Single-cell analysis of age-related changes in leukocytes of diabetic mouse hindpaws.

Complications associated with Type 1 and Type 2 diabetes, such as diabetic peripheral neuropathy and diabetic foot ulcers, are a growing health-care concern. In addition, this concern increases as diabetic patients age due to their increased susceptibility to complications. To address this growing problem, it is important to understand fluctuations in physiology which lead to pathological changes associated with the metabolic disturbances of diabetes. Our study explores dysregulation of immune cell populations in the hindpaws of healthy and diabetic mice at 12 and 21 weeks of age using single-cell RNA sequencing to provide insight into immune disruptions occurring in the distal limb during chronic diabetes. In 21-week-old Leprdb/db mice, increases were seen in mast cells/basophils, dermal γδ T cells, heterogeneous T cells, and Type 2 innate lymphoid cells. In addition, macrophages represented the largest cluster of immune cells and showed the greatest increase in genes associated with immune-specific pathways. Sub-clustering of macrophages revealed a bias toward angiogenic Lyve1+MHCIIlo macrophages in the hindpaws of 21-week-old diabetic mice, which corresponded to an increase in Lyve1+ macrophages in the hindpaws of 21-week-old diabetic mice on histology. Our results show that in Type 2 diabetes, the immunological function and phenotype of multiple immune cell types shift not only with metabolic disturbance, but also with duration of disease, which may explain the increased susceptibility to pathologies of the distal limb in patients with more chronic diabetes.

Xylem network connectivity and embolism spread in grapevine(Vitis vinifera L.).

Xylem networks are vulnerable to the formation and spread of gas embolisms that reduce water transport. Embolisms spread through interconduit pits, but the three-dimensional (3D) complexity and scale of xylem networks means that the functional implications of intervessel connections are not well understood. Here, xylem networks of grapevine (Vitis vinifera L.) were reconstructed from 3D high-resolution X-ray micro-computed tomography (microCT) images. Xylem network performance was then modeled to simulate loss of hydraulic conductivity under increasingly negative xylem sap pressure simulating drought stress conditions. We also considered the sensitivity of xylem network performance to changes in key network parameters. We found that the mean pit area per intervessel connection was constant across 10 networks from three, 1.5-m stem segments, but short (0.5 cm) segments fail to capture complete network connectivity. Simulations showed that network organization imparted additional resistance to embolism spread beyond the air-seeding threshold of pit membranes. Xylem network vulnerability to embolism spread was most sensitive to variation in the number and location of vessels that were initially embolized and pit membrane vulnerability. Our results show that xylem network organization can increase stem resistance to embolism spread by 40% (0.66 MPa) and challenge the notion that a single embolism can spread rapidly throughout an entire xylem network.

Analysis of HRCT-derived xylem network reveals reverse flow in some vessels.

Long distance water and nutrient transport in plants is dependent on the proper functioning of xylem networks, a series of interconnected pipe-like cells that are vulnerable to hydraulic dysfunction as a result of drought-induced embolism and/or xylem-dwelling pathogens. Here, flow in xylem vessels was modeled to determine the role of vessel connectivity by using three dimensional xylem networks derived from High Resolution Computed Tomography (HRCT) images of grapevine (Vitis vinifera cv. 'Chardonnay') stems. Flow in 4-27% of the vessel segments (i.e. any section of vessel elements between connection points associated with intervessel pits) was found to be oriented in the direction opposite to the bulk flow under normal transpiration conditions. In order for the flow in a segment to be in the reverse direction, specific requirements were determined for the location of connections, distribution of vessel endings, diameters of the connected vessels, and the conductivity of the connections. Increasing connectivity and decreasing vessel length yielded increasing numbers of reverse flow segments until a maximum value was reached, after which more interconnected networks and smaller average vessel lengths yielded a decrease in the number of reverse flow segments. Xylem vessel relays also encouraged the formation of reverse flow segments. Based on the calculated flow rates in the xylem network, the downward spread of Xylella fastidiosa bacteria in grape stems was modeled, and reverse flow was shown to be an additional mechanism for the movement of bacteria to the trunk of grapevine.

Automated analysis of three-dimensional xylem networks using high-resolution computed tomography.

Connections between xylem vessels represent important links in the vascular network, but the complexity of three-dimensional (3D) organization has been difficult to access. This study describes the development of a custom software package called TANAX (Tomography-derived Automated Network Analysis of Xylem) that automatically extracts vessel dimensions and the distribution of intervessel connections from high-resolution computed tomography scans of grapevine (Vitis vinifera) stems, although the method could be applied to other species. Manual and automated analyses of vessel networks yielded similar results, with the automated method generating orders of magnitude more data in a fraction of the time. In 4.5-mm-long internode sections, all vessels and all intervessel connections among 115 vessels were located, and the connections were analyzed for their radial distribution, orientation, and predicted shared wall area. Intervessel connections were more frequent in lateral than in dorsal/ventral zones. The TANAX-reconstructed network, in combination with commercial software, was used to visualize vessel networks in 3D. The 3D volume renderings of vessel networks were freely rotated for observation from any angle, and the 4.5 µm virtual serial sections were capable of being viewed in any plane, revealing aspects of vessel organization not possible with traditional serial sections.