Effects of blood-processing protocols on cell-free DNA fragmentomics in plasma: Comparisons of one- and two-step centrifugations.

BACKGROUND: Cell-free DNA (cfDNA) fragmentomic characteristics are promising analytes with abundant physiological signals for non-invasive disease diagnosis and monitoring. Previous studies on plasma cfDNA fragmentomics commonly employed a two-step centrifugation process for removing cell debris, involving a low-speed centrifugation followed by a high-speed centrifugation. However, the effects of centrifugation conditions on the analysis of cfDNA fragmentome remain uncertain. METHODS: We collected blood samples from 10 healthy individuals and divided each sample into two aliquots for plasma preparation with one- and two-step centrifugation processes. We performed whole genome sequencing (WGS) of the plasma cfDNA in the two groups and comprehensively compared the cfDNA fragmentomic features. Additionally, we reanalyzed the fragmentomic features of cfDNA from 16 healthy individuals and 16 COVID-19 patients, processed through one- and two-step centrifugation in our previous study, to investigate the impact of centrifugation on disease signals. RESULTS: Our results showed that there were no significant differences observed in the characteristics of nuclear cfDNA, including size, motif diversity score (MDS) of end motifs, and genome distribution, between plasma samples treated with one- and two-step centrifugation. The cfDNA size shortening in COVID-19 patients was observed in plasma samples with one- and two-step centrifugation methods. However, we observed a significantly higher relative abundance and longer size of cell-free mitochondrial DNA (mtDNA) in the one-step samples compared to the two-step samples. This difference in mtDNA caused by the one- and two-step centrifugation methods surpasses the pathological difference between COVID-19 patients and healthy individuals. CONCLUSIONS: Our findings indicate that one-step low-speed centrifugation is a simple and potentially suitable method for analyzing nuclear cfDNA fragmentation characteristics. These results offer valuable guidance for cfDNA research in various clinical scenarios.

Programming of Regulatory T Cells In Situ for Nerve Regeneration and Long-Term Patency of Vascular Grafts.

Rapid integration into the host tissue is critical for long-term patency after small diameter tissue engineering vascular grafts (sdTEVGs) transplantation. Neural recognition may be required for host integration and functionalization of the graft. However, immune rejection and inflammation hinder nerve regeneration of sdTEVGs. Here, a CRISPR/dCas9-nanocarrier was used for targeted programming of regulatory T cells (Treg cells) in situ to promote nerve regeneration of sdTEVGs by preventing excessive inflammation. Treg cells and (C-C chemokine receptor) CCR2+ macrophage recruitment occurred after transplantation. The nanodelivery system upregulated ten eleven translocation (TET2) in Treg cells in vitro. Reprogrammed Treg cells upregulated anti-inflammatory cytokines and decreased the proportion of CCR2+ macrophages. IL-6 concentrations decreased to the levels required for nerve regeneration. Implantation of CRISPR/dCas9 nanodelivery system-modified sdTEVGs in rats resulted in Treg cell editing, control of excessive inflammation, and promoted nerve regeneration. After 3 months, nerve regeneration was similar to that observed in normal blood vessels; good immune homeostasis, consistency of hemodynamics, and matrix regeneration were observed. Neural recognition promotes further integration of the graft into the host, with unobstructed blood vessels without intimal hyperplasia. Our findings provide new insights into vascular implant functionalization by the host.

Long non-coding RNA LINC00299 knockdown inhibits ox-LDL-induced T/G HA-VSMC injury by regulating miR-135a-5p/XBP1 axis in atherosclerosis.

BACKGROUND: Atherosclerosis (AS) is a highly relevant social problem. Long non-coding RNA (lncRNA) long intergenic non-coding00299 (LINC00299) participates in the regulation of AS development. Therefore, this study was to explore the potential role and mechanism of LINC00299 in AS. METHODS: Human aortic vascular smooth muscle cells (T/G HA-VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL). Cell viability and migration were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) and transwell assays, severally. The activities of SOD and MDA were detected by total superoxide dismutase assay kit and malondialdehyde assay kit. The protein levels of ki67, matrix metalloproteinase 9 (MMP9) and X-box binding protein 1 (XBP1) were detected by western blot assay. The expression levels of LINC00299, microRNA-135a-5p (miR-135a-5p) and XBP1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The binding relationship between miR-135a-5p and LINC00299 or XBP1 was predicted by miRcode and Starbase3.0 then verified by the dual-luciferase reporter assay. RESULTS: Ox-LDL induced cell viability, oxidative damage and migration of T/G HA-VSMCs. LINC00299 knockdown weakened ox-LDL-induced T/G HA-VSMCs injury. Mechanical analysis confirmed that LINC00299 improved XBP1 expression by interacting with miR-135a-5p. Furthermore, rescue assays showed that LINC00299 regulated ox-LDL-induced T/G HA-VSMCs injury through the miR-135a-5p/XBP1 axis. CONCLUSIONS: Our studies revealed the regulatory function of LINC00299/miR-135a-5p/XBP1 axis in AS development, suggesting a potential therapeutic strategy for AS treatment.

Programmable dual responsive system reconstructing nerve interaction with small-diameter tissue-engineered vascular grafts and inhibiting intimal hyperplasia in diabetes.

Small-diameter tissue-engineered vascular grafts (sdTEVGs) with hyperglycemia resistance have not been constructed. The intimal hyperplasia caused by hyperglycemia remains problem to hinder the patency of sdTEVGs. Here, inspired by bionic regulation of nerve on vascular, we found the released neural exosomes could inhibit the abnormal phenotype transformation of vascular smooth muscle cells (VSMCs). The transformation was a prime culprit causing the intimal hyperplasia of sdTEVGs. To address this concern, sdTEVGs were modified with an on-demand programmable dual-responsive system of ultrathin hydrogels. An external primary Reactive Oxygen Species (ROS)-responsive Netrin-1 system was initially triggered by local inflammation to induce nerve remolding of the sdTEVGs overcoming the difficulty of nerve regeneration under hyperglycemia. Then, the internal secondary ATP-responsive DENND1A (guanine nucleotide exchange factor) system was turned on by the neurotransmitter ATP from the immigrated nerve fibers to stimulate effective release of neural exosomes. The results showed nerve fibers grow into the sdTEVGs in diabetic rats 30 days after transplantation. At day 90, the abnormal VSMCs phenotype was not detected in the sdTEVGs, which maintained long-time patency without intima hyperplasia. Our study provides new insights to construct vascular grafts resisting hyperglycemia damage.

Discrete genetic loci in human gut Bacteroides thetaiotaomicron confer pectin metabolism.

Although the polysaccharide utilization loci (PULs) activated by pectin have been defined, due to the complex of side-chain structure, the degradative mechanisms still remain vague. Thus, we hypothesize that there may have other specific PULs to target pectin. Here, we characterize loci-encoded proteins expressed by Bacteroides thetaiotaomicron (BT) that are involved in the pectin capturing, importation, de-branching and degradation into monosaccharides. Totally, four PULs contain ten enzymes and four glycan binding proteins which including a novel surface enzyme and a surface glycan binding protein are identified. Notably, PUL2 and PUL3 have not been reported so far. Further, we show that the degradation products support the growth of other Bacteroides spp. and probiotics. In addition, genes involved in this process are conservative in other Bacteroides spp. Our results further highlight the contribution of Bacteroides spp. to metabolism the pectic network.

Review on the Vascularization of Organoids and Organoids-on-a-Chip.

The use of human cells for the construction of 3D organ models in vitro based on cell self-assembly and engineering design has recently increased in popularity in the field of biological science. Although the organoids are able to simulate the structures and functions of organs in vitro, the 3D models have difficulty in forming a complex vascular network that can recreate the interaction between tissue and vascular systems. Therefore, organoids are unable to survive, due to the lack of oxygen and nutrients, as well as the accumulation of metabolic waste. Organoids-on-a-chip provides a more controllable and favorable design platform for co-culture of different cells and tissue types in organoid systems, overcoming some of the limitations present in organoid culture. However, the majority of them has vascular networks that are not adequately elaborate to simulate signal communications between bionic microenvironment (e.g., fluid shear force) and multiple organs. Here, we will review the technological progress of the vascularization in organoids and organoids-on-a-chip and the development of intravital 3D and 4D bioprinting as a new way for vascularization, which can aid in further study on tissue or organ development, disease research and regenerative medicine.

Surface-Engineered Monocyte Inhibits Atherosclerotic Plaque Destabilization via Graphene Quantum Dot-Mediated MicroRNA Delivery.

Rupture-prone atherosclerotic plaque is the cause of the high mortality and morbidity rates that accompany atherosclerosis-associated diseases. MicroRNAs can regulate the expression of a variety of atherosclerotic inflammation-related genes in macrophages. There are currently no definitive methods for delivering microRNAs into the interior of plaque. Monocytes typically possess a pathological feature that allows them to be recruited to atherosclerotic plaque resulting in rupture-prone; however, whether monocytes can be modified to be gene carriers remains unclear. In this study, a novel monocyte surface-engineered gene-delivery system based on graphene quantum dots (GQDs) is developed. Briefly, GQDs-microRNA223 linked by disulfide bonds are grafted onto the monocyte membrane via a carefully designed C18-peptide (C18P) containing a hydrophobic end to afford the designed monocyte-C18P-GQDs-miR223 architecture. The system can reach and enter the interior of the plaque and release the GQDs-miRNA via C18P digestion. The released GQDs-miRNA are taken up by the macrophages in atherosclerotic plaques, and the disulfide linkages between the GQDs and the miRNA are cleaved through γ-interferon-inducible lysosomal thiol reductase (GILT) in the lysosome. Under the protection of GQDs, miRNA cargos are transfected into the cytosol and subsequently undergo nuclear translocation, allowing a significantly reduced plaque burden by regulating inflammatory response in vivo.

Engineered-Macrophage Induced Endothelialization and Neutralization via Graphene Quantum Dot-Mediated MicroRNA Delivery to Construct Small-Diameter Tissue-Engineered Vascular Grafts.

Rapid endothelialization of tissue-engineered blood vessels (TEBVs) is an essential strategy to inhibit thrombosis, chronic inflammation and intimal hyperplasia after transplantation into the body. Monocytes will be recruited to the transplantation site and converted to macrophages after TEBV implantation. Macrophages play an important role in angiogenesis; however, whether engineered macrophages can be utilized to promote rapid endothelialization of TEBVs remains unclear. Thus, a cell bioreactor that can engineer macrophages via graphene quantum dot (GQD)-mediated microRNA (miR) delivery was built in the TEBV. Briefly, GQD-miR-150 linked by disulfide bonds was adopted to functionalize both the inner and outer TEBVs. The GQD-miR-150 conjugation as an intracellular gene delivery system was taken up by macrophages. Under the protection of GQDs, miR-150 was transfected into the cytosol, allowing continuous secretion of vascular endothelial growth factor (VEGF) via upregulation of HIF-1α protein expression, and promoted the migration of endothelial cells (ECs) in vitro. An in vivo study showed a rapid endothelialization of the inner TEBVs after transplantation for 7 days, especially a holonomic endothelial layer after 30 days. For the outer TEBVs, neovascularization (vasa vasorum) accompanied by nerve growth was observed around the adventitia on day 90. In conclusion, the designed cell bioreactor consisting of GQD-miR-engineered macrophages can effectively promote endothelialization and neuralization in vivo for TEBVs.

Construction of a small-caliber tissue-engineered blood vessel using icariin-loaded ß-cyclodextrin sulfate for in situ anticoagulation and endothelialization.

The rapid endothelialization of tissue-engineered blood vessels (TEBVs) can effectively prevent thrombosis and inhibit intimal hyperplasia. The traditional Chinese medicine ingredient icariin is highly promising for the treatment of cardiovascular diseases. ß-cyclodextrin sulfate is a type of hollow molecule that has good biocompatibility and anticoagulation properties and exhibits a sustained release of icariin. We studied whether icariin-loaded ß-cyclodextrin sulfate can promote the endothelialization of TEBVs. The experimental results showed that icariin could significantly promote the proliferation and migration of endothelial progenitor cells; at the same time, icariin could promote the migration of rat vascular endothelial cells (RAVECs). Subsequently, we used an electrostatic force to modify the surface of the TEBVs with icariin-loaded ß-cyclodextrin sulfate, and these vessels were implanted into the rat common carotid artery. After 3 months, micro-CT results showed that the TEBVs modified using icariin-loaded ß-cyclodextrin sulfate had a greater patency rate. Scanning electron microscopy (SEM) and CD31 immunofluorescence results showed a better degree of endothelialization. Taken together, icariin-loaded ß-cyclodextrin sulfate can achieve anticoagulation and rapid endothelialization of TEBVs to ensure their long-term patency.

Development of a simple IgE-independent anaphylactic model using buckwheat antigen and B-type CpG oligodeoxynucleotide from Streptococcus thermophilus.

We developed a severe anaphylactic model in mice using buckwheat antigen and B-type CpG-oligodeoxynucleotides (CpG-ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5-week-old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B-type CpG-ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G2a production and interferon-γ positive cells were markedly increased in mice immunized with antigen mixed with B-type CpG-ODN, whereas serum IgE levels were decreased by B-type CpG-ODN. We also examined the effects of various ODNs (A, B and C-type CpG-ODNs) and antigens (buckwheat, α-casein, ß-lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B-type CpG-ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.

Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.

Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases.

Generation of dipeptidyl peptidase-IV-inhibiting peptides from ß-lactoglobulin secreted by Lactococcus lactis.

Previous studies showed that hydrolysates of ß-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.

[Syntheses and spectral study of novel metal zinc organic phosphonate].

Two novel Zn2+ organic phosphonate Zn(4,4'-bipy)(HBCP) 1 and [Zn2 (phen)2 (BCP)(H2 BCP)].H2O2 were hydrothermally synthesized. The compound 1 is with two-dimensional layer framework whereas the compound 2 is zero-dimensional structure. The Zn2+ ion is four-coordinated in compound 1 while five or six-coordinated in compound 2. The relationship between their properties and structures was studied by using FTIR, two-dimensional (2D) correlation infrared spectroscopy under thermal perturbation, fluorescence and UV-Vis DRS spectrum etc. Upon light excitation at 350 nm, the compound 1 exhibits luminescence with a strong wide emission at 412 nm and a weak emission at 520 nm, while the compound 2 exhibits luminescence with a wide emission only at 398 nm under light excitation at 336 nm.