Inhibition of the bone morphogenetic protein pathway suppresses tumor growth through downregulation of epidermal growth factor receptor in MEK/ERK-dependent colorectal cancer.

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, its role in colorectal cancer (CRC) is controversial, where it can act as context-dependent tumor promoter or tumor suppressor. Here we have found that CRC cells reside in a BMP-rich environment based on curation of two publicly available RNA-sequencing databases. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of select CRC organoids. Colorectal cancer organoids treated with LDN193189 showed a decrease in epidermal growth factor receptor, which was mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression. Among 18 molecularly characterized CRC organoids, suppression of growth by BMP inhibition correlated with induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, in CRC organoids, which are susceptible to growth suppression by LDN193189, simultaneous treatment with LDN193189 and trametinib, an FDA-approved MEK inhibitor, resulted in cooperative growth inhibition both in vitro and in vivo. Taken together, the simultaneous inhibition of BMP and MEK could be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders to this regimen.

Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity.

Dynamic changes in cell properties lead to intratumor heterogeneity; however, the mechanisms of nongenetic cellular plasticity remain elusive. When the fate of each cell from colorectal cancer organoids was tracked through a clonogenic growth assay, the cells showed a wide range of growth ability even within the clonal organoids, consisting of distinct subpopulations; the cells generating large spheroids and the cells generating small spheroids. The cells from the small spheroids generated only small spheroids (S-pattern), while the cells from the large spheroids generated both small and large spheroids (D-pattern), both of which were tumorigenic. Transition from the S-pattern to the D-pattern occurred by various extrinsic triggers, in which Notch signaling and Musashi-1 played a key role. The S-pattern spheroids were resistant to chemotherapy and transited to the D-pattern upon drug treatment through Notch signaling. As the transition is linked to the drug resistance, it can be a therapeutic target.

Anteromedial Tibial Attachment in Single-Bundle Anterior Cruciate Ligament Reconstruction Can Represent Normal Kinematics in Computer Simulation.

Tunnel position during anterior cruciate ligament (ACL) reconstruction is considered as an important factor to restore normal knee kinematics and to gain better clinical outcomes. It is still unknown where the optimal femoral and tibial tunnel position is located in single-bundle (SB) ACL reconstruction. The purposes of this study were to analyze the knee kinematics with various graft positions and to propose the optimal graft position during SB ACL reconstruction. A musculoskeletal computer simulation was used to analyze knee kinematics. Four attachments on the femoral side (anteromedial [AM], mid, posterolateral [PL], and over-the-top positions) and three attachments on the tibial side (AM, middle, and PL positions) were determined. The middle-bundle attachment was placed at the midpoint of the AM and PL bundle attachments for the femoral and tibial attachments. SB ACL reconstruction models were constructed to combine each of the four femoral attachments with each of three tibial attachments. Kinematic comparison was made among a double-bundle (DB) model and 12 SB reconstruction models during deep knee bend and stair descent activity. The tunnel position of the tibia had greater effect of knee kinematics than that of the femur. AM tibial attachment models showed similar medial and lateral anteroposterior positions to the DB model for both activities. Axial rotation in the AM tibial attachment models was similar to the DB model regardless of the femoral attachment, whereas greater maximum axial rotation was exhibited in the PL tibial attachment models, especially during stair descent activity. AM tibial attachment can represent normal knee kinematics, whereas the PL tibial attachment can induce residual rotational instability during high-demand activities. The AM tibial tunnel is recommended for SB ACL reconstruction.

How does asymmetric tibial insert affect tibiofemoral kinematics and contact stresses in total knee Arthroplasty?

BACKGROUND: Asymmetric tibial insert design is expected to restore normal knee kinematics better than symmetric design. A tri-condylar implant has asymmetric and symmetric tibial inserts with a ball-and-socket joint to replace the post-cam mechanism. The purpose of this study was to compare the knee kinematics of the two designs and to measure tibiofemoral contact stresses, including that of the ball-and-socket joint. METHODS: Using a computer simulation, the anteroposterior position and axial rotation of the femoral component were simulated during a weight-bearing deep knee bend for six validated models. Contact forces were simultaneously simulated in the medial, lateral, and ball-and-socket compartments. The relative position and the magnitude and direction of each contact force were applied to aforce/displacement control knee simulator. The contact stresses were measured individually using a pressure sensor. RESULTS: The asymmetric tibial insert demonstrated a more posterior position of the femoral component in the lateral compartment during the entire range of motion and greater external rotation of the femoral component, compared to the symmetrical tibial insert. The mean peak contact stress of the medial and lateral compartments was < 9 Mpa, with no significant differences between the two designs except at 0°. The contact stress of the ball-and-socket joint was < 5 MPa. CONCLUSIONS: Asymmetry of the tibial insert shows significant kinematic difference and has little influence on the peak contact stress, which is considerably lower than the yield strength of polyethylene. The asymmetric tibial insert can lead to clinical benefits owing to its kinematic and kinetic properties.

Novel cancer subtyping method based on patient-specific gene regulatory network.

The identification of cancer subtypes is important for the understanding of tumor heterogeneity. In recent years, numerous computational methods have been proposed for this problem based on the multi-omics data of patients. It is widely accepted that different cancer subtypes are induced by different molecular regulatory networks. However, only a few incorporate the differences between their molecular systems into the identification processes. In this study, we present a novel method to identify cancer subtypes based on patient-specific molecular systems. Our method realizes this by quantifying patient-specific gene networks, which are estimated from their transcriptome data, and by clustering their quantified networks. Comprehensive analyses of The Cancer Genome Atlas (TCGA) datasets applied to our method confirmed that they were able to identify more clinically meaningful cancer subtypes than the existing subtypes and found that the identified subtypes comprised different molecular features. Our findings also show that the proposed method can identify the novel cancer subtypes even with single omics data, which cannot otherwise be captured by existing methods using multi-omics data.

Dynamic changes in gene-to-gene regulatory networks in response to SARS-CoV-2 infection.

The current pandemic of SARS-CoV-2 has caused extensive damage to society. The characterization of SARS-CoV-2 profiles has been addressed by researchers globally with the aim of resolving this disruptive crisis. This investigation process is indispensable to understand how SARS-CoV-2 behaves in human host cells. However, little is known about the systematic molecular mechanisms involved in the effects of SARS-CoV-2 infection on human host cells. Here, we present gene-to-gene regulatory networks in response to SARS-CoV-2 using a Bayesian network. We examined the dynamic changes in the SARS-CoV-2-purturbated networks established by our proposed framework for gene network analysis, thus revealing that interferon signaling gradually switched to the subsequent inflammatory cytokine signaling cascades. Furthermore, we succeeded in capturing a COVID-19 patient-specific network in which transduction of these signals was concurrently induced. This enabled us to explore the local regulatory systems influenced by SARS-CoV-2 in host cells more precisely at an individual level. Our panel of network analyses has provided new insights into SARS-CoV-2 research from the perspective of cellular systems.

Miniaturized String Sampling Probe and Electrospray Extraction/Ionization within the Ion Inlet Tube for Mass Spectrometric Endoscopy.

A moving string sampling probe and a new ESI based ionization source that can be readily incorporated into the existing endoscopes are developed for performing in vivo mass spectrometry during the endoscopic procedure. The medical-grade silk suture driven by a stepping motor is used to perform the sampling on the region of interest when the probe head is brought gently into contact with the surface of the gastrointestinal tissue. The tissues and the compounds adhered to the sampling string are transported to an ionization region inside the ion inlet tube in which they are extracted and ionized by the charging droplets generated from an electrospray outside the ion inlet. Since the extraction/ionization and sampling processes are isolated, organic solvents, high voltage (HV), and heating can be used for the optimization of ionization without compromising the biocompatibility of the sampling probe. The demonstration of the in vivo analysis of the gastric mucosa of a mouse is performed using a 2 m long gastrointestinal endoscope.

Sequential peripheral enrichment of H2A.Zac and H3K9me2 during trophoblast differentiation in human embryonic stem cells.

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure, involving covalent modification of histone tails, use of histone variants and gene position changes with respect to the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone modifications for changes in nuclear peripheral enrichment during differentiation of human embryonic stem cells to a trophoblast-like lineage. We found two dynamic modifications at the nuclear periphery, acetylation of histone H2A.Z (H2A.Zac), and dimethylation of histone H3 at lysine 9 (H3K9me2). We demonstrate successive peripheral enrichment of these markers, with H2A.Zac followed by H3K9me2, over the course of 4 days. We find that H3K9me2 increases concomitantly with, but independently of, expression of lamin A, since deletion of lamin A did not affect H3K9me2 enrichment. We further show that inhibition of histone deacetylases causes persistent and increased H2A.Z acetylation at the periphery, delayed H3K9me2 enrichment and failure to differentiate. Our results show a concerted change in the nature of peripheral chromatin occurs upon differentiation into the trophoblast state.

Large medial proximal tibial angles cause excessively medial tibiofemoral contact forces and abnormal knee kinematics following open-wedge high tibial osteotomy.

BACKGROUND: Recurrent varus deformity and poor outcome sometimes occur following open-wedge high tibial osteotomy, but the mechanism remains unclear. The hypothesis of this study was that an excessively large medial proximal tibial angle with lateral joint surface inclination can worsen postoperative knee biomechanics. METHODS: A computer-simulated knee model was validated based on a volunteer knee. Osteotomy models with medial proximal tibial angles ranging from 90° to 97° in 1° increments were developed. Varus alignment correction of the distal femur was performed in each model to maintain identical coronal alignment passing through a point 62.5% lateral to the tibial plateau. The peak tibiofemoral contact forces and knee kinematics were compared in each model during walking and squatting. FINDINGS: All the osteotomy models demonstrated higher peak contact forces on the lateral tibiofemoral joints than on the medial tibiofemoral joints during walking. However, larger medial proximal tibial angles caused excessive increases in medial tibiofemoral contact forces, and the dominant tibiofemoral contact forces shifted to the medial side. Increased medial proximal tibial angles also caused progressive medial collateral ligament tension in knee flexion, but partial medial collateral ligament release effectively reduced medial tibiofemoral contact forces. Models with large medial proximal tibial angles showed nonphysiological roll-forward of the lateral femoral condyle during squatting and no screw-home movement around knee extension. INTERPRETATION: Excessively large medial proximal tibial angles following open-wedge high tibial osteotomy resulted in increased medial tibiofemoral contact forces and abnormal knee kinematics during knee flexion due to medial joint line elevation and ligament imbalance.

Abnormal knee kinematics caused by mechanical alignment in symmetric bicruciate-retaining total knee arthroplasty are alleviated by kinematic alignment.

BACKGROUND: Bicruciate-retaining total knee arthroplasty (BCR-TKA) was developed to maintain anterior cruciate ligament function and thus reproduce natural knee kinematics postoperatively. Traditional surgical techniques, however, may cause several complications secondary to kinematic conflict and ligament overtension. The objective of this study was to use a computer simulation of symmetric BCR-TKA to evaluate the effects of alternative surgical techniques on knee kinematics and ligaments. METHODS: A musculoskeletal computer model of a healthy knee was constructed and was used to simulate a BCR model with mechanical alignment (MA). Five adjusted models were investigated, characterized, respectively, by kinematic alignment (KA), two degrees increased tibial slope, two-millimeter distal setting of the tibial component, and an undersized femoral component with either MA or KA. RESULTS: All models exhibited a normal femoral position against the tibia at knee extension, with no anterior paradoxical motion during mid-flexion. The healthy knee model showed medial pivot motion and rollback. In contrast, the BCR MA model demonstrated abnormal bi-condylar rollback with excessive tensions of the lateral collateral ligament and posterior cruciate ligament during knee flexion, whereas the undersized femoral model with MA partly reduced both tensions. The BCR KA model retained relatively physiological kinematics and suppressed excessive ligament tensions. However, no adjusted model completely reproduced healthy knee conditions. CONCLUSIONS: The BCR MA model showed abnormal biomechanics due to kinematic conflict between the retained ligaments and the replaced joint surface. Surgeons using symmetric BCR-TKA should consider using the KA method to achieve sufficient ligament laxity throughout knee flexion.

Length of anterior cruciate ligament affects knee kinematics and kinetics using a musculoskeletal computer simulation model.

INTRODUCTION: The tension of anterior cruciate ligament (ACL) graft has an important role in antero-posterior (AP) and rotational stability of the knee. The purposes of this study were to analyze the kinematics and kinetics of normal knee models with loose and tight ACL tension, and to evaluate the effect of the tension of ACL on knee kinematics and kinetics. MATERIALS AND METHODS: Slack and tight ACL models were constructed in a musculoskeletal computer simulation. The effect of ACL tension on kinematics, and femorotibial contact force during various activities was analyzed. RESULTS: During stair descent activity in the slack ACL models, the lateral femoral condyles were positioned posterior, and more external rotation of the femur was observed in comparison with the normal model. The contact forces at the lateral compartment in the tight models increased during all activities, and the tension of the medial collateral ligament (MCL) in the slack models increased during the stair descent activity, compared with the normal knee model. CONCLUSION: AP and rotational instability and excessive MCL tension were observed in the ACL slack knees especially during stair descent movement, whereas the tibiofemoral contact force of the lateral compartment increased in the tight ACL knees.

Soluble Vegfr3 gene therapy suppresses multi-organ metastasis in a mouse mammary cancer model.

Accumulating evidence on the association of VEGF-C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti-cancer therapy. To evaluate whether blocking VEGF-C and VEGFR-3 signaling can inhibit multi-organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR-3 expression vector (psVEGFR-3). We showed that psVEGFR-3 significantly diminished cell growth in vitro with or without added VEGF-C, and significantly reduced primary tumor growth and tumor metastases to wide-spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR-3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR-3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real-time RT-PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR-3 group. Immunofluorescence staining indicated that phospho-tyrosine expression was considerably lower in tumor cells of psVEGFR-3-treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho-tyrosine+ /LYVE-1+ (a lymphatic vessel marker) tended to decrease in psVEGFR-3-treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF-C/VEGFR-3 axis. These findings showed that a blockade of VEGF-C/VEGFR-3 signaling caused by sVEGFR-3 sequestered VEGF-C and prevented the side-effects of anti-angiogenesis and suppressed overall metastases, suggesting their high clinical significance.

Intraoperative physiological lateral laxity in extension and flexion for varus knees did not affect short-term clinical outcomes and patient satisfaction.

PURPOSE: Medial release during total knee arthroplasty (TKA) is used to correct ligament imbalance in knees with varus deformity. However, questions remain on whether residual ligament imbalance would be related to inferior clinical results. The purposes of the present study were to measure the intraoperative joint gap and to evaluate the effect of intraoperative soft tissue condition on the new Knee Society Score (KSS 2011) at 2-year follow-up, without the maneuver of additional medial release to correct the asymmetrical gap balance. METHODS: Varus-valgus gap angle and joint gap were measured using a tensor device without medial release for 100 knees with preoperative varus deformity. The knees were categorized according to the varus-valgus gap angle and the laxity. The preoperative and postoperative clinical outcomes using KSS 2011 were compared between the groups. RESULTS: The average varus-valgus angles had a residual imbalance of 2.8° varus and 1.3° varus in extension and flexion, respectively. In comparison, according to varus-valgus joint gap angle and knee laxity in extension and flexion, no significant differences were found in postoperative range of motion and subscale of KSS 2011 among the groups. CONCLUSION: Intraoperative asymmetrical joint gap and physiological laxity do not affect early clinical results after TKA. LEVEL OF EVIDENCE: III.

System-Based Differential Gene Network Analysis for Characterizing a Sample-Specific Subnetwork.

Gene network estimation is a method key to understanding a fundamental cellular system from high throughput omics data. However, the existing gene network analysis relies on having a sufficient number of samples and is required to handle a huge number of nodes and estimated edges, which remain difficult to interpret, especially in discovering the clinically relevant portions of the network. Here, we propose a novel method to extract a biomedically significant subnetwork using a Bayesian network, a type of unsupervised machine learning method that can be used as an explainable and interpretable artificial intelligence algorithm. Our method quantifies sample specific networks using our proposed Edge Contribution value (ECv) based on the estimated system, which realizes condition-specific subnetwork extraction using a limited number of samples. We applied this method to the Epithelial-Mesenchymal Transition (EMT) data set that is related to the process of metastasis and thus prognosis in cancer biology. We established our method-driven EMT network representing putative gene interactions. Furthermore, we found that the sample-specific ECv patterns of this EMT network can characterize the survival of lung cancer patients. These results show that our method unveils the explainable network differences in biological and clinical features through artificial intelligence technology.

Triterpenoid saponins with hepatoprotective effects from the fresh leaves of Metapanax delavayi.

The fresh leaves of Metapanax delavayi (Araliaceae) have been used as a common wild vegetable for salad and soup, and also herbal tea by the local people living in its growing areas of Yunnan province, China. Detailed chemical investigation led to the identification of a new triterpenoid saponin, 3-O-α-L-arabinopyranosyl-28-O-ß-D-glucopyranosyl-(1→6)-O-ß-D-glucopyranosyl-3ß-hydroxyolean-12-ene-28,29-dioic acid (1) from the fresh leaves, together with 11 known compounds, including six triterpenoid saponins (2-7), two caffeoylquinic acid derivatives (8-9), and three flavonoid glycosides (10-12). Their structures were determined on the basis of spectroscopic analysis and acidic hydrolysis. Compounds 3-5 and 8-12 were isolated from M. delavayi for the first time. Moreover, the known saponins 3-O-ß-D-xylopyranosyl-3ß-hydroxyolean-12-ene-28,29-dioic acid (3) and yiyeliangwanoside IV (5) exhibited protective effects on HepG2 cells damaged by the alcohol intakes, at a concentration of 1.0 µg/mL. The results indicated M. delavayi is an ideal dietary vegetable and herbal tea with potential hepatoprotective activity.[Formula: see text].

Classical target coronal alignment in high tibial osteotomy demonstrates validity in terms of knee kinematics and kinetics in a computer model.

PURPOSE: The purpose of this study was to determine the ideal coronal alignment under dynamic conditions after open-wedge high tibial osteotomy (OWHTO). It was hypothesised that, although the classical target alignment was based on experimental evidence, it would demonstrate biomechanical validity. METHODS: Musculoskeletal computer models were analysed with various degrees of coronal correction in OWHTO during gait and squat, specifically with the mechanical axis passing through points at 40%, 50%, 60%, 62.5%, 70%, and 80% of the tibial plateau from the medial edge, defined as the weight-bearing line percentage (WBL%). The peak load on the lateral tibiofemoral (TF) joint, the medial collateral ligament (MCL), and anterior cruciate ligament (ACL) tensions, and knee kinematics with or without increased posterior tibial slope (PTS) were evaluated. RESULTS: The classical alignment with WBL62.5% achieved sufficient load on the lateral TF joint and maintained normal knee kinematics after OWHTO. However, over-correction with WBL80% caused an excessive lateral load and non-physiological kinematics. Increased WBL% resulted in increased MCL tension due to lateral femoral movement against the tibia. With WBL80%, abnormal contact between the medial femoral condyle and the medial intercondylar eminence of the tibia occurred at knee extension. The screw-home movement around knee extension and the TF rotational angle during flexion were reduced as WBL% increased. Increased PTS was associated with increased ACL tension and decreased TF rotation angle because of ligamentous imbalance. CONCLUSIONS: The classical target alignment demonstrated validity in OWHTO, and over-correction should be avoided as it negatively impacts clinical outcome. LEVEL OF EVIDENCE: IV.

Data of dynamic microscale strain distributions of Ti-6Al-4V alloys in dwell fatigue tests.

Dynamic microscale strain distributions with temporal resolution of 1 s in a smooth and a cracked Ti-6Al-4V alloys during one-cycle dwell fatigue tests are illustrated in videos (URL: https://drive.google.com/drive/folders/1pit_VV2apGOpETVfaJAAtL5Xl2CNOiJ3?usp=sharing). The tensile strain distributions were measured by the video sampling moiré method from the 1-µm-pitch grid images in a scanning electron microscope. The strain concentration factors of the smooth and the cracked specimens are 1.96 and 2.65, respectively. The plastic strain increment is 0.0007 during the displacement holding time of 591s in the smoothed specimen at maximum stress of 900 MPa., and 0.0008 during the displacement holding time of 593s in the cracked specimen at maximum stress of 870 MPa. The typical strain results are analyzed in 1-s-resolved strain mapping in Ti-6Al-4V alloys during dwell fatigue in SEM by video sampling moiré [1].

Impact of intraoperative adjustment method for increased flexion gap on knee kinematics after posterior cruciate ligament-sacrificing total knee arthroplasty.

BACKGROUND: In general, the flexion gap is larger than the extension gap with posterior cruciate ligament-sacrificing total knee arthroplasty. Several methods compensate for an excessive flexion gap, but their effects are unknown. The purpose of this study was to compare three methods to compensate for an increased flexion gap. METHODS: In this study, squatting in knees with excessive (4 mm) and moderate (2 mm) flexion gaps was simulated in a computer model. Differences in knee kinematics and kinetics with joint line elevation, setting the femoral component in flexion, and using a larger femoral component as compensatory methods were investigated. FINDINGS: The rotational kinematics during flexion with setting the femoral component in flexion were opposite to those in the other models. Using a larger femoral component resulted in the most physiological motion. The peak anterior translation was 10 mm in the joint line elevation model compared with approximately 6 mm in the other models. In the joint line elevation model, patellofemoral contact stress was excessively increased at 90° of knee flexion. In contrast, tibiofemoral contact stress was higher during knee extension with setting the femoral component in flexion due to anterior impingement. There were few differences in the effect of the three compensatory methods with a moderate flexion gap. INTERPRETATION: A larger femoral component should be used to compensate for an excessive flexion gap because it has less negative impact on posterior cruciate ligament-sacrificing total knee arthroplasty, whereas any compensation method might be acceptable for a moderate flexion gap.

Delta-like 3 is silenced by HBx via histone acetylation in HBV-associated HCCs.

Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis. We previously showed that expression of Delta-like 3 (DLL3), a member of the family of Delta/Serrate/Lag2 ligands for the Notch receptor, is silenced by aberrant DNA methylation and that overexpression of DLL3 in an HCC cell line induces cellular apoptosis. However, how DLL3 expression is regulated during hepatocarcinogenesis is still unclear. Here, we show that silencing of DLL3 during hepatocarcinogenesis is closely related to viral infection, especially hepatitis B virus (HBV) infection (p = 0.005). HepG2.2.15 cells, which are stably transformed with the HBV genome, showed lower DLL3 expression than the parent cell line, HepG2 cells. Treatment with Hepatitis B virus X protein (HBx) small interfering RNA upregulated DLL3 expression in HepG2.2.15 cells, and overexpression of HBx in HepG2 cells downregulated DLL3 expression. Treatment of cells with a histone deacetylase inhibitor induced DLL3 expression in HepG2.2.15 cells. These data suggest that DLL3 expression is silenced during hepatocarcinogenesis in association with HBV infection via an epigenetic mechanism.