SUN5 interacts with nuclear membrane LaminB1 and cytoskeletal GTPase Septin12 mediating the sperm head-and-tail junction.

Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated, and malformed sperm, resulting in male infertility. Nuclear envelope protein SUN5 localizes to the junction between the sperm head and tail. Mutations in the SUN5 gene have been identified most frequently (33-47%) in ASS cases, and its molecular mechanism of action is yet to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1 and cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 by immunoprecipitation-mass spectrometry in mouse testis. Further studies demonstrated that SUN5 connected the nucleus by interacting with LaminB1 and connected the proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted their aggregation together in the sperm neck. The disruption of the LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12-proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.

A combination of a TLR7/8 agonist and an epigenetic inhibitor suppresses triple-negative breast cancer through triggering anti-tumor immune.

BACKGROUND: Combination therapy involving immune checkpoint blockade (ICB) and other drugs is a potential strategy for converting immune-cold tumors into immune-hot tumors to benefit from immunotherapy. To achieve drug synergy, we developed a homologous cancer cell membrane vesicle (CM)-coated metal-organic framework (MOF) nanodelivery platform for the codelivery of a TLR7/8 agonist with an epigenetic inhibitor. METHODS: A novel biomimetic codelivery system (MCM@UN) was constructed by MOF nanoparticles UiO-66 loading with a bromodomain-containing protein 4 (BRD4) inhibitor and then coated with the membrane vesicles of homologous cancer cells that embedding the 18 C lipid tail of 3M-052 (M). The antitumor immune ability and tumor suppressive effect of MCM@UN were evaluated in a mouse model of triple-negative breast cancer (TNBC) and in vitro. The tumor immune microenvironment was analyzed by multicolor immunofluorescence staining. RESULTS: In vitro and in vivo data showed that MCM@UN specifically targeted to TNBC cells and was superior to the free drug in terms of tumor growth inhibition and antitumor immune activity. In terms of mechanism, MCM@UN blocked BRD4 and PD-L1 to prompt dying tumor cells to disintegrate and expose tumor antigens. The disintegrated tumor cells released damage-associated molecular patterns (DAMPs), recruited dendritic cells (DCs) to efficiently activate CD8+ T cells to mediate effective and long-lasting antitumor immunity. In addition, TLR7/8 agonist on MCM@UN enhanced lymphocytes infiltration and immunogenic cell death and decreased regulatory T-cells (Tregs). On clinical specimens, we found that mature DCs infiltrating tumor tissues of TNBC patients were negatively correlated with the expression of BRD4, which was consistent with the result in animal model. CONCLUSION: MCM@UN specifically targeted to TNBC cells and remodeled tumor immune microenvironment to inhibit malignant behaviors of TNBC.

Combination therapy based on dual-target biomimetic nano-delivery system for overcoming cisplatin resistance in hepatocellular carcinoma.

Strategies to overcome toxicity and drug resistance caused by chemotherapeutic drugs for targeted therapy against hepatocellular carcinoma (HCC) are urgently needed. Previous studies revealed that high oxidored-nitro domain-containing protein 1(NOR1) expression in HCC was associated with cisplatin (DDP) resistance. Herein, a novel dual-targeting nanocarrier system AR-NADR was generated for the treatment of DDP resistance in HCC. The core of the nanocarrier system is the metal-organic frameworks (MOF) modified with nuclear location sequence (NLS), which loading with DDP and NOR1 shRNA (R). The shell is an A54 peptide inserted into the erythrocyte membrane (AR). Our results show that AR-NADR efficiently internalized by tumor cells due to its specific binding to the A54 receptors that are abundantly expressed on the surface of HCC cells and NLS peptide-mediated nuclear entry. Additionally, DDP is more likely to be released due to the degradation of Ag-MOF in the acidic tumor microenvironment. Moreover, by acting as a vector for gene delivery, AR-NADR effectively inhibits tumor drug resistance by suppressing the expression of NOR1, which induces intracellular DDP accumulation and makes cells sensitive to DDP. Finally, the anti-HCC efficacy and mechanisms of AR-NADR were systematically elucidated by a HepG2/DDP cell model as well as a tumor model. Therefore, AR-NADR constitutes a key strategy to achieve excellent gene silencing and antitumor efficacy, which provides effective gene therapy and precise treatment strategies for cisplatin resistance in HCC.

Bioengineered stem cell membrane functionalized nanoparticles combine anti-inflammatory and antimicrobial properties for sepsis treatment.

BACKGROUND: Sepsis is a syndrome of physiological, pathological and biochemical abnormalities caused by infection. Although the mortality rate is lower than before, many survivors have persistent infection, which means sepsis calls for new treatment. After infection, inflammatory mediators were largely released into the blood, leading to multiple organ dysfunction. Therefore, anti-infection and anti-inflammation are critical issues in sepsis management. RESULTS: Here, we successfully constructed a novel nanometer drug loading system for sepsis management, FZ/MER-AgMOF@Bm. The nanoparticles were modified with LPS-treated bone marrow mesenchymal stem cell (BMSC) membrane, and silver metal organic framework (AgMOF) was used as the nanocore for loading FPS-ZM1 and meropenem which was delivery to the infectious microenvironments (IMEs) to exert dual anti-inflammatory and antibacterial effects. FZ/MER-AgMOF@Bm effectively alleviated excessive inflammatory response and eliminated bacteria. FZ/MER-AgMOF@Bm also played an anti-inflammatory role by promoting the polarization of macrophages to M2. When sepsis induced by cecal ligation and puncture (CLP) challenged mice was treated, FZ/MER-AgMOF@Bm could not only reduce the levels of pro-inflammatory factors and lung injury, but also help to improve hypothermia caused by septic shock and prolong survival time. CONCLUSIONS: Together, the nanoparticles played a role in combined anti-inflammatory and antimicrobial properties, alleviating cytokine storm and protecting vital organ functions, could be a potential new strategy for sepsis management.

T and B cell Epitope analysis of SARS-CoV-2 S protein based on immunoinformatics and experimental research.

COVID-19 caused by SARS-CoV-2 is pandemic with a severe morbidity and mortality rate across the world. Despite the race for effective vaccine and drug against further expansion and fatality rate of this novel coronavirus, there is still lack of effective antiviral therapy. To this effect, we deemed it necessary to identify potential B and T cell epitopes from the envelope S protein. This can be used as potential targets to develop anti-SARS-CoV-2 vaccine preparations. In this study, we used immunoinformatics to identify conservative B and T cell epitopes for S proteins of SARS-CoV-2, which might play roles in the initiation of SARS-CoV-2 infection. We identified the B cell and T cell peptide epitopes of S protein and their antigenicity, as well as the interaction between the peptide epitopes and human leucocyte antigen (HLA). Among the B cell epitopes, 'EILDITPCSFGGVS' has the highest score of antigenicity and great immunogenicity. In T cell epitopes, MHC-I peptide 'KIADYNYKL' and MHC-II peptide 'LEILDITPC' were identified as high antigens. Besides, docking analysis showed that the predicted peptide 'KIADYNYKL' was closely bound to the HLA-A*0201. The results of molecular dynamics simulation through GROMACS software showed that 'HLA-A*0201~peptide' complex was very stable. And the peptide we selected could induce the T cell response similar to that of SARS-CoV-2 infection. Moreover, the predicted peptides were highly conserved in different isolates from different countries. The antigenic epitopes presumed in this study were effective new vaccine targets to prevent SARS-CoV-2 infection.

Platelet membrane-camouflaged silver metal-organic framework drug system against infections caused by methicillin-resistant Staphylococcus aureus.

BACKGROUND: Due to the intelligent survival strategy and self-preservation of methicillin-resistant Staphylococcus aureus (MRSA), many antibiotics are ineffective in treating MRSA infections. Nano-drug delivery systems have emerged as a new method to overcome this barrier. The aim of this study was to construct a novel nano-drug delivery system for the treatment of MRSA infection, and to evaluate the therapeutic effect and biotoxicity of this system. We prepared a nano silver metal-organic framework using 2-methylimidazole as ligand and silver nitrate as ion provider. Vancomycin (Vanc) was loaded with Ag-MOF, and nano-sized platelet vesicles were prepared to encapsulate Ag-MOF-Vanc, thus forming the novel platelet membrane-camouflaged nanoparticles PLT@Ag-MOF-Vanc. RESULTS: The synthesized Ag-MOF particles had uniform size and shape of radiating corona. The mean nanoparticle size and zeta potential of PLT@Ag-MOF-Vanc were 148 nm and - 25.6 mV, respectively. The encapsulation efficiency (EE) and loading efficiency (LE) of vancomycin were 81.0 and 64.7 %, respectively. PLT@Ag-MOF-Vanc was shown to be a pH-responsive nano-drug delivery system with good biocompatibility. Ag-MOF had a good inhibitory effect on the growth of three common clinical strains (Escherichia coli, Pseudomonas aeruginosa, and S. aureus). PLT@Ag-MOF-Vanc showed better antibacterial activity against common clinical strains in vitro than free vancomycin. PLT@Ag-MOF-Vanc killed MRSA through multiple approaches, including interfering with the metabolism of bacteria, catalyzing reactive oxygen species production, destroying the integrity of cell membrane, and inhibiting biofilm formation. Due to the encapsulation of the platelet membrane, PLT@Ag-MOF-Vanc can bind to the surface of the MRSA bacteria and the sites of MRSA infection. PLT@Ag-MOF-Vanc had a good anti-infective effect in mouse MRSA pneumonia model, which was significantly superior to free vancomycin, and has no obvious toxicity. CONCLUSIONS: PLT@Ag-MOF-Vanc is a novel effective targeted drug delivery system, which is expected to be used safely in anti-infective therapy of MRSA.

A fluorometric aptamer nanoprobe for alpha-fetoprotein by exploiting the FRET between 5-carboxyfluorescein and palladium nanoparticles.

Alpha-fetoprotein (AFP) is a reliable clinical marker of hepatocellular carcinoma (HCC). A highly sensitive fluorometric aptamer nanoprobe is described for AFP detection. It is based on fluorescence resonance energy transfer (FRET) between AFP aptamer labelled with 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs). The PdNPs quench the green fluorescence of the FAM-AFP aptamer via interactions between nitrogen functional groups of the AFP aptamer and PdNPs. When AFP was introduced into the FAM-AFP aptamer-PdNPs FRET system, the AFP aptamer preferentially combines with AFP. This results in a conformational change and weakens the interaction between the aptamer and the PdNPs. Thus, the fluorescence of FAM recovers. The fluorescence recovery of FAM increases linearly in the 5.0-150 ng·mL-1 AFP concentration range and has a 1.4 ng·mL-1 detection limit. The assay was applied to the analysis of spiked diluted human serum. The recovery values ranged from 98.3 to 112.9%, with relative standard deviations of <1.1%. This biosensing strategy provides a reliable and ultrasensitive protocol for the quantification of biomarkers with relevant antigens and aptamers. Graphical abstract Schematic presentation of a fluorometric aptamer nanoprobe for AFP assay based on fluorescence resonance energy transfer (FRET) between AFP aptamer labelled with 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs).

Meropenem-induced immune thrombocytopenia and the diagnostic process of laboratory testing.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) is a serious, life-threatening clinical syndrome, the diagnosis of which is consistently difficult. In this report, we present a case of DITP caused by meropenem that was confirmed by laboratory tests. CASE REPORT: A 59-year-old male patient developed severe thrombocytopenia 8 days after the administration of meropenem and cefoperazone-sulbactam. After other causes were ruled out, DITP was suspected. Drug-induced platelet (PLT) antibodies were detected by enzyme immunoassay, flow cytometry, and monoclonal antibody immobilization of PLT antigens (MAIPA). All these tests were performed in the presence and absence of the associated drugs. RESULTS: PLT antibodies were detected in the patient's serum only in the presence of meropenem. MAIPA experiments demonstrated that glycoprotein IIb/IIIa was the binding site of the meropenem-induced PLT antibodies. CONCLUSIONS: Drug-induced immune thrombocytopenia should be considered in cases of acute thrombocytopenia in patients undergoing meropenem treatment. Clinicians should be cognizant of DITP, and a definitive diagnosis should be pursued, if feasible.

A turn-on fluorescent strategy for alkaline phosphatase detection based on enzyme-assisted signal amplification.

As a representative biochemical indicator, alkaline phosphatase (ALP) is of great importance in indicating and diagnosing clinical diseases. Herein, we developed a signal-on fluorescence sensing method for sensitive ALP activity detection based on the enzyme-assisted target recycling (EATR) technique. In this method, a two-step signal amplification process is designed. In the presence of ALP, the 3' phosphate group of an ss-DNA is removed explicitly by ALP, thus releasing free 3'-OH. Terminal deoxynucleotidyl transferase (TdT) can subsequently extend this substrate to generate poly(A) tails, converting the trace-level ALP information into multiple sequences and achieving the first-time amplification. A poly(T) Taqman probe labeled with FAM and BHQ1 provides the second one under the assistance of T7 exonuclease (T7 Exo) through alternate hybridization and degradation of ds-DNA regions. The previously quenched fluorescence is recovered due to the departure of FAM/BHQ1 during the cleavage of T7 Exo. Thus, taking advantage of template-free TdT-mediated polymerization and T7 Exo-based EATR, this strategy shows a sensitive LOD at 0.0074 U/L (S/N = 3) and a linear range of 0.01-8 U/L between ALP concentration and fluorescence intensity. To further verify the specificity and accuracy in practical application, we challenged it in a set of co-existing interference and biological environments and have gained satisfying results. The proposed method successfully quantified the ALP levels in clinical human serum samples, suggesting its applicability in practical application. Moreover, we have used this method to investigate the inhibition effects of Na3VO4. Above all, the proposed assay is sensitive, facile, and cost-effective for ALP determining, holding a promising perspective and excellent potential in clinical diagnosis and drug screening.

Erythrocyte membrane-camouflaged DNA-functionalized upconversion nanoparticles for tumor-targeted chemotherapy and immunotherapy.

A synergistic combination of treatment with immunogenic cell death (ICD) inducers and immunoadjuvants may be a practical way to boost the anticancer response and successfully induce an immune response. The use of HR@UCNPs/CpG-Apt/DOX, new biomimetic drug delivery nanoparticles generated to combat breast cancer, is reported here as a unique strategy to produce immunogenicity and boost cancer immunotherapy. HR@UCNPs/CpG-Apt/DOX (HR-UCAD) consists of two parts. The core is composed of an immunoadjuvant CpG (a toll-like receptor 9 agonist) fused with a dendritic cell-specific aptamer sequence (CpG-Apt) to decorate upconversion nanoparticles (UCNPs) with the successful intercalation of doxorubicin (DOX) into the consecutive base pairs of Apt-CpG to construct an immune nanodrug UCNPs@CpG-Apt/DOX. The targeting molecule hyaluronic acid (HA) was inserted into a red blood cell membrane (RBCm) to form the shell (HR). HR-UCAD possessed a strong capacity to specifically induce ICD. Following DOX-induced ICD of cancer cells, sufficient exposure to tumor antigens and UCNPs@CpG-Apt (UCA) activated the tumor-specific immune response and reversed the immunosuppressive tumor microenvironment. In addition, HR-UCAD has good biocompatibility and increases the active tumor-targeting effect. Furthermore, HR-UCAD exhibits excellent near-infrared upconversion luminescence emission at 804 nm under irradiation with a 980 nm laser, which has great potential in biomedical imaging. Thus, the RBCm-camouflaged drug delivery system is a promising targeted chemotherapy and immunotherapy nanocomplex that could be used for effective targeted breast cancer treatment.

Study on the Role of an Erythrocyte Membrane-Coated Nanotheranostic System in Targeted Immune Regulation of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the elderly population. Despite significant advances in studies of the pathobiology on AD, there is still no effective treatment. Here, an erythrocyte membrane-camouflaged nanodrug delivery system (TR-ZRA) modified with transferrin receptor aptamers that can be targeted across the blood-brain barrier to ameliorate AD immune environment is established. Based on metal-organic framework (Zn-CA), TR-ZRA is loaded with CD22shRNA plasmid to silence the abnormally high expression molecule CD22 in aging microglia. Most importantly, TR-ZRA can enhance the ability of microglia to phagocytose Aß and alleviate complement activation, which can promote neuronal activity and decrease inflammation level in the AD brain. Moreover, TR-ZRA is also loaded with Aß aptamers, which allow rapid and low-cost monitoring of Aß plaques in vitro. After treatment with TR-ZRA, learning, and memory abilities are enhanced in AD mice. In conclusion, the biomimetic delivery nanosystem TR-ZRA in this study provides a promising strategy and novel immune targets for AD therapy.

Multifunctional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-Based Nanobomb against Carbapenem-Resistant Acinetobacter baumannii Infection through Cascade Reaction and Amplification Synergistic Effect.

Carbapenems have been considered to be the preferred antibiotics against Acinetobacter baumannii thus far. However, carbapenem-resistant Acinetobacter baumannii (CRAB) has gradually escalated worldwide, and it frequently causes respiratory and bloodstream infections. Its resistance may lead to high mortality. Thus, there is an urgent need to develop antibacterial drugs. In our research, the pH-sensitive sgRNA-I/L@ZS nanosystem delivered imipenem and better released it in infected tissues to synergistically damage bacteria with nanoparticles. Gene editing of the CRISPR-Cas9 nanosystem amplified the synergistic effect by reversing the drug-resistance of imipenem. Nitric oxide, which l-arginine reacted with ROS to produce in cascade reaction and bacterial infection sites, was beneficial to heal the infected tissues and induce bacteria death for further enhancing antibacterial effects. In addition, this nanocomposite influenced host-bacteria interactions and restrained and destroyed biofilms. The sgRNA-I/L@ZS nanosystem, similar to a nanobomb, was a high-efficiency bactericide against CRAB. Eventually, in acute pneumonia and peritonitis mouse models, the sgRNA-I/L@ZS nanosystem could combat bacteria and protect tissues from infection. It had marked suppressive effects on inflammation and promoted healing and proliferation of infected tissues. This multifunctional nanosystem is expected to be an effective antibacterial agent in the clinic based on good biocompatibility and no toxic side effects. Therefore, developing the nanocomposites will take a favorable step toward solving intractable public health issues.

[Analysis of the relationship between the MecA gene and resistance of ß-lactam antibiotics].

OBJECTIVE: To investigate the mechanisms by which MecA gene expression leads to ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), and to study the resistance mechanism of MRSA at the molecular level. METHODS: A variety of molecular biological techniques were employed, including screening MRSA using cefoxitin paper disk method, extraction of MRSA mRNA, reverse transcription into cDNA, real-time fluorescence PCR for quantitation of MecA gene expression, and agar dilution method for assessment of minimum inhibitory concentrations in MRSA treated with cefoxitin, oxacillin, vancomycin, or linezolid. RESULTS: According to the level of resistance of MRSA to cefoxitin, 40 MRSA strains were divided into a low resistance group (n=12), a middle resistance group (n=15), and a high resistance group (n=13). The expression level of the MecA gene in the low resistance group, the middle resistance group, and the high resistance group was 58.87±30.30, 363.37±200.05, and 1257.72±446.63, respectively. MRSA resistance to cefoxitin and oxacillin was 100%; MRSA resistance to vancomycin or linezolid could not be detected. For all 40 MRSA strains the MIC90 for vancomycin was 2.0 µg/mL. CONCLUSION: MecA gene expression levels may correlate with the MRSA level of resistance to cefoxitin within a certain range of concentration.

CircRNA-011235 Counteracts The Deleterious Effect of Irradiation Treatment on Bone Mesenchymal Stem Cells by Regulating The miR-741-3p/CDK6 Pathway.

OBJECTIVE: The present work was aimed at uncovering the effect of circRNA-011235 (circ-011235) on irradiation-induced bone mesenchymal stem cells (BMSCs) injury and its regulatory mechanism, with a view to establish a scientific basis for its possible medical applications. MATERIALS AND METHODS: In this experimental study, after irradiation with different doses (0, 2, 4, 6 GY), the relative expression levels of circ-011235, miR-741-3p, and cyclin-dependent kinases 6 (CDK6) were detected in the BMSCs, using the real time-quantitative polymerase chain reaction (RT-qPCR). The overexpression effects of circ-011235 and CDK6 on the cell proliferation in irradiation-treated BMSCs were measured by the Cell Counting Kit-8 (CCK8) assay. And also, their effects on the cell cycle were evaluated by flow cytometry. RT-qPCR and immunoblotting were performed to detect the effects of pcDNA-circ-011235 and pcDNA-CDK6 on the expression of cyclin D1 and cyclindependent kinases 4 (CDK6) at the gene and protein levels, respectively. RESULTS: Irradiation treatment elevated the expression of circ-011235 and CDK6, but reduced miR-741-3p expression in the BMSCs with a dose-dependent effect. The proliferation of BMSCs was significantly inhibited in the irradiation treatment group, while the overexpression of circ-011235 and CDK6 effectively attenuated this inhibition. Also, overexpression of circ-011235 and CDK6 elevated the expression of cyclin D1 in irradiation-treated BMSCs, but had no significant effect on the CDK4 expression. CONCLUSION: Our results demonstrated that circ-011235 up-regulated the expression of cyclin D1 via miR-741-3p/ CDK6 signal pathway, thereby promoting cell cycle progression and proliferation of irradiation-treated BMSCs. This finding suggested circ-011235/ miR-741-3p/CDK6 pathway exerted a protective role in the response to irradiation and will be a potential new target for future research on the mechanism involved in the resistance of BMSCs to radiation.

SUN5 Interacting With Nesprin3 Plays an Essential Role in Sperm Head-to-Tail Linkage: Research on Sun5 Gene Knockout Mice.

Acephalic spermatozoa syndrome is a rare genetic and reproductive disease. Recent studies have shown that approximately 33-47% of patients with acephalic spermatozoa syndrome have SUN5 mutations, but the molecular mechanism underlying this phenomenon has not been elucidated. In this study, we generated Sun5 knockout mice and found that the head-to-tail linkage was broken in Sun5-/- mice, which was similar to human acephalic spermatozoa syndrome. Furthermore, ultrastructural imaging revealed that the head-tail coupling apparatus (HTCA) and the centrosome were distant from the nucleus at steps 9-10 during spermatid elongation. With the manchette disappearing at steps 13-14, the head and the tail segregated. To explore the molecular mechanism underlying this process, bioinformatic analysis was performed and showed that Sun5 may interact with Nesprin3. Further coimmunoprecipitation (Co-IP) and immunofluorescence assays confirmed that Sun5 and Nesprin3 were indeed bona fide interaction partners that formed the linker of the nucleoskeleton and cytoskeleton (LINC) complex participating in the connection of the head and tail of spermatozoa. Nesprin3 was located posterior and anterior to the nucleus during spermiogenesis in wild-type mice, whereas it lost its localization at the implantation fossa of the posterior region in Sun5-/- mice. Without correct localization of Nesprin3 at the nuclear membrane, the centrosome, which is the originator of the flagellum, was distant from the nucleus, which led to the separation of the head and tail. In addition, isobaric tag for relative and absolute quantitation results showed that 47 proteins were upregulated, and 56 proteins were downregulated, in the testis in Sun5-/- mice, and the downregulation of spermatogenesis-related proteins (Odf1 and Odf2) may also contribute to the damage to the spermatozoa head-to-tail linkage. Our findings suggested that Sun5 is essential for the localization of Nesprin3 at the posterior nuclear membrane, which plays an essential role in the sperm head-tail connection.

CircRNA-016901 silencing attenuates irradiation-induced injury in bone mesenchymal stem cells via regulating the miR-1249-5p/HIPK2 axis.

Currently, bone marrow transplantation remains the basic treatment for various hematological tumors and irradiation is one of the most important pretreatment methods. However, irradiation pretreatment may result in damage to bone mesenchymal stem cells (BMSCs). The present study aimed to investigate the effect of circular RNA-016901 (circ-016901) on the injury of irradiation-induced BMSCs and the underlying mechanism. The expression levels of circ-016901, microRNA-1249-5p (miR-1249-5p) and homeodomain interacting protein kinase 2 (HIPK2) in irradiation-induced mouse BMSCs at various irradiation doses were detected via reverse transcription-quantitative PCR (RT-qPCR). The effect of circ-016901 on cell proliferation was examined using Cell Counting Kit-8 assays following silencing or overexpression of circ-016901. Cell apoptosis was detected by flow cytometry and caspase-3/7 activity. The expression of autophagy-related markers, including Beclin-1 and LC3-II/I, was detected at the mRNA and protein levels by RT-qPCR and western blotting, respectively. Irradiation treatment upregulated the expression of circ-016901 and HIPK2 and downregulated miR-1249-5p expression. The expression levels of LC3-II/I and Beclin-1 in BMSCs were downregulated in a dose-dependent manner. Silencing of circ-016901 promoted proliferation of irradiation-induced BMSCs and attenuated irradiation-induced apoptosis. Moreover, silencing of circ-016901 elevated the expressions of LC3-II/I and Beclin-1 in irradiation-induced BMSCs. Similar results were obtained with miR-1249-5p overexpression and HIPK2 silencing. These results demonstrated that circ-016901 silencing attenuated injury in irradiation-induced mouse BMSCs by regulating the miR-1249-5p/HIPK2 axis, providing a novel target for future research on the mechanism of radiation resistance in BMSCs.

Zirconia Nanoparticles Induce HeLa Cell Death Through Mitochondrial Apoptosis and Autophagy Pathways Mediated by ROS.

Zirconia nanoparticles (ZrO2 NPs) are commonly used in the field of biomedical materials, but their antitumor activity and mechanism is unclear. Herein, we evaluated the anti-tumor activity of ZrO2 NPs and explored the anti-tumor mechanism. The results of in vitro and in vivo experiments showed that the level of intracellular reactive oxygen species (ROS) in HeLa cells was elevated after ZrO2 NPs treatment. Transmission electron microscopy (TEM) showed that after treatment with ZrO2 NPs, the mitochondria of HeLa cells were swollen, accompanied with the induction of autophagic vacuoles. In addition, flow cytometry analysis showed that the apoptotic rate of HeLa cells increased significantly by Annexin staining after treatment with ZrO2 NPs, and the mitochondrial membrane potential (MMP) was reduced significantly. The proliferation of HeLa cells decreased as indicated by reduced Ki-67 labeling. In contrast, TUNEL-positive cells in tumor tissues increased after treatment with ZrO2 NPs, which is accompanied by increased expression of mitochondrial apoptotic proteins including Bax, Caspase-3, Caspase-9, and Cytochrome C (Cyt C) and increased expression of autophagy-related proteins including Atg5, Atg12, Beclin-1, and LC3-II. Treating HeLa cells with N-acetyl-L-cysteine (NAC) significantly reduced ROS, rate of apoptosis, MMP, and in vivo anti-tumor activity. In addition, apoptosis- and autophagy-related protein expressions were also suppressed. Based on these observations, we conclude that ZrO2 NPs induce HeLa cell death through ROS mediated mitochondrial apoptosis and autophagy.

Novel Multifunctional Silver Nanocomposite Serves as a Resistance-Reversal Agent to Synergistically Combat Carbapenem-Resistant Acinetobacter baumannii.

In the face of the abundant production of various types of carbapenemases, the antibacterial efficiency of imipenem, seen as "the last line of defense", is weakening. Following, the incidence of carbapenem-resistant Acinetobacter baumannii (CRAB), which can generate antibiotic-resistant biofilms, is increasing. Based on the superior antimicrobial activity of silver nanoparticles against multifarious bacterial strains compared with common antibiotics, we constructed the IPM@AgNPs-PEG-NOTA nanocomposite (silver nanoparticles were coated with SH-PEG-NOTA as well as loaded by imipenem) whose core was a silver nanoparticle to address the current challenge, and IPM@AgNPs-PEG-NOTA was able to function as a novel smart pH-sensitive nanodrug system. Synergistic bactericidal effects of silver nanoparticles and imipenem as well as drug-resistance reversal via protection of the ß-ring of carbapenem due to AgNPs-PEG-NOTA were observed; thus, this nanocomposite confers multiple advantages for efficient antibacterial activity. Additionally, IPM@AgNPs-PEG-NOTA not only offers immune regulation and accelerates tissue repair to improve therapeutic efficacy in vivo but also can prevent the interaction of pathogens and hosts. Compared with free imipenem or silver nanoparticles, this platform significantly enhanced antibacterial efficiency while increasing reactive oxygen species (ROS) production and membrane damage, as well as affecting cell wall formation and metabolic pathways. According to the results of crystal violet staining, LIVE/DEAD backlight bacterial viability staining, and real-time quantitative polymerase chain reaction (RT-qPCR), this silver nanocomposite downregulated the levels of ompA expression to prevent formation of biofilms. In summary, this research demonstrated that the IPM@AgNPs-PEG-NOTA nanocomposite is a promising antibacterial agent of security, pH sensitivity, and high efficiency in reversing resistance and synergistically combatting carbapenem-resistant A. baumannii. In the future, various embellishments and selected loads for silver nanoparticles will be the focus of research in the domains of medicine and nanotechnology.

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus - Worldwide, 2021.

Background: COVID-19 infection is a major public health problem worldwide, and the D614G mutation enhances the infectivity of COVID-19.Methods: A probe-directed recombinase amplification (PDRA) assay was discussed to detect the D614G mutation at 39 ℃ for 30 min. The sensitivity, specificity, and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids. The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples. Direct sequencing was carried out in parallel for comparison.Result: With good reproducibility and specificity, the PDRA assay worked well with the concentration in the range of 103-107 copies/reaction. Compared with direct sequencing as a reference, the recombinase-aided amplification (RAA) assay obtained 100% sensitivity and 100% specificity using clinical samples.Conclusions: A rapid, convenient, sensitive, and specific method to detect D614G mutation was developed, which offers a useful tool to monitor mutations in COVID-19 virus RNA.