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1.
Methods Mol Biol ; 2762: 309-328, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315374

RESUMO

Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.


Assuntos
Escherichia coli , Glicoproteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Sistema Livre de Células/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Polissacarídeos/metabolismo
2.
Genet Med ; 26(1): 101006, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37869996

RESUMO

PURPOSE: Copy-number variants (CNVs) and other non-single nucleotide variant/indel variant types contribute an important proportion of diagnoses in individuals with suspected genetic disease. This study describes the range of such variants detected by genome sequencing (GS). METHODS: For a pediatric cohort of 1032 participants undergoing clinical GS, we characterize the CNVs and other non-single nucleotide variant/indel variant types that were reported, including aneuploidies, mobile element insertions, and uniparental disomies, and we describe the bioinformatic pipeline used to detect these variants. RESULTS: Together, these genetic alterations accounted for 15.8% of reported variants. Notably, 67.9% of these were deletions, 32.9% of which overlapped a single gene, and many deletions were reported together with a second variant in the same gene in cases of recessive disease. A retrospective medical record review in a subset of this cohort revealed that up to 6 additional genetic tests were ordered in 68% (26/38) of cases, some of which failed to report the CNVs/rare variants reported on GS. CONCLUSION: GS detected a broad range of reported variant types, including CNVs ranging in size from 1 Kb to 46 Mb.


Assuntos
Genoma , Genômica , Humanos , Criança , Estudos Retrospectivos , Mapeamento Cromossômico , Nucleotídeos , Variações do Número de Cópias de DNA/genética , Polimorfismo de Nucleotídeo Único/genética
3.
NPJ Genom Med ; 8(1): 5, 2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36788231

RESUMO

Universal newborn screening (NBS) is a highly successful public health intervention. Archived dried bloodspots (DBS) collected for NBS represent a rich resource for population genomic studies. To fully harness this resource in such studies, DBS must yield high-quality genomic DNA (gDNA) for whole genome sequencing (WGS). In this pilot study, we hypothesized that gDNA of sufficient quality and quantity for WGS could be extracted from archived DBS up to 20 years old without PCR (Polymerase Chain Reaction) amplification. We describe simple methods for gDNA extraction and WGS library preparation from several types of DBS. We tested these methods in DBS from 25 individuals who had previously undergone diagnostic, clinical WGS and 29 randomly selected DBS cards collected for NBS from the California State Biobank. While gDNA from DBS had significantly less yield than from EDTA blood from the same individuals, it was of sufficient quality and quantity for WGS without PCR. All samples DBS yielded WGS that met quality control metrics for high-confidence variant calling. Twenty-eight variants of various types that had been reported clinically in 19 samples were recapitulated in WGS from DBS. There were no significant effects of age or paper type on WGS quality. Archived DBS appear to be a suitable sample type for WGS in population genomic studies.

4.
Nat Commun ; 13(1): 6325, 2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36280670

RESUMO

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.


Assuntos
Glicosiltransferases , Polissacarídeos , Humanos , Glicosiltransferases/metabolismo , Proteínas de Membrana , Água , Anticorpos Monoclonais , Trastuzumab
5.
Adv Mater ; 34(12): e2106110, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34933395

RESUMO

2D van der Waals (vdW) materials have been considered as potential building blocks for use in fundamental elements of electronic and optoelectronic devices, such as electrodes, channels, and dielectrics, because of their diverse and remarkable electrical properties. Furthermore, two or more building blocks of different electronic types can be stacked vertically to generate vdW heterostructures with desired electrical behaviors. However, such fundamental approaches cannot directly be applied practically because of issues such as precise alignment/positioning and large-quantity material production. Here, these limitations are overcome and wafer-scale vdW heterostructures are demonstrated by exploiting the lateral and vertical assembly of solution-processed 2D vdW materials. The high exfoliation yield of the molecular intercalation-assisted approach enables the production of micrometer-sized nanosheets in large quantities and its lateral assembly in a wafer-scale via vdW interactions. Subsequently, the laterally assembled vdW thin-films are vertically assembled to demonstrate various electronic device applications, such as transistors and photodetectors. Furthermore, multidimensional vdW heterostructures are demonstrated by integrating 1D carbon nanotubes as a p-type semiconductor to fabricate p-n diodes and complementary logic gates. Finally, electronic devices are fabricated via inkjet printing as a lithography-free manner based on the stable nanomaterial dispersions.

6.
Med Sci Monit ; 27: e928987, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097670

RESUMO

BACKGROUND Abnormal posture can affect the alignment of the cervical spine, which can lead to various physical problems. There are many ways to solve the problem by limiting the area around the neck to restore abnormal neck condition. However, there is a need to look at these problems from an enlarged perspective through the relationship between the cervical spine and trunk. This study aimed to investigate the significance of the thickness of the transverse abdominis, internal oblique, and external oblique muscles in patients with forward head posture and reduced cranio-vertebral angle. MATERIAL AND METHODS We included 24 healthy adult males in their 20s without lower back pain. The cranio-vertebral angle (CVA) in all the subjects was measured with the help of pictures taken in the sagittal plane using a digital camera. The thickness of muscles, including transverse abdominis (TrA), internal oblique (IO), and external oblique (EO), was measured using a diagnostic ultrasound device. Pearson's correlation analysis was used to identify the correlation between the variables measured. RESULTS CVA showed a statistically significant correlation with TrA thickness (r=0.506/p=0.012), and among the abdominal muscles, there was a significant correlation between IO and EO thickness (r=0.663/p=0.000). CONCLUSIONS A reduced CVA due to FHP was significantly associated with reduced TrA thickness. Therefore, increasing the bulk of the abdominal muscles with restoration of the abnormal CVA is a potential treatment approach and requires further study.


Assuntos
Vértebras Cervicais/fisiologia , Postura/fisiologia , Tronco/fisiologia , Músculos Abdominais/fisiologia , Músculos Abdominais Oblíquos/fisiologia , Adulto , Vértebras Cervicais/diagnóstico por imagem , Cabeça/fisiologia , Humanos , Masculino , Pescoço/fisiologia , Ultrassonografia
7.
Neural Regen Res ; 11(1): 126-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26981100

RESUMO

This study investigated the effect of transcranial direct current stimulation (tDCS) polarity depending on lateralized function of task property in normal individuals performing visuomotor and simple repetitive tasks. Thirty healthy participants with no neurological disorders were recruited to participate in this study. Participants were randomly allocated into active or control condition. For the active condition, tDCS intensity was 2 mA with stimulation applied for 15 minutes to the right hemisphere (tDCS condition). For the sham control, electrodes were placed in the same position, but the stimulator was turned off after 30 seconds (sham condition). The tapping and tracking task tests were performed before and after for both conditions. Univariate analysis revealed significant difference only in the tracking task. For direct comparison of both tasks within each group, the tracking task had significantly higher Z score than the tapping task in the tDCS group (P < 0.05). Thus, our study indicates that stimulation of the right hemisphere using tDCS can effectively improve visuomotor (tracking) task over simple repetitive (tapping) task.

8.
Am J Phys Med Rehabil ; 95(8): 580-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26829086

RESUMO

OBJECTIVE: We attempted to investigate postural instability in patients with injury of the corticoreticular pathway (CRP) after mild traumatic brain injury. METHODS: The CRP was reconstructed, and the fractional anisotropy value, apparent diffusion coefficient value, and fiber volume of the CRP were measured. For evaluation of postural instability, both the Balance Error Scoring System score and the displacement of center of pressure were measured. RESULT: Significantly lower tract volume of the CRP was observed in the patient group than in the control group with no significant difference in fractional anisotropy and apparent diffusion coefficient values(P > 0.05). The results of the Balance Error Scoring System shown on a firm and foam surface were significantly higher in the patient group than in the control group (P < 0.05). Significant increments in displacement of center of pressure for 3 stances of double-leg, single-leg, and tandem stances in distance, maximum distance, and path length were observed in the patient group compared with the control group (P < 0.05). By contrast, no significant difference in the double stance on the x axis of the distance was observed between the patient and control groups (P > 0.05). CONCLUSION: We demonstrated postural instability in patients with injury of the CRP following mild traumatic brain injury.


Assuntos
Concussão Encefálica/fisiopatologia , Formação Reticular Mesencefálica/lesões , Equilíbrio Postural/fisiologia , Adulto , Estudos de Casos e Controles , Imagem de Tensor de Difusão , Feminino , Humanos , Masculino , Formação Reticular Mesencefálica/diagnóstico por imagem , Pessoa de Meia-Idade , Debilidade Muscular/fisiopatologia , Substância Branca/diagnóstico por imagem , Adulto Jovem
9.
Neurosci Lett ; 610: 213-7, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26598020

RESUMO

The cingulum, a major structure of the limbic system, is closely associated with memory function. In the current study, we investigated aging of the cingulum according to the location of the cingulum in each part of the cingulum after dividing the cingulum into five parts in normal subjects, using DTT parameters (fractional anisotropy (FA) and fiber number (FN)). Ninety healthy subjects (males: 44, females: 46, mean age: 49.0 years; range: 20-78 years) were enrolled in this study. Subjects were categorized according to six groups by age intervals of 10 years; each age group consisted of 15 subjects. The cingulum was divided into five parts (anterior, anterior superior, posterior superior cingulum, posterior, and inferior cingulum). The FA and FN of each part were measured. The FA value indicates the degree of directionality and integrity of white matter microstructures such as axons, myelin, and microtubules, and the FN reflects the total number of fibers in a neural tract. Age-related decline in the FA value may indicate demyelination, and a decline in the number of myelinated fibers of a neural tract can also lead to a decline of the FN. Significant differences in the FA value of the anterior cingulum and anterior superior cingulum, and the FN of the inferior cingulum were observed between age groups (AVOVA, p<0.05). A significant decrease was observed in the FA values of the anterior and anterior superior cingulum of the 60s and 70s age groups compared with those of the 20s and 30s age groups, and in the FN of the inferior cingulum of the 60s and 70s age groups compared with that of the 20s age group (LSD post hoc test, p<0.05). Aging of the cingulum began at both ends of the cingulum in the 20s or 30s, and progressed steadily at a near continuous rate over the lifespan and a significant degenerative aging effect at both ends of the cingulum occurred into the 60s, compared with the 20s or 30s.


Assuntos
Envelhecimento , Giro do Cíngulo/anatomia & histologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Imagem de Tensor de Difusão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
J Phys Ther Sci ; 27(3): 769-72, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25931727

RESUMO

[Purpose] The purpose of the current study was to compare the effectiveness of motor sequential learning according to two different types of practice schedules, distributed practice schedule (two 12-hour inter-trial intervals) and massed practice schedule (two 10-minute inter-trial intervals) using a serial reaction time (SRT) task. [Subjects and Methods] Thirty healthy subjects were recruited and then randomly and evenly assigned to either the distributed practice group or the massed practice group. All subjects performed three consecutive sessions of the SRT task following one of the two different types of practice schedules. Distributed practice was scheduled for two 12-hour inter-session intervals including sleeping time, whereas massed practice was administered for two 10-minute inter-session intervals. Response time (RT) and response accuracy (RA) were measured in at pre-test, mid-test, and post-test. [Results] For RT, univariate analysis demonstrated significant main effects in the within-group comparison of the three tests as well as the interaction effect of two groups × three tests, whereas the between-group comparison showed no significant effect. The results for RA showed no significant differences in neither the between-group comparison nor the interaction effect of two groups × three tests, whereas the within-group comparison of the three tests showed a significant main effect. [Conclusion] Distributed practice led to enhancement of motor skill acquisition at the first inter-session interval as well as at the second inter-interval the following day, compared to massed practice. Consequentially, the results of this study suggest that a distributed practice schedule can enhance the effectiveness of motor sequential learning in 1-day learning as well as for two days learning formats compared to massed practice.

11.
J Phys Ther Sci ; 27(4): 997-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25995541

RESUMO

[Purpose] The purpose of this study was to investigate the effect of aging on respiratory synergy, through the comparison of an elderly group and a young group, to help further understanding of postural control in the elderly. [Subjects and Methods] Ten community-dwelling elderly subjects and ten young subjects performed standing under two different respiratory conditions: quiet breathing and apnea. Center of foot pressure displacement and joint angular movements of the head, trunk, pelvis, hips, knees and ankles were measured. [Results] The results of this study showed that the elderly group had a respiratory synergy different from that of the young group. The elderly group in quiet stance used significantly more hip and pelvis movements when compensating for respiratory disturbance than standing with apnea, while the young group used significantly more whole body segments. There were no differences in angular displacements in the quiet stance between the elderly and the young groups. [Conclusion] The elderly group demonstrated a respiratory synergy pattern different from that of the young group. The findings indicate that aging changes the respiratory synergy pattern and this change is not due to decreased functioning of the ankle joint alone.

12.
Neural Regen Res ; 10(3): 463-6, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25878597

RESUMO

Transcranial direct current stimulation (tDCS), an emerging technique for non-invasive brain stimulation, is increasingly used to induce changes in cortical excitability and modulate motor behavior, especially for upper limbs. The purpose of this study was to investigate the effects of tDCS of the primary motor cortex on visuomotor coordination based on three levels of task difficulty in healthy subjects. Thirty-eight healthy participants underwent real tDCS or sham tDCS. Using a single-blind, sham-controlled crossover design, tDCS was applied to the primary motor cortex. For real tDCS conditions, tDCS intensity was 1 mA while stimulation was applied for 15 minutes. For the sham tDCS, electrodes were placed in the same position, but the stimulator was turned off after 5 seconds. Visuomotor tracking task, consisting of three levels (levels 1, 2, 3) of difficulty with higher level indicating greater difficulty, was performed before and after tDCS application. At level 2, real tDCS of the primary motor cortex improved the accurate index compared to the sham tDCS. However, at levels 1 and 3, the accurate index was not significantly increased after real tDCS compared to the sham tDCS. These findings suggest that tasks of moderate difficulty may improve visuomotor coordination in healthy subjects when tDCS is applied compared with easier or more difficult tasks.

13.
J Phys Ther Sci ; 27(2): 401-3, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25729178

RESUMO

[Purpose] The purpose of this study was to determine differences in respiratory pressure and pulmonary function among children with spastic diplegic and hemiplegic cerebral palsy (CP) in comparison with children with normal development. [Subjects and Methods] Fourteen children with spastic diplegic CP, 11 children with hemiplegic CP, and 14 children with normal development were recruited. Respiratory pressure was measured and the pulmonary function test (PFT) was performed to evaluate the strength of the respiratory muscles and lung volumetric capacity. [Results] Regarding respiratory pressure, children with spastic diplegic and hemiplegic CP showed significantly lower functions in terms of MIP and MEP compared with children with normal development, although no significant differences were found between children with the two types of CP. In the pulmonary function test, children with spastic diplegic CP showed significantly higher pulmonary function than children with normal development in terms of only FVC and FEV1. [Conclusion] Children with CP showed relatively lower function in terms of respiratory pressure and lung capacity, in comparison with children with normal development. Therefore, respiratory function in children with CP should be carefully evaluated and should receive more attention in a rehabilitation setting.

14.
Percept Mot Skills ; 119(3): 799-810, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25387036

RESUMO

This study investigated the prevalence of pain in the ipsilateral upper-limb in stroke patients. 229 stroke patients (133 men, 96 women; M age = 59.0 yr., SD = 12.4) were assessed with the Pain Behaviors Scales and their motor weakness was measured with the Motricity Index. Results indicated that over 27% of patients experienced pain in at least one joint of the ipsilateral upper limb. Shoulder pain was the most common. Further analysis indicated that the occurrence of pain in the ipsilateral upper limb was higher among women, among patients who used a cane, and among patients with a greater weakness of the affected lower limb.


Assuntos
Artralgia/epidemiologia , Medição da Dor/métodos , Dor/epidemiologia , Acidente Vascular Cerebral/epidemiologia , Extremidade Superior/fisiopatologia , Atividades Cotidianas , Artralgia/diagnóstico , Comorbidade , Articulação do Cotovelo/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dor/diagnóstico , Dor/fisiopatologia , Prevalência , República da Coreia/epidemiologia , Articulação do Ombro/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Articulação do Punho/fisiopatologia
16.
Neural Regen Res ; 9(4): 436-9, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25206832

RESUMO

This study was designed to observe and compare the circadian fluctuations in tactile sense, joint reposition sense and two-point discrimination in healthy subjects. Twenty-one healthy adult subjects received perceptual ability tests through these three different sensory modules at approximately 9:00, 13:00 and 18:00 in a day. The distribution of ranking for perceptual ability was significantly different among the three different time points in each individual, with highest perceptual ability in the evening compared with noon and morning, in terms of tactile sense and two-point discrimination. These findings suggest that the perceptual ability of healthy subjects fluctuates according to the time points in a day.

18.
J Phys Ther Sci ; 26(3): 389-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24707090

RESUMO

[Purpose] The current study was designed to investigate the difference in lung capacity and muscle strengthening related to respiration depending on the level of the Gross Motor Function Classification System (GMFCS) in children with cerebral palsy (CP) through tests of respiratory function and respiratory pressure. [Subjects and Methods] A total of 49 children with CP who were classified as below level III of the GMFCS were recruited for this study. They were divided into three groups (i.e., GMFCS level I, GMFCS level II, and GMFCS level III). All children took the pulmonary function test (PFT) and underwent respiratory pressure testing for assessment of respiratory function in terms of lung capacity and respiratory muscle strength. [Results] The GMFCS level III group showed significantly lower scores for all tests of the PFT (i.e., forced vital capacity (FVC), forced expiratory volume at one second (FEV1), and slow vital capacity (SVC)) and testing for respiratory pressures (maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP)) compared with the other two groups. The results of post hoc analysis indicated that the GMFCS level III group differed significantly from the other two groups in terms of FVC, FEV1, MIP, and MEP. In addition, a significant difference in SVC was observed between GMFCS level II and III. [Conclusion] Children with CP who had relatively low motor function showed poor pulmonary capacity and respiratory muscle weakness. Therefore, clinical manifestations regarding lung capacity and respiratory muscle will be required in children with CP who demonstrate poor physical activity.

19.
J Phys Ther Sci ; 26(3): 409-12, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24707094

RESUMO

[Purpose] We attempted to determine whether static and dynamic postural control ability fluctuated depending on the influence of the time of day (9 am, 1 pm, and 5 pm), and at which time point postural balance performance was best in healthy individuals. [Subjects and Methods] Twenty-four healthy subjects participated in this study. The static and dynamic postural balance test was conducted during three sessions (i.e., at 9 am, 1 pm, and 5 pm) with a counterbalanced order for prevention of learning effects. As outcome measurements, AP distance, ML distance, and velocity moment were adopted in the static balance test, and the performance time and total distance were measured in the dynamic balance test. [Results] For the static postural balance test, COP distance was shorter and COP velocity was slower at 9 am compared with those at 1 and 5 pm. In particular, the COP distance at 9 am was statistically different from that at 13 pm. During the dynamic postural balance test, performance time and total distance were influenced by the time of day, as the best performance was observed in the morning. [Conclusion] This study found that static and dynamic postural balance abilities were greatest in the morning and worst at 1 pm. Understanding of the mechanism of the time-of-day effect on postural balance will be helpful for assessment and treatment of postural balance by physical therapists and in making desirable clinical decisions.

20.
J Phys Ther Sci ; 25(5): 567-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-24259803

RESUMO

[Purpose] Previous studies have reported on motor deficits in the ipsilateral upper limbs (UL) of a damaged brain hemisphere in motor tasks. However, little is known about sensory deficits on the ipsilateral side. Therefore, we investigated whether both motor and sensory function of the ipsilateral UL are affected in patients with stroke. [Subjects and Methods] Fifty patients with unilateral stroke and 40 age- and sex- matched normal subjects participated in this study. Subjects were evaluated on performance of a tracking task for motor function, and by the joint reposition test for integrity of proprioceptive sense in the ipsilateral UL. [Result] The comparison of the stroke group and the control group showed significant differences in performance of the tracking task and the joint reposition test. The accuracy index for the tracking task showed significant correlation with the error score for the joint reposition test in the stroke group. [Conclusion] These results suggest that the ipsilateral UL of stroke patients has impairment in sensory function which is related to proprioceptive sense, along with motor deficits. Therefore, we think that the difficulty stroke patients experience with motor tasks for the ipsilateral UL is induced by diminished integrity of sensorimotor function due to both sensory and motor deficits.

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