Plasma ctDNA increases tissue NGS-based detection of therapeutically targetable mutations in lung cancers.

BACKGROUND: Circulating tumor DNA (ctDNA) has been becoming a novel convenient and noninvasive method for dynamically monitoring landscape of genomic information to guild personalized cancer treatment. In this study we comprehensively evaluated the additional value of plasma ctDNA to routine tissue next generation sequencing (NGS) of therapeutically targetable mutations in lung cancers. METHODS: The tumor tissues and peripheral blood samples from 423 cases of patients with lung cancer were subjected to NGS of mutations in oncodrivers (EGFR, ERBB2, ALK, ROS1, C-MET, KRAS, BRAF, RET, BRCA1 and BRCA2). RESULTS: One hundred and ninety-seven cases showed both plasma and tissue positive and 96 showed both negative. The concordance for tissue and blood detection was 69.27% (293/423). 83 (19.62%) cases showed positive by tissue NGS alone and 47 (11.11%) positive by plasma ctDNA alone. The sensitivity of tissue and plasma detection was 85.63%, and 74.62%, respectively. Plasma had lower detection and sensitivity than tissue, but plasma additionally detected some important mutations which were omitted by tissue NGS. Plasma plus tissue increased the detection rate of 66.19% by tissue alone to 77.30% as well as the sensitivity of 85.63-100%. Similar results were also observed when the cases were classified into subpopulations according to different stages (IV vs. III vs. I-II), grades (low vs. middle grade) and metastatic status (metastasis vs. no metastasis). CONCLUSION: Plasma ctDNA shares a high concordance with tissue NGS, and plasma plus tissue enhances the detection rate and sensitivity by tissue alone, implying that the tissue and plasma detection should be mutually complementary in the clinical application.

Increasing coverage of mono-layer graphene grown on hexagonal boron nitride.

Graphene sitting on hexagonal boron nitride (h-BN) always exhibits excellent electrical properties. And the properties of graphene onh-BN are often dominated by its domain size and boundaries. Chemical vapor deposition (CVD) is a promising approach to achieve large size graphene crystal. However, the CVD growth of graphene onh-BN still faces challenges in increasing coverage of monolayer graphene because of a weak control on nucleation and vertical growth. Here, an auxiliary source strategy is adapted to increase the nucleation density of graphene onh-BN and synthesis continuous graphene films. It is found that both silicon carbide and organic polymer e.g. methyl methacrylate can assist the nucleation of graphene, and then increases the coverage of graphene onh-BN. By optimizing the growth temperature, vertical accumulation of graphitic materials can be greatly suppressed. This work provides an effective approach for preparing continuous graphene film onh-BN, and may bring a new sight for the growth of high quality graphene.

Towards chirality control of graphene nanoribbons embedded in hexagonal boron nitride.

The integrated in-plane growth of graphene nanoribbons (GNRs) and hexagonal boron nitride (h-BN) could provide a promising route to achieve integrated circuitry of atomic thickness. However, fabrication of edge-specific GNRs in the lattice of h-BN still remains a significant challenge. Here we developed a two-step growth method and successfully achieved sub-5-nm-wide zigzag and armchair GNRs embedded in h-BN. Further transport measurements reveal that the sub-7-nm-wide zigzag GNRs exhibit openings of the bandgap inversely proportional to their width, while narrow armchair GNRs exhibit some fluctuation in the bandgap-width relationship. An obvious conductance peak is observed in the transfer curves of 8- to 10-nm-wide zigzag GNRs, while it is absent in most armchair GNRs. Zigzag GNRs exhibit a small magnetic conductance, while armchair GNRs have much higher magnetic conductance values. This integrated lateral growth of edge-specific GNRs in h-BN provides a promising route to achieve intricate nanoscale circuits.

Dual-Scale Stick-Slip Friction on Graphene/h-BN Moiré Superlattice Structure.

Using atomic force microscopy, we have shown that friction on graphene/h-BN superlattice structures may exhibit unusual moiré-scale stick slip in addition to the regular ones observed at the atomic scale. Such dual-scale slip instability will lead to unique length-scale dependent energy dissipation when the different slip mechanisms are sequentially activated. Assisted by an improved theoretical model and comparative experiments, we find that accumulation and unstable release of the in-plane strain of the graphene layer is the key mechanism underlying the moiré-scale behavior. This work highlights the distinct role of the internal state of the van der Waals interfaces in determining the rich dynamics and energy dissipation of layer-structured materials.

Imaging Dual-Moiré Lattices in Twisted Bilayer Graphene Aligned on Hexagonal Boron Nitride Using Microwave Impedance Microscopy.

Moiré superlattices (MSLs) formed in van der Waals materials have become a promising platform to realize novel two-dimensional electronic states. Angle-aligned trilayer structures can form two sets of MSLs which could potentially interfere. In this work, we directly image the moiré patterns in both monolayer and twisted bilayer graphene aligned on hexagonal boron nitride (hBN), using combined scanning microwave impedance microscopy and conductive atomic force microscopy. Correlation of the two techniques reveals the contrast mechanism for the achieved ultrahigh spatial resolution (<2 nm). We observe two sets of MSLs with different periodicities in the trilayer stack. The smaller MSL breaks the 6-fold rotational symmetry and exhibits abrupt discontinuities at the boundaries of the larger MSL. Using a rigid atomic-stacking model, we demonstrate that the hBN layer considerably modifies the MSL of twisted bilayer graphene. We further analyze its effect on the reciprocal space spectrum of the dual-moiré system.

Weak localization in graphene sandwiched by aligned h-BN flakes.

Charge carriers in graphene exhibit distinct characteristics from those in other two-dimensional materials because of their chiral nature. Additionally, multiple Dirac cones that emerge in graphene superlattices have been regarded as an interesting point in condensed-matter physics in recent years. Here, we report an investigation of the magneto-conductance in graphene encapsulated on the top and bottom by aligned h-BN. The bottom h-BN is precisely aligned with graphene, while the top h-BN is rotated a very small angle relative to it. Such a heterostructure could spoil the commensurate state existing in precisely aligned graphene while the giant moiré superlattice remains. A clear signature of weak localization and weak anti-localization is observed at multiple Dirac cones. Both the weak (anti)localization and the universal conductance fluctuations exhibit strong dependencies on the carrier density, temperature and channel length. This artificial heterostructure allows one to explore quantum interference in graphene with a wide spectrum of electronic properties.

Nrf2/ARE pathway activation is involved in negatively regulating heat-induced apoptosis in non-small cell lung cancer cells.

Hyperthermia, particularly in combination with chemoradiotherapy, is widely used to treat various cancers. However, hyperthermia treatment is often insufficient due to thermo-tolerance. To date, the detailed mechanism underlying thermo-tolerance has not been clarified. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway is an important cellular cytoprotective defense system that is activated by various stresses. In this study, using immunocytochemistry and western blot analysis, we demonstrated that heat stress induced Nrf2/ARE activation through the nuclear translocation of Nrf2 in non-small cell lung cancer cells. Luciferase activity was also increased. Additionally, antioxidant enzymes were increased through Nrf2 activation after heat stress. Transfection of lung cancer cells with siRNA directed against Nrf2 increased heat cytotoxicity and cell apoptosis. Heat stress could induce reactive oxygen species (ROS) accumulation, while the antioxidant NAC obviously reduced cell apoptosis ratio, indicating that heat stress induced cell apoptosis in a ROS-dependent manner. Knockdown of Nrf2 led to an abnormal elevation of ROS, and the antioxidant NAC could increase Nrf2 activation, indicating that ROS and Nrf2 act within a negative feedback loop. Taken together, these results demonstrated that Nrf2 pathway is important for maintaining resistance to heat stress, and we postulated that Nrf2 may represent a potential therapeutic target for hyperthermia in lung cancer.

Bombesin receptor-activated protein regulates neutrophil elastase-induced mucin5AC hypersecretion in human bronchial epithelial cells.

Bombesin receptor-activated protein (BRAP) is highly expressed in human bronchial epithelial cells. Recent studies have shown that BRAP reduces oxidative stress, inhibits airway inflammation and suppresses nuclear factor kappaB (NF-κB) activity. Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. Neutrophil elastase (NE) is a potent inducer of mucin5AC (MUC5AC), which is considered the predominant mucin secreted by human airway epithelial cells. Here, we hypothesize that BRAP may regulate NE-induced MUC5AC hypersecretion in a bronchial epithelial cell line (HBE16). We also investigated the underlying mechanism involved in the process. In this study, we found that BRAP was present in HBE16 human bronchial epithelial cells and was significantly increased by NE. Next, we found that the up-regulation of BRAP by pEGFP-N1-BRAP caused a significant decrease in the increased levels of MUC5AC expression, NF-κB activity, and the phosphorylation of extracellular signal-regulated kinases (ERK) and epidermal growth factor receptor (EGFR) induced by NE. Meanwhile, there was a significant decrease in ROS, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) levels when BRAP was up-regulated by pEGFP-N1-BRAP. Moreover, when cells were transfected with pEGFP-N1-BRAP and pretreated with NF-κB, ERK or EGFR inhibitors before the NE stimulation, there were further decreased in MUC5AC expression, NF-κB activity, and the phosphorylation of ERK and EGFR. These results suggest that BRAP plays an important role in airway inflammation and its overexpression may regulate NE-induced MUC5AC hypersecretion in HBE16 cells via the EGFR/ERK/NF-κB signaling pathway.

Molecular network-based analysis of the mechanism of liver injury induced by volatile oils from Artemisiae argyi folium.

BACKGROUND: Volatile oils from Artemisiae argyi folium (VOAAF) is reported with hepatotoxicity, but the underlying mechanism is still unclear. METHODS: In the present study this molecular mechanism was explored with the Ingenuity Pathway Analysis (IPA). The chemical components of the VOAAF were searched in the database, and their target proteins were all identified in the PubChem, while drug-induced liver injury (DILI) genes were searched in the PubMed gene databases. The molecular network of protein targets for VOAAF and DILI genes was built with the IPA. The canonical pathways between the 2 networks were compared to decipher the molecular mechanisms of the liver injury induced by VOAAF. RESULTS: There were 159 target proteins for VOAAF and 338 genes related to DILI identified, which were further analyzed in the IPA. The canonical pathway comparison showed that VOAAF and DILI both worked on aryl hydrocarbon receptor (AHR), lipopolysaccharide (LPS)/interleukin 1 (IL-1) mediated inhibition of retinoid X receptor (RXR) function, pregnane X receptor (PXR)/RXR activation, xenobiotic metabolism, peroxisome proliferator-activated receptor (PPAR), hepatic cholestasis, farnesoid X receptor (FXR)/RXR activation, and glucocorticoid receptor. CONCLUSION: VOAAF-induced liver injury may be involved in many pathways in which the AHR signaling and LPS/IL-1 mediated inhibition of RXR function pathways could be the most vital.

Inhibition of autophagy enhances heat-induced apoptosis in human non-small cell lung cancer cells through ER stress pathways.

The occurrence and mechanisms of autophagy induced by heat stress are not well known in lung cancer cells. Here, we have demonstrated that heat stress induces autophagy in A549 and NCI-H460 cells through morphological and biochemical analyses. The inhibition of autophagy by chloroquine, 3-methyladenine and Beclin 1 siRNA enhanced heat-induced apoptosis. Moreover, the combination of chloroquine and heat stress inhibited tumor growth and enhanced apoptosis in vivo experiments. In addition, heat-induced autophagy involved the ER stress pathway (PERK- or IRE1-dependent). Further, heat treatment led to the increased phosphorylation of AMPK and the decreased phosphorylation of mTOR in vitro and in vivo. Knockdown of GRP78 inhibited the AMPK-mTOR pathway, and the AMPK inhibitor compound C decreased heat-induced autophagy, suggesting that activation of ER stress was involved in autophagy induction and promotion of the AMPK-mTOR pathway. In conclusion, our data suggested that the heat treatment of lung cancer cells triggered protective autophagy, as mediated by ER stress. Thus, inhibition of autophagy can be a promising strategy to enhance hyperthermia in the treatment of lung cancer patients.

Cold-inducible RNA binding protein regulates mucin expression induced by cold temperatures in human airway epithelial cells.

Mucus overproduction is an important manifestation of chronic airway inflammatory diseases, however, the mechanisms underlying the association between cold air and mucus overproduction remain unknown. We found that the expression of the cold-inducible RNA binding protein (CIRP) was increased in patients with chronic obstructive pulmonary disease (COPD). In the present study, we tested whether CIRP was involved in inflammatory factors and mucin5AC (MUC5AC) expression after cold stimulation and investigated the potential signaling pathways involved in this process. We found that CIRP was highly expressed in the bronchi of COPD patients. The expression of CIRP, interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were increased, and the CIRP was localized in cytoplasm after cold stimulation. MUC5AC mRNA and protein expression levels were elevated in a temperature- and time-dependent manner after cold stimulation and were associated with the phosphorylation of ERK and NF-κB, which reflected their activation. These responses were suppressed by knockdown of CIRP with a specific siRNA or the ERK and NF-κB inhibitors. These results demonstrated that CIRP was expressed in the bronchi of human COPD patients and was involved in inflammatory factors and MUC5AC expression after cold stimulation through the ERK and NF-κB pathways.

Acid-induced autophagy protects human lung cancer cells from apoptosis by activating ER stress.

An acidic tumor microenvironment exists widely in solid tumors. However, the detailed mechanism of cell survival under acidic stress remains unclear. The aim of this study is to clarify whether acid-induced autophagy exists and to determine the function and mechanism of autophagy in lung cancer cells. We have found that acute low pH stimulated autophagy by increasing LC3-positive punctate vesicles, increasing LC3 II expression levels and reducing p62 protein levels. Additionally, autophagy was inhibited by the addition of Baf or knockdown of Beclin 1, and cell apoptosis was increased markedly. In mouse tumors, the expression of cleaved caspase3 and p62 was enhanced by oral treatment with sodium bicarbonate, which can raise the intratumoral pH. Furthermore, the protein levels of ER stress markers, including p-PERK, p-eIF2α, CHOP, XBP-1s and GRP78, were also increased in response to acidic pH. The antioxidant NAC, which reduces ROS accumulation, alleviated acid-mediated ER stress and autophagy, and knocking down GRP78 reduced autophagy activation under acidic conditions, which suggests that autophagy was induced by acidic pH through ER stress. Taken together, these results indicate that the acidic microenvironment in non-small cell lung cancer cells promotes autophagy by increasing ROS-ER stress, which serves as a survival adaption in this setting.

Exploring Urban Green Space Optimization of the Urban Walking Life Circle in Fuzhou, China.

The spatial distribution of urban green spaces (UGS) is closely related to the health of residents and the ecological pattern of cities. Exploring the equity of UGSs plays an important role in urban planning and also provides guidance for urban development. Taking the main urban area of Fuzhou City as an example, this study uses network big data and census data to pinpoint the population demand, evaluates the accessibility and equity of UGS within the basic living circle, neighborhood living circle and daily living circle of residents at the scale of residential and sub-districts. Based on the G2SFCA model, we also quantify the actual effective UGS's service capacity. Then, using the scale and travel range as the entry point, we further discuss the similarities and differences under different scales and different travel ranges. Finally, optimization strategies are proposed for the construction status. The results show that: (1) The spatial allocation of urban green space resources varies significantly, and there is a serious inequity in the spatial distribution of urban green space under pedestrian conditions; (2) The results of UGS accessibility, equity, and service capacity in Fuzhou at both residential and sub-district scales are consistent; (3) Urban construction should be multi-level overall planning, combined with local economic and social development factors in accordance with local conditions to take measures. The results of the study can provide a scientific reference for the optimization of the spatial distribution of UGS.

Minimizing the Programming Power of Phase Change Memory by Using Graphene Nanoribbon Edge-Contact.

Nonvolatile phase-change random access memory (PCRAM) is regarded as one of the promising candidates for emerging mass storage in the era of Big Data. However, relatively high programming energy hurdles the further reduction of power consumption in PCRAM. Utilizing narrow edge-contact of graphene can effectively reduce the active volume of phase change material in each cell, and therefore realize low-power operation. Here, it demonstrates that the power consumption can be reduced to ≈53.7 fJ in a cell with ≈3 nm-wide graphene nanoribbon (GNR) as edge-contact, whose cross-sectional area is only ≈1 nm2 . It is found that the polarity of the bias pulse determines its cycle endurance in the asymmetric structure. If a positive bias is applied to the graphene electrode, the endurance can be extended at least one order longer than the case with a reversal of polarity. In addition, the introduction of the hexagonal boron nitride (h-BN) multilayer leads to a low resistance drift and a high programming speed in a memory cell. The work represents a great technological advance for the low-power PCRAM and can benefit in-memory computing in the future.

Cold-inducible RNA-binding protein migrates from the nucleus to the cytoplasm under cold stress in normal human bronchial epithelial cells via TRPM8-mediated mechanism.

BACKGROUND: Cold-inducible RNA-binding protein (CIRP or hnRNP A18) is a multifunctional stress-responsive protein. Our previous study demonstrated that cold stress increased CIRP expression and migrated from the nucleus to the cytoplasm in airway epithelial cells. However, the mechanism through which CIRP migrates from the nucleus to the cytoplasm upon cold stress remains unknown. METHODS: The expression of CIRP in the bronchial epithelium was examined using immunofluorescence, real-time polymerase chain reaction (RT-PCR), and Western blotting. The expression of inflammatory factors interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) were detected by ELISA and RT-PCR. Transient receptor potential melastatin 8 (TRPM8) receptor function was characterized by Ca2+ imaging. RESULTS: Cold stress upregulated the expression of CIRP, inflammatory factors and promoted the translocation of CIRP from the nucleus to the cytoplasm in normal human bronchial epithelial (NHBE) cells. Cold stress activated the TRPM8/(Ca2+)/PKCα/glycogen synthase kinase 3ß (GSK3ß) signaling cascade, and that inhibition of this signaling pathway attenuated the migration of CIRP from the nucleus to cytoplasm but did not decrease its overexpression induced by cold stress. Knocked down CIRP expression or blocked CIRP migration between the nucleus and cytoplasm significantly decreased inflammatory factor expression. CONCLUSIONS: These results indicate that cold stress leads to the migration of CIRP from the nucleus to the cytoplasm with alteration of expression, which are involved in the expression of inflammatory factors (IL-1ß, IL-6, IL-8 and TNF-α) induced by cold air, through TRPM8/Ca2+/PKCα/GSK3ß signaling cascade.

The Degradation of Airway Epithelial Tight Junctions in Asthma Under High Airway Pressure Is Probably Mediated by Piezo-1.

Full functioning of the airway physical barrier depends on cellular integrity, which is coordinated by a series of tight junction (TJ) proteins. Due to airway spasm, edema, and mucus obstruction, positive end-expiratory alveolar pressure (also termed auto-PEEP) is a common pathophysiological phenomenon, especially in acute asthma attack. However, the influence of auto-PEEP on small airway epithelial TJs is currently unclear. We performed studies to investigate the effect of extra pressure on small airway epithelial TJs and its mechanism. The results first confirmed that a novel mechanosensitive receptor, piezo-1, was highly expressed in the airway epithelium of asthmatic mice. Extra pressure induced the degradation of occludin, ZO-1 and claudin-18 in primary human small airway epithelial cells (HSAECs), resulting in a decrease in transepithelial electrical resistance (TER) and an increase in cell layer permeability. Through in vitro investigations, we observed that exogenous pressure stimulation could elevate the intracellular calcium concentration ([Ca2+] i ) in HSAECs. Downregulation of piezo-1 with siRNA and pretreatment with BAPTA-AM or ALLN reduced the degradation of TJs and attenuated the impairment of TJ function induced by exogenous pressure. These findings indicate the critical role of piezo-1/[Ca2+] i /calpain signaling in the regulation of small airway TJs under extra pressure stimulation.

miR-939-3p promotes epithelial-mesenchymal transition and may be used as a prognostic marker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide with a high morbidity and mortality rate. An increasing number of studies have demonstrated that microRNAs (miRNAs) serve an important role in HCC. The present study investigated the role of miR-939-3p in HCC. It was demonstrated that miR-939-3p was upregulated in HCC cell lines and HCC tissues compared with normal liver cell lines and paired normal tissues, respectively. It was also found that upregulation of miR-939-3p expression levels in HCC tissues was associated with a less favorable prognosis. Moreover, the overexpression of miR-939-3p in LM3 cells enhanced the metastatic capacity of these cells and promoted epithelial-mesenchymal transition (EMT). In contrast, miR-939-3p inhibition decreased the invasive capacity of HCC cells and EMT. Potential binding target of miR-939-3p to estrogen receptor 1 (ESR1) were predicted using TargetScan. The expression levels of miR-939-3p were negatively associated with ESR1 in HCC tissues based on data from The Cancer Genome Atlas. A luciferase reporter assay was used to confirm ESR1 as a direct downstream target of miR-393-3p. The miR-939-3p/ESR1 axis may be a potential novel target for the treatment of HCC.

Abnormal conductivity in low-angle twisted bilayer graphene.

Controlling the interlayer twist angle offers a powerful means for tuning the electronic properties of two-dimensional (2D) van der Waals materials. Typically, the electrical conductivity would increase monotonically with decreasing twist angle owing to the enhanced coupling between adjacent layers. Here, we report a nonmonotonic angle-dependent vertical conductivity across the interface of bilayer graphene with low twist angles. More specifically, the vertical conductivity enhances gradually with decreasing twist angle up to a crossover angle at θc ≈ 5°, and then it drops notably upon further decrease in the twist angle. Revealed by density functional theory calculations and scanning tunneling microscopy, the abnormal behavior is attributed to the unusual reduction in average carrier density originating from local atomic reconstruction. The impact of atomic reconstruction on vertical conductivity is unique for low-angle twisted 2D van der Waals materials and provides a strategy for designing and optimizing their electronic performance.

Isolating hydrogen in hexagonal boron nitride bubbles by a plasma treatment.

Atomically thin hexagonal boron nitride (h-BN) is often regarded as an elastic film that is impermeable to gases. The high stabilities in thermal and chemical properties allow h-BN to serve as a gas barrier under extreme conditions. Here, we demonstrate the isolation of hydrogen in bubbles of h-BN via plasma treatment. Detailed characterizations reveal that the substrates do not show chemical change after treatment. The bubbles are found to withstand thermal treatment in air, even at 800 °C. Scanning transmission electron microscopy investigation shows that the h-BN multilayer has a unique aligned porous stacking nature, which is essential for the character of being transparent to atomic hydrogen but impermeable to hydrogen molecules. In addition, we successfully demonstrated the extraction of hydrogen gases from gaseous compounds or mixtures containing hydrogen element. The successful production of hydrogen bubbles on h-BN flakes has potential for further application in nano/micro-electromechanical systems and hydrogen storage.

Tree growth traits and social status affect the wood density of pioneer species in secondary subtropical forest.

Wood density (WD) is not only an important parameter to estimate aboveground biomass but also an indicator of timber quality and plant adaptation strategies to stressful conditions (i.e., windthrow, pests, and pathogens). This study had three objectives: (1) to compare WD among seven subtropical tree species; (2) to determine how tree growth traits may influence possible differences in WD between the pioneer and shade-tolerant species; and (3) to examine whether or not WD differs by tree social status (dominant vs. suppressed trees) within species. To do this, 70 trees were destructively harvested. From each tree, disks at different stem heights were obtained and subjected to a method of stem analysis to measure whole tree level WD. The results showed that WD differed significantly among the seven species (p < .001). Their average WD was 0.537 g/cm3, ranging from 0.409 g/cm3 for Choerospondias axillaris to 0.691 g/cm3 for Cyclobalanopsis glauca. The average WD of the four pioneer species (0.497 ± 0.13 g/cm3) was significantly lower (p < .01) than that of the three shade-tolerant species (0.589 ± 0.12 g/cm3). The WD of the pioneers had a significant positive correlation with their stem diameter at breast height (DBH), tree height (H), and tree age, but WD had a significant negative correlation with relative growth rate (RGR). In contrast, the WD of the shade-tolerant tree species had no significant relationships with DBH, H, tree age, or RGR. The dominant trees of the pioneer species had a higher WD than the suppressed trees, whereas the shade-tolerant species had a lower WD for dominant trees than the suppressed trees. However, the differences in WD between dominant and suppressed trees were not significant. Taken together, the results suggest that classifying species into pioneer and shade-tolerant groups to examine the effects of tree growth traits and social status could improve our understanding of intra- and interspecific variation in WD among subtropical tree species.