressed in pollen grains and tubes. Subcellular localization showed that FLA3 anchors tightly to the plasma membrane, and its glycosylphosphatidylinositol anchor may affect its localization. FLA3-RNA interference transgenic plants had approximately 50% abnormal pollen grains (including shrunken and wrinkled phenotypes) which lacked viability. Cytological observations revealed that pollen abortion occurred during the transition from uninucleate microspores to bicellular pollens, with abnormal cellulose distribution seen by calcofluor white staining. Transmission electron microscopy showed that the basic structure of the exine layer in aberrant pollen was normal, but the intine layer appeared to have some abnormalities. Taken together, these results suggest that FLA3 is involved in microspore development and may affect pollen intine formation, possibly by participating in cellulose deposition. In FLA3-overexpressing transgenic plants, defective elongation of the stamen filament and reduced female fertility led to short siliques with low seed set, which suggested that ectopic expression of FLA3 in tissues may reduce or disrupt cell growth and then result in defects throughout the plant.

Structure-based design of peptides against G3BP with cytotoxicity on tumor cells.

Herein, we report a successful application of molecular modeling techniques to design two novel peptides with cytotoxicity on tumor cells. First, the interactions between the nuclear transport factor 2 (NTF2)-like domain of G3BP and the SH3 domain of RasGAP were studied by a well-designed protocol, which combines homology modeling, protein/protein docking, molecular dynamics simulations, molecular mechanics/generalized born surface area (MM/GBSA) free energy calculations, and MM/GBSA free energy decomposition analysis together. Then, based on the theoretical predictions, two novel peptides were designed and synthesized for biological assays, and they showed an obvious sensitizing effect on cis-platin. Furthermore, the designed peptides had no significant effects on normal cells, while cis-platin did. Our results demonstrate that it is feasible to use the peptides to enhance the efficacy of clinical drugs and to kill cancer cells selectively. We believe that our work should be very useful for finding new therapies for cancers.

Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction.

Cockayne syndrome (CS) is a rare autosomal recessive inherited disorder characterized by a variety of clinical features, including increased sensitivity to sunlight, progressive neurological abnormalities, and the appearance of premature aging. However, the pathogenesis of CS remains unclear due to the limitations of current disease models. Here, we generate integration-free induced pluripotent stem cells (iPSCs) from fibroblasts from a CS patient bearing mutations in CSB/ERCC6 gene and further derive isogenic gene-corrected CS-iPSCs (GC-iPSCs) using the CRISPR/Cas9 system. CS-associated phenotypic defects are recapitulated in CS-iPSC-derived mesenchymal stem cells (MSCs) and neural stem cells (NSCs), both of which display increased susceptibility to DNA damage stress. Premature aging defects in CS-MSCs are rescued by the targeted correction of mutant ERCC6. We next map the transcriptomic landscapes in CS-iPSCs and GC-iPSCs and their somatic stem cell derivatives (MSCs and NSCs) in the absence or presence of ultraviolet (UV) and replicative stresses, revealing that defects in DNA repair account for CS pathologies. Moreover, we generate autologous GC-MSCs free of pathogenic mutation under a cGMP (Current Good Manufacturing Practice)-compliant condition, which hold potential for use as improved biomaterials for future stem cell replacement therapy for CS. Collectively, our models demonstrate novel disease features and molecular mechanisms and lay a foundation for the development of novel therapeutic strategies to treat CS.

Chemical screen identifies a geroprotective role of quercetin in premature aging.

Aging increases the risk of various diseases. The main goal of aging research is to find therapies that attenuate aging and alleviate aging-related diseases. In this study, we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established. Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects. Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs. RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C. Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs. Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

[Expression of MicroRNA-15a in Children with Primary Immune Thrombocytopenia and Its Significance].

OBJECTIVE: To analyze the expression of MicroRNA-15a (miR-15a) in children with primary immune thrombocytopenia (ITP) and its significance. METHODS: The peripheral blood monomuclear cells (PBMNC) were isolated and cultured from ITP patients and healthy volunteers. The expression level of miR-15a was measured by real-time PCR. After miR-15a mimic was transfected into PBMNC, the levels of INF-γ, IL-2, IL-4 and IL-10 were measured by ELISA. RESULTS: The expression of miR-15a was significantly decreased in PBMNC. The production of IFN-γ and IL-2 was dramatically increased, and the level of IL-4, IL-10 was decreased in PBMNC. Moreover, the expression of miR-15a was negatively correlated with IFN-γ and IL-2, and positively with IL-4 and IL-10. Furthermore, the results showed that the overexpression of miR-15a could decrease the production of IFN-γ and IL-2, and increase the production of IL-4 and IL-10. CONCLUSION: miR-15a is significantly down-regulated in PBMNC of children with primary ITP and involved in the regulation of Th1/Th2 imbalance. It is suggested that miR-15a may be a potential therapeutic target for ITP.

Chiral Nanoparticle as a New Efficient Antimicrobial Nanoagent.

d-type functionalized nanoparticles (NPs) can bind to MurD ligase with high affinity and inhibit its peptidoglycan synthetic enzyme activity, and finally cause bacterial killing. In contrast, its L-type counterpart displays a negligible effect, indicating that the chiral structure of the functionalized NPs plays an essential role in their binding interaction with MurD and therefore the antibacterial activity.