NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia.

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

The novel role of ER protein TXNDC5 in the pathogenesis of organ fibrosis: mechanistic insights and therapeutic implications.

Fibrosis-related disorders account for an enormous burden of disease-associated morbidity and mortality worldwide. Fibrosis is defined by excessive extracellular matrix deposition at fibrotic foci in the organ tissue following injury, resulting in abnormal architecture, impaired function and ultimately, organ failure. To date, there lacks effective pharmacological therapy to target fibrosis per se, highlighting the urgent need to identify novel drug targets against organ fibrosis. Recently, we have discovered the critical role of a fibroblasts-enriched endoplasmic reticulum protein disulfide isomerase (PDI), thioredoxin domain containing 5 (TXNDC5), in cardiac, pulmonary, renal and liver fibrosis, showing TXNDC5 is required for the activation of fibrogenic transforming growth factor-ß signaling cascades depending on its catalytic activity as a PDI. Moreover, deletion of TXNDC5 in fibroblasts ameliorates organ fibrosis and preserves organ function by inhibiting myofibroblasts activation, proliferation and extracellular matrix production. In this review, we detailed the molecular and cellular mechanisms by which TXNDC5 promotes fibrogenesis in various tissue types and summarized potential therapeutic strategies targeting TXNDC5 to treat organ fibrosis.

Hard X-ray ptychography at Taiwan Photon Source at 11-20†nm spatial resolution.

X-ray ptychography, a technique based on scanning and processing of coherent diffraction patterns, is a non-destructive imaging technique with a high spatial resolution far beyond the focused beam size. Earlier demonstrations of hard X-ray ptychography at Taiwan Photon Source (TPS) using an in-house program successfully recorded the ptychographic diffraction patterns from a gold-made Siemens star as a test sample and retrieved the finest inner features of 25 nm. Ptychography was performed at two beamlines with different focusing optics: a pair of Kirkpatrick-Baez mirrors and a pair of nested Montel mirrors, for which the beam sizes on the focal planes were 3 µm and 200 nm and the photon energies were from 5.1 keV to 9 keV. The retrieved spatial resolutions are 20 nm to 11 nm determined by the 10-90% line-cut method and half-bit threshold of Fourier shell correlation. This article describes the experimental conditions and compensation methods, including position correction, mixture state-of-probe, and probe extension methods, of the aforementioned experiments. The discussions will highlight the criteria of ptychographic experiments at TPS as well as the opportunity to characterize beamlines by measuring factors such as the drift or instability of beams or stages and the coherence of beams. Besides, probe functions, the full complex fields illuminated on samples, can be recovered simultaneously using ptychography. Theoretically, the wavefield at any arbitrary position can be estimated from one recovered probe function undergoing wave-propagating. The verification of probe-propagating has been carried out by comparing the probe functions obtained by ptychography and undergoing wave-propagating located at 0, 500 and 1000 µm relative to the focal plane.

Synchrotron X-Ray Analysis and Morphology Evidence for Stereo-Assemblies of Periodic Aggregates in Poly(3-hydroxybutyrate) with Unusual Photonic Iridescence.

3D morphology of poly(3-hydroxybutyrate) (PHB), crystallized in the presence of diluents of poly(1,3-trimethylene adipate) and poly(ethylene oxide), is probed using a novel approach coupled with selective etching. For interpreting the mechanisms of crystal periodic aggregation, various microscopic techniques and synchrotron microbeam X-ray analysis are used to observe the top surface in connection with the 3D crystal assemblies. Periodic grating architectures, with the cross-bar pitch exactly matching with the optical band spacing, are proved in banded PHB. The crystals under the ridge branch out to spawn finer crystals orienting/bending horizontally underneath the valley band, repeating till species drainage or impingement. The grating structure in the banded PHB resembles many nature's iridescence crystals and is further proved by photonic reflection results as a critical breakthrough novel finding.

Integrated optical chip for a high-resolution, single-resonance-mode x-ray monochromator system.

An integrated optical chip that minimizes the size of the energy-tuning single-resonance-mode x-ray monochromator system into a 3cm×5cm silicon wafer is proposed. A Fabry-Perot x-ray resonator and two back-reflecting Si mirrors are employed on the wafer as the optical components, where Si(12 4 0) back reflection is used for both Fabry-Perot resonance and re-diffraction of the x-ray beams from the resonator in the incident direction. We can achieve an energy bandwidth of 3.4 meV in single-mode x rays and tune the energy by temperature variation. Such Si chips can be readily employed at the synchrotron beamlines and conventional x-ray laboratories for high-resolution investigations.

Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia.

Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell-autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. We further show that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2-dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the sty 3' untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.

Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization.

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1α/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion.

Time-resolved X-ray reflection phases of the nearly forbidden Si(222) reflection under laser excitation.

The covalent electron density, which makes Si(222) measurable, is subject to laser excitation. The three-wave Si(222)/(13 {\overline 1}) diffraction at 7.82 keV is used for phase measurements. It is found that laser excitation causes a relative phase change of around 4° in Si(222) in the first 100 ps of excitation and this is gradually recovered over several nanoseconds. This phase change is due to laser excitation of covalent electrons around the silicon atoms in the unit cell and makes the electron density deviate further from the centrosymmetric distribution.

Design, fabrication, and characterization of a multidimensional prism.

In this research, a novel multidimensional prism with three distinct 45°, 135°, and 225° inclined optical surfaces were realized by using inclined exposure technology and SU-8 polymer. To obtain a smooth surface, the solvent loss percentage and temperature of post-exposure bake (PEB) are two key factors that need to be well controlled during fabrication. The experimental results showed that surface roughness can achieve 42.9±7.6 nm, which is one-tenth of high-precision machining or molding processes, under the processes parameter combinations of PEB temperature, and solvent loss percentage is 95°C and 82.86%, respectively. The surface reflectivity of the prism was measured by using a He-Ne laser, and reflectivity of the prism surface without and with aluminum metal film was 90.8% and 91.8%, respectively. The slight difference of reflectivity means that a prism with a high-quality inclined surface can be realized. The functionality of the prism for three lasers was also verified with RGB lasers, and it successfully demonstrated the feasibility of application of a multidimensional prism on the optical system. Finally, the utilization of inclined exposure technology not only monolithically integrates three 45° inclined surfaces into one prism without precision assembly but also greatly simplifies the fabrication processes to further reduce the cost. This component and technology can also be applied to medical endoscope systems if the SU-8 is replaced by PDMS or other biocompatible materials using a molding process. These results provide the potential for mass manufacturing, which is of considerable value to the optical markets.

High-resolution monochromator using a high-efficiency single-mode x-ray resonator at Laue incidence.

We report a high-resolution and high-efficiency monochromator with energy resolution, ΔE/E∼2.08*10-7, utilizing a hard x-ray single-mode Fabry-Perot (FP) resonator at Laue incidence at 14.4388 keV. Instead of using multiple-crystal diffraction via several asymmetric-cut crystals, a 3 meV single-peak is generated from the interference of a Si-made two-plate cavity with a thickness of 70 µm and a gap of 45 µm, where a (0-40) Laue reflection is used to excite the backreflection (12 4 0) for the enhancement of the FP efficiency. Due to the large angular acceptance of (12 4 0) and (0-40), the energy tunability can be achieved by changing the incident angle into the resonator. The application of x-ray resonators at Laue incidence as a monochromator can be further implemented to x-ray optics.

Revisiting La0.5Sr1.5MnO4 lattice distortion and charge ordering with multi-beam resonant diffraction.

Sinusoidal wave type distortions of La0.5Sr1.5MnO4 in the low-temperature orthorhombic phase were observed using multi-beam resonant X-ray diffraction (MRXD) with (7/4 7/4 0) fractional primary diffraction. Two four-beam diffractions with opposite asymmetry were measured at 6.5545 keV and compared with the curves simulated by the dynamical X-ray diffraction theory. This approach provides the possibility of resolving the distortion modes which are perpendicular to the momentum transfer by a single azimuthal scan. The paper also demonstrates the sensitivity of MRXD profiles versus incident X-ray energy in the vicinity of the Mn K edge to the charge disproportion between the two manganese sites, reconfirming the small charge disproportion feature.

Sapphire hard X-ray Fabry-Perot resonators for synchrotron experiments.

Hard X-ray Fabry-Perot resonators (FPRs) made from sapphire crystals were constructed and characterized. The FPRs consisted of two crystal plates, part of a monolithic crystal structure of Al2O3, acting as a pair of mirrors, for the backward reflection (0 0 0 30) of hard X-rays at 14.3147 keV. The dimensional accuracy during manufacturing and the defect density in the crystal in relation to the resonance efficiency of sapphire FPRs were analyzed from a theoretical standpoint based on X-ray cavity resonance and measurements using scanning electron microscopic and X-ray topographic techniques for crystal defects. Well defined resonance spectra of sapphire FPRs were successfully obtained, and were comparable with the theoretical predictions.

High-resolution interference-monochromator for hard X-rays.

An X-ray interference-monochromator combining a Fabry-Perot resonator (FPR) and a double-crystal monochromator (DCM) is proposed and realized for obtaining single-mode X-rays with 3.45 meV energy resolution. The monochromator is based on the generation of cavity interference fringes from a FPR and single-mode selection of the transmission spectrum by a DCM of a nearly backward symmetric reflection geometry. The energy of the monochromator can be tuned within 2500 meV(= ΔE) by temperature control of the FPR and the DCM crystals in the range of ΔT = 70 K at room temperature. The diffraction geometry and small size of the optical components used make the interference-monochromator very easy to be adapted in modern synchrotron beamlines and X-ray optics applications.

Single-mode selection for hard x-ray cavity resonance.

Single-mode selection is realized for hard x-ray cavity resonance using a three-mirror crystal device. The developed device consists of two coupled Si Fabry-Perot resonators (FPRs) and uses (12 4 0) backward diffraction to reflect back and forth the incident 14.4388 keV x-ray beam. The coupling between the two cavities gives an effective single-mode spectrum with a bandwidth of 0.81 meV. This method can be used to enhance the longitudinal coherent length without affecting transverse coherence, and is potentially useful in generating nearly total coherent beams in synchrotron or free-electron laser facilities.

Post ischemia intermittent hypoxia induces hippocampal neurogenesis and synaptic alterations and alleviates long-term memory impairment.

Adult hippocampal neurogenesis is important for learning and memory, especially after a brain injury such as ischemia. Newborn hippocampal neurons contribute to memory performance by establishing functional synapses with target cells. This study demonstrated that the maturation of hippocampal neurons is enhanced by postischemia intermittent hypoxia (IH) intervention. The effects of IH intervention in cultured neurons were mediated by increased synaptogenesis, which was primarily regulated by brain-derived neurotrophic factor (BDNF)/PI3K/AKT. Hippocampal neo-neurons expressed BDNF and exhibited enhanced presynaptic function as indicated by increases in the pSynapsin expression, synaptophysin intensity, and postsynapse density following IH intervention after ischemia. Postischemia IH-induced hippocampal neo-neurons were affected by presynaptic activity, which reflected the dynamic plasticity of the glutamatergic receptors. These alterations were also associated with the alleviation of ischemia-induced long-term memory impairment. Our results suggest that postischemia IH intervention rescued ischemia-induced spatial learning and memory impairment by inducing hippocampal neurogenesis and functional synaptogenesis via BDNF expression.

Design and fabrication of a novel prism for micro-optical system.

This study presents a special k-type (45°, 135°) microprism structure design for the lateral-type semiconductor laser of optical systems. This study combines front- and back-side inclined exposure technology to develop a k-type microprism structure and replaces the optical prism manufactured through traditional machine processing or injection molding with a new polymer (thick-film negative photoresists) micro-optical prism structure. To obtain the optimal structural surface roughness (12-15 nm at 400 µm×400 µm area), the percentage of polymer material loss caused by the solvent was controlled. This roughness level meets Blu-ray specifications (λ/10, λ=405 nm).

Effects of hyperbaric oxygenation on oxidative stress in acute transient focal cerebral ischemic rats.

The aim of this study was to investigate the effects of hyperbaric oxygenation (HBO) after brain ischemia. Middle cerebral artery occlusion (MCAO) procedure was used to induce the brain ischemia. Rats were assigned to control or HBO group after brain ischemia. In order to examine the role of glutathione after HBO treatment, another group of brain ischemic rats were included to receive the glutathione synthesis inhibitor and HBO treatment. HBO was administered at a pressure of 3 atmospheres absolute for 1 h with 100% oxygen, starting at 3 h post brain ischemia in HBO groups. Animals in control group were placed in their home cage and exposed to normobaric room air. The infarct volume (IV), activation of astrocyte, and level of total glutathione and lipid peroxidation (LP) were assessed 24 h post-reperfusion. Significant reduction in IV was noted in HBO group when compared with control group. The activation of astrocyte was significantly increased in the right cerebral cortex and right striatum in the HBO group when compared with those of the control group. The glutathione level was higher with lower LP level in right cortex and right striatum after HBO as compared with those of the control. However, such effects of HBO treatment were markedly reduced by glutathione synthesis inhibitor administration. These results show that inhibiting glutathione synthesis dramatically reduces the effectiveness of HBO in acute transient focal cerebral ischemia.

Intermittent hypoxia after transient focal ischemia induces hippocampal neurogenesis and c-Fos expression and reverses spatial memory deficits in rats.

BACKGROUND: Memory impairment is a frequent complication of brain ischemia. Neurogenesis is implicated in learning and memory and is regulated by the transcription factor c-Fos. Preconditioning intermittent hypoxia (IH) attenuates ischemia-related memory impairments, but it is not known whether post-ischemia IH intervention has a similar effect. We investigated the effects of post-ischemia IH on hippocampal neurogenesis and c-Fos expression as well as spatial learning and memory in rats. METHODOLOGY/PRINCIPAL FINDINGS: Focal cerebral ischemia was induced in some rats by middle cerebral artery occlusion (MCAO), while other rats received sham MCAO surgery. Beginning a week later, half of the rats of each group received IH interventions (12% oxygen concentration, 4 hrs/d, for 7 d) and half received sham IH sessions. An additional group of rats received MCAO, IH, and injections of the neurogenesis-impairing agent 3'-AZT. Spatial learning and memory was measured in the Morris water maze, and hippocampal neurogenesis and c-Fos expression were examined. Hypoxia-inducible factor 1α (HIF-1α) and phosphorylated mitogen-activated protein kinase (pMAPK) were considered as possible mediators of IH-induced changes in neurogenesis and c-Fos expression. IH intervention following MCAO resulted in recovered spatial memory, increased hippocampal neurogenesis, and increased expression of c-Fos in newborn hippocampal cells. These effects were blocked by 3'-AZT. IH intervention following MCAO also was associated with increased hippocampal pMAPK and HIF-1α expression. CONCLUSIONS/SIGNIFICANCE: IH intervention following MCAO rescued ischemia-induced spatial learning and memory impairments, likely by inducing hippocampal neurogenesis and c-Fos expression through mediators including pMAPK and HIF-1α.

The time window of intermittent hypoxia intervention after middle cerebral artery occlusion.

It was known that preconditioning hypoxia can reduce the damage caused by ischemia. However, there was no study investigating the effects of intermittent hypoxia post ischemia. The purpose of this study was to investigate the time window for administering the hypoxia for beneficial effects after cerebral ischemic damage. According to the recovery days post transient middle cerebral occlusion (MCAO), the rats were randomly assigned to one of the 4 groups (n = 20 for each group, I, II, III, and IV). Rats were then assigned to one of the 2 subgroups (a and b). Rats in group Ia, IIa, IIIa, and IVa were exposed to 7 days of intermittent hypoxia (12% O2 for 4 hours per day) after recovery 1, 2, 3, and 7 days from MCAO, respectively. Rats in group Ib, IIb, IIIb, and IVb rested for 7 days in the same hypoxia chamber without hypoxia exposure after 1, 2, 3, and 7 days post MCAO, respectively. The mortality rate of rats received hypoxia after 1 day' and 2 days' recovery post MCAO was 40% (P = 0.087) and 10% (P = 0.5), respectively. The mean infarct volume of rats received hypoxia after 7 days' recovery was significantly less than that of the comparable control group (9.23 +/- 0.71% vs. 13.32 +/- 1.26%; P = 0.013), and no rats died in this group. In summary, intermittent hypoxia intervention for 7 days after 7 days of recovery post ischemia can reduce the infract volume, and does not increase the mortality rate. According to our results, we suggest that 7 days post ischemia may be the suitable time to begin the intermittent hypoxia intervention to enhance the recovery from cerebral ischemia.