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The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions. We analyzed HTLV-1-positive asymptomatic carriers (ACs) and AC samples at high risk for developing ATL due to high proviral load (ATL high-risk (HR) ACs) using dPCR. Deletions were identified in 11.8 % (4/34, all Type 1) of ACs and 33.3 % (7/21, Type 1:1, Type 2:6) of ATL HR ACs. dPCR analysis revealed that in three ATL samples, all exhibited Type 1 defective characteristics, and two showed extremely low ratios in the pol region. Clonality analysis of these two samples revealed high monoclonality, indicating monoclonal expansion of ATL cells with defective proviruses. These findings demonstrate that our method effectively detects defective proviruses in both ACs and ATL, providing a valuable tool for understanding the genomic characteristics of proviruses in these conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: This study conducted a comprehensive glycan analysis of serum to determine how glycan biomarkers are associated with the pathophysiology of chronic inflammatory demyelinating polyneuropathy (CIDP) and the effects of its treatment., Methods: We comparatively analyzed N- and O-glycans in the pretreatment serum of 27 treatment-naïve patients with typical CIDP and 20 age- and sex-matched healthy controls (HC) using mass spectrometry. We determined the association between clinical parameters and glycans. The serum glycan and neurofilament light-chain (NfL) levels were assessed at the baseline, and treatment response was defined according to the degree of improvement in the modified Rankin scale 12 weeks after the first dose of intravenous immunoglobulin (IVIg)., Results: Compared with the HC, the CIDP group demonstrated significantly lower levels of serum total N-glycans (CIDP, median 973.3 [IQR 836.2-1131.3] pmol/μL; HC, 1125.0 [1005.0-1236.2] pmol/μL; p < 0.05), especially sialylated N-glycans (CIDP, 898.0 [752.2-1037.2] pmol/μL; HC, 1064.4 [942.7-1189.8] pmol/μL; p < 0.01). In contrast, the O-glycan levels did not differ significantly between the two groups. The treatment response was associated with low N-glycan levels, but not with the serum NfL levels. Low levels of sialylated N-glycans were associated with resistance to treatment over 12 weeks, with an area under the curve of 0.822 (p < 0.01)., Conclusions: Low levels of sialylated N-glycans could potentially serve as a novel biomarker, reflecting pathophysiology and therapeutic resistance in typical CIDP., (© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ideonella sakaiensis is a bacterium that can degrade and consume polyethylene terephthalate (PET), a plastic material that was previously considered non-biodegradable. The degradation of PET requires two enzymes, namely poly (ethylene terephthalate) hydrolase (PETase) and mono (2-hydroxyethyl) terephthalate hydrolase (MHETase), which break down PET into terephthalate (TPA) and ethylene glycol (EG), which serve as carbon sources for the bacterium. Previous studies have focused on the enzymatic properties, structure, and mechanism of action of PETase and MHETase. However, the regulation of PETase and MHETase gene expression has not been investigated. This study identified a protein that binds to the MHETase promoter DNA, MHETase gene-regulating protein (MRP) in I. sakaiensis. PET or TPA induced the expression of PETase and MHETase genes. Furthermore, the induction of the MHETase gene was abolished by the deletion of the mrp gene, while the expression of the PETase gene was maintained. In addition, the genes involved in TPA metabolism were not induced in the mrp mutant. Furthermore, the growth of the PET and TPA deteriorated due to mrp mutation. Also, MRP binds to the promoter regions of the MHETase gene and TPA metabolizing genes, but not to the PETase gene promoter. These results suggest that MRP is a transcription factor that activates MHETase and TPA-metabolizing genes., (© 2024 Federation of European Biochemical Societies.)

Comorbid Alzheimer's disease (AD) neuropathology is common in Lewy body disease (LBD); however, AD comorbidity in the prodromal phase of LBD remains unclear. This study investigated AD comorbidity in the prodromal and symptomatic phases of LBD by analyzing plasma biomarkers in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and individuals at risk of LBD (NaT-PROBE cohort). Patients with PD (PD group, n = 84) and DLB (DLB group, n = 16) and individuals with LBD with ≥ 2 (high-risk group, n = 82) and without (low-risk group, n = 37) prodromal symptoms were enrolled. Plasma amyloid-beta (Aβ) composite was measured using immunoprecipitation-mass spectrometry assays. Plasma phosphorylated tau 181 (p-tau181), neurofilament light chain (NfL), and alpha-synuclein (aSyn) were measured using a single-molecule array. Plasma p-tau181 levels were higher in the PD and DLB groups than in the low-risk group. Aβ composite level was higher in the DLB group than in the high-risk group. AD-related biomarker levels were not elevated in the high-risk group. NfL levels were higher in the high-risk, PD, and DLB groups than in the low-risk group. In the PD group, Aβ composite was associated with cognitive function, p-tau181 with motor function and non-motor symptoms, and NfL with cognitive and motor functions and non-motor symptoms. In the high-risk group, NfL was associated with metaiodobenzylguanidine scintigraphy abnormalities. The PD and DLB groups exhibited comorbid AD neuropathology, though not in the prodromal phase. Elevated plasma NfL levels, even without elevated AD-related plasma biomarker levels, may indicate aSyn-induced neurodegeneration in the LBD prodromal phase., (© 2024. The Author(s).)

Background: Simian T-cell leukemia virus type 1 (STLV-1) is a retrovirus closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia (ATL). It has been shown that Japanese macaques (Macaca fuscata, JMs) are one of the main hosts of STLV-1 and that a high percentage of JMs (up to 60%) are infected with STLV-1; however, the molecular epidemiology of STLV-1 in JMs has not been examined., Methods: In this study, we analyzed full-length STLV-1 genome sequences obtained from 5 independent troops including a total of 68 JMs., Results: The overall nucleotide heterogeneity was 4.7%, and the heterogeneity among the troops was 2.1%, irrespective of the formation of distinct subclusters in each troop. Moreover, the heterogeneity within each troop was extremely low (>99% genome homology) compared with cases of STLV-1 in African non-human primates as well as humans. It was previously reported that frequent G-to-A single-nucleotide variants (SNVs) occur in HTLV-1 proviral genomes in both ATL patients and HTLV-1 carriers, and that a G-to-A hypermutation is associated with the cellular antiviral restriction factor, Apobec3G. Surprisingly, these SNVs were scarcely observed in the STLV-1 genomes in JMs., Conclusions: Taken together, these results indicate that STLV-1 genomes in JMs are highly homologous, at least in part due to the lack of Apobec3G-dependent G-to-A hypermutation., (© 2024. The Author(s).)

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan., (© 2024 Wiley-VCH GmbH.)

Objective: To evaluate the status of lupus nephritis treatment particularly regarding the need for intensification of treatment in Japan from 2010 to 2019 using a large-scale claims database., Methods: This descriptive study included adult lupus nephritis patients who were administered glucocorticoid equivalent to ≥15 mg prednisolone as the initial dose. After summarizing patient characteristics, we assessed the rate of treatment intensification using the Kaplan-Meier method among six groups based on initial dose of glucocorticoid., Results: We identified 403 patients (mean age, 42.7 years; 68.5% women) with the median initial glucocorticoid dose of 30 mg/day prednisolone equivalent. We observed 56 treatment intensifications; the incidence rate was 71.3 per 1,000 person-years (95% confidence interval: 52.6-90.0). The rate in higher glucocorticoid dose groups was higher than that in lower glucocorticoid dose groups., Conclusions: We found that the rate of treatment intensification was higher in the high-dose glucocorticoid groups than in the low-dose glucocorticoid groups. Further studies are needed to clarify the relationship between the initial dose of glucocorticoids and the prognosis of lupus nephritis patients.

Importance: The World Health Organization estimated that 5-10 million people are infected with human T-cell leukemia virus type 1 (HTLV-1). This number is likely to be underestimated because reliable endemic data are available for only approximately 1.5 billion people worldwide. The point-of-care test is a powerful tool for the easy and quick detection of infections without the requirement for expensive instruments and laboratory equipment. Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test that was evaluated in this study, might significantly advance our understanding of the global epidemiology of HTLV-1 infection., Competing Interests: The authors declare no conflict of interest.

Objective: Many models for predicting various disease prognoses have achieved high performance without laboratory test results. However, whether laboratory test results can improve performance remains unclear. This study aimed to investigate whether laboratory test results improve the model performance for coronavirus disease 2019 (COVID-19)., Methods: Prediction models were developed using data from the electronic healthcare record database in Japan. Patients aged ≥18 years hospitalized for COVID-19 after February 11, 2020, were included. Their age, sex, comorbidities, laboratory test results, and number of days from February 11, 2020, were collected. We developed a logistic regression, XGBOOST, random forest, and neural network analysis and compared the performance with and without laboratory test results. The performance of predicting in-hospital death was evaluated using the area under the curve (AUC)., Results: Data from 8,288 hospitalized patients (females, 46.5%) were analyzed. The median patient age was 71 years. A total of 6,630 patients were included in the training dataset, and 312 (4.7%) died. In the logistic regression model, the area under the curve was 0.88 (95% confidence interval [CI] = 0.83-0.93) and 0.75 (95% CI = 0.68-0.81) with and without laboratory test results, respectively. The performance was not fundamentally different between the model types, and the laboratory test results improved the performance in all cases. The variables useful for prediction were blood urea nitrogen, albumin, and lactate dehydrogenase., Conclusions: Laboratory test results, such as blood urea nitrogen, albumin, and lactate dehydrogenase levels, along with background information, helped estimate the prognosis of patients hospitalized for COVID-19.

4-Amino-4-deoxychorismate synthase (ADCS), a chorismate-utilizing enzyme, is composed of two subunits: PabA and PabB. PabA is a glutamine amidotransferase that hydrolyzes glutamine into glutamate and ammonia. PabB is an aminodeoxychorismate synthase that converts chorismate to 4-amino-4-deoxychorismate (ADC) using the ammonia produced by PabA. ADCS functions under allosteric regulation between PabA and PabB. However, the allosteric mechanism remains unresolved because the structure of the PabA-PabB complex has not been determined. Here, the crystal structure and characterization of PapA from Streptomyces venezuelae (SvPapA), a bifunctional enzyme comprising the PabA and PabB domains, is reported. SvPapA forms a unique dimer in which PabA and PabB domains from different monomers complement each other and form an active structure. The chorismate-bound structure revealed that recognition of the C1 carboxyl group by Thr501 and Gly502 of the 498-PIKTG-502 motif in the PabB domain is essential for the catalytic Lys500 to reach the C2 atom, a reaction-initiation site. SvPapA demonstrated ADCS activity in the presence of Mg 2+ when glutamate or NH + 4 was used as the amino donor. The crystal structure indicated that the Mg 2+ -binding position changed depending on the binding of chorismate. In addition, significant structural changes were observed in the PabA domain depending on the presence or absence of chorismate. This study provides insights into the structural factors that are involved in the allosteric regulation of ADCS.

Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideβ ( 6 ), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo . We demonstrate that cholicamideβ ( 6 ), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideβ ( 6 ) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideβ ( 6 ) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideβ ( 6 ) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.

Introduction: There are approximately 13 million patients with osteoporosis in Japan; however, only 20% of them receive treatment. This study compared the 4-year continuation rates of osteoporosis drugs at different dosing intervals across throughout Japan., Materials and Methods: Receipt data were analyzed to determine the continuation of osteoporosis medication in patients who began treatment at 210 hospitals throughout Japan between October 2016 and September 2017. Continuation rates were compared using Kaplan-Meier curves and log-rank tests., Results: The 4-year continuation rates of daily, weekly, monthly, semi-annual, and yearly medications nationwide were 8.6%, 16.5%, 13.5%, 31.0%, and 26.0%, respectively. The 4-year continuation rates for semi-annual and yearly drugs were significantly higher than daily, weekly, monthly drugs., Conclusion: Throughout Japan, the 4-year treatment continuation rates of daily, weekly, monthly, semi-annual, and yearly osteoporosis drugs all decreased over time. However, the continuation rates of semi-annual and yearly drugs in the fourth year were significantly higher than those for the other dosing regimens. Therefore, injectable drugs with dosing intervals ≥6 months may improve continuation rates of osteoporosis drugs. Geriatr Gerontol Int 2023; 23: 665-670., (© 2023 Japan Geriatrics Society.)

Introduction: Patients with Parkinson's disease (PD) encounter a variety of speech-related problems, including dysarthria and language disorders. To elucidate the pathophysiological mechanisms for linguistic alteration in PD, we compared the utterance of patients and that of healthy controls (HC) using automated morphological analysis tools., Methods: We enrolled 53 PD patients with normal cognitive function and 53 HC, and assessed their spontaneous speech using natural language processing. Machine learning algorithms were used to identify the characteristics of spontaneous conversation in each group. Thirty-seven features focused on part-of-speech and syntactic complexity were used in this analysis. A support-vector machine (SVM) model was trained with ten-fold cross-validation., Results: PD patients were found to speak less morphemes on one sentence than the HC group. Compared to HC, the speech of PD patients had a higher rate of verbs, case particles (dispersion), and verb utterances, and a lower rate of common noun utterances, proper noun utterances, and filler utterances. Using these conversational changes, the respective discrimination rates for PD or HC were more than 80%., Conclusions: Our results demonstrate the potential of natural language processing for linguistic analysis and diagnosis of PD., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Individuals with prodromal symptoms of Lewy body disease (LBD), such as rapid eye movement sleep behavior disorder (RBD), often showed imaging defects similar to patients with Parkinson's disease and dementia with Lewy bodies. We examined dopamine transporter (DaT) single-photon-emission computed tomography (SPECT) and metaiodobenzylguanidine (MIBG) scintigraphy in 69 high-risk subjects with ≥2 prodromal symptoms (dysautonomia, hyposmia, and probable RBD) and 32 low-risk subjects without prodromal symptoms, whom were identified through a questionnaire survey of health checkup examinees. The high-risk subjects had significantly worse scores on Stroop test, line orientation test, and the Odor Stick Identification Test for Japanese than the low-risk subjects. The prevalence of abnormalities on DaT-SPECT was higher in the high-risk group than in the low-risk group (24.6% vs. 6.3%, p = 0.030). A decreased uptake on DaT-SPECT was associated with motor impairment, and MIBG scintigraphy defects were associated with hyposmia. The simultaneous evaluation of DaT-SPECT and MIBG scintigraphy may capture a wide range of individuals with prodromal LBD., (© 2023. The Author(s).)

Background: Developmental venous anomalies (DVAs) are congenital anatomical variants of the normal deep parenchymal veins. DVAs are occasionally found incidentally on brain imaging, and most cases are asymptomatic. However, they rarely cause central nervous disorders. Herein, a case of mesencephalic DVA that caused aqueduct stenosis and hydrocephalus and discuss its diagnosis and treatment is reported., Observations: The patient was a 48-year-old female who presented with depression. Computed tomography and magnetic resonance imaging (MRI) of the head revealed obstructive hydrocephalus. Contrast-enhanced MRI revealed an abnormally distended linear region with enhancement on the top of the cerebral aqueduct, which was confirmed as a DVA by digital subtraction angiography. An endoscopic third ventriculostomy (ETV) was performed to improve the patient's symptoms. Intraoperative endoscopic imaging showed obstruction of the cerebral aqueduct by the DVA., Lessons: This report describes a rare case of obstructive hydrocephalus caused by DVA. It highlights the usefulness of contrast-enhanced MRI for diagnosing cerebral aqueduct obstructions due to DVAs and the effectiveness of ETV as a treatment option.

Non-motor symptoms (NMS) are common among patients with Parkinson's disease and reduce patients' quality of life (QOL). However, there remain considerable unmet needs for NMS management. Three monoamine oxidase B inhibitors (MAO-BIs), selegiline, rasagiline, and safinamide, have become commercially available in many countries. Although an increasing number of studies have reported potential beneficial effects of MAO-BIs on QOL and NMS, there has been no consensus. Thus, the primary objective of this study was to provide an up-to-date systematic review of the QOL and NMS outcomes from the available clinical studies of MAO-BIs. We conducted a literature search using the PubMed, Scopus, and Cochrane Library databases in November 2021. We identified 60 publications relevant to this topic. Overall, rasagiline and safinamide had more published evidence on QOL and NMS changes compared with selegiline. This was likely impacted by selegiline being introduced many years prior to the field embarking on the study of NMS. The impact of MAO-BIs on QOL was inconsistent across studies, and this was unlikely to be clinically meaningful. MAO-BIs may potentially improve depression, sleep disturbances, and pain. In contrast, cognitive and olfactory dysfunctions are likely unresponsive to MAO-BIs. Given the paucity of evidence and controlled, long-term studies, the effects of MAO-BIs on fatigue, autonomic dysfunctions, apathy, and ICD remain unclear. The effects of MAO-BIs on static and fluctuating NMS have never been investigated systematically. More high-quality studies will be needed and should enable clinicians to provide personalized medicine based on a non-motor symptom profile., (© 2022. The Author(s).)

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identified and characterized the ans operon required for L-Asn catabolism in Corynebacterium glutamicum R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5'-end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1- 13 C]Gly- d 2 -NMe 2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence 1-3 . Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents 4,5 . Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic 6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: The impact of deep brain stimulation (DBS) on speech rhythm and its mechanism remains unclear. We investigated speech rhythm characteristics of patients with Parkinson's disease (PD) treated with subthalamic nucleus (STN) DBS to understand the underlying pathophysiology better., Methods: We enrolled a total of 105 participants and evaluated speech rhythm performances among patients with PD who had undergone STN-DBS (the PD-DBS group), patients with PD treated only with medication (the PD-Med group), patients with cerebellar ataxia (the CA group), and healthy controls (the HC group). Each participant was asked to repeat the syllable/pa/at a comfortable self-chosen steady pace. A widely-used software (the Motor Speech Profile) program performed an acoustic analysis., Results: Compared to the PD-Med and HC groups, speech rate instability (DDKjit) was significantly higher in the PD-DBS and CA groups (p < 0.01). However, after DBS was turned off, the DDKjit of the PD-DBS group improved to a level comparable to that of the PD-Med and HC groups. In contrast to the significantly higher variability of speech volume (DDKcvi) in the CA group, the PD-DBS group showed similar DDKcvi to the PD-Med and HC groups., Conclusions: STN-DBS affects the speech rate stability of patients with PD. Speech rhythm disorders caused by STN-DBS were phenotypically similar to that in CA in terms of interval variability but different regarding amplitude variability. Further studies are warranted to elucidate the underlying pathophysiology of speech rhythm disorders in PD patients treated with DBS., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

Few reports have described the biological degradation or utilization of poly(ethylene terephthalate) (PET) to support microbial growth. We screened environmental samples from a PET bottle recycling site and identified the microbial consortium no. 46, which degraded amorphous PET at ambient temperature; thereafter, we isolated the resident Ideonella sakaiensis 201-F6 strain responsible for the degradation. We further identified two hydrolytic enzymes from I. sakaiensis, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase), which synergistically converted PET into its monomeric building blocks. Here, we provide original methods of microbial screening and isolation of PET degrading microbe(s). These novel approaches can be adapted for exploring microorganisms that degrade PET and other plastics. Furthermore, our enzyme assay protocols to characterize PETase and MHETase can be applied to evaluate new enzymes that target PET and its hydrolysates., (© 2021 Elsevier Inc. All rights reserved.)

Individual cell shape and integrity must precisely be orchestrated during morphogenesis. Here, we determine function of type II cadherins, Cdh6, Cdh8, and Cdh11, whose expression combinatorially demarcates the mouse neural plate/tube. While CRISPR/Cas9-based single type II cadherin mutants show no obvious phenotype, Cdh6/8 double knockout (DKO) mice develop intermingled forebrain/midbrain compartments as these two cadherins' expression opposes at the nascent boundary. Cdh6/8/11 triple, Cdh6/8 or Cdh8/11 DKO mice further cause exencephaly just within the cranial region where mutated cadherins' expression merges. In the Cdh8/11 DKO midbrain, we observe less-constricted apical actin meshwork, ventrally-directed spreading, and occasional hyperproliferation among dorsal neuroepithelial cells as origins for exencephaly. These results provide rigid evidence that, by conferring distinct adhesive codes to each cell, redundant type II cadherins serve essential and shared roles in compartmentalization and neurulation, both of which proceed under the robust control of the number, positioning, constriction, and fluidity of neuroepithelial cells.

Background: Volunteers are expected to play a key role in children's hospice. However, there is a lack of information about how to cultivate effective volunteer training programs., Objective: To verify the effect of a training program on volunteers' confidence in providing learning support and sharing experiences with children with life-threatening conditions and their families in a children's hospice., Methods: In this pre-post study, participants were 48 undergraduate and graduate students from 3 universities in Japan. They received 5 lectures on children's hospice learning support. They evaluated the training program by rating their self-confidence in meeting each of the 15 program goals on a questionnaire., Results: An exploratory factor analysis of the questionnaire yielded 12 goals in 4 factors: understanding of one's own and others' mental state, accommodating the learning needs of children with life-threatening conditions, understanding and accommodating the physical state of children with life-threatening conditions, and understanding the significance of children's hospice. A paired t test revealed that participants' self-confidence had increased significantly in 3 of these 4 factors after the program. However, the score for accommodating the learning needs of children with life-threatening conditions decreased but not significantly., Conclusion: Although it needs some improvements, the program was effective for improving volunteers' self-confidence in and understanding of learning support and sharing experiences with children with life-threatening conditions.

Comprehensive analysis is required for the accurate diagnosis of MV2-type sporadic Creutzfeldt-Jakob disease (sCJD) because it shows a wide clinicopathological spectrum. Here, we describe the clinical findings and neuropathologic observations of an autopsy-confirmed MV2K-type sCJD case with extensive spongiform changes of the cerebral cortex. In the early disease stages, the patient exhibited gait disturbance with ataxia and gradually showed cognitive dysfunction. Diffusion-weighted magnetic resonance images revealed hyperintense regions in the cerebral cortex, basal ganglia, and particularly in the thalamus. Prion protein (PrP) gene analysis revealed no mutations, and polymorphic codon 129 exhibited methionine and valine heterozygosity. During the course of the disease, a startle reaction was observed, whereas myoclonus was not observed. Electroencephalography showed no periodic sharp wave complexes. The patient died at age 61 years with 13 months total disease duration and did not reach the akinetic mutism state. Pathologic investigation revealed extensive fine vacuole-type spongiform change in the cerebral cortex, and the appearance of vacuolation tended to be more pronounced in the deeper layers. Numerous kuru plaques were observed in the cerebellum. PrP immunostaining revealed extensive diffuse synaptic-type PrP deposition in the cerebral cortex, and the finding was prominent in the deeper layer with perineuronal-type PrP deposition. In the limbic system, basal ganglia, and thalamus, mixed small plaque-type PrP with synaptic-type PrP deposition was observed. In the cerebellar cortex, diffuse synaptic-type PrP depositions were observed with numerous strongly immunopositive plaques. Western blot analysis of examined brain samples revealed mixed type 2 PrP Sc (scrapie type) and intermediate-type PrP Sc ., (© 2019 Japanese Society of Neuropathology.)