Rear cortex contraction aids in nuclear transit during confined migration by increasing pressure in the cell posterior.

As cells migrate through biological tissues, they must frequently squeeze through micron-sized constrictions in the form of interstitial pores between extracellular matrix fibers and/or other cells. Although it is now well recognized that such confined migration is limited by the nucleus, which is the largest and stiffest organelle, it remains incompletely understood how cells apply sufficient force to move their nucleus through small constrictions. Here, we report a mechanism by which contraction of the cell rear cortex pushes the nucleus forward to mediate nuclear transit through constrictions. Laser ablation of the rear cortex reveals that pushing forces behind the nucleus are the result of increased intracellular pressure in the rear compartment of the cell. The pushing forces behind the nucleus depend on accumulation of actomyosin in the rear cortex and require Rho kinase (ROCK) activity. Collectively, our results suggest a mechanism by which cells generate elevated intracellular pressure in the posterior compartment to facilitate nuclear transit through three-dimensional (3D) constrictions. This mechanism might supplement or even substitute for other mechanisms supporting nuclear transit, ensuring robust cell migrations in confined 3D environments.

3D Traction Force Microscopy in Biological Gels: From Single Cells to Multicellular Spheroids.

Cell traction force plays a critical role in directing cellular functions, such as proliferation, migration, and differentiation. Current understanding of cell traction force is largely derived from 2D measurements where cells are plated on 2D substrates. However, 2D measurements do not recapitulate a vital aspect of living systems, that is, cells actively remodel their surrounding extracellular matrix (ECM), and the remodeled ECM, in return, can have a profound impact on cell phenotype and traction force generation. This reciprocal adaptivity of living systems is encoded in the material properties of biological gels. In this review, we summarize recent progress in measuring cell traction force for cells embedded within 3D biological gels, with an emphasis on cell-ECM cross talk. We also provide perspectives on tools and techniques that could be adapted to measure cell traction force in complex biochemical and biophysical environments. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 26 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Knockdown of circZMIZ1 enhances the anti-tumor activity of CD8+ T cells to alleviate hepatocellular carcinoma.

ZMIZ1 acts as an oncogene in hepatocellular carcinoma (HCC). circZMIZ1 (hsa_circ_0018964) derives from ZMIZ1; its underlying mechanism in HCC has not been reported. Peripheral blood and peripheral blood mononuclear cells (PBMCs) were obtained from HCC patients and healthy volunteers. CD8+ T cells were sorted from PBMCs of HCC patients. Applying flow cytometry, cell apoptosis and the proportion of KCNJ2/CD8+ T cells were examined. The cytotoxicity of CD8+ T cells against HCC cells was evaluated. The interaction among circZMIZ1, miR-15a-5p, and KCNJ2 was investigated by dual luciferase assay, RNA immunoprecipitation, and RNA pull-down assay. An orthotopic mouse model of HCC was constructed by intrahepatic injection of H22 cells. Upregulation of circZMIZ1 and KCNJ2 and downregulation of miR-15a-5p were observed in peripheral blood and PBMCs of HCC patients. The proportion of KCNJ2/CD8+ T cells was also increased in HCC patients. circZMIZ1 knockdown restrained apoptosis of CD8+ T cells and elevated cytotoxicity of CD8+ T cells. Mechanically speaking, circZMIZ1 elevated KCNJ2 expression by sponging miR-15a-5p. miR-15a-5p inhibitor reversed circZMIZ1 silencing-mediated inhibition of apoptosis and promotion of cytotoxicity in CD8+ T cells. In vivo, orthotopic mice of HCC exhibited increased expression of circZMIZ1 and KCNJ2, elevated proportion of KCNJ2/CD8+ T cells, and decreased expression of miR-15a-5p. This work demonstrated that circZMIZ1 inhibited the anti-tumor activity of CD8+ T cells in HCC by regulating the miR-15a-5p/KCNJ2 axis. This provides a theoretical basis for the development of effective circZMIZ1 in tumor immunotherapy.

Impact of baseline adipose tissue characteristics on change in adipose tissue volume during a low calorie diet in people with obesity-results from the LION study.

BACKGROUND/OBJECTIVES: Weight loss outcomes vary individually. Magnetic resonance imaging (MRI)-based evaluation of adipose tissue (AT) might help to identify AT characteristics that predict AT loss. This study aimed to assess the impact of an 8-week low-calorie diet (LCD) on different AT depots and to identify predictors of short-term AT loss using MRI in adults with obesity. METHODS: Eighty-one adults with obesity (mean BMI 34.08 ± 2.75 kg/m², mean age 46.3 ± 10.97 years, 49 females) prospectively underwent baseline MRI (liver dome to femoral head) and anthropometric measurements (BMI, waist-to-hip-ratio, body fat), followed by a post-LCD-examination. Visceral and subcutaneous AT (VAT and SAT) volumes and AT fat fraction were extracted from the MRI data. Apparent lipid volumes based on MRI were calculated as approximation for the lipid contained in the AT. SAT and VAT volumes were subdivided into equidistant thirds along the craniocaudal axis and normalized by length of the segmentation. T-tests compared baseline and follow-up measurements and sex differences. Effect sizes on subdivided AT volumes were compared. Spearman Rank correlation explored associations between baseline parameters and AT loss. Multiple regression analysis identified baseline predictors for AT loss. RESULTS: Following the LCD, participants exhibited significant weight loss (11.61 ± 3.07 kg, p < 0.01) and reductions in all MRI-based AT parameters (p < 0.01). Absolute SAT loss exceeded VAT loss, while relative apparent lipid loss was higher in VAT (both p < 0.01). The lower abdominopelvic third showed the most significant SAT and VAT reduction. The predictor of most AT and apparent lipid losses was the normalized baseline SAT volume in the lower abdominopelvic third, with smaller volumes favoring greater AT loss (p < 0.01 for SAT and VAT loss and SAT apparent lipid volume loss). CONCLUSIONS: The LCD primarily reduces lower abdominopelvic SAT and VAT. Furthermore, lower abdominopelvic SAT volume was detected as a potential predictor for short-term AT loss in persons with obesity.

Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices (ECMs) on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D ECMs. Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlight the importance of tumor mechanics and invasion.

Silicone implant and fibrous capsule assessment based on water-fat-silicone chemical shift encoding-based species separation in breast MRI.

BACKGROUND: With rising breast augmentations worldwide, there is an increasing clinical need for an early and accurate detection of implant complications. PURPOSE: To compare the quality of chemical shift encoding-based (CSE) water-fat-silicone separation compared to double inversion recovery (DIR) silicone-only imaging in breast magnetic resonance imaging (MRI). MATERIAL AND METHODS: This retrospective, single-center study included women with silicone implants subjected to 3-T MRI between January 2021 and March 2022. MRI included (i) two-dimensional silicone-only T2-weighted turbo spin echo DIR acquisition and (ii) three-dimensional CSE imaging based on multi-echo gradient-echo sequence enabling water-, fat-, and silicone-image separation. Images were evaluated and compared by three independent radiologists using a clinically established rating including differentiability of the silicone implant, visibility and contouring of the adjacent fibrous capsule, and accuracy of intralesional folds in a ranking of 1-5. The apparent contrast-to-noise (aCNR) was calculated. RESULTS: In 71 women, the average quality of water-fat-silicone images from CSE imaging was assessed as "good" (assessment 4 ± 0.9). In 68 (96%) patients, CSE imaging achieved a concise delineation of the silicone implant and precise visualization of the fibrous capsule that was not distinguishable in DIR imaging. Implant ruptures were more easily detected in CSE imaging. The aCNR was higher in CSE compared to DIR imaging (18.43 ± 9.8 vs. 14.73 ± 2.5; P = 0.002). CONCLUSION: Intrinsically co-registered water-fat-silicone-separated CSE-based images enable a reliable assessment of silicone implants. The simultaneously improved differentiability of the implant and fibrous capsule may provide clinicians with a valuable tool for an accurate evaluation of implant integrity and early detection of potential complications.

Identification and Functional Analysis of KH Family Genes Associated with Salt Stress in Rice.

Salinity stress has a great impact on crop growth and productivity and is one of the major factors responsible for crop yield losses. The K-homologous (KH) family proteins play vital roles in regulating plant development and responding to abiotic stress in plants. However, the systematic characterization of the KH family in rice is still lacking. In this study, we performed genome-wide identification and functional analysis of KH family genes and identified a total of 31 KH genes in rice. According to the homologs of KH genes in Arabidopsis thaliana, we constructed a phylogenetic tree with 61 KH genes containing 31 KH genes in Oryza sativa and 30 KH genes in Arabidopsis thaliana and separated them into three major groups. In silico tissue expression analysis showed that the OsKH genes are constitutively expressed. The qRT-PCR results revealed that eight OsKH genes responded strongly to salt stresses, and OsKH12 exhibited the strongest decrease in expression level, which was selected for further study. We generated the Oskh12-knockout mutant via the CRISPR/Cas9 genome-editing method. Further stress treatment and biochemical assays confirmed that Oskh12 mutant was more salt-sensitive than Nip and the expression of several key salt-tolerant genes in Oskh12 was significantly reduced. Taken together, our results shed light on the understanding of the KH family and provide a theoretical basis for future abiotic stress studies in rice.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Paracrine secretion of IL8 by breast cancer stem cells promotes therapeutic resistance and metastasis of the bulk tumor cells.

BACKGROUND: Breast tumors consist of heterogeneous cellular subpopulations that differ in molecular properties and functional attributes. Cancer stem cells (CSCs) play pivotal roles in cancer therapeutic failure and metastasis. However, it remains indeterminate how CSCs determine the progression of the bulk cancer cell population. METHODS: Co-culture systems in vitro and co-implantation systems in vivo were designed to characterize the interactions between breast cancer stem cells (BCSCs) and bulk cancer cells. RNA sequencing was performed to study the functional and mechanistic implications of the BCSC secretome on bulk cancer cells. A cytokine antibody array was employed to screen the differentially secreted cytokines in the BCSC secretome. Tail vein injection metastatic models and orthotopic xenograft models were applied to study the therapeutic potential of targeting IL8. RESULTS: We identified that the BCSC secretome potentiated estrogen receptor (ER) activity in the bulk cancer cell population. The BCSC secretome rendered the bulk cancer cell population resistant to anti-estrogen and CDK4/6 inhibitor therapy; as well as increased the metastatic burden attributable to bulk cancer cells. Screening of the BCSC secretome identified IL8 as a pivotal factor that potentiated ERα activity, endowed tamoxifen resistance and enhanced metastatic burden by regulation of bulk cancer cell behavior. Pharmacological inhibition of IL8 increased the efficacy of fulvestrant and/or palbociclib by reversing tamoxifen resistance and abrogated metastatic burden. CONCLUSION: Taken together, this study delineates the mechanism by which BCSCs determine the therapeutic response and metastasis of bulk cancer cells; and thereby suggests potential therapeutic strategies to ameliorate breast cancer outcomes. Video Abstract.

Assessing breast density using the chemical-shift encoding-based proton density fat fraction in 3-T MRI.

OBJECTIVES: There is a clinical need for a non-ionizing, quantitative assessment of breast density, as one of the strongest independent risk factors for breast cancer. This study aims to establish proton density fat fraction (PDFF) as a quantitative biomarker for fat tissue concentration in breast MRI and correlate mean breast PDFF to mammography. METHODS: In this retrospective study, 193 women were routinely subjected to 3-T MRI using a six-echo chemical shift encoding-based water-fat sequence. Water-fat separation was based on a signal model accounting for a single T2* decay and a pre-calibrated 7-peak fat spectrum resulting in volumetric fat-only, water-only images, PDFF- and T2*-values. After semi-automated breast segmentation, PDFF and T2* values were determined for the entire breast and fibroglandular tissue. The mammographic and MRI-based breast density was classified by visual estimation using the American College of Radiology Breast Imaging Reporting and Data System categories (ACR A-D). RESULTS: The PDFF negatively correlated with mammographic and MRI breast density measurements (Spearman rho: -0.74, p < .001) and revealed a significant distinction between all four ACR categories. Mean T2* of the fibroglandular tissue correlated with increasing ACR categories (Spearman rho: 0.34, p < .001). The PDFF of the fibroglandular tissue showed a correlation with age (Pearson rho: 0.56, p = .03). CONCLUSION: The proposed breast PDFF as an automated tissue fat concentration measurement is comparable with mammographic breast density estimations. Therefore, it is a promising approach to an accurate, user-independent, and non-ionizing breast density assessment that could be easily incorporated into clinical routine breast MRI exams. KEY POINTS: • The proposed PDFF strongly negatively correlates with visually determined mammographic and MRI-based breast density estimations and therefore allows for an accurate, non-ionizing, and user-independent breast density measurement. • In combination with T2*, the PDFF can be used to track structural alterations in the composition of breast tissue for an individualized risk assessment for breast cancer.

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique.

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a a machine learning-assisted FRET technique to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.

Comparative Transcriptome Analysis of the Heterosis of Salt Tolerance in Inter-Subspecific Hybrid Rice.

Soil salinity is one of the major abiotic stresses limiting rice growth. Hybrids outperform their parents in salt tolerance in rice, while its mechanism is not completely understood. In this study, a higher seedling survival was observed after salt treatment in an inter-subspecific hybrid rice, Zhegengyou1578 (ZGY1578), compared with its maternal japonica Zhegeng7A (ZG7A) and paternal indica Zhehui1578 (ZH1578). A total of 2584 and 3061 differentially expressed genes (DEGs) with at least twofold changes were identified between ZGY1578 and ZG7A and between ZGY1578 and ZH1578, respectively, in roots under salt stress using the RNA sequencing (RNA-Seq) approach. The expressions of a larger number of DEGs in hybrid were lower or higher than those of both parents. The DEGs associated with transcription factors, hormones, and reactive oxygen species (ROS)-related genes might be involved in the heterosis of salt tolerance. The expressions of the majority of transcription factors and ethylene-, auxin-, and gibberellin-related genes, as well as peroxidase genes, were significantly higher in the hybrid ZGY1578 compared with those of both parents. The identified genes provide valuable clues to elucidate the heterosis of salt tolerance in inter-subspecific hybrid rice.

Glutaredoxin in Rice Growth, Development, and Stress Resistance: Mechanisms and Research Advances.

Rice (Oryza sativa L.) is a staple food for more than half of the global population. Various abiotic and biotic stresses lead to accumulation of reactive oxygen species in rice, which damage macromolecules and signaling pathways. Rice has evolved a variety of antioxidant systems, including glutaredoxin (GRX), that protect against various stressors. A total of 48 GRX gene loci have been identified on 11 of the 12 chromosomes of the rice genome; none were found on chromosome 9. GRX proteins were classified into four categories according to their active sites: CPYC, CGFS, CC, and GRL. In this paper, we summarized the recent research advances regarding the roles of GRX in rice development regulation and response to stresses, and discussed future research perspectives related to rice production. This review could provide information for rice researchers on the current status of the GRX and serve as guidance for breeding superior varieties.

LncRNA H19 Regulates Breast Cancer DNA Damage Response and Sensitivity to PARP Inhibitors via Binding to ILF2.

Although DNA damage repair plays a critical role in cancer chemotherapy, the function of lncRNAs in this process remains largely unclear. In this study, in silico screening identified H19 as an lncRNA that potentially plays a role in DNA damage response and sensitivity to PARP inhibitors. Increased expression of H19 is correlated with disease progression and with a poor prognosis in breast cancer. In breast cancer cells, forced expression of H19 promotes DNA damage repair and resistance to PARP inhibition, whereas H19 depletion diminishes DNA damage repair and increases sensitivity to PARP inhibitors. H19 exerted its functional roles via direct interaction with ILF2 in the cell nucleus. H19 and ILF2 increased BRCA1 stability via the ubiquitin-proteasome proteolytic pathway via the H19- and ILF2-regulated BRCA1 ubiquitin ligases HUWE1 and UBE2T. In summary, this study has identified a novel mechanism to promote BRCA1-deficiency in breast cancer cells. Therefore, targeting the H19/ILF2/BRCA1 axis might modulate therapeutic approaches in breast cancer.

Effect of dietary supplemental vitamin C and betaine on the growth performance, humoral immunity, immune organ index, and antioxidant status of broilers under heat stress.

Heat stress (HS) has become one of the important factors affecting the development of animal husbandry. The purpose of this experiment was to investigate whether vitamin C (Vc) and betaine (Bet) improve immune organ index and humoral immunity by enhancing the antioxidant status of immune organs, thus protecting broilers from HS-induced injuries. A total of 200 28-day-old Ross 308 broilers were randomly assigned into 5 groups (n = 4 replicates/group, 10 broilers/replicate) which were reared at different ambient temperatures (24 ± 1°C or 33 ± 1°C). The control group fed basal diet, while high-temperature groups were either fed a basal diet (HS group) or a basal diet supplemented with 250-mg Vc/kg diet (HSVc group), 1000-mg Bet/kg diet (HSBet group), and 250-mg Vc plus 1000 mg Bet/kg diet (HSVcBet group), respectively. On day 42, growth performance, humoral immune function, immune organ index, and antioxidant capacity were measured. HS reduced the productive performance of broilers, antibody potency against the Newcastle disease virus (NDV) and sheep red blood cells (SRBC), indices of thymus and bursa, and antioxidant capacity of immune organs. Adding Vc alone or in combination with Bet improved performance, NDV and SRBC antibody potency, thymus and bursa indices, and antioxidant capacity of immune organs in heat-stressed broilers, with the most effective being combination. In summary, HS reduces the antioxidant capacity and immune organ development status of broiler immune organs. Vc and/or Bet can improve the development of immune organs and restore part of the production performance by regulating the antioxidant status of immune organs, among which the combined addition of Vc and Bet has the best effect.

Oriented Ultrathin π-complexation MOF Membrane for Ethylene/Ethane and Flue Gas Separations.

Rational design and engineering of high-performance molecular sieve membranes towards C2 H4 /C2 H6 and flue gas separations remain a grand challenge to date. In this study, through combining pore micro-environment engineering with meso-structure manipulation, highly c-oriented sub-100 nm-thick Cu@NH2 -MIL-125 membrane was successfully prepared. Coordinatively unsaturated Cu ions immobilized in the NH2 -MIL-125 framework enabled high-affinity π-complexation interactions with C2 H4 , resulting in an C2 H4 /C2 H6 selectivity approaching 13.6, which was 9.4 times higher than that of pristine NH2 -MIL-125 membrane; moreover, benefiting from π-complexation interactions between CO2 and Cu(I) sites, our membrane displayed superior CO2 /N2 selectivity of 43.2 with CO2 permeance of 696 GPU, which far surpassed the benchmark of other pure MOF membranes. The above multi-scale structure optimization strategy is anticipated to present opportunities for significantly enhancing the separation performance of diverse molecular sieve membranes.

Susceptibility artifact correction in MR thermometry for monitoring of mild radiofrequency hyperthermia using total field inversion.

PURPOSE: MR temperature monitoring of mild radiofrequency hyperthermia (RF-HT) of cancer exploits the linear resonance frequency shift of water with temperature. Motion-induced susceptibility distribution changes cause artifacts that we correct here using the total field inversion (TFI) approach. METHODS: The performance of TFI was compared to two background field removal (BFR) methods: Laplacian boundary value (LBV) and projection onto dipole fields (PDF). Data sets with spatial susceptibility change and B0 -drift were simulated, phantom heating experiments were performed, four volunteer data sets at thermoneutral conditions as well as data from one cervical cancer, two sarcoma, and one seroma patients undergoing mild RF-HT were corrected using the proposed methods. RESULTS: Simulations and phantom heating experiments revealed that using BFR or TFI preserves temperature-induced phase change, while removing susceptibility artifacts and B0 -drift. TFI resulted in the least cumulative error for all four volunteers. Temperature probe information from four patient data sets were best depicted by TFI-corrected data in terms of accuracy and precision. TFI also performed best in case of the sarcoma treatment without temperature probe. CONCLUSION: TFI outperforms previously suggested BFR methods in terms of accuracy and robustness. While PDF consistently overestimates susceptibility contribution, and LBV removes valuable pixel information, TFI is more robust and leads to more accurate temperature estimations.

Subunit E isoform 1 of vacuolar H+-ATPase OsVHA enables post-Golgi trafficking of rice seed storage proteins.

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.

Tumor spheroid invasion in epidermal growth factor gradients revealed by a 3D microfluidic device.

Epidermal growth factor (EGF), a potent cytokine, is known to promote tumor invasion bothin vivoandin vitro. Previously, we observed that single breast tumor cells (MDA-MB-231 cell line) embedded within a 3D collagen matrix displayed enhanced motility but no discernible chemotaxis in the presence of linear EGF gradients using a microfluidic platform. Inspired by a recent theoretical development that clustered mammalian cells respond differently to chemical gradients than single cells, we studied tumor spheroid invasion within a 3D extracellular matrix (ECM) in the presence of EGF gradients. We found that EGF gradients promoted tumor cell detachment from the spheroid core, and the position of the tumor spheroid core showed a mild chemotactic response towards the EGF gradients. For those tumor cells detached from the spheroids, they showed an enhanced motility response in contrast to previous experimental results using single cells embedded within an ECM. No discernible chemotactic response towards the EGF gradients was found for the cells outside the spheroid core. This work demonstrates that a cluster of tumor cells responds differently than single tumor cells towards EGF gradients and highlights the importance of a tumor spheroid platform for tumor invasion studies.

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression.

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.