Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson's disease.

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

Nanopore Signatures of Nucleoside Drugs.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

3D-printed LEGO®-inspired titanium scaffolds for patient-specific regenerative medicine.

Despite the recent advances in 3D-printing, it is often difficult to fabricate implants that optimally fit a defect size or shape. There are some approaches to resolve this issue, such as patient-specific implant/scaffold designs based on CT images of the patients, however, this process is labor-intensive and costly. Especially in developing countries, affordable treatment options are required, while still not excluding these patient groups from potential material and manufacturing advances. Here, a selective laser melting (SLM) 3D-printing strategy was used to fabricate a hierarchical, LEGO®-inspired Assemblable Titanium Scaffold (ATS) system, which can be manually assembled in any shape or size with ease. A surgeon can quickly create a scaffold that would fit to the defect right before the implantation during the surgery. Additionally, the direct inclusion of micro- and macroporous structures via 3D-printing, as well as a double acid-etched surface treatment (ST) in the ATS, ensure biocompatibility, sufficient nutrient flow, cell migration and enhanced osteogenesis. Three different structures were designed (non-porous:NP, semi-porous:SP, ultra-porous:UP), 3D-printed with the SLM technique and then surface treated for the ST groups. After analyzing characteristics of the ATS such as printing quality, surface roughness and interconnected porosity, mechanical testing and finite element analysis (FEA) demonstrated that individual and stacked ATS have sufficient mechanical properties to withstand loading in a physiological system. All ATS showed high cell viability, and the SP and UP groups demonstrated enhanced cell proliferation rates compared to the NP group. Furthermore, we also verified that cells were well-attached and spread on the porous structures and successful cell migration between the ATS units was seen in the case of assemblies. The UP and SP groups exhibited higher calcium deposition and RT-qPCR proved higher osteogenic gene expression compared to NP group. Finally, we demonstrate a number of possible medical applications that reveal the potential of the ATS through assembly.

A 3D-Printed Assemblable Bespoke Scaffold as a Versatile Microcryogel Carrier for Site-Specific Regenerative Medicine.

Advances in additive manufacturing have led to diverse patient-specific implant designs utilizing computed tomography, but this requires intensive work and financial implications. Here, Digital Light Processing is used to fabricate a hive-structured assemblable bespoke scaffold (HIVE). HIVE can be manually assembled in any shape/size with ease, so a surgeon can create a scaffold that will best fit a defect before implantation. Simultaneously, it can have site-specific treatments by working as a carrier filled with microcryogels (MC) incorporating different biological factors in different pockets of HIVE. After characterization, possible site-specific applications are investigated by utilizing HIVE as a versatile carrier with incorporated treatments such as growth factors (GF), bioceramic, or cells. HIVE as a GF-carrier shows a controlled release of bone morphogenetic protein/vascular endothelial growth factor (BMP/VEGF) and induced osteogenesis/angiogenesis from human mesenchymal stem cells (hMSC)/human umbilical vein endothelial cells (HUVECs). Furthermore, as a bioceramic-carrier, HIVE demonstrates enhanced mineralization and osteogenesis, and as a HUVEC carrier, it upregulates both osteogenic and angiogenic gene expression of hMSCs. HIVE with different combinations of MCs yields a distinct local effect and successful cell migration is confirmed within assembled HIVEs. Finally, an in vivo rat subcutaneous implantation demonstrates site-specific osteogenesis and angiogenesis.

Single-Molecule Interconversion between Chiral Configurations of Boronate Esters Observed in a Nanoreactor.

Isomers of some chemical compounds may be dynamically interconvertible. Due to a lack of sensing methods with a sufficient resolution, however, direct monitoring of such processes can be difficult. Engineered Mycobacterium smegmatis porin A (MspA) nanopores can be applied as nanoreactors so that chemical reactions can be directly monitored. Here, an MspA modified with a phenylboronic acid (PBA) adapter was prepared and was used to observe dynamic interconversion between chiral configurations of boronate esters, which appears as telegraphic switching on top of nanopore events. The mechanism of this behavior was further confirmed by trials with different halogenated catechols, dopamine, adenosine, 1,2-propanediol, and (2R,3R)-2,3-butanediol, and its generality has been demonstrated. These results suggest that an engineered MspA possesses an exceptional resolution in its monitoring of chemical reaction processes and may inspire the future design of nanopore small-molecule sensors.

Discrimination of Ribonucleoside Mono-, Di-, and Triphosphates Using an Engineered Nanopore.

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.

Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore.

A large collection of unique molecular barcodes is useful in the simultaneous sensing or screening of molecular analytes. Though the sequence of DNA has been widely applied to encode for molecular barcodes, decoding of these barcodes is normally assisted by sequencing. We here demonstrate a barcode system based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. Each molecular barcode is composed of "n" distinct information nodes in a non-binary manner and can be sequentially scanned and decoded by a Mycobacterium smegmatis porin A (MspA) nanopore. Nanopore events containing step-shaped features were consistently reported. 14 unique information nodes were developed which in principle could encode for 14n unique molecular barcodes in a barcode containing "n" information nodes. These barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications.

Thermo/glutathione-sensitive release kinetics of heterogeneous magnetic micro-organogel prepared by sono-catalysis.

To improve the loading and delivery for hydrophobic drugs and optimize the release efficiency in tumor microenvironment, a novel core-shell magnetic micro-organogel carrier was successfully prepared by a sono-catalysis process in the study. As-synthesized magnetic micro-organogel had an appropriate dispersibility in water owing to the hydrophilicity of protein shell and could be kept steadily with a well-defined spherical morphology owing to the three-dimensional gel structure of oil core, and it promised an accessible targeted drug delivery owing to its good magnetism-mediated motion ability. Moreover, the magnetic micro-organogel showed a high loading efficiency up to 94.22% for coumarin 6 which was dissolved into the micro-organogel as a model hydrophobic drug. More importantly, the release kinetics revealed that the magnetic micro-organogel had a thermo-sensitive and glutathione (GSH)-sensitive ability to control the drug release, and proved that its release mechanisms referred to the combination of erosion, diffusion and degradation.

Nanopore Sequencing Accurately Identifies the Cisplatin Adduct on DNA.

Cisplatin, which selectively binds to N7 atoms of purines to inhibit normal replication and transcription, is a widely applied chemotherapeutic drug in the treatment of cancer. Though direct identification of cisplatin lesions on DNA is of great significance, existing sequencing methods have issues such as complications of preamplification or enrichment-induced false-positive reports. Direct identification of cisplatin lesions by nanopore sequencing (NPS) is in principle feasible. However, relevant investigations have never been reported. By constructing model sequences (83 nucleotides in length) containing a sole cisplatin lesion, identification of corresponding lesions by NPS is achieved with <10 ng of input sequencing library. Moreover, characteristic high-frequency noises caused by cisplatin lesions are consistently observed during NPS, clearly identifiable in corresponding high-pass filtered traces. This feature is, however, never observed in any other combinations of natural DNA bases and could be taken as a reference to identify cisplatin lesions on DNA. Further investigations demonstrate that cisplatin stalls the replication of phi29 DNA polymerase, which appears as a ∼5 pA level fluctuation in the single-molecule resolution. These results have confirmed the feasibility of NPS to identify cisplatin lesions at the genomic level and may provide new insights into understanding the molecular mechanism of platinum-based drugs.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors.

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

NPRL2 reduces the niraparib sensitivity of castration-resistant prostate cancer via interacting with UBE2M and enhancing neddylation.

In this study, we explored the regulatory effects of nitrogen permease regulator 2-like (NPRL2) on niraparib sensitivity, a PARP inhibitor (PARPi) in castrate-resistant prostate cancer (CRPC). Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program were retrospectively examined. Gene-set enrichment analysis (GSEA) was conducted between high and low NRPL2 expression prostate adenocarcinoma (PRAD) cases in TCGA. CCK-8 assay, Western blot analysis of apoptotic proteins, and flow cytometric analysis of apoptosis were applied to test niraparib sensitivity. Immunofluorescent (IF) staining and co-immunoprecipitation (co-IP) were conducted to explore the proteins interacting with NPRL2. Results showed that the upregulation of a canonical protein-coding transcript of NPRL2 (ENST00000232501.7) is associated with an unfavorable prognosis. Bioinformatic analysis predicts a physical interaction between NPRL2 and UBE2M, which is validated by a following Co-IP assay. This interaction increases NPRL2 stability by reducing polyubiquitination and proteasomal degradation. Depletion of NPRL2 or UBE2M significantly increases the niraparib sensitivity of CRPC cells and enhances niraparib-induced tumor growth inhibition in vivo. NPRL2 cooperatively enhances UBE2M-mediated neddylation and facilitates the degradation of multiple substrates of Cullin-RING E3 ubiquitin ligases (CRLs). In conclusion, this study identified a novel NPRL2-UBE2M complex in modulating neddylation and niraparib sensitivity of CRPC cells. Therefore, targeting NPRL2 might be considered as an adjuvant strategy for PARPi therapy.

Tb3+-Doped Ag-MOFs for fluorescent detection of formaldehyde in a novel smartphone platform and its removal applications in milk products and wastewater.

As one kind of reactive carbonyl species (RCS), formaldehyde (FA) with a high concentration could be extremely toxic to living bodies as well as the environment. This paper reports a three-dimensional (3D) Tb3+@Ag-MOFs-based fluorescent probe for fast sensing of FA, which uses a novel turn-on mechanism based on the luminescence induced by Tb3+. The MOF sensor shows broad dynamic ranges of 0.1-1 mM for FA with the detection limit of 1.9 µM. For online and real-time detection of FA, a portable smartphone platform was employed to analyze the RGB values of the fluorescence by a smartphone application. By incorporating this probe into a polyacrylonitrile (PAN) layer, we synthesized a film composite that could effectively remove FA in real samples including milk and chemical factory wastewater, and the removal rate reached 98.52% and 95.38% respectively. Moreover, the potential of the film to remove gaseous FA was confirmed by experiments as well.

Sestrin 2, a potential star of antioxidant stress in cardiovascular diseases.

Physiological reactive oxygen species (ROS) play an important role in cellular signal transduction. However, excessive ROS is an important pathological mechanism in most cardiovascular diseases (CVDs), such as myocardial aging, cardiomyopathy, ischemia/reperfusion injury (e.g., myocardial infarction) and heart failure. Programmed cell death, hypertrophy and fibrosis may be due to oxidative stress. Sestrin 2 (Sesn2), a stress-inducible protein associated with various stress conditions, is a potential antioxidant. Sesn2 can suppress the process of heart damage caused by oxidative stress, promote cell survival and play a key role in a variety of CVDs. This review discusses the effect of Sesn2 on the redox signal, mainly via participation in the signaling pathway of nuclear factor erythroid 2-related factor 2, activation of adenosine monophosphate-activated protein kinase and inhibition of mammalian target of rapamycin complex 1. It also discusses the effect of Sesn2's antioxidant activity on different CVDs. We speculate that Sesn2 plays an important role in CVDs by stimulating the process of antioxidation and promoting the adaptation of cells to stress conditions and/or the environment, opening a new avenue for related therapeutic strategies.

Sestrin 2 controls the cardiovascular aging process via an integrated network of signaling pathways.

As an inevitable biological process, cardiovascular aging is the greatest risk factor for cardiovascular diseases (CVDs). Sestrin 2 (Sesn2), a stress-inducible and age-related protein associated with various stress conditions, plays a pivotal role in slowing this process. It acts as an anti-aging agent, mainly through its antioxidant enzymatic activity and regulation of antioxidant signaling pathways, as well as by activating adenosine monophosphate-activated protein kinase and inhibiting mammalian target of rapamycin complex 1. In this review, we first introduce the biochemical functions of Sesn2 in the cardiovascular aging process, and describe how Sesn2 expression is regulated under various stress conditions. Next, we emphasize the role of Sesn2 signal transduction in a series of age-related CVDs, including hypertension, myocardial ischemia and reperfusion, atherosclerosis, and heart failure, as well as provide potential mechanisms for the association of Sesn2 with CVDs. Finally, we present the potential therapeutic applications of Sesn2-directed therapy and future prospects.

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur.

The antineoplastic drug carmofur is shown to inhibit the SARS-CoV-2 main protease (Mpro). Here, the X-ray crystal structure of Mpro in complex with carmofur reveals that the carbonyl reactive group of carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 µM) and is a promising lead compound to develop new antiviral treatment for COVID-19.

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Clonal copy-number mosaicism in autoreactive T lymphocytes in diabetic NOD mice.

Concordance for type 1 diabetes (T1D) is far from 100% in monozygotic twins and in inbred nonobese diabetic (NOD) mice, despite genetic identity and shared environment during incidence peak years. This points to stochastic determinants, such as postzygotic mutations (PZMs) in the expanding antigen-specific autoreactive T cell lineages, by analogy to their role in the expanding tumor lineage in cancer. Using comparative genomic hybridization of DNA from pancreatic lymph-node memory CD4+ T cells of 25 diabetic NOD mice, we found lymphocyte-exclusive mosaic somatic copy-number aberrations (CNAs) with highly nonrandom independent involvement of the same gene(s) across different mice, some with an autoimmunity association (e.g., Ilf3 and Dgka). We confirmed genes of interest using the gold standard approach for CNA quantification, multiplex ligation-dependent probe amplification (MLPA), as an independent method. As controls, we examined lymphocytes expanded during normal host defense (17 NOD and BALB/c mice infected with Leishmania major parasite). Here, CNAs found were fewer and significantly smaller compared to those in autoreactive cells (P = 0.0019). We determined a low T cell clonality for our samples suggesting a prethymic formation of these CNAs. In this study, we describe a novel, unexplored phenomenon of a potential causal contribution of PZMs in autoreactive T cells in T1D pathogenesis. We expect that exploration of point mutations and studies in human T cells will enable the further delineation of driver genes to target for functional studies. Our findings challenge the classical notions of autoimmunity and open conceptual avenues toward individualized prevention and therapeutics.

3D printing of layered mesoporous bioactive glass/sodium alginate-sodium alginate scaffolds with controllable dual-drug release behaviors.

Scaffolds with controlled drug release are valuable for bone tissue engineering, but constructing the scaffolds with controllable dual-drug release behaviors is still a challenge. In this study, layered mesoporous bioactive glass/sodium alginate-sodium alginate (MBG/SA-SA) scaffolds with controllable dual-drug release behaviors were fabricated by 3D printing. The porosity and compressive strength of three-dimensional (3D) printed MBG/SA-SA scaffolds by cross-linking are about 78% and 4.2 MPa, respectively. As two model drugs, bovine serum albumin (BSA) and ibuprofen (IBU) were separately loaded in SA layer and MBG/SA layer, resulting in a relatively fast release of BSA and a sustained release of IBU. Furthermore, layered MBG/SA-SA scaffolds were able to stimulate human bone mesenchymal stem cells (hBMSCs) adhesion, proliferation and osteogenic differentiation than SA scaffolds. Hence, the 3D printed MBG/SA-SA scaffolds would be prospective for the treatment of bone defects.

3D printing of mesoporous bioactive glass/silk fibroin composite scaffolds for bone tissue engineering.

The fabrication of bone tissue engineering scaffolds with high osteogenic ability and favorable mechanical properties is of huge interest. In this study, a silk fibroin (SF) solution of 30 wt% was extracted from cocoons and combined with mesoporous bioactive glass (MBG) to fabricate MBG/SF composite scaffolds by 3D printing. The porosity, compressive strength, degradation and apatite forming ability were evaluated. The results illustrated that MBG/SF scaffolds had superior compressive strength (ca. 20 MPa) and good biocompatibility, and stimulated bone formation ability compared to mesoporous bioactive glass/polycaprolactone (MBG/PCL) scaffolds. We subcutaneously transplanted hBMSCs-loaded MBG/SF and MBG/PCL scaffolds into the back of nude mice to evaluate heterotopic bone formation assay in vivo, and the results revealed that the gene expression levels of common osteogenic biomarkers on MBG/SF scaffolds were significantly better than MBG/PCL scaffolds. These results showed that 3D-printed MBG/SF composite scaffolds are great promising for bone tissue engineering.