Mark4 ablation attenuates pathological phenotypes in a mouse model of tauopathy.

Accumulation of abnormally phosphorylated tau proteins is linked to various neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. Microtubule affinity-regulating kinase 4 (MARK4) has been genetically and pathologically associated with Alzheimer's disease and reported to enhance tau phosphorylation and toxicity in Drosophila and mouse traumatic brain-injury models but not in mammalian tauopathy models. To investigate the role of MARK4 in tau-mediated neuropathology, we crossed P301S tauopathy model (PS19) and Mark4 knockout mice. We performed behaviour, biochemical and histology analyses to evaluate changes in PS19 pathological phenotype with and without Mark4. Here, we demonstrated that Mark4 deletion ameliorated the tau pathology in a mouse model of tauopathy. In particular, we found that PS19 with Mark4 knockout showed improved mortality and memory compared with those bearing an intact Mark4 gene. These phenotypes were accompanied by reduced neurodegeneration and astrogliosis in response to the reduction of pathological forms of tau, such as those phosphorylated at Ser356, AT8-positive tau and thioflavin S-positive tau. Our data indicate that MARK4 critically contributes to tau-mediated neuropathology, suggesting that MARK4 inhibition may serve as a therapeutic avenue for tauopathies.

[Clinical characteristics of seizure-predominant autoimmune encephalitis and utility of anti-neuronal antibody scores for early treatment].

We analyzed 20 patients diagnosed with autoimmune neurological diseases with seizure predominance. In these patients, we examined the usefulness of Antibody Prevalence in Epilepsy and Encephalopathy (APE2) score and Antibodies Contributing to Focal Epilepsy Signs and Symptoms (ACES) score in autoimmune encephalitis (AE) for facilitating early treatment. APE2 score was positive in 19 of 20 patients. ACES score was positive in 15 of 20 patients, and 4 of 5 of the patients with negative ACES score did not have AE. Comprehensive assessment including the use of the above scores is desirable in the early stage of AE.

Microtubule-affinity regulating kinase family members distinctively affect tau phosphorylation and promote its toxicity in a Drosophila model.

Accumulation of abnormally phosphorylated tau and its aggregation constitute a significant hallmark of Alzheimer's disease (AD). Tau phosphorylation at Ser262 and Ser356 in the KXGS motifs of microtubule-binding repeats plays a critical role in its physiological function and AD disease progression. Major tau kinases to phosphorylate tau at Ser262 and Ser356 belong to the Microtubule Affinity Regulating Kinase family (MARK1-4), which are considered one of the major contributors to tau abnormalities in AD. However, whether and how each member affects tau toxicity in vivo is unclear. We used transgenic Drosophila as a model to compare the effect on tau-induced neurodegeneration among MARKs in vivo. MARK4 specifically promotes tau accumulation and Ser396 phosphorylation, which yields more tau toxicity than was caused by other MARKs. Interestingly, MARK1, 2, and 4 increased tau phosphorylation at Ser262 and Ser356, but only MARK4 caused tau accumulation, indicating that these sites alone did not cause pathological tau accumulation. Our results revealed MARKs are different in their effect on tau toxicity, and also in tau phosphorylation at pathological sites other than Ser262 and Ser356. Understanding the implementation of each MARK into neurodegenerative disease helps to develop more target and safety therapies to overcome AD and related tauopathies.

Integration of the Generalist Predator Nabis americoferus (Hemiptera: Nabidae) in a Greenhouse Strawberry Biocontrol Program with Phytoseiid Predatory Mites and the Entomopathogenic Fungus Beauveria bassiana.

In strawberry production, western flower thrips (WFT) and two-spotted spider mites (TSSM) inflict feeding damage and reduce the yield. Biological control for these pests often includes phytoseiid predatory mites and entomopathogenic fungi. The hemipteran family Nabidae have been reported as prominent predators in open-field strawberry. Nabis americoferus Carayon is a new biocontrol agent developed in Canada. This study examined if this species was a good candidate for integration with biological control for greenhouse strawberry production. The laboratory trials showed that Phytoseiulus persimilis Athias-Henriot and Amblyseius swirskii Athias-Henriot were compatible with N. americoferus, especially when alternative food was available. In contrast, the nabid was not compatible with the Beauveria bassiana (Balsamo) GHA strain. A greenhouse cage study was conducted to determine if it was beneficial to add N. americoferus to the phytoseiid-mites-based biological control program for WFT and TSSM in greenhouse strawberry. The release of N. americoferus on a banker plant together with the placement of sachets of Neoseiulus cucumeris (Oudemans) and Neoseiulus californicus (McGregor) was beneficial, not only potentially reducing the number of sachet applications, but also providing better pest control than phytoseiid mites alone. Neither the phytoseiids nor the N. americoferus numbers were significantly affected by the presence of each other.

Rapid and high throughput assessment of cellular immunity against SARS-CoV-2 based on the ex vivo activation of genes in leukocyte assay with whole blood.

During the novel coronavirus outbreak and vaccine development, antibody production garnered major focus as the primary immunogenic response. However, cellular immunity's recent demonstration of comparable or greater significance in controlling infection demands the re-evaluation of the importance of T-cell immunity in SARS-CoV-2 infection. Here, we developed a novel assay, the ex vivo activation of genes in leukocytes (EAGL), which employs short-term whole blood stimulation with the LeukoComplete™ system, to measure ex vivo SARS-CoV-2-specific T cell responses (cellular immunity). This assay measures upregulated mRNA expression related to leukocyte activation 4 h after antigen stimulation. LeukoComplete™ system uses whole blood samples, eliminating the need for pretreatment before analysis. Furthermore, this system's high reproducibility is ensured through a series of operations from mRNA extraction to cDNA synthesis on a 96-well plate. In the performance evaluation using fresh blood from previously SARS-CoV-2-infected and COVID-19-vaccinated individuals, the EAGL assay had a comparable sensitivity and specificity to the ELISpot assay (EAGL: 1.000/1.000; ELISpot: 0.900/0.973). As a simple, high-throughput assay, the EAGL assay is also a quantitative test that is useful in studies with large sample numbers, such as monitoring new vaccine efficacies against novel coronaviruses or epidemiologic studies that require cellular immune testing during viral infection.

A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity.

Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78-95). Extracellular supplementation of TAT-Orf9b10-18_78-95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78-95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78-95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200 µg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20 µg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila.

Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.

Endoscopic drainage with a metallic stent for obstructive jaundice caused by bile duct metastasis of breast cancer: A case report.

Key Clinical Message: Bile duct metastasis of breast cancer is rare. It often causes obstructive jaundice which makes the patient interrupt state of treatment. Endoscopic drainage for obstructive jaundice is effective and less invasive treatment option also in this case. Abstract: A 66-year-old breast ductal carcinoma patient developed obstructive jaundice, presenting with epigastric discomfort and dark-colored urine. Computed tomography and endoscopic retrograde cholangiopancreatography revealed bile duct stenosis. Brushing cytology and tissue biopsy confirmed bile duct metastasis, a self-expandable metallic stent was placed/replaced endoscopically, and chemotherapy was continued, extending the patient's life.

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring.

Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1-100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.

Cerebral Tuberculoma Diagnosed by Nested Polymerase Chain Reaction of a Formalin-fixed Paraffin-embedded Brain Biopsy Sample.

A 38-year-old man was taken to hospital with generalized clonic seizure. Brain magnetic resonance imaging (MRI) showed multiple ring-enhancing lesions centered in the left frontoparietal lobe. A histopathological examination of a brain biopsy sample revealed granulomatous lesions with caseous necrosis. We extracted DNA from a formalin-fixed paraffin-embedded (FFPE) brain specimen, and nested polymerase chain reaction (PCR) of the DNA sample detected the Mycobacterium tuberculosis-specific insertion sequence IS6110. The lesions worsened after anti-tuberculosis drugs were administered, which we considered to be a paradoxical response and continued treatment. A genetic diagnosis of M. tuberculosis using FFPE specimens is useful for diagnosing tuberculoma.

Inhibitory Effect of a Late Sodium Current Blocker, NCC-3902, on the Automaticity of the Guinea Pig Pulmonary Vein Myocardium.

The effect of blocking the persistent component of the sodium channel current (late INa) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late INa, but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late INa induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca2+ transients, but had no effect on Ca2+ sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late INa like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.

Anti-signal Recognition Particle Antibody-positive Immune-mediated Myopathy after mRNA-1273 SARS-CoV-2 Vaccination.

A 26-year-old Japanese woman developed a fever, myalgia and gait disturbance one day after receiving the second dose of the mRNA-1273 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. A neurological examination revealed symmetrical weakness and myalgia in proximal lower limbs, and a blood examination showed prominent elevation of creatinine kinase. Magnetic resonance imaging (MRI) revealed a high signal intensity in the thigh muscles on short-tau inversion recovery images, and antibody testing revealed positive findings for anti-signal recognition particle (SRP) antibody. Thus, anti-SRP antibody-positive immune-mediated myopathy was diagnosed. We initiated immunotherapy, and she was ultimately able to walk stably.

Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells.

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

The generalist predatory mite Anystis baccarum (Acari: Anystidae) as a new biocontrol agent for western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae).

A generalist predatory mite, Anystis baccarum (L.), was evaluated as a biological control agent against western flower thrips (WFT), Frankliniella occidentalis (Pergande). Laboratory assays showed A. baccarum was able to kill a mean of five WFT adult females or nine WFT larvae in 24 h, out-performing both Neoseiulus cucumeris (Oudemans) and Amblyseius swirskii Athias-Henriot. Next, a greenhouse assay was conducted to assess the performance of A. baccarum on potted chrysanthemums, comparing their efficacy to that provided by N. cucumeris slow-release sachets which represented the commercial standard in Canada. A combined treatment which incorporated both predatory mite species was also included to assess compatibility and potential additive effects of using both species together for WFT management. Introduction of two A. baccarum per pot was as efficacious as 125 N. cucumeris in terms of WFT control; however, despite the lack of significance between the level of WFT control obtained in the single predatory species treatments and the combined treatment, only the combination treatment suppressed WFT populations to levels that were almost unchanged over 8 weeks. There was no significant difference between the number of N. cucumeris recovered from plants in the single-species and the combination treatments, demonstrating the functional compatibility of the two predators. Additionally, WFT feeding damage was significantly lower on the A. baccarum-treated plants than on the untreated control and the N. cucumeris treatment. This study, together with our development of a prototype mass rearing method, shows that A. baccarum could be successfully used as a biocontrol agent for WFT.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of ß-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE1). The ß-cleavage of APP by BACE1 is the initial and rate-limiting step in Aß generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The ß-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

5-Aminolevulinic acid and sodium ferrous citrate ameliorate muscle aging and extend healthspan in Drosophila.

Declines in mitochondrial functions are associated with aging. The combination of 5-aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5-ALA and SFC (5-ALA/SFC) on the healthspan and life span of Drosophila melanogaster. Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5-ALA/SFC. Locomotor functions, life span, muscle architecture, and age-associated changes in mitochondrial function were analyzed. We found that feeding 5-ALA/SFC mitigated age-associated declines in locomotor functions and extended organismal life span. Moreover, 5-ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5-ALA phosphate/SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age-related declines in muscle functions, prevent age-associated clinical conditions such as frailty, and extend healthspan and life span.

Efficacy of Anystis baccarum against Foxglove Aphids, Aulacorthum solani, in Laboratory and Small-Scale Greenhouse Trials.

A generalist predatory mite, Anystis baccarum (L.), has been identified as a key predator of small, soft-bodied pest species in various agroecosystems around the world. The foxglove aphid Aulacorthum solani (Keltenbach) is a new problematic pest in Canadian greenhouses. Laboratory colonies of A. baccarum were established and its predatory efficacy against A. solani was assessed. In laboratory trials, A. baccarum ate approximately one adult aphid or seven first instar aphids in 24 h. In a greenhouse bench trial on sweet peppers with the free-flying aphid parasitoid, Aphidius ervi Haliday, the population dynamics of A. solani in the presence or absence of A. baccarum was evaluated. Although the parasitoid alone successfully eradicated A. solani, when A. baccarum were present on the plants, the aphid population was eradicated more rapidly. Fruit yield was also 15% higher from plants where A. baccarum was released than the control (without A. baccarum). Furthermore, plants were naturally infested by Frankliniella occidentalis (Pergande) during the trial, which caused visible feeding damage to the fruits. Anystis baccarum also predates on thrips and thrips' feeding damage to the fruits was reduced on plants where A. baccarum was released. Anystis baccarum was able to establish in sweet peppers and was determined to be complementary to the current practice of using A. ervi for the biological control of A. solani.

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo.

Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.