Analysis of aquaporins in northern grasses reveal functional importance of Puccinellia nuttalliana PIP2;2 in salt tolerance.

To better understand the roles of aquaporins in salt tolerance, we cloned PIP2;1, PIP2;2, PIP2;3, PIP1;1, PIP1;3, and TIP1;1 aquaporins from three northern grasses varying is salt tolerance including the halophytic grass Puccinellia nuttalliana, moderately salt tolerant Poa juncifolia, and relatively salt sensitive Poa pratensis. We analysed aquaporin expression in roots by exposing the plants to 0 and 150 mM for 6 days in hydroponic culture. NaCl treatment upregulated several PIP transcripts in P. nuttalliana while decreasing PnuTIP1;1. The PnuPIP2;2 transcripts increased by about six-fold in P. nuttalliana, two-fold in Poa juncifolia, and did not change in Poa pratensis. The NaCl treatment enhanced the rate of water transport in yeast expressing PnuPIP2;2 by 56% compared with control. PnuPIP2,2 expression also resulted in a higher Na+ uptake in yeast cells compared with an empty vector suggesting that PnuPIP2;2 may have both water and ion transporting functions. Structural analysis revealed that the transport properties of PnuPIP2;2 could be affected by its unique pore characteristics, which include a combination of hourglass, cylindrical, and increasing diameter conical entrance shape with pore hydropathy of -0.22.

The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit.

The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm), or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of a lack of PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than wild-type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional plasma membrane aquaporin AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes in humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

105° field of view non-contact handheld swept-source optical coherence tomography.

We demonstrate a handheld swept-source optical coherence tomography (OCT) system with a 400 kHz vertical-cavity surface-emitting laser (VCSEL) light source, a non-contact approach, and an unprecedented single shot 105° field of view (FOV). We also implemented a spiral scanning pattern allowing real-time visualization with improved scanning efficiency. To the best of our knowledge, this is the widest FOV achieved in a portable non-contact OCT retinal imaging system to date. Improvements to the FOV may aid the evaluation of retinal diseases such as retinopathy of prematurity, where important vitreoretinal changes often occur in the peripheral retina.

Overexpression of Nicotiana tabacum PIP1;3 enhances root aeration and oxygen metabolism in canola (Brassica napus) plants exposed to root hypoxia.

High mortality and reduced growth due to root hypoxia are commonly observed in plants impacted by flooding or soil compaction. Since earlier research suggested that Nicotiana tabacum PIP1;3 may facilitate cell-to-cell oxygen transport, we overexpressed NtPIP1;3 in canola (Brassica napus) and studied the effects on growth, physiological parameters, root oxygen concentrations, and energy metabolism in plants subjected to waterlogging. Compared with wild-type plants (WT), the waterlogged plants overexpressing NtPIP1;3 (OE) maintained higher dry biomass, gas exchange, root hydraulic conductivity, root oxygen concentrations, leaf water potentials, root respiration rates, and root ATP concentrations. Metabolic profiling revealed that overexpressing plants responded to root hypoxia by altering the glycolysis, pyruvate metabolism, and TCA cycle in roots. Moreover, the differences in expression patterns of RAP2.12, RAP2.2, PCO1, and PCO2 in WT and OE canola plants exposed to root hypoxia point to increased oxygen supply to OE roots, which was confirmed by direct measurements of root O2 concentrations. Our results demonstrate that the overexpression of NtPIP1;3 affected plant responses to hypoxia by enhancing their aerobic metabolism and strengthened the notion that some of the plant aquaporins may facilitate oxygen transport.

Optical Assessment of Photoreceptor Function Over the Macula.

Purpose: The purpose of this study was to develop next-generation functional photoreceptor imaging using ultrahigh-speed swept-source optical coherence tomography (UHS-SS-OCT) and split-spectrum amplitude-decorrelation optoretinography (SSADOR) algorithm. The advancement enables rapid surveying of large retinal areas, promising non-contact, objective, and quantifiable measurements of macular visual function. Methods: We designed and built a UHS-SS-OCT prototype instrument using a wavelength tunable laser with 1 MHz A-scan rate. The functional scanning protocol records 5 repeated volumes in 3 seconds. A flash pattern selectively exposes the imaged retina area. SSADOR quantifies photoreceptor light response by extracting optical coherence tomography (OCT) signal changes within the photoreceptor outer segment before and after the flash. Results: The study prospectively enrolled 16 eyes from 8 subjects, demonstrating the ability to measure photoreceptor light response over a record field of view (3 × 3 mm2) with high topographical resolution (approximately 100 µm). The measured SSADOR signal corresponds to the flashed pattern, whose amplitude also correlates with flash strength, showing consistency and reproducibility across subjects. Conclusions: The integration of high-performance UHS-SS-OCT and SSADOR enables characterizing photoreceptor function over a clinically meaningful field of view, while maintaining a workflow that can be integrated into routine clinical tests and trials. The new approach allows detecting changes in photoreceptor light response with high sensitivity and can detect small focal impairments. Translational Relevance: This innovative advance can enable us to detect early photoreceptor abnormalities, as well as help to stage and monitor degenerative retinal diseases, potentially providing a surrogate visual function marker for retinal diseases and accelerating therapeutic development through a safe and efficient outcome endpoint.

Segmental biomechanics of the normal and glaucomatous human aqueous outflow pathway.

The conventional aqueous outflow pathway, encompassing the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and inner wall endothelium of Schlemm's canal (SC), governs intraocular pressure (IOP) regulation. This study targets the biomechanics of low-flow (LF) and high-flow (HF) regions within the aqueous humor outflow pathway in normal and glaucomatous human donor eyes, using a combined experimental and computational approach. LF and HF TM/JCT/SC complex tissues from normal and glaucomatous eyes underwent uniaxial tensile testing. Dynamic motion of the TM/JCT/SC complex was recorded using customized green-light optical coherence tomography during SC pressurization in cannulated anterior segment wedges. A hyperviscoelastic model quantified TM/JCT/SC complex properties. A fluid-structure interaction model simulated tissue-aqueous humor interaction. FluoSpheres were introduced into the pathway via negative pressure in the SC, with their motion tracked using two-photon excitation microscopy. Tensile test results revealed that the elastic moduli of the LF and HF regions in glaucomatous eyes are 3.5- and 1.5-fold stiffer than the normal eyes, respectively. The FE results also showed significantly larger shear moduli in the TM, JCT, and SC of the glaucomatous eyes compared to the normal subjects. The LF regions in normal eyes demonstrated larger elastic moduli compared to the HF regions in glaucomatous eyes. The resultant strain in the outflow tissues and velocity of the aqueous humor in the FSI models were in good agreement with the digital volume correlation and 3D particle image velocimetry data, respectively. This study uncovers stiffer biomechanical responses in glaucomatous eyes, with LF regions stiffer than HF regions in both normal and glaucomatous eyes. STATEMENT OF SIGNIFICANCE: This study delves into the biomechanics of the conventional aqueous outflow pathway, a crucial regulator of intraocular pressure and ocular health. By analyzing mechanical differences in low-flow and high-flow regions of normal and glaucomatous eyes, this research unveils the stiffer response in glaucomatous eyes. The distinction between regions' properties offers insights into aqueous humor outflow regulation, while the integration of experimental and computational methods enhances credibility. These findings have potential implications for disease management and present a vital step toward innovative ophthalmic interventions. This study advances our understanding of glaucoma's biomechanical basis and its broader impact on ocular health.

Real-time line-field optical coherence tomography for cellular resolution imaging of biological tissue.

A real-time line-field optical coherence tomography (LF-OCT) system is demonstrated with image acquisition rates of up to 5000 B-frames or 2.5 million A-lines per second for 500 A-lines per B-frame. The system uses a high-speed low-cost camera to achieve continuous data transfer rates required for real-time imaging, allowing the evaluation of future applications in clinical or intraoperative environments. The light source is an 840 nm super-luminescent diode. Leveraging parallel computing with GPU and high speed CoaXPress data transfer interface, we were able to acquire, process, and display OCT data with low latency. The studied system uses anamorphic beam shaping in the detector arm, optimizing the field of view and sensitivity for imaging biological tissue at cellular resolution. The lateral and axial resolution measured in air were 1.7 µm and 6.3 µm, respectively. Experimental results demonstrate real-time inspection of the trabecular meshwork and Schlemm's canal on ex vivo corneoscleral wedges and real-time imaging of endothelial cells of human subjects in vivo.

Developing an experimental-computational workflow to study the biomechanics of the human conventional aqueous outflow pathway.

The aqueous humor actively interacts with the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm's canal (SC) through a dynamic fluid-structure interaction (FSI) coupling. Despite the fact that intraocular pressure (IOP) undergoes significant fluctuations, our understanding of the hyperviscoelastic biomechanical properties of the aqueous outflow tissues is limited. In this study, a quadrant of the anterior segment from a normal human donor eye was dynamically pressurized in the SC lumen, and imaged using a customized optical coherence tomography (OCT). The TM/JCT/SC complex finite element (FE) with embedded collagen fibrils was reconstructed based on the segmented boundary nodes in the OCT images. The hyperviscoelastic mechanical properties of the outflow tissues' extracellular matrix with embedded viscoelastic collagen fibrils were calculated using an inverse FE-optimization method. Thereafter, the 3D microstructural FE model of the TM, with adjacent JCT and SC inner wall, from the same donor eye was constructed using optical coherence microscopy and subjected to a flow load-boundary from the SC lumen. The resultant deformation/strain in the outflow tissues was calculated using the FSI method, and compared to the digital volume correlation (DVC) data. TM showed larger shear modulus (0.92 MPa) compared to the JCT (0.47 MPa) and SC inner wall (0.85 MPa). Shear modulus (viscoelastic) was larger in the SC inner wall (97.65 MPa) compared to the TM (84.38 MPa) and JCT (56.30 MPa). The conventional aqueous outflow pathway is subjected to a rate-dependent IOP load-boundary with large fluctuations. This necessitates addressing the biomechanics of the outflow tissues using hyperviscoelastic material-model. STATEMENT OF SIGNIFICANCE: While the human conventional aqueous outflow pathway is subjected to a large-deformation and time-dependent IOP load-boundary, we are not aware of any studies that have calculated the hyperviscoelastic mechanical properties of the outflow tissues with embedded viscoelastic collagen fibrils. A quadrant of the anterior segment of a normal humor donor eye was dynamically pressurized from the SC lumen with relatively large fluctuations. The TM/JCT/SC complex were OCT imaged and the mechanical properties of the tissues with embedded collagen fibrils were calculated using the inverse FE-optimization algorithm. The resultant displacement/strain in the FSI outflow model was validated versus the DVC data. The proposed experimental-computational workflow may significantly contribute to understanding of the effects of different drugs on the biomechanics of the conventional aqueous outflow pathway.

Volumetric directional optical coherence tomography.

Photoreceptor loss and resultant thinning of the outer nuclear layer (ONL) is an important pathological feature of retinal degenerations and may serve as a useful imaging biomarker for age-related macular degeneration. However, the demarcation between the ONL and the adjacent Henle's fiber layer (HFL) is difficult to visualize with standard optical coherence tomography (OCT). A dedicated OCT system that can precisely control and continuously and synchronously update the imaging beam entry points during scanning has not been realized yet. In this paper, we introduce a novel imaging technology, Volumetric Directional OCT (VD-OCT), which can dynamically adjust the incident beam on the pupil without manual adjustment during a volumetric OCT scan. We also implement a customized spoke-circular scanning pattern to observe the appearance of HFL with sufficient optical contrast in continuous cross-sectional scans through the entire volume. The application of VD-OCT for retinal imaging to exploit directional reflectivity properties of tissue layers has the potential to allow for early identification of retinal diseases.

Corneal imaging with blue-light optical coherence microscopy.

Corneal imaging is important for the diagnostic and therapeutic evaluation of many eye diseases. Optical coherence tomography (OCT) is extensively used in ocular imaging due to its non-invasive and high-resolution volumetric imaging characteristics. Optical coherence microscopy (OCM) is a technical variation of OCT that can image the cornea with cellular resolution. Here, we demonstrate a blue-light OCM as a low-cost and easily reproducible system to visualize corneal cellular structures such as epithelial cells, endothelial cells, keratocytes, and collagen bundles within stromal lamellae. Our blue-light OCM system achieved an axial resolution of 12 µm in tissue over a 1.2 mm imaging depth, and a lateral resolution of 1.6 µm over a field of view of 750 µm × 750 µm.

Widefield Optical Coherence Tomography in Pediatric Retina: A Case Series of Intraoperative Applications Using a Prototype Handheld Device.

Optical coherence tomography (OCT) has changed the standard of care for diagnosis and management of macular diseases in adults. Current commercially available OCT systems, including handheld OCT for pediatric use, have a relatively narrow field of view (FOV), which has limited the potential application of OCT to retinal diseases with primarily peripheral pathology, including many of the most common pediatric retinal conditions. More broadly, diagnosis of all types of retinal detachment (exudative, tractional, and rhegmatogenous) may be improved with OCT-based assessment of retinal breaks, identification of proliferative vitreoretinopathy (PVR) membranes, and the pattern of subretinal fluid. Intraocular tumors both benign and malignant often occur outside of the central macula and may be associated with exudation, subretinal and intraretinal fluid, and vitreoretinal traction. The development of wider field OCT systems thus has the potential to improve the diagnosis and management of myriad diseases in both adult and pediatric retina. In this paper, we present a case series of pediatric patients with complex vitreoretinal pathology undergoing examinations under anesthesia (EUA) using a portable widefield (WF) swept-source (SS)-OCT device.

Transcriptome and Metabolome Analyses Reveal Potential Salt Tolerance Mechanisms Contributing to Maintenance of Water Balance by the Halophytic Grass Puccinellia nuttalliana.

Elevated soil salinity exacerbated by human activities and global climate change poses serious threats to plant survival. Although halophytes provide many important clues concerning salt tolerance in plants, some unanswered questions remain to be addressed, including the processes of water and solute transport regulation. We performed high-throughput RNA-sequencing in roots and metabolome characterizations in roots and leaves of Puccinellia nuttalliana halophytic grass subjected to 0 (control) and 150 mM NaCl. In RNAseq, a total of 31 Gb clean bases generated were de novo assembled into 941,894 transcripts. The PIP2;2 and HKT1;5 transcript levels increased in response to the NaCl treatment implying their roles in water and ion homeostasis. Several transcription factors, including WRKY39, DEK3, HY5, and ABF2, were also overexpressed in response to NaCl. The metabolomic analysis revealed that proline and dopamine significantly increased due to the upregulation of the pathway genes under salt stress, likely contributing to salt tolerance mechanisms. Several phosphatidylcholines significantly increased in roots suggesting that the alterations of membrane lipid composition may be an important strategy in P. nuttalliana for maintaining cellular homeostasis and membrane integrity under salt stress. In leaves, the TCA cycle was enriched suggesting enhanced energy metabolism to cope with salt stress. Other features contributing to the ability of P. nuttalliana to survive under high salinity conditions include salt secretion by the salt glands and enhanced cell wall lignification of the root cells. While most of the reported transcriptomic, metabolomics, and structural alterations may have consequences to water balance maintenance by plants under salinity stress, the key processes that need to be further addressed include the role of the changes in the aquaporin gene expression profiles in the earlier reported enhancement of the aquaporin-mediated root water transport.

Advantages of Widefield Optical Coherence Tomography in the Diagnosis of Retinopathy of Prematurity.

Recent advances in portable optical coherence tomography (OCT) and OCT angiography (OCTA) have resulted in wider fields of view (FOV) and shorter capture times, further expanding the potential clinical role of OCT technology in the diagnosis and management of retinopathy of prematurity (ROP). Using a prototype, handheld OCT device, retinal imaging was obtained in non-sedated infants in the neonatal intensive care unit (NICU) as well as sedated infants in the operating room of Oregon Health & Science University (OHSU) Hospital. In this observational study, we provide an overview of potential advantages of OCT-based disease assessment in ROP. We observed that next-generation OCT imaging (a) may be sufficient for objective diagnosis and zone/stage/plus disease categorization, (b) allows for minimally-invasive longitudinal monitoring of disease progression and post-treatment course, (c) provides three-dimensional mapping of the vitreoretinal interface, and (d) with OCTA, enables dye-free visualization of normal and pathologic vascular development.

Variation in Aquaporin and Physiological Responses Among Pinus contorta Families Under Different Moisture Conditions.

A population of eight open pollinated families of Pinus contorta was selected from sites varying in precipitation regimes and elevation to examine the possible role of aquaporins in adaptation to different moisture conditions. Five Pinus contorta aquaporins encoding PiconPIP2;1, PiconPIP2;2, PiconPIP2;3, PiconPIP1;2, and PiconTIP1;1 were cloned and detailed structural analyses were conducted to provide essential information that can explain their biological and molecular function. All five PiconAQPs contained hydrophilic aromatic/arginine selective filters to facilitate the transport of water. Transcript abundance patterns of PiconAQPs varied significantly across the P. contorta families under varying soil moisture conditions. The transcript abundance of five PiconPIPs remained unchanged under control and water-stress conditions in two families that originated from the sites with lower precipitation levels. These two families also displayed a different adaptive strategy of photosynthesis to cope with drought stress, which was manifested by reduced sensitivity in photosynthesis (maintaining the same rate) while exhibiting a reduction in stomatal conductance. In general, root:shoot ratios were not affected by drought stress, but some variation was observed between families. The results showed variability in drought coping mechanisms, including the expression of aquaporin genes and plant biomass allocation among eight families of Pinus contorta.

Plant water transport and aquaporins in oxygen-deprived environments.

Oxygen deprivation commonly affects plants exposed to flooding and soil compaction. The resulting root hypoxia has an immediate effect on plant water relations and upsets water balance. Hypoxia inhibits root water transport and triggers stomatal closure. The processes contributing to the inhibition of root hydraulic conductivity and conductance (hydraulic conductivity of the whole root system) are complex and involve changes in root morphology and the functions of aquaporins. Aquaporins (AQPs) comprise a group of membrane intrinsic proteins that are responsible for the transport of water, as well as some small neutral solutes and ions. They respond to a wide range of environmental stresses including O2 deprivation, but the underlying functional mechanisms are still elusive. The aquaporin-mediated water transport is affected by the acidification of the cytoplasm and depletion of ATP that is required for aquaporin phosphorylation and membrane functions. Cytoplasmic pH, phosphorylation, and intracellular Ca2+ concentration directly control AQP gating, all of which are related to O2 deprivation. This review addresses the structural determinants that are essential for pore conformational changes in AQPs, to highlight the underlying mechanisms triggered by O2 deprivation stress. Gene expression of AQPs is modified in hypoxic plants, which may constitute an important, yet little explored, mechanism of hypoxia tolerance. In addition to water transport, AQPs may contribute to hypoxia tolerance by transporting O2, H2O2, and lactic acid. Responses of plants to O2 deprivation, and especially those that contribute to maintenance of water transport, are highly complex and entail the signals originating in roots and shoots that lead to and follow the stomatal closure. These complex responses may involve ethylene, abscisic acid, and possibly other hormonal factors and signaling molecules in ways that remain to be elucidated.