Circulating cell-free DNA sequencing for early detection of lung cancer.

INTRODUCTION: Lung cancer is a leading cause of death in patients with cancer. Early diagnosis is crucial to improve the prognosis of patients with lung cancer. Plasma circulating cell-free DNA (cfDNA) contains comprehensive genetic and epigenetic information from tissues throughout the body, suggesting that early detection of lung cancer can be done non-invasively, conveniently, and cost-effectively using high-sensitivity techniques such as sequencing. AREAS COVERED: In this review, we summarize the latest technological innovations, coupled with next-generation sequencing (NGS), regarding genomic alterations, methylation, and fragmentomic features of cfDNA for the early detection of lung cancer, as well as their clinical advances. Additionally, we discuss the suitability of study designs for diagnostic accuracy evaluation for different target populations and clinical questions. EXPERT OPINION: Currently, cfDNA-based early screening and diagnosis of lung cancer faces many challenges, such as unsatisfactory performance, lack of quality control standards, and poor repeatability. However, the progress of several large prospective studies employing epigenetic features has shown promising predictive performance, which has inspired cfDNA sequencing for future clinical applications. Furthermore, the development of multi-omics markers for lung cancer, including genome-wide methylation and fragmentomics, is expected to play an increasingly important role in the future.

Norepinephrine inhibits CD8+ T-cell infiltration and function, inducing anti-PD-1 mAb resistance in lung adenocarcinoma.

BACKGROUND: Mental stress-induced neurotransmitters can affect the immune system in various ways. Therefore, a better understanding of the role of neurotransmitters in the tumour immune microenvironment is expected to promote the development of novel anti-tumour therapies. METHODS: In this study, we analysed the plasma levels of neurotransmitters in anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb)-resistance patients and sensitive patients, to identify significantly different neurotransmitters. Subsequently, animal experiments and experiments in vitro were used to reveal the specific mechanism of norepinephrine's (NE) effect on immunotherapy. RESULTS: The plasma NE levels were higher in anti-PD-1 mAb-resistance patients, which may be the main cause of anti-PD-1 mAb resistance. Then, from the perspective of the immunosuppressive microenvironment to explore the specific mechanism of NE-induced anti-PD-1 mAb resistance, we found that NE can affect the secretion of C-X-C Motif Chemokine Ligand 9 (CXCL9) and adenosine (ADO) in tumour cells, thereby inhibiting chemotaxis and function of CD8+ T cells. Notably, the WNT7A/ß-catenin signalling pathway plays a crucial role in this progression. CONCLUSION: NE can affect the secretion of CXCL9 and ADO in tumour cells, thereby inhibiting chemotaxis and the function of CD8+ T cells and inducing anti-PD-1 mAb resistance in lung adenocarcinoma (LUAD).

A 12-week personalised physical activity and dietary protein intervention for older adults undergoing peritoneal dialysis: A feasibility study.

This single-arm observational study explored the feasibility and efficacy of a 12-week personalised physical activity and dietary protein intervention programme for older adults undergoing peritoneal dialysis. Older adults undergoing peritoneal dialysis received eight individualised nutrition and physical activity advice sessions provided by trained nurses. Protein intake and physical activity were regarded as primary outcomes. All data were collected at baseline and at week 12. The enrolment rate was 78.4%. Twenty-nine patients participated in the study. Of these, 86.2% (25/29) completed the intervention. There was a significant increase in protein intake (t = -4.453, P< 0.001) and physical activity levels (Z = -2.929, P = 0.004). Of the participants, 56.0% achieved the targeted protein goal, and 41.4% met the physical activity goal. The timed up-and-go performance (t = 4.135, P = 0.001) increased after intervention. Trained nurses can successfully implement personalised diet and physical activity advice, and achieve promising patient outcomes.

circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves the Repair of Damaged Endometrium.

Background: Human umbilical cord mesenchymal stem cells- (HuMSCs-) based therapy has shown promising results in the treatment of intrauterine adhesions (IUA). In this study, we aimed to construct a HuMSCs-seeded silk fibroin small-intestinal submucosa (SF-SIS) scaffold and evaluate its ability to repair the damaged endometrium in an IUA mouse model. Methods: To identify the functional effect of HuMSCs-SF-SIS scaffolds on the repair of damaged endometrium, a mouse IUA model was established. Uterine morphology and fibrosis were evaluated by hematoxylin-eosin staining and Masson staining. CircRNA sequencing, real-time PCR, and RNA fluorescence in situ hybridization were used to screen and verify the potential circRNAs involved in the repair of damaged endometrium by HuMSCs. Real-time integrated cellular measurement of oxygen consumption rate was performed using the Seahorse XF24 Extracellular Flux Analyzer. The potential downstream miRNAs and proteins of circRNAs were analyzed by dual-luciferase reporter assay and western blot. Results: HuMSCs-SF-SIS not only increased the number of glands but also reduced the ulcer area in the IUA model. circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and was targeted by miR-330-5p-PDK2. It also stabilized the mitochondrial metabolism of HuMSCs. Moreover, miR-330-5p was found to inhibit PDK2 expression through the 3' UTR target region. A rescue experiment further showed that circPTP4A2-miR-330-5p-PDK2 signaling was critical to HuMSCs-SF-SIS in decreasing the fibrosis area and increasing the number of glands in the IUA model. Conclusion: We demonstrated that circPTP4A2 was elevated in HuMSCs-seeded on SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSCs-seeded SF-SIS scaffolds have potential for the treatment of IUA.

Mechanical stretch induces osteogenesis through the alternative activation of macrophages.

For reconstructive surgeons, critically skeletal damage represents a major challenge. Growing evidence indicate that bone repair is dynamically regulated by the mesenchymal stem cell (MSC)-macrophage interaction. Mechanical strain plays a fundamental role in bone repair and regeneration by influencing MSCs differentiation. Recently, a few findings indicate that macrophages may be mechanically sensitive and their phenotype can be regulated, in part, by mechanical cues. However, how macrophages subjected mechanical stretch influence the osteogenic differentiation of MSCs remain unclear. Thus, the purpose of this study is to explore the effect of macrophages stimulated with mechanical stretch on MSCs osteogenesis. By using a coculture system, we discover that macrophages efficiently induce osteogenic differentiation of MSCs under specific stretch conditions. A synergy mechanism between M2 polarization and YAP/BMP2 axis are identified through molecular and genetic analyses. Macrophages are activated by cyclic stretch and polarized to M2 phenotype that produce anti-inflammatory cytokines such as IL-10 and TGF-ß to regulate the local inflammatory microenvironment. Furthermore, mechanical stretch induces YAP activation and nuclear translocation, subsequently regulates downstream BMP2 expression to facilitate MSCs osteogenesis. These findings not only advance our understanding of the complex influence among the mechanical strain, macrophage inflammatory response as well as the osteogenic differentiation of MSCs, but also reveal a control system from mechanical signals to chemical response then to cell behaviors during bone repair and regeneration.

Polycaprolactone nanofiber scaffold enhances the osteogenic differentiation potency of various human tissue-derived mesenchymal stem cells.

BACKGROUND: Polycaprolactone (PCL) has been regarded as a promising synthetic material for bone tissue engineering application. Owing to its unique biochemical properties and great compatibility, PCL fibers have come to be explored as a potential delivering scaffold for stem cells to support bone regeneration during clinical application. METHODS: The human derived mesenchymal stem cells (MSCs) were obtained from umbilical cord (UC), bone marrow (BM), and adipose tissue (AD), respectively. The osteogenic differentiation potency of various human MSCs on this novel synthetic biomaterial was also investigated in vitro. RESULTS: Here, we illustrated that those human UC-, BM-, and AD-derived MSCs exhibited fibroblast-like morphology and expressed characteristic markers. Impressively, PCL nanofiber scaffold could support those MSC adhesion and proliferation. Long-term culture on PCL nanofiber scaffold maintained the viability as well as accelerated the proliferation of those three different kinds of human MSCs. More importantly, the osteogenic differentiation potency of those human MSCs was increased significantly by culturing on PCL nanofiber scaffold. Of note, BM-derived MSCs demonstrated greater differentiation potency among the three kinds of MSCs. The Wnt/ß-catenin and Smad3 signaling pathways contributed to the enhanced osteogenesis of human MSCs, which was activated consistently by PCL nanofiber scaffold. CONCLUSIONS: The utilization of PCL nanofiber scaffold would provide a great application potential for MSC-based bone tissue repair by enhancing the osteogenic differentiation of human MSCs.

Expression of CD11c+HLA-DR+dendritic cells and related cytokines in the follicular fluid might be related to pathogenesis of ovarian hyperstimulation syndrome.

OBJECTIVE: To explore the expressions of CD11c+HLA-DR+dentritic cells in the follicular fluid of patients with OHSS and their significances. SUBJECTS: 100 individuals. TREATMENT: embryos were observed. The distribution of dentritic cells in follicular fluid and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected. METHODS: There were ovarian hyperstimulation syndrome (OHSS) group and control group in this study. The OHSS group consisted of 50 patients with OHSS and the control group consisted of 50 patients who underwent in vitro fertilization-embryo transfer (IVF-ET) only due to male factors. The statuses of embryos were compared between the two groups. The distribution of dentritic cells in follicular fluid was determined with flow cytometry, and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected with enzyme-linked immunosorbent assay (ELISA) in all patients. RESULTS: The two-pronuclear (2PN) fertility rate, high-quality embryo rate and available embryo rate were all significantly lower in OHSS group than in control group (all P<0.05). The number of CD11c+HLA-DR+dentritic cells (P<0.05) and the levels of IL-10, IL-12, IL-18 and IL-23 were all significantly higher in OHSS group than in control group (all P<0.01). CONCLUSION: The follicular fluid of the patients with OHSS is in an inflammatory status, the inflammatory status may be involved in OHSS and the microenvironment of follicular fluid may affects oocyte quality and embryo development.

Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation.

Human mesenchymal stem cells (hMSCs) can differentiate into various cell types, including osteogenic and chondrogenic cells. The matrix elasticity and cell seeding density are important factors in hMSCs differentiation. We cultured hMSCs at different seeding densities on polyacrylamide hydrogels with different stiffness corresponding to Young's moduli of 1.6 ± 0.3 and 40 ± 3.6 kPa. The promotion of osteogenic marker expression by hard gel is overridden by a high seeding density. Cell seeding density, however, did not influence the chondrogenic marker expressions induced by soft gel. These findings suggest that interplays between cell-matrix and cell-cell interactions contribute to hMSCs differentiation. The promotion of osteogenic differentiation on hard matrix was shown to be mediated through the Ras pathway. Inhibition of Ras (RasN17) significantly decreased ERK, Smad1/5/8 and AKT activation, and osteogenic markers expression. However, constitutively active Ras (RasV12) had little effect on osteogenic marker expression, suggesting that the Ras pathways are necessary but not sufficient for osteogenesis. Taken together, our results indicate that matrix elasticity and cell density are important microenvironmental cues driving hMSCs proliferation and differentiation.

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells.

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.

Combined effects of TNF-α, IL-1ß, and HIF-1α on MMP-2 production in ACL fibroblasts under mechanical stretch: an in vitro study.

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra-articular joint, are very complex after injury. Injury to intra-articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro-inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix-metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP-2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor-1α (HIF-1α) on induction of MMP-2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF-α and IL-1ß elevate the activity of MMP-2 in a dose- and time-dependent manner. In addition, mechanical stretch further enhances the MMP-2 protein levels with TNF-α, IL-1ß, and their mixture. CoCl(2) -induced HIF-1α (100 and 500 µM) also increases the levels and activity of MMP-2. Mechanical stretch has a strong additional effect on MMP-2 production with HIF-1α. Our results conclude that mechanical injury, HIF-1α and inflammatory factors collectively induce increased MMP-2 production in ACL fibroblasts, which was inhibited by NF-κB pathway inhibitor (Bay-11-7082).

TGF-ß1 promoted MMP-2 mediated wound healing of anterior cruciate ligament fibroblasts through NF-κB.

The adult human anterior cruciate ligament (ACL) has poor functional healing response. Transforming growth factor (TGF)-ß1 enhances the wound repair by stimulating matrix proteins deposition as well as the proliferation and migration of cells. However, the function of the TGF-ß1-induced matrix metalloproteinases' (MMPs) activities in the wound healing process is poorly understood. In this study, exogenous MMP-2 is added to mimic the TGF-ß1-induced MMP-2 expression. Role of NF-κB pathway is further examined. Our results show that TGF-ß1 induces dramatic elevation of MMP-2 activities and the MMP-2/tissue inhibitors of metalloproteinases ratio. Furthermore, the exogenous MMP-2 significantly promoted in vitro wound healing abilities of ACL fibroblasts that are significantly blocked with the addition of its inhibitors. TGF-ß1 also increases the proliferation of ACL fibroblasts whereas MMP-2 alone does not, indicating that MMP-2 activities are not involved in the proliferation. TGF-ß1-induced MMP-2 activity is inhibited by Bay11-7082 and Bay11-7085 (NF-κB inhibitors). Our results demonstrate that increased TGF-ß1 facilitates the ACL healing process by promoting the fibroblasts migration and proliferation. The migration process is mediated by MMP-2 and NF-κB pathway is involved in TGF-ß1-mediated MMP-2 release.

The profile of MMP and TIMP in injured rat ACL.

Human anterior cruciate ligament (ACL) has poor healing ability after injury. The devastating effects of matrix metalloproteinases (MMPs) excess expression are regarded as the main reason. Tissue inhibitor metalloproteinases (TIMPs) may be independent of MMPs inhibition. In this paper, a rat ACL rotating injury apparatus was designed to produce ACL injury. After inducing injury, joint fluids and ACL tissue total proteins were immediately extracted. In addition, ACL tissue was isolated in a culture plate with 1%FBS medium for the ex vivo study. We found MMP-2 in joint fluids increased significantly by 4 folds after ACL injury as a function of time. Ex vivo study showed MMP-2 in the medium and ACL cultured tissue increased significantly respectively to 3 folds and to 6 folds. The joint fluids global MMP increased to 3.5 folds with non-treatment and APMA-treatment in day three. On the gene expression level, the changes in MMP-1 and CD147 have the similar tendency. The ratio of MMP-1/TIMP-1 increased with time after ACL injury. We conclude that MMP-2 increased significantly in the early phase in the joint cavity after ACL injury. The ex vivo study demonstrated the same tendency. Generic MMP Activity Assay (global MMP assay) an dzymography also showed significant increase in MMP activity in joint fluids. These results showed ACL having poor healing ability after injury may not be only due to ACL release of large quantities of MMPs. The important factor may be the alterations in the whole joint cavity's internal environment.

Differential MMP-2 activity induced by mechanical compression and inflammatory factors in human synoviocytes.

The anterior cruciate ligament, posterior cruciate ligament, cartilage and meniscus in human knee joint have poor healing ability. Accumulation of MMPs in the joint fluids due to knee injury has been considered as the main reason. Our previous experiments showed that synovium may be the major regulator of MMPs in joint cavity after injury. In this paper, we used human synoviocytes harvested from synovium to determine whether mechanical injury and inflammatory factors will induce MMP-2 production in synoviocytes. With zymography, we found that mechanical compression increased the MMP-2 production by 23% under 6% compressions, 61% under 12% compression and 109% under 14% compressions. In addition, TNF-alpha can also elevate the activity of MMP-2 in a dose dependent manner, while IL-1alpha does not. However, mixture of these two factors dramatically increased MMP-2 production by 201%. In addition, mechanical injury had a strong synergistic effect on MMP-2 production with TNF-alpha, IL-1alpha and their mixture, increasing by 207%, 354% and 468% individually. The generic MMP activity assayrevealed that mechanical compression increased the generic activity. APMA treatment increased the generic activity of MMPs induced by compres-

Coordinated expression of MMPs and TIMPs in rat knee intra-articular tissues after ACL injury.

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.

Differential expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in anterior cruciate ligament and medial collateral ligament fibroblasts after a mechanical injury: involvement of the p65 subunit of NF-kappaB.

The anterior cruciate ligament (ACL) is known to have a poor healing ability, especially in comparison with the medial collateral ligament, which can heal relatively well. In this study, we detected significant increases in the mRNA levels of multiple matrix metalloproteinases (MMPs) (MMP-1, -2, -7, -9, -11, -14, -17, -21, -23A, -24, -25, -27, and -28) and tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, -2, -3, and -4) in ACL fibroblasts after an in vitro injury with an equi-biaxial stretch chamber. However, only some MMPs (MMP-7, -9, -14, -21, and -24) showed increases in injured medial collateral ligament fibroblasts, and to a much lesser degree than that observed in the injured ACL fibroblasts. Zymography revealed a 6.3-fold increase of MMP-2 activity in injured ACL but not medial collateral ligament fibroblasts, which agrees with the global MMP activities assay. Bay-11 and curcumin can significantly decrease MMP-2 activities to 13% and 29% in injured ACL fibroblasts, respectively, which implies the involvement of p65 subunits of nuclear factor kappaB and AP-1 pathways. Furthermore, Bay-11 can decrease the global MMP activity released from injured ACL fibroblasts in a dose-dependent manner. In summary, the differential expression and activities of MMPs might help to explain the poor healing ability of ACL, and the p65 subunit of nuclear factor kappaB might be a potential target to facilitate the ACL repair.

Expression of MMPs and TIMPs family in human ACL and MCL fibroblasts.

The human ACL (anterior cruciate ligament) is susceptible to injury but has poor healing response, whereas an injured MCL (medial collateral ligament) can be repaired relatively well. Since MMPs (matrix metalloproteases) and TIMPs (tissue inhibitor of metalloproteases) are involved in this tissue remodeling process, investigation of different response of MMPs and TIMPs family in ACL and MCL fibroblasts might lead to understanding the differential matrix remodeling process as well as their different healing ability. The first step would be determination of whether these tissue remodeling effectors are present in ligaments. In this study, we designed primers for real-time RT-PCR and determined the expression of MMPs and TIMPs family in ACL and MCL fibroblasts with synovium as a positive control. Semiquantitative RT-PCR revealed that multiple MMPs and TIMPs expressed in human ACL and MCL fibroblasts except MMP-8, 10, 12, 13, 15, 16, 20, and 26. MMP-7 was present in MCL but not in ACL fibroblast. Quantitative real-time RT-PCR showed that mRNA levels of MMP-1, 2, 14, 17, 23A, and 23B and TIMP-4 are significantly higher in MCL than in ACL fibroblasts. However, MMP-3 is higher in ACL than in MCL fibroblasts. We conclude that numerous MMPs and TIMPs family members that are differentially expressed in ACL and MCL might be involved in the differential matrix remodeling process as well as the differential healing ability of ACL and MCL.

Contributions of different intraarticular tissues to the acute phase elevation of synovial fluid MMP-2 following rat ACL rupture.

Inflammation and accumulation of matrix metalloproteinases (MMPs) in synovial fluids may be involved in the poor healing ability of the anterior cruciate ligament (ACL) after injury. With a rat ACL rotating injury model, we found that levels of IL-1beta, IL-6, and TNF-alpha were significantly higher in synovial fluids after ACL injury. MMP-2 activity and global MMP activity in synovial fluids also increased significantly in a time-dependent manner. Ex vivo studies showed that all tissues contributed to the elevation of MMP-2 in synovial fluids, especially synovium and the injured ACL. We concluded that although the regular wound-healing mechanism also occurs after ACL injury, accumulation of MMP activity in the synovial fluids, due to all of the intraarticular tissues, may be at least one of the important reasons why an injured ACL cannot be repaired.