We report that the Arabidopsis thaliana mutant sensitive to ABA and drought2 (sad2), which harbors a T-DNA insertion in an importin b-like gene, is more tolerant to UV-B radiation than the wild type. Analysis of cyclobutane pyrimidine dimer accumulation revealed that less DNA damage occurred in sad2 than in the wild type during UV-B treatment. No significant growth difference was observed between sad2 and the wild type when treated with the genotoxic drug methyl methanesulfonate, suggesting that SAD2 functions in UV-B protection rather than in DNA damage repair. Whereas the R2R3-type transcription repressor MYB4 has previously been shown to negatively regulate the transcription of cinnamate 4-hydroxylase (C4H) and thus to regulate the synthesis of sinapate esters, expression of both MYB4 and C4H and accumulation of UVabsorbing compounds were significantly higher in sad2 than in the wild type. MYB4 did not localize to the nucleus in the sad2 mutant, suggesting that SAD2 is required for MYB4 nuclear trafficking. SAD2 and MYB4 coimmunoprecipitated, indicating that these proteins localize in the same complex in vivo. MYB4 protein specifically bound to its own promoter in gel shift assays and repressed its own expression, demonstrating that MYB4 protein and mRNA are part of a negative autoregulatory loop. This feedback loop is altered in the sad2 mutant due to the absence of MYB4 protein in the nucleus, leading to the constitutive expression of MYB4 and C4H and resulting in accumulation of UV-absorbing pigments that shield the plant from UV-B radiation.

The blood-brain barrier (BBB) restricts the uptake of many neuro-therapeutic molecules, presenting a formidable hurdle to drug development in brain diseases. We proposed a new and dynamic in vivo-like three-dimensional microfluidic system that replicates the key structural, functional and mechanical properties of the blood-brain barrier in vivo. Multiple factors in this system work synergistically to accentuate BBB-specific attributes–permitting the analysis of complex organ-level responses in both normal and pathological microenvironments in brain tumors. The complex BBB microenvironment is reproduced in this system via physical cell-cell interaction, vascular mechanical cues and cell migration. This model possesses the unique capability to examine brain metastasis of human lung, breast and melanoma cells and their therapeutic responses to chemotherapy. The results suggest that the interactions between cancer cells and astrocytes in BBB microenvironment might affect the ability of malignant brain tumors to traverse between brain and vascular compartments. Furthermore, quantification of spatially resolved barrier functions exists within a single assay, providing a versatile and valuable platform for pharmaceutical development, drug testing and neuroscientific research.

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H 2 O 2 -, and CaCl 2 -regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.

Rapid changes in turgor pressure (P:) and temperature (T:) are giving new information about the mechanisms of plant growth. In the present work, single internode cells of the large-celled alga Chara corallina were used as a model for plant growth. P was changed without altering the chemical environment of the wall while observing growth without elastic changes. When P: was measured before any changes, the original growth rate bore no relationship to the original P. However, if P of growing cells was decreased, growth responded immediately without evidence for rapid changes in wall physical properties. Growth occurred only above a 0.3 MPa threshold, and increasing P caused small increases in growth that became progressively larger as P rose, resulting in a curvilinear response overall. The small changes in growth close to the threshold may explain early failures to detect these responses. When T was lowered, the elastic properties of the cell were unaffected, but growth was immediately inhibited. The lower T caused P to decrease, but returning P to its original value did not return growth to its original rate. The decreased P at low T occurred because of T effects on the osmotic potential of the cell. At above-normal P, growth partially resumed at low T Therefore, growth required a P-sensitive process that was also T-sensitive. Because elastic properties were little affected by T, but growth was markedly affected, the process is likely to involve metabolism. The rapidity of its response to P and T probably excludes the participation of changes in gene expression.

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The nematode Caenorhabditis elegans (C. elegans) has been widely used as a multicellular organism in developmental research due to its simplicity, short lifecycle, and its relevance to human genetics and biology. Droplet microfluidics is an attractive platform for the study of C. elegans in integrated mode with flexibility at the single animal resolution. However, it is still challenging to conduct the developmental study of worms within droplets initiating at the L1 larval stage, due to the small size, active movement, and the difficulty in achieving effective substance exchange within the droplets. Here, we present a multifunctional droplet microchip to address these issues and demonstrate the usefulness of this device for investigating post-embryonic development in individual C. elegans initiating at the larval L1 stage. The key components of this device consist of multiple functional units that enable parallel worm loading, droplet formation/trapping, and worm encapsulation in parallel. In particular, it exhibits superior functions in encapsulating and trapping individual larval L1 worms into droplets in a controlled way. Continuous food addition and expulsion of waste by mixing the static worm-in-droplet with moving medium plugs allows for the long-term culture of worms under a variety of conditions. We used this device to investigate the development processes of C. elegans in transgenic strains with deletion and overexpression of the hypoxia-inducible factor (HIF-1), a highly conserved transcript factor in regulating an organism's response to hypoxia. This microdevice may be a useful tool for the high throughput analysis of individual worms starting at the larval stage, and facilitates the study of developmental worms in response to multiple drugs or environmental toxins.

The rapidly self-renewing epithelium in the mammalian intestine is maintained by multipotent intestinal stem cells (ISCs) located at the bottom of the intestinal crypt that are interspersed with Paneth cells in the small intestine and Paneth-like cells in the colon. The ISC compartment is also closely associated with a sub-epithelial compartment that contains multiple types of mesenchymal stromal cells. With the advances in single cell and gene editing technologies, rapid progress has been made for the identification and characterization of the cellular components of the niche microenvironment that is essential for self-renewal and differentiation of ISCs. It has become increasingly clear that a heterogeneous population of mesenchymal cells as well as the Paneth cells collectively provide multiple secreted niche signals to promote ISC self-renewal. Here we review and summarize recent advances in the regulation of ISCs with a main focus on the definition of niche cells that sustain ISCs.

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