High-resolution structural and functional retinal imaging in the awake behaving mouse.

The laboratory mouse has provided tremendous insight to the underpinnings of mammalian central nervous system physiology. In recent years, it has become possible to image single neurons, glia and vascular cells in vivo by using head-fixed preparations combined with cranial windows to study local networks of activity in the living brain. Such approaches have also succeeded without the use of general anesthesia providing insights to the natural behaviors of the central nervous system. However, the same has not yet been developed for the eye, which is constantly in motion. Here we characterize a novel head-fixed preparation that enables high-resolution adaptive optics retinal imaging at the single-cell level in awake-behaving mice. We reveal three new functional attributes of the normal eye that are overlooked by anesthesia: 1) High-frequency, low-amplitude eye motion of the mouse that is only present in the awake state 2) Single-cell blood flow in the mouse retina is reduced under anesthesia and 3) Mouse retinae thicken in response to ketamine/xylazine anesthesia. Here we show key benefits of the awake-behaving preparation that enables study of retinal physiology without anesthesia to study the normal retinal physiology in the mouse.

In Vivo Capillary Structure and Blood Cell Flux in the Normal and Diabetic Mouse Eye.

Purpose: To characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2Akita mouse. Methods: A custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2Akita positive (hyperglycemic) and 9 Ins2Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually. Retinal thickness and fundus photos were captured monthly using a commercial scanning laser ophthalmoscope/optical coherence tomography. Retinal thickness was calculated using a custom algorithm. Blood glucose and weight were tracked throughout the duration of the study. Results: Elevated blood glucose (>250 mg/dL) was observed at 4 to 5 weeks of age in Ins2Akita mice and remained elevated throughout the study, whereas euglycemic littermates maintained normal glucose levels. There was no significant difference in red blood cell flux, capillary anatomy, lumen diameter, or occurrence of stalled capillaries between hyperglycemic and euglycemic mice between postnatal weeks 5 and 18. Hyperglycemic mice had a thinner retina than euglycemic littermates (p < 0.001), but retinal thickness did not change with duration of hyperglycemia despite glucose levels that were more than twice times normal. Conclusions: In early stages of hyperglycemia, retinal microvasculature structure (lumen diameter, capillary anatomy) and function (red blood cell flux, capillary perfusion) were not impaired despite 3 months of chronically elevated blood glucose. These findings suggest that hyperglycemia alone for 3 months does not alter capillary structure or function in profoundly hyperglycemic mice.

Self-Organization Formation of Multicellular Spheroids Mediated by Mechanically Tunable Hydrogel Platform: Toward Revealing the Synergy of Chemo- and Noninvasive Photothermal Therapy against Colon Microtumor.

Three-dimensional (3D) tumor cell culture offers a more tissue-recapitulating model in cancer treatment evaluation. However, conventional models based on cell-substrate adhesion deprivation are still of insufficient real tumor mimic. In this work, a novel method is proposed for inducing multicellular spheroids (MCSs) formation based on hydrogel with tunable microenvironmental properties. Colon tumor cells DLD1 cultured on hydrogel substrate with proper physical stimulation form MCSs via self-organization. Chemotherapy based on clinical drug and far-infrared photothermal therapy is evaluated with DLD1 MCSs obtained by this method. The synergism of chemotherapy and noninvasive photothermal therapy based on graphene device is further verified in MCSs model and it is believed this method holds potential in in vitro anti-tumor strategies evaluation for precision medicine.

Lipid transporter LSR1 positively regulates leaf senescence in Arabidopsis.

Senescence is the final stage in the life history of a leaf, whereby plants relocate nutrients from leaves to other developing organs. Recent efforts have begun to focus on understanding the network-based molecular mechanism that incorporates various environmental signals and leaf age information and involves a complex process with the coordinated actions of multiple pathways. Here, we identified a novel participant, named LSR1 (Leaf Senescence Related 1), that involved in the regulation of leaf senescence. Loss-of-function lsr1-1 mutant showed delayed leaf senescence whereas the overexpression of LSR1 accelerated senescence. LSR1 encodes a lipid transfer protein, and the results show that the protein is located in chloroplast and intercellular space. The LSR1 may be involved in the regulation of leaf senescence by transporting lipids in plants.

Octaketide Synthase from Polygonum cuspidatum Implements Emodin Biosynthesis in Arabidopsis thaliana.

Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding OKS of Polygonum cuspidatum was isolated using a homology-based cloning strategy. When produced in Escherichia coli, P. cuspidatum octaketide synthase (PcOKS) catalyzed the condensation of eight molecules of malonyl-CoA to yield a mixture of unphysiologically folded aromatic octaketides. However, when the ORF for PcOKS was expressed in Arabidopsis thaliana, the anthranoid emodin was detected in the roots of transgenic lines. No emodin was found in the roots of wild-type A. thaliana. This result indicated that OKS is the key enzyme of plant anthranoids biosynthesis. In addition, the root growth of the transgenic A. thaliana lines was inhibited to an extent that resembled the inhibitory effect of exogenous emodin on the root growth of wild-type A. thaliana. Immunochemical studies of P. cuspidatum plants detected PcOKS mainly in roots and rhizome, in which anthranoids accumulate. Co-incubation of E. coli - produced PcOKS and cell-free extract of wild-type A. thaliana roots did not form a new product, suggesting an alternative, physiological folding of PcOKS and its possible interaction with additional factors needed for anthranoids assembling in transgenic A. thaliana. Thus, transgenic A. thaliana plants producing PcOKS provide an interesting system for elucidating the route of plant anthranoid biosynthesis.

Label-free imaging of immune cell dynamics in the living retina using adaptive optics.

Our recent work characterized the movement of single blood cells within the retinal vasculature (Joseph et al. 2019) using adaptive optics ophthalmoscopy. Here, we apply this technique to the context of acute inflammation and discover both infiltrating and tissue-resident immune cells to be visible without any labeling in the living mouse retina using near-infrared light alone. Intravital imaging of immune cells can be negatively impacted by surgical manipulation, exogenous dyes, transgenic manipulation and phototoxicity. These confounds are now overcome, using phase contrast and time-lapse videography to reveal the dynamic behavior of myeloid cells as they interact, extravasate and survey the mouse retina. Cellular motility and differential vascular responses were measured noninvasively and in vivo across hours to months at the same retinal location, from initiation to the resolution of inflammation. As comparable systems are already available for clinical research, this approach could be readily translated to human application.

Sensitivity of spatially offset Raman spectroscopy (SORS) to subcortical bone tissue.

The development of spatially offset Raman spectroscopy (SORS) has enabled deep, non-invasive chemical characterization of turbid media. Here, we use SORS to measure subcortical bone tissue and depth-resolved biochemical variability in intact, exposed murine bones. We also apply the technique to study a mouse model of the genetic bone disorder osteogenesis imperfecta. The results suggest that SORS is more sensitive to disease-related biochemical differences in subcortical trabecular bone and marrow than conventional Raman measurements.

Deacetylated Konjac Glucomannan Is Less Effective in Reducing Dietary-Induced Hyperlipidemia and Hepatic Steatosis in C57BL/6 Mice.

Konjac gel foods that mainly consist of deacetylated konjac glucomannan (Da-KGM) are considered to have the same health benefits as native konjac glucomannan (KGM); however, no definitive data support this notion. The objective of this study was to compare the effects of Da-KGM and KGM on the hyperlipidemia and liver steatosis induced by high-fat diet feeding and to investigate the underlying molecular mechanisms. C57BL/6 mice were fed (1) normal chow diet, (2) high-fat diet, (3) HFD with KGM, or (4) HFD with Da-KGM for 10 weeks. KGM, but not Da-KGM, showed decreased fat accumulation, improved blood and liver lipid profiles, and prevention of liver lipid droplet deposition compared with HFD. Compared with Da-KGM, KGM increased the outputs of fecal bile acid (KGM 22.5 ± 2.34 mg/g vs Da-KGM 19.3 ± 1.87 mg/g), fat (KGM 5.56 ± 0.68 mg/g vs Da-KGM 4.42 ± 0.57 mg/g) and cholesterol (KGM2.67 ± 0.43 mg/g vs Da-KGM 1.78 ± 0.28 mg/g), fecal concentrations of total short-chain fatty acids (KGM 103 ± 14.8 µmol/g vs Da-KGM 74.5 ± 8.49 µmol/g), and improved hepatic antioxidant status and upregulated CYP7A1 and LDLR gene expression. These findings suggest that deacetylation of KGM negatively affects its fermentation characteristics and its inhibition of lipid absorption, which thereby reduces Da-KGM's health benefits.

Full-field functional optical angiography.

We propose full-field functional optical angiography for a live biological specimen based on the absorption intensity fluctuation modulation (AIFM) effect. Because of the difference in absorption between red blood cells (RBCs) and the background tissue under low-coherence light illumination, the moving RBCs, which discontinuously pass though the capillary vessels, generate an AIFM effect. This effect offers high contrast of absorption imaging and sensitivity of low-coherence interference between RBCs and the background tissue. It is used to distinguish the signal of RBCs from that of the background tissue. The averaged and real-time modulation depths are computed to obtain full-field label-free optical angiography and measure blood flow velocity simultaneously. The AIFM method could potentially be applied to study the physiological mechanisms of blood circulation systems of near-transparent live biologic samples.

The Arabidopsis EIN2 restricts organ growth by retarding cell expansion.

The growth of plant organ to its characteristic size is a fundamental developmental process, but the mechanism is still poorly understood. Plant hormones play a great role in organ size control by modulating cell division and/or cell expansion. Ethylene insensitve 2 (EIN2) was first identified by a genetic screen for ethylene insensitivity and is regarded as a central component of ethylene signaling, but its role in cell growth has not been reported. Here we demonstrate that changed expression of EIN2 led to abnormity of cell expansion by morphological and cytological analyses of EIN2 loss-of-function mutants and the overexpressing transgenic plant. Our findings suggest that EIN2 controls final organ size by restricting cell expansion.

The Arabidopsis ORGAN SIZE RELATED 2 is involved in regulation of cell expansion during organ growth.

BACKGROUND: In plants, the growth of an aerial organ to its characteristic size relies on the coordination of cell proliferation and expansion. These two different processes occur successively during organ development, with a period of overlap. However, the mechanism underlying the cooperative and coordinative regulation of cell proliferation and expansion during organ growth remains poorly understood. RESULTS: This study characterized a new Arabidopsis ORGAN SIZE RELATED (OSR) gene, OSR2, which participates in the regulation of cell expansion process during organ growth. OSR2 was expressed primarily in tissues or organs undergoing growth by cell expansion, and the ectopic expression of OSR2 resulted in enlarged organs, primarily through enhancement of cell expansion. We further show that OSR2 functions redundantly with ARGOS-LIKE (ARL), another OSR gene that regulates cell expansion in organ growth. Moreover, morphological and cytological analysis of triple and quadruple osr mutants verified that the four OSR members differentially but cooperatively participate in the regulation of cell proliferation and cell expansion and thus the final organ size. CONCLUSIONS: Our results reveal that OSR2 is functional in the regulation of cell expansion during organ growth, which further implicates the involvement of OSR members in the regulation of both cell proliferation and expansion and thus the final organ size. These findings, together with our previous studies, strongly suggest that OSR-mediated organ growth may represent an evolutionary mechanism for the cooperative regulation of cell proliferation and expansion during plant organogenesis.

Laser Doppler projection tomography.

We propose a laser Doppler projection tomography (LDPT) method to obtain visualization of three-dimensional (3D) flowing structures. With LDPT, the flowing signal is extracted by a modified laser Doppler method, and the 3D flowing image is reconstructed by the filtered backprojection algorithm. Phantom experiments are performed to demonstrate that LDPT is able to obtain 3D flowing structure with higher signal-to-noise ratio and spatial resolution. Our experiment results display its potentially useful application to develop 3D label-free optical angiography for the circulation system of live small animal models or microfluidic experiments.

In vivo label-free microangiography by laser speckle imaging with intensity fluctuation modulation.

We present the theory of laser speckle imaging improved with intensity fluctuation modulation, where the dynamic speckle pattern can be isolated from its stationary counterpart. A series of in vivo experiments demonstrate the effectiveness of our method in achieving microangiography and monitoring vascular self-recovering process. All results show the convincing performance of our imaging method in both structural and functional imaging of blood flow, which may have potential applications in biological research and disease diagnosis.

Laser speckle projection tomography.

We propose a laser speckle projection tomography (LSPT) method to obtain a three-dimensional (3D) flowing image. The method combines the advantages of optical projection tomography and laser speckle imaging to reconstruct the visualization of 3D flowing structure. With LSPT, the flowing signal is extracted by laser speckle contrast method and the 3D flowing image is reconstructed by the filtered back-projection algorithm. A phantom experiment is performed to demonstrate that LSPT is able to obtain 3D flowing structure, influenced by concentration and the flow speed.

Broadband focusing ultrasonic transducers based on dimpled LiNbO3 plate with inversion layer.

A high-frequency broadband focusing transducer based on dimpled LiNbO(3) inversion layer plate has been fabricated and characterized. A spherical surface with a curvature radius of 6 mm is formed on the half-thickness LiNbO(3) inversion layer plate of Y36° cut orientation. The domain structure in the cross section is observed after a hydrofluoric acid etching process. For transducer fabrication, conductive epoxy is used as the backing material and polymer is deposited on the front face as the matching layer. The center frequency, bandwidth, and insertion loss of the focused transducer are measured to be 72 MHz, 136%, and -32 dB, respectively. The focused transducer has been successfully used for rabbit eyeball imaging and a better imaging capability compared with the planar transducer has been demonstrated. These promising results prove that the dimpled LiNbO(3) inversion layer plate has great potential for fabrication of high-frequency broadband focusing ultrasonic transducers.

Arabidopsis ORGAN SIZE RELATED1 regulates organ growth and final organ size in orchestration with ARGOS and ARL.

• The growth of a plant organ to its characteristic size is regulated by an elaborate developmental program involving both internal and external signals. Here, we identify a novel Arabidopsis gene, ORGAN SIZE RELATED1 (OSR1), that is involved in regulation of organ growth and overall organ size. • A combination of genetic, cytological and molecular approaches was used to characterize the expression profile, subcellular localization and roles of OSR1 during organ growth. • Ectopic expression of OSR1 in Arabidopsis resulted in enlarged organs, as a consequence of increases in both cell number and cell size. OSR1 shares a conserved OSR domain with ARGOS and ARGOS-LIKE (ARL), which is sufficient for their functions in promoting organ growth. OSR1 is a plant hormone-responsive gene and appears to act redundantly with ARGOS and ARL during organ growth. The OSR proteins are localized to the endoplasmic reticulum. • Our results suggest that three co-evolved members of the OSR family may act coordinately to orchestrate growth signals and cell proliferation and expansion, thereby affecting organ growth and final organ size.

Optimal electrode shape and size of lateral-field-excited piezoelectric crystal resonators.

We determine optimal electrode shape and size of lateral-field-excited (LFE) thickness-shear resonators. The determined electrodes are optimal in that they satisfy the criterion for Bechmann's number in every direction. Numerical and graphical results are provided for AT-cut quartz, (yxl)- 45°langasite, and (yxl)-16.5°LiTaO(3) LFE resonators. The optimal electrodes of AT-cut quartz LFE resonators are also compared with those of AT-cut quartz thickness-field-excited (TFE) resonators.

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth.

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle-related genes and over-accumulation of cells expressing CYCB1;1:beta-glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G(2)-M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2-mediated cellular events are required for proper progression of cell division in plant growth and development.

Effect of transverse force on the performance of quartz resonator force sensors.

This paper discusses the effect of transverse force on the performance of quartz crystal resonator (QXR) force sensors that use symmetrical incomplete circular QXRs as sensing elements. Based on Lee's vibration theory and the transverse force-induced stress distribution, frequency changes are derived. This result and finite element method (FEM) are applied to investigate the performance of two metal-quartz combined sensors under the action of transverse force. Analytical and experimental results show that transverse force affects the sensitivity and linearity of QXR force sensors.