Pyrenoid-core CO2-evolving machinery is essential for diatom photosynthesis in elevated CO2.

Marine diatoms are responsible for up to 20% of the annual global primary production by performing photosynthesis in seawater where CO2 availability is limited while HCO3- is abundant. Our previous studies have demonstrated that solute carrier 4 proteins at the plasma membrane of the diatom Phaeodactylum tricornutum facilitate the use of the abundant seawater HCO3-. There has been an unconcluded debate as to whether such HCO3- use capacity may itself supply enough dissolved inorganic carbon (DIC) to saturate the enzyme Rubisco. Here, we show that the θ-type carbonic anhydrase, Ptθ-CA1, a luminal factor of the pyrenoid-penetrating thylakoid membranes, plays an essential role in saturating photosynthesis of P. tricornutum. We isolated and analyzed genome-edited mutants of P. tricornutum defective in Ptθ-CA1. The mutants showed impaired growth in seawater aerated with a broad range of CO2 levels, from atmospheric to 1%. Independently of growth CO2 conditions, the photosynthetic affinity measured as K0.5 for DIC in mutants reached around 2 mm, which is about 10 times higher than K0.5[DIC] of high-CO2-grown wild-type cells that have repressed CO2-concentrating mechanism levels. The results clearly indicate that diatom photosynthesis is not saturated with either seawater-level DIC or even under a highly elevated CO2 environment unless the CO2-evolving machinery is at the core of the pyrenoid.

The Supercarbonate Apatite-MicroRNA Complex Inhibits Dextran Sodium Sulfate-Induced Colitis.

The incidence of inflammatory bowel disease (IBD) is increasing. Nucleic acid-based medicine has potential as a next-generation treatment, but it is rarely successful with IBD. The aim of this study was to establish a microRNA-based therapy in an IBD model. For this purpose, we used microRNA-29 (miR-29) and a supercarbonate apatite (sCA) nanoparticle as a drug delivery system. Injection of sCA-miR-29a-3p or sCA-miR-29b-3p into mouse tail veins markedly prevented and restored inflammation because of dextran sulfate sodium (DSS)-induced colitis. RNA sequencing analysis revealed that miR-29a and miR-29b could inhibit the interferon-associated inflammatory cascade. Subcutaneous injection of sCA-miR-29b also potently inhibited inflammation, and it efficiently targeted CD11c+ dendritic cells (DCs) among various types of immune cells in the inflamed mucosa. RT-PCR analysis indicated that the miR-29 RNAs in CD11c+ DCs suppressed the production of interleukin-6 (IL-6), transforming growth factor ß (TGF-ß), and IL-23 subunits in DSS-treated mice. This may inhibit Th17 differentiation and subsequent activation, which is critical in IBD pathogenesis. In vivo experiments using a non-natural artificial microRNA sequence revealed that targeting of DCs in the inflamed colon is an exceptional feature of sCA. This study suggests that sCA-miR-29s may open a new avenue in nucleic acid-based medicine for IBD treatment.

Identification of caffeoylquinic acid derivatives as natural protein tyrosine phosphatase 1B inhibitors from Artemisia princeps.

Considerable attention has been paid to protein tyrosine phosphatase 1B (PTP1B) inhibitors as a potential therapy for diabetes, obesity, and cancer. Ten caffeoylquinic acid derivatives (1-10) from leaves of Artemisia princeps Pamp. (Asteraceae) were identified as natural PTP1B inhibitors. Among them, chlorogenic acid (3) showed the most potent inhibitory activity (IC50 11.1 µM). Compound 3 was demonstrated to be a noncompetitive inhibitor by a kinetic analysis. Molecular docking simulation suggested that compound 3 bound to the allosteric site of PTP1B. Furthermore, compound 3 showed remarkable selectivity against four homologous PTPs. According to these findings, compound 3 might be potentially valuable for further drug development.

Effect of rivastigmine on plasma butyrylcholine esterase activity and plasma ghrelin levels in patients with dementia in Alzheimer's disease.

AIM: Alzheimer's disease causes loss of appetite, resulting in bodyweight reduction. This, in turn, causes progression of cognitive dysfunction and physical complications that hasten death. Earlier care for loss of appetite is essential in Alzheimer's disease management. Rivastigmine is a therapeutic agent for Alzheimer's disease that has dual inhibition effects on acetylcholine esterase and butyrylcholine esterase. Butyrylcholine esterase is known to degrade the gastric hormone, ghrelin, which regulates appetite; therefore, we considered that rivastigmine might have an effect on appetite. The present study aimed to evaluate the hypothesis that rivastigmine improves appetite in Alzheimer's disease patients. METHODS: Rivastigmine was given to mild-to-moderate Alzheimer's disease patients for 16 weeks. We evaluated the effects of rivastigmine on food intake, bodyweight, motivation (estimated by the vitality index), cognition function (estimated by the Hasegawa Dementia Scale-Revised), plasma butyrylcholine esterase activity, active ghrelin and inactive ghrelin. RESULTS: Plasma butyrylcholine esterase activity significantly decreased over time (percent change: -18.9 ± 27.0%, P < 0.05 at week 8; percent change: -33.4 ± 45.4%, P < 0.05 at week 16). Negative correlations were detected between percent changes in butyrylcholine esterase activity and active ghrelin (rs = -0.62, P = 0.033) or active/inactive ghrelin ratio (rs = -0.73, P = 0.007). Furthermore, motivation (including appetite) improved significantly (percent change: 17.9 ± 18.6%, P < 0.05 at week 16). CONCLUSIONS: The present study suggests that rivastigmine might improve appetite in mild-to-moderate Alzheimer's disease patients by suppressing degradation of plasma active ghrelin through the inhibition of plasma butyrylcholine esterase. Geriatr Gerontol Int 2018; 18: 886-891.

Isolation, Structural Elucidation, and Liquid Chromatography-Mass Spectrometry Analysis of Steroidal Glycosides from Polygonatum odoratum.

The rhizomes of Polygonatum odoratum represent a traditional Chinese medicine and functional food. A phytochemical investigation resulted in the isolation of eight steroidal glycosides (1-8), including two new compounds, polygonatumosides F (1) and G (2). The structures were elucidated by spectroscopic data and chemical reactions. Compound 7 showed antiproliferation activity against human hepatocellular carcinoma cell line HepG2 (IC50 of 3.2 µM). The chemical profile and contents of steroidal glycosides of P. odoratum rhizomes collected at different dates and geographical locations were also investigated, indicating that the rational harvest of P. odoratum in spring and autumn is preferable to obtain higher levels of steroidal glycosides. Compounds 1 and 7 showed the highest contents in all P. odoratum samples and have potential to serve as chemotaxonomic and chemical markers for quality control of this important plant material. 14-Hydroxylation may be a key step for the biosynthesis of compounds 1-7.

Identification of microRNA-487b as a negative regulator of liver metastasis by regulation of KRAS in colorectal cancer.

Recent studies have shown that microRNAs (miRNAs) are involved in the progression of colorectal cancer (CRC). The aim of this study is to identify a novel miRNA that especially relates to liver metastasis and to explore the underlying mechanism. Differentially expressed miRNAs were analyzed using microarray, in primary CRC tumors without metastasis (n=16), those with liver metastasis (n=12), and liver metastatic lesions (n=8). We found that miR-487b level decreased in liver metastatic lesions, and qRT-PCR confirmed the results in the validating cohort (n=134). Survival analysis indicated that high expression of miR-487b was associated with better prognosis. In vitro studies were also performed to investigate the functional significance of miR-487b in human CRC cell lines. miR-487b showed an inhibitory effect on cell proliferation and invasion of CRC cells. miR-487b downregulated KRAS and inhibited its downstream signal pathways, and the luciferase reporter assay revealed that miR-487b directly targeted LRP6, a receptor for WNT/ß-catenin signaling. These findings showed that decrease in miR-487b was related with liver metastasis. Our data suggest a possibility that miR-487b may suppress metastasis of CRC progression through inhibition of KRAS.

Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted in Vivo Gene Expression.

Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into a nanosized toroidal structure or folding it into a rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 or 0 mM NaCl solutions, respectively. Compared with the rod-like structure, the toroidal structure possessed superior biological functions capable not only of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest in this ordered nanostructure as a possible inherent structure for DNA.

Muscle-targeted hydrodynamic gene introduction of insulin-like growth factor-1 using polyplex nanomicelle to treat peripheral nerve injury.

The recovery of neurologic function after peripheral nerve injury often remains incomplete because of the prolonged reinnervation process, which leads to skeletal muscle atrophy and articular contracture from disuse over time. To rescue the skeletal muscle and promote functional recovery, insulin-like growth factor-1 (IGF-1), a potent myogenic factor, was introduced into the muscle by hydrodynamic injection of IGF-1-expressing plasmid DNA using a biocompatible nonviral gene carrier, a polyplex nanomicelle. In a mouse model of sciatic nerve injury, the introduction of IGF-1 into the skeletal muscle of the paralyzed limb effectively alleviated a decrease in muscle weight compared with that in untreated control mice. Histologic analysis of the muscle revealed the IGF-1-expressing plasmid DNA (pDNA) to have a myogenic effect, inducing muscle hypertrophy with the upregulation of the myogenic regulatory factors, myogenin and MyoD. The evaluation of motor function by walking track analysis revealed that the group that received the hydrodynamic injection of IGF-1-expressing pDNA using the polyplex nanomicelle had significantly early recovery of motor function compared with groups receiving negative control pDNA and untreated controls. Early recovery of sensation in the distal area of sciatic nerve injury was also induced by the introduction of IGF-1-expressing pDNA, presumably because of the effect of secreted IGF-1 protein in the vicinity of the injured sciatic nerve exerting a synergistic effect with muscle hypertrophy, inducing a more favorable prognosis. This approach of introducing IGF-1 into skeletal muscle is promising for the treatment of peripheral nerve injury by promoting early motor function recovery.

Low-level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro.

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N-methyl-D-aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm(2) delivered at 25 mW/cm(2) over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies.

Low-level laser therapy (LLLT) reduces oxidative stress in primary cortical neurons in vitro.

Low-level laser (light) therapy (LLLT) involves absorption of photons being in the mitochondria of cells leading to improvement in electron transport, increased mitochondrial membrane potential (MMP), and greater ATP production. Low levels of reactive oxygen species (ROS) are produced by LLLT in normal cells that are beneficial. We exposed primary cultured murine cortical neurons to oxidative stressors: hydrogen peroxide, cobalt chloride and rotenone in the presence or absence of LLLT (3 J/cm², CW, 810 nm wavelength laser, 20 mW/cm²). Cell viability was determined by Prestoblue™ assay. ROS in mitochondria was detected using Mito-sox, while ROS in cytoplasm was detected with CellRox™. MMP was measured with tetramethylrhodamine. In normal neurons LLLT elevated MMP and increased ROS. In oxidatively-stressed cells LLLT increased MMP but reduced high ROS levels and protected cultured cortical neurons from death. Although LLLT increases ROS in normal neurons, it reduces ROS in oxidatively-stressed neurons. In both cases MMP is increased. These data may explain how LLLT can reduce clinical oxidative stress in various lesions while increasing ROS in cells in vitro.

Bioactive polymeric metallosomes self-assembled through block copolymer-metal complexation.

Spontaneous formation of polymeric metallosomes with uniform size (~100 nm) was found to occur in aqueous medium through the reaction of an anticancer agent, (1,2-diaminocyclohexane)platinum(II) (DACHPt), with a Y-shaped block copolymer of ω-cholesteroyl-poly(L-glutamic acid) and two-armed poly(ethylene glycol) (PEGasus-PLGA-Chole). Circular dichroism spectrum measurements revealed that the PLGA segment forms an α-helix structure within the metallosomes, suggesting that secondary-structure formation of metallocomplexed PLGA segment may drive the self-assembly of the system into vesicular structure. These metallosomes can encapsulate water-soluble fluorescent macromolecules into their inner aqueous phase and eventually deliver them selectively into tumor tissues in mice, owing to the prolonged blood circulation. Accordingly, fluorescent imaging of the tumor was successfully demonstrated along with an appreciable antitumor activity by DACHPt moieties retained in the vesicular wall of the metallosomes, indicating the potential of metallosomes as multifunctional drug carriers.

Newly developed endoscopic instruments for the removal of intracerebral hematoma.

Ultra-early surgical treatment in which associated brain injury is minimized and maximal volume of hematoma is removed shortly after onset with secure hemostasis is expected to be established. We developed a transparent guiding sheath and other surgical instruments for endoscopic surgery and established a novel, ultra-early stage surgical procedure using those instruments. This procedure has the following characteristics: (a) burr hole opening under local anesthesia is possible; (b) a transparent sheath improves the visualization of the surgical field in the parenchyma and the hematoma; (c) free-hand surgery without fixing an endoscope and a sheath to a frame facilitates three-dimensional operation; (d) secure hemostasis by electric coagulation is possible; (e) relatively simple surgical instruments are easy to prepare. We have performed this procedure in 82 patients with intracerebral or intraventricular hemorrhage (44 with putaminal hemorrhage, 12 with thalamic hemorrhage, 8 with subcortical hemorrhage, 8 with cerebellar hemorrhage, 10 with intraventricular hemorrhage). Twenty-four of those patients received our treatment in the ultra-early stage (within 3 hours after onset). The mean duration of surgery was 63 minutes, the mean hematoma reduction rate was 96%, and no peri-operative hemorrhage with deterioration of symptoms and/or signs occurred. Therefore, we believe that endoscopic hematoma evacuation with our surgical procedure is a promising ultra-early stage treatment for intracerebral hemorrhage and that it may improve the long-term prognosis in patents with intracerebral hemorrhage.

Role of the bloodstream impacting force and the local pressure elevation in the rupture of cerebral aneurysms.

BACKGROUND AND PURPOSE: Inertial force of the bloodstream results in the local elevation of intravascular pressure secondary to flow impact. Previous studies suggest that this "impacting force" and the local pressure elevation at the aneurysm may have a large contribution to the development of cerebral aneurysms. The goal of the present study is to evaluate how the bloodstream impacting force and the local pressure elevation at the aneurysm influences the rupture of cerebral aneurysms. METHODS: A total of 29 aneurysms were created in 26 patient-specific vessel models, and computer simulations were used to calculate pressure distributions around the vessel branching points and the aneurysms. RESULTS: Direct impact of the parent artery bloodstream resulted in local elevation in pressure at branch points, and bends in arteries (231.2+/-198.1 Pa; 100 Pa=0.75 mm Hg). The bloodstream entered into the aneurysm with a decreased velocity after it impacted on the branching points or bends. Thus, the flow impact at the aneurysm occurred usually weakly. At the top or the rupture point of the aneurysm, the flow velocity was always delayed. The local pressure elevation at the aneurysm was 119.3+/-91.2 Pa. CONCLUSIONS: The pressure elevation at the area of flow impact and at the aneurysm constituted only 1% to 2% of the peak intravascular pressure. The results suggest that the bloodstream impacting force and the local pressure elevation at the aneurysm may have less contribution to the rupture of cerebral aneurysms than was expected previously.

Effectiveness of the head-shaking method combined with cisternal irrigation with urokinase in preventing cerebral vasospasm after subarachnoid hemorrhage.

OBJECT: The head-shaking method combined with cisternal irrigation has been proposed to be effective in preventing cerebral vasospasm after subarachnoid hemorrhage (SAH) by facilitating rapid washout of the clot from the subarachnoid space. This study was conducted to evaluate the effectiveness of this method. METHODS: The inclusion criteria included the following: 1) Fisher Grade 3 SAH on admission computerized tomography (CT) scans; 2) aneurysm secured within 48 hours of SAH onset; and 3) no focal deficit and ability to obey commands within 24 hours postsurgery. Two hundred thirty patients treated between 1994 and 2002 fulfilled the criteria. Because only one machine was available and it required I month of maintenance every other month, 114 patients underwent irrigation combined with the head-shaking method (head-shaking group), whereas the remaining 116 patients received cisternal irrigation alone (control group). There were no significant differences in sex, age, site of aneurysm, or preoperative grade between the two groups. The incidence of symptomatic vasospasm with or without infarction, cerebral infarction on CT scans, and permanent ischemic neurological deficit was 25.7, 17.7, and 8.8%, respectively, in the control group and 15.2, 4.5, and 2.7% in the head-shaking group. The difference was statistically significant for symptomatic vasospasm, cerebral infarction, and permanent ischemic neurological deficit (p < 0.05). In a multivariate backward stepwise logistic regression analysis, absence of head shaking was the only variable that was predictive of permanent ischemic neurological deficit (p = 0.061). The outcomes evaluated using the modified Rankin Scale were better in the head-shaking group (p = 0.051). CONCLUSIONS: The head-shaking method significantly reduced the incidence of symptomatic vasospasm, cerebral infarction, and permanent ischemic neurological deficit and improved the clinical outcomes in patients who underwent cisternal irrigation therapy after aneurysmal SAH.

Expression of myosin heavy chain isoforms by smooth muscle cells in cerebral arteriovenous malformations.

We have characterised the blood vessels found in normal cerebral vasculature and in arteriovenous malformations (AVMs), based on the expression of smooth muscle cell (SMC)-specific proteins. The marker proteins used were smooth muscle alpha-actin and four myosin heavy chain isoforms (SM1, SM2, SMemb and NMHC-A). Specimens of AVM obtained during surgery, and normal cerebral vessels from autopsy cases were studied immunohistochemically and compared. The arterial components of AVM contained an abundance of SMCs of the contractile phenotype, which were positive for alpha-actin, SM1 and SM2, but not for SMemb and NMHC-A. These components showed the same staining pattern as mature normal arteries. Two different types of abnormal veins were found in the AVM specimens: large veins with a thick and fibrous wall (so-called 'arterialised' veins) and intraparenchymal thin-walled sinusoidal veins. The former expressed alpha-actin, SM1, SM2, and SMemb, the latter expressed alpha-actin, SM1, and SM2. These marker expression patterns resembled those of normal cerebral arteries, and the results were compatible with arterialisation of the cerebral veins caused by arteriovenous shunting. However, the expression of SMemb was found only in the arterialised type of veins, not in the sinusoidal type or the arteries that had sustained abnormal blood flow in the AVMs. The thick-walled veins in the AVMs showed the same staining pattern as normal veins of dural plexus origin (large subarachnoid veins and dural sinuses). It is therefore possible to assume that they originated from the dural plexus, and extended into the brain during the formation of AVMs.

Oxidation-induced dynamic changes in morphology reflected on freeze-fractured surface of gamma-irradiated ultra-high molecular weight polyethylene components.

Oxidative degradation of ultra-high molecular weight polyethylene (UHMWPE) attributed to sterilization by gamma-radiation in the presence of air has been cited as one of the major causes of premature wear in total joint arthroplasty. For example, in retrieved UHMWPE tibial bearings, not only adhesive and abrasive wear, but also fatigue wear characterized by delamination and fracture is frequently observed. In this study, we examined the effects of gamma radiation on the microstructural morphology of UHMWPE tibial bearings, and propose a severe fatigue wear mechanism. Scanning electron microscopic observations were conducted on freeze-fractured surface of retrieved UHMWPE components that had been sterilized with gamma radiation in air before implantation, unused components that had been stored on the shelf for several years (5-11) after sterilization, and disc specimens given an accelerated aging protocol after gamma radiation. Scanning electron microscopic observations showed that the freeze-fractured surface of these components had a double layer, which was bordered below the surface. A closer observation of the subsurface layer below the border revealed an extremely rough and porous honeycomb-like structure. Fourier transform infrared analysis demonstrated that the honeycomb-like region in the subsurface had a high oxidation level. The internal morphology of oxidized UHMWPE was classified into four categories based on the level of the oxidation. According to these results, the morphological changes with oxidative degradation of gamma-irradiated UHMWPEs in the presence of air could consistently be explained as the result of major chemical and physical changes such as increased crystallinity, elevated density, and reduced mechanical strength. We relate the morphological changes caused by oxidative degradation in the subsurface to the location of the origin of fatigue wear in total knee joints.

[Treatment for the ruptured bilateral vertebral dissecting aneurysms].

We report a case of a bilateral vertebral dissecting aneurysm associated with subarachnoid hemorrhage. Proximal ligation of the vertebral artery on the ruptured side combined with wrapping of the contralateral dissection failed to prevent fatal rebleeding. Since enlargement of the contralateral dissection was observed by postoperative angiography, rupture of the growing contralateral dissecting aneurysm may have caused rebleeding. Hemodynamic changes following the occlusion of one vertebral artery might have led to enlargement and subsequent rupture of the contralateral dissection. Direct wrapping was unable to prevent enlargement of the dissection, so radical surgery including bilateral vertebral artery occlusion combined with vascular reconstruction may be the treatment of choice for this type of lesion.