Lineage frequency time series reveal elevated levels of genetic drift in SARS-CoV-2 transmission in England.

Genetic drift in infectious disease transmission results from randomness of transmission and host recovery or death. The strength of genetic drift for SARS-CoV-2 transmission is expected to be high due to high levels of superspreading, and this is expected to substantially impact disease epidemiology and evolution. However, we don't yet have an understanding of how genetic drift changes over time or across locations. Furthermore, noise that results from data collection can potentially confound estimates of genetic drift. To address this challenge, we develop and validate a method to jointly infer genetic drift and measurement noise from time-series lineage frequency data. Our method is highly scalable to increasingly large genomic datasets, which overcomes a limitation in commonly used phylogenetic methods. We apply this method to over 490,000 SARS-CoV-2 genomic sequences from England collected between March 2020 and December 2021 by the COVID-19 Genomics UK (COG-UK) consortium and separately infer the strength of genetic drift for pre-B.1.177, B.1.177, Alpha, and Delta. We find that even after correcting for measurement noise, the strength of genetic drift is consistently, throughout time, higher than that expected from the observed number of COVID-19 positive individuals in England by 1 to 3 orders of magnitude, which cannot be explained by literature values of superspreading. Our estimates of genetic drift suggest low and time-varying establishment probabilities for new mutations, inform the parametrization of SARS-CoV-2 evolutionary models, and motivate future studies of the potential mechanisms for increased stochasticity in this system.

Distinct phosphorylation states of mammalian CaMKIIß control the induction and maintenance of sleep.

The reduced sleep duration previously observed in Camk2b knockout mice revealed a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII)ß as a sleep-promoting kinase. However, the underlying mechanism by which CaMKIIß supports sleep regulation is largely unknown. Here, we demonstrate that activation or inhibition of CaMKIIß can increase or decrease sleep duration in mice by almost 2-fold, supporting the role of CaMKIIß as a core sleep regulator in mammals. Importantly, we show that this sleep regulation depends on the kinase activity of CaMKIIß. A CaMKIIß mutant mimicking the constitutive-active (auto)phosphorylation state promotes the transition from awake state to sleep state, while mutants mimicking subsequent multisite (auto)phosphorylation states suppress the transition from sleep state to awake state. These results suggest that the phosphorylation states of CaMKIIß differently control sleep induction and maintenance processes, leading us to propose a "phosphorylation hypothesis of sleep" for the molecular control of sleep in mammals.

BayesianSSA: a Bayesian statistical model based on structural sensitivity analysis for predicting responses to enzyme perturbations in metabolic networks.

BACKGROUND: Chemical bioproduction has attracted attention as a key technology in a decarbonized society. In computational design for chemical bioproduction, it is necessary to predict changes in metabolic fluxes when up-/down-regulating enzymatic reactions, that is, responses of the system to enzyme perturbations. Structural sensitivity analysis (SSA) was previously developed as a method to predict qualitative responses to enzyme perturbations on the basis of the structural information of the reaction network. However, the network structural information can sometimes be insufficient to predict qualitative responses unambiguously, which is a practical issue in bioproduction applications. To address this, in this study, we propose BayesianSSA, a Bayesian statistical model based on SSA. BayesianSSA extracts environmental information from perturbation datasets collected in environments of interest and integrates it into SSA predictions. RESULTS: We applied BayesianSSA to synthetic and real datasets of the central metabolic pathway of Escherichia coli. Our result demonstrates that BayesianSSA can successfully integrate environmental information extracted from perturbation data into SSA predictions. In addition, the posterior distribution estimated by BayesianSSA can be associated with the known pathway reported to enhance succinate export flux in previous studies. CONCLUSIONS: We believe that BayesianSSA will accelerate the chemical bioproduction process and contribute to advancements in the field.

Applications and Limitations of Equilibrium Density Gradient Analytical Ultracentrifugation for the Quantitative Characterization of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

Diversity in bread and durum wheat stigma morphology and linkage of increased stigma length to dwarfing gene Rht14.

KEY MESSAGE: The dwarfing allele Rht14 of durum wheat associates with greater stigma length, an important trait for hybrid breeding, whilst major dwarfing alleles Rht-B1b and Rht-D1b showed little to no effect. Although much understudied in wheat, the stigma is a crucial component for attaining grain set, the fundamental basis for yield, particularly in hybrid production systems where successful grain set relies on wind-driven pollen dispersal by the male parent and effective pollen capture by the female parent. Females with long stigma that exsert early are thought to be advantageous. Using glasshouse-grown lines, we examined variation in Total Stigma Length (TSL) across diverse panels comprising 27 durum and 116 bread wheat genotypes. Contrasting genotypes were selected for population development and genetic analysis. Quantitative trait loci (QTL) analysis was performed on a durum F2 population and a bread wheat recombinant inbred line (RIL) population. Contrasting with studies of anther length, we found no large effect on TSL of the GA-insensitive semi-dwarfing genes Rht-B1 and Rht-D1 in either durum or bread wheat. However, in durum cultivar Italo, we identified a region on chromosome 6A which is robustly associated with larger TSL and contains the Rht14 allele for reduced plant height, a trait that is favourable for female line development in hybrid systems. This dual effect locus explained 25.2 and 19.2% of TSL phenotypic variation in experiments across two growing seasons, with preliminary results suggesting this locus may increase TSL when transferred to bread wheat. In a bread wheat, RIL population minor QTL on 1A and 2A was indicated, but the strongest association was with Ppd-B1. Methods developed here, and the identification of a TSL-enhancing locus provides advances and further opportunities in the study of wheat stigma.

Oxytocin-induced increases in cytokines and clinical effect on the core social features of autism: Analyses of RCT datasets.

Although oxytocin may provide a novel therapeutics for the core features of autism spectrum disorder (ASD), previous results regarding the efficacy of repeated or higher dose oxytocin are controversial, and the underlying mechanisms remain unclear. The current study is aimed to clarify whether repeated oxytocin alter plasma cytokine levels in relation to clinical changes of autism social core feature. Here we analyzed cytokine concentrations using comprehensive proteomics of plasmas of 207 adult males with high-functioning ASD collected from two independent multi-center large-scale randomized controlled trials (RCTs): Testing effects of 4-week intranasal administrations of TTA-121 (A novel oxytocin spray with enhanced bioavailability: 3U, 6U, 10U, or 20U/day) and placebo in the crossover discovery RCT; 48U/day Syntocinon or placebo in the parallel-group verification RCT. Among the successfully quantified 17 cytokines, 4 weeks TTA-121 6U (the peak dose for clinical effects) significantly elevated IL-7 (9.74, 95 % confidence interval [CI] 3.59 to 15.90, False discovery rate corrected P (PFDR) < 0.001), IL-9 (56.64, 20.46 to 92.82, PFDR < 0.001) and MIP-1b (18.27, 4.96 to 31.57, PFDR < 0.001) compared with placebo. Inverted U-shape dose-response relationships peaking at TTA-121 6U were consistently observed for all these cytokines (IL-7: P < 0.001; IL-9: P < 0.001; MIP-1b: P = 0.002). Increased IL-7 and IL-9 in participants with ASD after 4 weeks TTA-121 6U administration compared with placebo was verified in the confirmatory analyses in the dataset before crossover (PFDR < 0.001). Furthermore, the changes in all these cytokines during 4 weeks of TTA-121 10U administration revealed associations with changes in reciprocity score, the original primary outcome, observed during the same period (IL-7: Coefficient = -0.05, -0.10 to 0.003, P = 0.067; IL-9: -0.01, -0.02 to -0.003, P = 0.005; MIP-1b: -0.02, -0.04 to -0.007, P = 0.005). These findings provide the first evidence for a role of interaction between oxytocin and neuroinflammation in the change of ASD core social features, and support the potential role of this interaction as a novel therapeutic seed. Trial registration: UMIN000015264, NCT03466671/UMIN000031412.

Thrombolysis for Acute Wake-Up and Unclear-Onset Strokes with Alteplase at 0.6 mg/kg in Clinical Practice: THAWS2 Study.

INTRODUCTION: The aim of this study was to determine the safety and efficacy of intravenous (IV) alteplase at 0.6 mg/kg for patients with acute wake-up or unclear-onset strokes in clinical practice. METHODS: This multicenter observational study enrolled acute ischemic stroke patients with last-known-well time >4.5 h who had mismatch between DWI and FLAIR and were treated with IV alteplase. The safety outcomes were symptomatic intracranial hemorrhage (sICH) after thrombolysis, all-cause deaths, and all adverse events. The efficacy outcomes were favorable outcome defined as an mRS score of 0-1 or recovery to the same mRS score as the premorbid score, complete independence defined as an mRS score of 0-1 at 90 days, and change in NIHSS at 24 h from baseline. RESULTS: Sixty-six patients (35 females; mean age, 74 ± 11 years; premorbid complete independence, 54 [82%]; median NIHSS on admission, 11) were enrolled at 15 hospitals. Two patients (3%) had sICH. Median NIHSS changed from 11 (IQR, 6.75-16.25) at baseline to 5 (3-12.25) at 24 h after alteplase initiation (change, -4.8 ± 8.1). At discharge, 31 patients (47%) had favorable outcome and 29 (44%) had complete independence. None died within 90 days. Twenty-three (35%) also underwent mechanical thrombectomy (no sICH, NIHSS change of -8.5 ± 7.3), of whom 11 (48%) were completely independent at discharge. CONCLUSIONS: In real-world clinical practice, IV alteplase for unclear-onset stroke patients with DWI-FLAIR mismatch provided safe and efficacious outcomes comparable to those in previous trials. Additional mechanical thrombectomy was performed safely in them.

Hand Grip Strength Assessment Based on Sarcopenia Diagnostic Criteria Predicts Swallowing Function.

The purpose of this study is to clarify whether swallowing function can be inferred from grip strength. Based on the diagnostic criteria of sarcopenia, patients were divided into two groups according to grip strength, and it was analyzed whether there was a difference in the evaluation index for swallowing function between the two groups. Among the cases requesting evaluation of swallowing function from June 10, 2020 to October 28, 2020, 83 cases (mean age: 71.7 years, 59 males and 24 females) who received assessment tests and swallowing endoscopy were included. According to the diagnostic criteria for grip strength in the Asian working group in Sarcopenia, less than 28 kgf and 18 kgf were defined as the weak group for men and women, respectively. Hyodo scores, repeated salivary swallowing tests (RSST), maximum vocalization time (MPT), and dysphagia severity classification (DSS) were compared between the two groups. Of the 83 patients, 29 and 54 were in the normal group and weak group, respectively. In all indicators, the normal group showed significantly better results than the weak group: Hyodo score (2.4 vs. 4.0, p < 0.01), RSST (4.1 times vs. 2.4 times, p < 0.01), MPT (12.1 s vs. 5.9 s, p < 0.001), DSS (4.5 vs. 5.9, p < 0.001), respectively. In multiple regression analysis with DSS as the dependent variable, grip strength was a significant independent variable of DSS even after adjusting for age, gender, and body mass index. Grip strength assessment based on sarcopenia criteria can be a useful tool for estimating swallowing function.

Large-scale purification of functional AAV particles packaging the full genome using short-term ultracentrifugation with a zonal rotor.

Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method for full-genome AAV vectors remains crucial to increase productivity and reduce cost of GMP production. In this study, we developed a large-scale short-term purification method for functional full-genome AAV particles by using 2-step cesium chloride (CsCl) density-gradient ultracentrifugation with a zonal rotor. The 2-step CsCl method with a zonal rotor improves separation between empty and full-genome AAV particles, reducing the ultracentrifugation time (4-5 h) and increasing the AAV volume for purification. The highly purified full-genome AAV particles were confirmed by analytical ultracentrifugation (AUC), droplet digital PCR (ddPCR) in the whole region of the AAV vector genome, transduction efficiency in target cells, and transmission electronic microscopy (TEM). The high-purity AAV9 particles were obtained using culture supernatant during vector preparation rather than cell lysate. CsCl could be simply removed by a hydroxyapatite column. Interestingly, ddPCR analysis revealed that "empty" AAV particles contain small fragments of the inverted terminal repeat (ITR), probably due to unexpected packaging of Rep-mediated ITR fragments. This large-scale functional AAV vector purification with ultracentrifugation would be effective for gene therapy.

The role of mtDNA in oocyte quality and embryo development.

The mitochondrial genome resides in the mitochondria present in nearly all cell types. The porcine (Sus scrofa) mitochondrial genome is circa 16.7 kb in size and exists in the multimeric format in cells. Individual cell types have different numbers of mitochondrial DNA (mtDNA) copy number based on their requirements for ATP produced by oxidative phosphorylation. The oocyte has the largest number of mtDNA of any cell type. During oogenesis, the oocyte sets mtDNA copy number in order that sufficient copies are available to support subsequent developmental events. It also initiates a program of epigenetic patterning that regulates, for example, DNA methylation levels of the nuclear genome. Once fertilized, the nuclear and mitochondrial genomes establish synchrony to ensure that the embryo and fetus can complete each developmental milestone. However, altering the oocyte's mtDNA copy number by mitochondrial supplementation can affect the programming and gene expression profiles of the developing embryo and, in oocytes deficient of mtDNA, it appears to have a positive impact on the embryo development rates and gene expression profiles. Furthermore, mtDNA haplotypes, which define common maternal origins, appear to affect developmental outcomes and certain reproductive traits. Nevertheless, the manipulation of the mitochondrial content of an oocyte might have a developmental advantage.

Development of a novel purification method for AAV vectors using tangential flow filtration.

Adeno-associated virus (AAV) vector can efficiently transduce therapeutic genes in various tissue types with less side effects; however, owing to complex multistep processes during manufacture, there have been surges in the pricing of recently approved AAV vector-based gene therapy products. This study aimed to develop a simple and efficient method for high-quality purification of AAV vector via tangential flow filtration (TFF), which is commonly used for concentration and diafiltration of solutions during AAV vector purification. We established a novel purification method using TFF and surfactants. Treatment with two classes of surfactants (anionic and zwitterionic) successfully inhibited the aggregation of residual proteins separated from the AAV vector in the crude product by TFF, obtaining a clearance of 99.5% residual proteins. Infectivity of the AAV vector purified using the new method was confirmed both in vitro and in vivo, and no remarkable inflammation or tissue damage was observed in mouse skeletal muscle after local administration. Overall, our proposed method could be used to establish a platform for the purification of AAV vector.

Effect of a novel nasal oxytocin spray with enhanced bioavailability on autism: a randomized trial.

Although intranasal oxytocin is expected to be a novel therapy for the core symptoms of autism spectrum disorder, which has currently no approved medication, the efficacy of repeated administrations was inconsistent, suggesting that the optimal dose for a single administration of oxytocin is not optimal for repeated administration. The current double-blind, placebo-controlled, multicentre, crossover trial (ClinicalTrials.gov Identifier: NCT03466671) was aimed to test the effect of TTA-121, a new formulation of intranasal oxytocin spray with an enhanced bioavailability (3.6 times higher than Syntocinon® spray, as assessed by area under the concentration-time curve in rabbit brains), which enabled us to test a wide range of multiple doses, on autism spectrum disorder core symptoms and to determine the dose-response relationship. Four-week administrations of TTA-121, at low dose once per day (3 U/day), low dose twice per day (6 U/day), high dose once per day (10 U/day), or high dose twice per day (20 U/day), and 4-week placebo were administered in a crossover manner. The primary outcome was the mean difference in the reciprocity score (range: 0-14, higher values represent worse outcomes) on the Autism Diagnostic Observation Schedule between the baseline and end point of each administration period. This trial with two administration periods and eight groups was conducted at seven university hospitals in Japan, enrolling adult males with high-functioning autism spectrum disorder. Enrolment began from June 2018 and ended December 2019. Follow-up ended March 2020. Of 109 males with high-functioning autism spectrum disorder who were randomized, 103 completed the trial. The smallest P-value, judged as the dose-response relationship, was the contrast with the peak at TTA-121 6 U/day, with inverted U-shape for both the full analysis set (P = 0.182) and per protocol set (P = 0.073). The Autism Diagnostic Observation Schedule reciprocity score, the primary outcome, was reduced in the TTA-121 6 U/day administration period compared with the placebo (full analysis set: P = 0.118, mean difference = -0.5; 95% CI: -1.1 to 0.1; per protocol set: P = 0.012, mean difference = -0.8; 95% CI: -1.3 to -0.2). The per protocol set was the analysis target population, consisting of all full analysis set participants except those who deviated from the protocol. Most dropouts from the full analysis set to the per protocol set occurred because of poor adherence to the test drug (9 of 12 in the first period and 8 of 15 in the second period). None of the secondary clinical and behavioural outcomes were significantly improved with the TTA-121 compared with the placebo in the full analysis set. A novel intranasal spray of oxytocin with enhanced bioavailability enabled us to test a wide range of multiple doses, revealing an inverted U-shape dose-response curve, with the peak at a dose that was lower than expected from previous studies. The efficacy of TTA-121 shown in the current exploratory study should be verified in a future large-scale, parallel-group trial.

Hip flexion angle affects longitudinal muscle activity of the rectus femoris in leg extension exercise.

PURPOSE: We investigated the effect of the hip flexion angle (HFA) on the longitudinal muscle activity of the rectus femoris (RF) during leg extension exercise (LEE). METHODS: We conducted an acute study in a specific population. Nine male bodybuilders performed isotonic LEE using a leg extension machine at three different HFAs: 0°, 40°, and 80°. Participants extended their knees from 90° to 0° at each HFA setting for four sets of ten repetitions at 70% of the one-repetition maximum. The transverse relaxation time (T2) of the RF was measured before and after LEE using magnetic resonance imaging. We analyzed the rate of change in the T2 value in the proximal, middle, and distal regions of the RF. The subjective sensation of muscle contraction of the quadriceps was measured using a numerical rating scale (NRS) and compared with the T2 value which was the objective index. RESULTS: At 80°, the T2 value in the middle RF was lower than that in the distal RF (p < 0.05). The T2 values at 0° and 40° HFA were higher than those at 80° HFA in the proximal (p < 0.05, p < 0.01) and middle RF (p < 0.01, p < 0.01). The NRS scores were inconsistent with the objective index. CONCLUSION: These results suggest that the 40° HFA is practical for region-specific strengthening of the proximal RF, and subjective sensation alone as an indication of training may not activate the proximal RF. We conclude that activation of each longitudinal section of the RF is possible depending on the hip joint angle.

Mitochondrial DNA Deficiency and Supplementation in Sus scrofa Oocytes Influence Transcriptome Profiles in Oocytes and Blastocysts.

Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell division. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.

Mitochondrial DNA Supplementation of Oocytes Has Downstream Effects on the Transcriptional Profiles of Sus scrofa Adult Tissues with High mtDNA Copy Number.

Oocytes can be supplemented with extra copies of mitochondrial DNA (mtDNA) to enhance developmental outcome. Pigs generated through supplementation with mtDNA derived from either sister (autologous) or third-party (heterologous) oocytes have been shown to exhibit only minor differences in growth, physiological and biochemical assessments, and health and well-being do not appear affected. However, it remains to be determined whether changes in gene expression identified during preimplantation development persisted and affected the gene expression of adult tissues indicative of high mtDNA copy number. It is also unknown if autologous and heterologous mtDNA supplementation resulted in different patterns of gene expression. Our transcriptome analyses revealed that genes involved in immune response and glyoxylate metabolism were commonly affected in brain, heart and liver tissues by mtDNA supplementation. The source of mtDNA influenced the expression of genes associated with oxidative phosphorylation (OXPHOS), suggesting a link between the use of third-party mtDNA and OXPHOS. We observed a significant difference in parental allele-specific imprinted gene expression in mtDNA-supplemented-derived pigs, with shifts to biallelic expression with no effect on expression levels. Overall, mtDNA supplementation influences the expression of genes in important biological processes in adult tissues. Consequently, it is important to determine the effect of these changes on animal development and health.

Origin of diverse phosphorylation patterns in the ERBB system.

Intercellular signals induce various cellular responses, including growth, proliferation, and differentiation, via the dynamic processes of signal transduction pathways. For cell fate decisions, ligand-binding induces the phosphorylation of ERBB receptors, which in turn activate downstream molecules. The ERBB family includes four subtypes, which diverged through two gene duplications from a common ancestor. Differences in the expression patterns of the subtypes have been reported between different organs in the human body. However, how these different expression properties influence the diverse phosphorylation levels of ERBB proteins is not well understood. Here we study the origin of the phosphorylation responses by experimental and mathematical analyses. The experimental measurements clarified that the phosphorylation levels heavily depend on the ERBB expression profiles. We developed a mathematical model consisting of the four subtypes as monomers, homodimers, and heterodimers and estimated the rate constants governing the phosphorylation responses from the experimental data. To understand the origin of the diversity, we analyzed the effects of the expression levels and reaction rates of the ERBB subtypes on the diversity. The difference in phosphorylation rates between ERBB subtypes showed a much greater contribution to the diversity than did the dimerization rates. This result implies that divergent evolution in phosphorylation reactions rather than in dimerization reactions after whole genome duplications was essential for increasing the diversity of the phosphorylation responses.

A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging.

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

Utility of computed tomography findings as a predictor of carotid artery invasion by metastatic lymph nodes in head and neck squamous cell carcinoma.

PURPOSE: Carotid artery invasion by metastatic lymph nodes in head and neck squamous cell carcinoma (HNSCC) is one of the diagnostic criteria for unresectable tumors. However, to date, the diagnostic criteria for carotid artery invasion have not been well documented. This study investigated the utility of computed tomography (CT) findings as a predictor of carotid artery invasion by metastatic lymph nodes in HNSCC. METHODS: Twenty-eight patients who had metastatic lymph nodes of HNSCC attached to the carotid artery as seen on CT images before neck dissection from January 2011 to November 2017 were included. Five imaging parameters (angle of contact [AC], length of contact [LC], haziness of the carotid artery wall [HW], size of the lymph node, and involvement of the bifurcation of the carotid artery [IB]) were assessed using CT to predict carotid artery invasion. Furthermore, the utility of the combination of these five parameters was evaluated. RESULTS: There were significant differences in AC, LC, and IB between patients with and without carotid artery invasion. There were significant differences in all combinations of the two image findings between patients with and without carotid artery invasion. In particular, the combinations of LC and HW, and LC and IB could clearly predict carotid artery invasion. CONCLUSION: AC, LC, and IB were useful predictors of carotid artery invasion of metastatic lymph nodes in HNSCC. This study is the first to report that IB is a useful predictor of carotid artery invasion in HNSCC.

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin-glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis-circulating RNA molecules-has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.

Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14).

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.