Impact of a novel pre-hospital stroke notification programme on acute stroke care key performance indicators in Hong Kong: a multicentre prospective cohort study with historical controls.

INTRODUCTION: Early identification and initiation of reperfusion therapy is essential for suspected acute ischaemic stroke. A pre-hospital stroke notification (PSN) protocol using FASE (facial drooping, arm weakness, speech difficulties, and eye palsy) was implemented to improve key performance indicators (KPIs) in acute stroke care delivery. We assessed KPIs and clinical outcomes before and after PSN implementation in Hong Kong. METHODS: This prospective cohort study with historical controls was conducted in the Accident and Emergency Departments of four public hospitals in Hong Kong. Patients were screened using the PSN protocol between August 2021 and February 2022. Suspected stroke patients between August 2020 and February 2021 were included as historical controls. Door-to-needle (DTN) and door-to-computed tomography (DTC) times before and after PSN implementation were compared. Clinical outcomes including National Institutes of Health Stroke Scale score at 24 hours and modified Rankin Scale score at 3 months after intravenous recombinant tissue-type plasminogen activator (IV-rtPA) were also assessed. RESULTS: Among the 715 patients (266 PSN and 449 non-PSN) included, 50.8% of PSN patients and 37.7% of non-PSN patients had a DTC time within 25 minutes (P<0.001). For the 58 PSN and 134 non-PSN patients given IV-rtPA, median DTN times were 67 and 75.5 minutes, respectively (P=0.007). The percentage of patients with a DTN time within 60 minutes was higher in the PSN group than in the non-PSN group (37.9% vs 21.6%; P=0.019). No statistically significant differences in clinical outcomes were observed. CONCLUSION: Although the PSN protocol shortened DTC and DTN times, clinical outcomes did not significantly differ.

Neutral particles pushed or pulled by laser pulses: erratum.

We present an erratum to our Letter [Opt. Lett.41, 230 (2016)10.1364/OL.41.000230]. This erratum corrects three typing errors. The corrections have no influence on the results and conclusions of the original Letter.

Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of HEK293 adherent cultures.

Transfection of surface adherent cells remain as a standard methodology for lentiviral production for early phase clinical studies and research purposes. Production today is based on transient co-transfection of three or four plasmids, where the viral elements are encoded separately for safety reasons. Assembly of functional lentiviral particles requires all plasmids to be efficiently transfected into each cell, a notable challenge with many currently available methods for transient transfection. We have previously demonstrated the significant improvement of cationic polymer-based transfection in various cell types using a combination of fusogenic lipids and histone deacetylase 6 inhibitor (Enhancers). In this report, we focused on the transfection step and the feasibility of improving lentiviral production using the Enhancers. After optimization of DNA amount and N/P ratio, transfection using seven commercial gene carriers showed comparable maximal efficiency of production with high cell viability. In the presence of Enhancers, the production of functional lentivirus using LPEI was increased by as much as tenfold and outperformed lentiviral production using Lipofectamine 3000. We demonstrate a scalable and optimized workflow where the use of the Enhancers significantly improved the lentiviral particle production in various HEK293 cell lines.

Neutral particles pushed or pulled by laser pulses.

Acceleration of neutral particles is of great importance in many areas, such as controlled chemical reactions, atomic nanofabrication, and atom optics. Recent experimental studies have shown that pulsed lasers can be used to push neutral Rydberg atoms forward [Nature 461, 1261 (2009)10.1038/nature08481; Nat. Photonics 6, 386 (2012)10.1038/nphoton.2012.87]. Our simulation shows that pulsed lasers can also be used to pull Rydberg atoms back toward a light source. In particular, we proposed a method of using two laser pulses on a neutral atom, then selective operations on the neutral atom (pushing or pulling) can be performed by adjusting the delay time between the two laser pulses.

A selective inhibitor of the immunoproteasome subunit LMP2 induces apoptosis in PC-3 cells and suppresses tumour growth in nude mice.

BACKGROUND: Although the proteasome is a validated anticancer target, the clinical application of its inhibitors has been limited because of inherent systemic toxicity. To broaden clinical utility of proteasome inhibitors as anticancer agents, it is critical to develop strategies to selectively target proteasomes in cancer cells. The immunoproteasome is an alternative form of the constitutive proteasome that is expressed at high levels in cancer tissues, but not in most normal cells in the body. METHODS: To validate the immunoproteasome as a chemotherapeutic target, an immunoproteasome catalytic subunit LMP2-targeting inhibitor and siRNA were used. The sensitivity of PC-3 prostate cancer cells to these reagents was investigated using viability assays. Further, a xenograft model of prostate cancer was studied to test the in vivo effects of LMP2 inhibition. RESULTS: A small molecule inhibitor of the immunoproteasome subunit LMP2, UK-101, induced apoptosis of PC-3 cells and resulted in significant inhibition (~50-60%) of tumour growth in vivo. Interestingly, UK-101 did not block degradation of IκBα in PC-3 cells treated with TNF-α, suggesting that its mode of action may be different from that of general proteasome inhibitors, such as bortezomib, which block IκBα degradation. CONCLUSION: These results strongly suggest that the immunoproteasome has important roles in cancer cell growth and thus provide a rationale for targeting the immunoproteasome in the treatment of prostate cancer.

Binding, internalization, and hydrolysis of low density lipoprotein in long-term lymphoid cell lines from a normal subject and a patient with homozygous familial hypercholesterolemia.

Long-term established human lymphoid cells were shown to possess high affinity cell surface receptors for low density lipoprotein (LDL), the major cholesterol-carrying protein in human plasma. Binding of LDL to these receptors was followed by internalization of the lipoprotein and hydrolysis of its protein and cholesteryl ester components. Cultured lymphocytes from a patient with the homozygous form of familial hypercholesterolemia lacked cell surface LDL receptors and therefore failed to take up and degrade the lipoprotein with high affinity. Cultured human lymphocytes should prove useful for further studies of: (a) the relation between cholesterol metabolism and cellular function and (b) the mechanism by which LDL binding at the cell surface leads to internalization of the lipoprotein.

Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes. Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia.

The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.

Simulations of vacuum laser acceleration: hidden errors from particle's initial positions.

Simulation of vacuum laser acceleration, because of its scheme's simplicity, attracts many people involved in. However, how to put the particle in the initial positions in the field has not been considered seriously in some such schemes. An inattentive choice of electron's initial conditions may lead to misleading results. Here we show that arbitrarily placing the particle within the laser field leads to an overestimation of its energy gain, and offer suggestions for selecting appropriate initial conditions.

The SREBP pathway in Drosophila: regulation by palmitate, not sterols.

In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. Here, we show that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the two proteases that process SREBP at sites 1 and 2 to release the nuclear fragment. In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis.

Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.

Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding.

A cDNA encoding a cytoplasmic oxysterol binding protein was expressed at high levels by transfection in animal cells. This protein binds oxysterols such as 25-hydroxycholesterol that regulate sterol metabolism by transcriptional and posttranscriptional effects. In the transfected cells, some of the oxysterol binding protein (OSBP) was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus, as revealed by indirect immunofluorescence. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in large perinuclear structures that stained with lentil lectin, a protein that stains the Golgi apparatus. The structures that contained OSBP were disrupted by brefeldin A, confirming their identification as Golgi. A mutant OSBP lacking the COOH-terminal oxysterol binding domain localized to the Golgi spontaneously, suggesting that this domain normally occludes the domain that binds to the Golgi and that sterols relieve this occlusion. The previously noted potential leucine zipper sequence in OSBP was not required for Golgi localization, nor was it essential for homodimer formation. We conclude that OSBP is triggered to bind extrinsically to Golgi membranes when it binds oxysterols and speculate that this translocation may play a role in the transport, metabolism, or regulatory actions of oxysterols.

Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes.

Circulating human lymphocytes freshly isolated from venous blood of 15 normal subjects exhibited a low capacity to bind, take up, and degrade 125I-labeled low density lipoprotein (LDL). However, when these cells were incubated for 72 h in the absence of lipoproteins, they gradually acquired in increased number of high affinity cell surface receptors for LDL. The increase in the number of LDL receptors was associated with a 16-fold increase in the rate at which the cells were able to take up and degrade the lipoprotein. The LDL binding and degradation processes that developed in normal lymphocytes exhibited the following characteristics; (a) high affinity (saturation was achieved at LDL concentrations below 50 mug protein/ml); (b) specificity (unlabeled LDL was much more effective than human high density lipoprotein or other plasma proteins in competing with 125I-LDL for binding to the LDL receptor); and(c) feedback regulation (the increase in the number of LDL receptors that appeared after incubation of freshly isolated lymphocytes in lipoprotein-deficient medium was prevented by exposure of the cells to either LDL or a mixture of 25-hydroxycholesterol plus cholesterol but not to HDL). Freshly isolated lymphocytes obtaine from three subjects with the homozygous form of familial hypercholesterolemia failed to develop normal amounts of LDL receptor activity when incubated in medium devoid of lipoproteins. The current data indicate: (a) that the LDL receptors that appear on the surface of cholesterol-deprived, normal human lymphocytes are genetically identical to the previously characterized LDL receptors of cultured human fibroblasts and long-term lymphoid cells and (b) that at least one cell type in the human body, the circulating human lymphocyte, has the capacity to produce a high affinity LDL receptor that mediates the cellular uptake and degradation of plasma LDL.

Genetics of the low density lipoprotein receptor. Diminished receptor activity in lymphocytes from heterozygotes with familial hypercholesterolemia.

Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.

Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Watanabe Heritable Hyperlipidemic (WHHL) rabbits, like humans with familial hypercholesterolemia, have a genetic defect in the low density lipoprotein (LDL) receptor. WHHL fibroblasts produce a low molecular weight precursor form of the receptor that is not glycosylated normally and is not transported to the cell surface at a normal rate. In the current studies, we have used a monoclonal antibody that reacts with the rabbit LDL receptor to extend these findings to intact rabbits. We have made the following observations: (a) In normal rabbits the liver and adrenal glands synthesize high molecular weight mature LDL receptors like those in fibroblasts. (b) In WHHL rabbits the adrenals express only the low molecular weight receptor precursor, and the liver expresses no detectable receptors. (c) When injected intravenously, the radioiodinated anti-LDL receptor monoclonal antibody is cleared from plasma 6-10-fold faster in normal than in WHHL rabbits, with specific uptake demonstrable in livers and adrenals of normal but not WHHL rabbits. The latter finding raises the general possibility that the total number of cell surface receptors expressed by an animal or human in vivo can be estimated by measuring the rate of clearance of an intravenously injected monoclonal antibody directed against the receptor of interest.

Vacuum electron acceleration and bunch compression by a flat-top laser beam.

The field intensity distribution and phase velocity characteristics of a flat-top laser beam are analyzed and discussed. The dynamics of electron acceleration in this kind of beam are investigated using three-dimensional test particle simulations. Compared with the standard (i.e., TEM(00) mode) Gaussian beam, a flat-top laser beam has a stronger longitudinal electric field and a larger diffraction angle. These characteristics make it easier for electrons to be trapped and accelerated by the beam. With a flat-top shape, the laser beam is also applicable to the acceleration of low energy electron and bunch compression.

Phosducin induces a structural change in transducin beta gamma.

BACKGROUND: Phosducin binds tightly to the beta gamma subunits (Gt beta gamma) of the heterotrimeric G protein transducin, preventing Gt beta gamma reassociation with Gt alpha-GDP and thereby inhibiting the G-protein cycle. Phosducin-like proteins appear to be widely distributed and may play important roles in regulating many heterotrimeric G-protein signaling pathways. RESULTS: The 2.8 A crystal structure of a complex of bovine retinal phosducin with Gt beta gamma shows how the two domains of phosducin cover one side and the top of the seven-bladed beta propeller of Gt beta gamma. The binding of phosducin induces a distinct structural change in the beta propeller of Gt beta gamma, such that a small cavity opens up between blades 6 and 7. Electron density in this cavity has been assigned to the farnesyl moiety of the gamma subunit. CONCLUSIONS: beta gamma subunits of heterotrimeric G proteins can exist in two distinct conformations. In the R (relaxed) state, corresponding to the structure of the free beta gamma or the structure of beta gamma in the alpha beta gamma heterotrimer, the hydrophobic farnesyl moiety of the gamma subunit is exposed, thereby mediating membrane association. In the T (tense) state, as observed in the phosducin-Gt beta gamma structure, the farnesyl moiety of the gamma subunit is effectively buried in the cavity formed between blades 6 and 7 of the beta subunit. Binding of phosducin to Gt beta gamma induces the formation of this cavity, resulting in a switch from the R to the T conformation. This sequesters beta gamma from the membrane to the cytosol and turns off the signal-transduction cascade. Regulation of this membrane association/dissociation switch of Gt beta gamma by phosducin may be a general mechanism for attenuation of G protein coupled signal transduction cascades.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.