Non–Destructive Imaging of Phytosulfokine Trafficking Using a Fiber–Optic Fluorescence Microscope

Plants secrete peptide ligands and use receptor signaling to respond to stress and control development. Understanding the signaling mechanisms and associated molecular trafficking is key to improving plant health and productivity for food, fiber and energy applications. However, one of the challenges to elucidating communication pathways in plants is to study the trafficking of molecules and signals iteratively and non-destructively. This study focuses on using fiber-optic fluorescence microscopy to image live plants iteratively and non-destructively after delivering both labeled and unlabeled phytosulfokine (PSK) into the plant. PSK is a sulfated peptide hormone involved in the regulation of plant cell division and growth via specific receptors, PSKRs. It also plays a role in regulating how plants are able to tolerate stress conditions. The microscope provides two-color (FITC/TRITC) optics and provides high-resolution (3–5 µm) epifluorescence micrographs via a 1-m coherent imaging fiber and a GRIN objective lens. To obtain high-quality images, the fiber was mounted either to a conventional upright microscope body equipped with a leaf compressor, or to a leaf clip with 5-axis positioning (X–Y–Z plus pitch and yaw) mounted on an extensible arm. PSK and TAMRA-labelled PSK were delivered into the roots of various Arabidopsis thaliana genotypes (wt; receptor-deficient: pskr1/pskr2; and tagged receptor overproducing: PSKR1‑GFP), and their movement in roots and leaves was tracked with the fiber-optic fluorescence microscope. Peptide trafficking was successfully observed in live plants non- destructively, confirming that PSK is mobile in both wt and receptor-deficient plants. Preliminary results suggest that the level of receptor PSKR1 may change in response to PSK, and that levels of PSKR1, PSKR2 or both may impact the trafficking of PSK. Understanding how PSK is trafficked in plants will offer insights into how we can improve plants health and productivity

Identification of a lysosomal pathway regulating degradation of the bone morphogenetic protein receptor type II.

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease

Software Citation Checklist for Developers

This document provides a minimal, generic checklist that developers of software (either open or closed source) used in research can use to ensure they are following good practice around software citation. This will help developers get credit for the software they create, and improve transparency, reproducibility, and reuse

Caveolae protect endothelial cells from membrane rupture during increased cardiac output.

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo

An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species

Endogenous sodium pump inhibitors and ageassociated increases in salt sensitivity of blood pressure in normotensives

AY, Lakatta EG. Endogenous sodium pump inhibitors and age-associated increases in salt sensitivity of blood pressure in normotensives. Am J Physiol Regul Integr Comp Physiol 294: R1248-R1254, 2008. First published February 20, 2008 doi:10.1152/ajpregu.00782.2007.-Factors that mediate increases in salt sensitivity of blood pressure with age remain to be clarified. The present study investigated 1) the effects of high-NaCl intake on two Na pump inhibitors, endogenous ouabain (EO) and marinobufagenin (MBG), in middle-aged and older normotensive Caucasian women; and 2) whether individual differences in EO and MBG are linked to variations in sodium excretion or salt sensitivity. A change from 6 days of a lower (0.7 mmol ⅐ kg Ϫ1 ⅐ day Ϫ1 )-to 6 days of a higher (4 mmol ⅐ kg Ϫ1 ⅐ day Ϫ1 )-NaCl diet elicited a sustained increase in MBG excretion that directly correlated with an increase in the fractional Na excretion and was inversely related to age and to an age-dependent increase in salt sensitivity. In contrast, EO excretion increased only transiently in response to NaCl loading and did not vary with age or correlate with fractional Na excretion or salt sensitivity. A positive correlation of both plasma and urine levels of EO and MBG during salt loading may indicate a casual link between two Na pump inhibitors in response to NaCl loading, as observed in animal models. A linear mixed-effects model demonstrated that age, dietary NaCl, renal MBG excretion, and body mass index were each independently associated with systolic blood pressure. Thus, a sustained increase in MBG in response to acutely elevated dietary NaCl is inversely linked to salt sensitivity in normotensive middle-aged and older women, and a relative failure of MBG elaboration by these older persons may be involved in the increased salt sensitivity with advancing age. marinobufagenin; ouabain; hypertension NUMEROUS STUDIES HAVE REPORTED that the magnitude of the systolic blood pressure (SBP) response to acute changes in dietary NaCl intake, i.e., salt sensitivity of blood pressure, increases with advancing age NaCl ingestion results in an increase in plasma volume and natriuresis. It has been postulated for some time that endogenous substances [sodium pump inhibitors (SPI)] are stimulated by increased Na intake and increase natriuresis by inhibiting renal tubular Na pumps to prevent renal reabsorption of filtered Na More recently, SPI assays have improved and become more specific That age-associated differences in circulating endogenous Na pump inhibitors may be implicated in the age-associated increase in SBP and increased NaCl sensitivity of SBP in older humans has been suggested previously (21) but never tested. The goal of the present study was to investigate effects of a subacute change in dietary NaCl on urinary and plasma EO and MBG in middle-aged and older normotensive subjects, and to determine whether NaCl-induced individual differences in the levels of these substances are linked to variations in renal sodium excretion or salt sensitivity of SBP. METHODS Subjects: inclusion and exclusion criteria. MBG and EO were measured in a series of healthy Caucasian women, ages 40 -70 yr, who, as part of another study, were salt restricted and then salt loaded to determine interactions among breathing patterns, PCO 2, and arterial pressure (2). Determination of subject eligibility involved telephone interview, physical examination, and informed consent. Inclusion and exclusion criteria also include the following: all qualified candidates were normotensive (resting SBP, Ͻ139 mmHg and resting diastolic blood pressure, Ͻ89 mmHg); had no history of respiratory, cardiovascular, liver or kidney disease, or diabetes; were free of cardiac organ damage determined by electrocardiogram; and were free of kidney dysfunction as determined by plasma creatinine Ͼ1.5. Subjects treated with estrogens or nonsteroidal anti-inflammatory agents, or medications affecting sodium, potassium, calcium, water, hemodynamic, or neural regulation (including diuretics, steroids, major tranquilizers, narcotics, or benzodiazepines) were also excluded, as were smokers or those with body mass index (BMI) of Ͻ19 or Ͼ30. The protocol of the study was approved by the Medstar Research Institute Institutional Review Board, and all study subjects signed informed consents. Experimental design. The experiment consisted of a 12-day, outpatient, dietary intervention, including 6 days on a low-sodium (0.7 mmol⅐kg Ϫ1 ⅐day Ϫ1 ), low-potassium (0.7 mmol⅐kg Ϫ1 ⅐day Ϫ1 ) diet, followed immediately by 6 days on a high-sodium (4 mmol⅐kg Ϫ1 ⅐day Ϫ1 ), low-potassium (0.7 mmol⅐kg Ϫ1 ⅐day Ϫ1 ) diet. The average recommended dietary sodium intake in the American diet for an average-sized adult i

Reducing Voltage Hysteresis in Li-rich Sulfide Cathodes by Tuning the Metal-ligand Covalency

Conventional intercalation-based cathode materials in Li-ion batteries are based on charge compensation of the redox active cation and can only intercalate one mole of electron per formula unit. Anion redox, which employs the anion sublattice to compensate charge, is a promising way to achieve multielectron cathode materials. Most anion redox materials still face the problems of slow kinetics and large voltage hysteresis. One potential solution to reduce voltage hysteresis is to increase the covalency of the metal-ligand bonds. By substituting Mn into the electrochemically inert Li1.33Ti0.67S2 (Li2TiS3), anion redox can be activated in the Li1.33–2y/3Ti0.67–y/3MnyS2 (y = 0 – 0.5) series. Not only do we observe substantial anion redox, but the voltage hysteresis is significantly reduced, and the rate capability is dramatically enhanced. The y = 0.3 phase exhibits excellent rate and cycling performance, maintaining 90% of the C/10 capacity at 1C, which indicates fast kinetics for anion redox. X-ray absorption spectroscopy (XAS) shows that both the cation and anion redox processes contribute to the charge compensation. We attribute the drop in hysteresis and increase in rate performance to the increased covalency between the metal and the anion. Electrochemical signatures suggest the anion redox mechanism resembles holes on the anion, but the S K-edge XAS data confirm persulfide formation. The mechanism of anion redox suggests that even if persulfides are formed, the hysteresis and rate performances can still be improved. This work provides insights on how to design cathode materials with anion redox to achieve fast kinetics and low voltage hysteresis