Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase (nsp13) that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds double-stranded DNA. Our data reveal nsp13's single-nucleotide steps, translocating at ∼1000 nt/s or unwinding at ∼100 bp/s. Nanopore tweezers' high spatiotemporal resolution enables detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13's motion in the presence of the ATPase inhibitor ATPγS. We construct a detailed picture of inhibition in which ATPγS has multiple mechanisms of inhibition. The dominant mechanism of inhibition depends on the application of assisting force. This lays the groundwork for future single-molecule inhibition studies with viral helicases.

Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.

We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA. We show how PcrA's kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an 'active helicase,' capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores.

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter "hachimoji" expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.

Effects of Assistive Technology Application in Dementia Intervention for People with Mild Cognitive Impairment & Mild Alzheimer Type Dementia and Caregiver.

Background: Dementia, a degenerative disease, requires alternative treatment to maintain function, but previous studies suggest only the therapeutic effect of a temporary program. Primary Study Objective: The current study aimed to examine the effects of assistive technologies on cognitive function, daily living ability, and psychosocial symptoms in elderlies with mild cognitive impairment, elderlies with mild dementia and their caregivers. Design: The research team designed an experimental study that used application as the intervention. Setting: To recruit participants living in the local community, research participation was supported through local public health centers, welfare centers, and social welfare organizations. Evaluation and intervention were conducted by visiting the participant's home. Participant: The study participants were 29 Mild Cognitive Impairment (MCI) and 16 mild Alzheimer type dementia (AD) patients over the age of 75 with a total of 45 patients, 10 MCI caregivers and 11 AD caregivers with a total of 21 caregivers. Intervention: The assistive technologies used for intervention are 3 area (8 daily living assistive devices, 7 safety assistive technologies, and 7 cognitive assistive technologies). Up to 5 assistive technologies were provided to one subject, and they were instructed to use them every day for 8 weeks. Outcome measure: Participants were evaluated at baseline and postintervention using specific scales appropriate to an area: cognitive function, activities of daily living, depression, anxiety, quality of life, satisfaction. Results: Cognitive function showed statistically significant changes in the MCI group. Basic activities of daily living, depression, anxiety, quality of life, satisfaction showed statistically significant positive effects in both MCI and AD groups. Instrumental activities of daily living did not show any statistically significant differences. Conclusion: As an alternative to dementia care in the future, the application and management of assistive technologies for each area should be provided at the government level.

Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with noncanonical amino acids.

A Novel Method Using Kinesiology Taping for the Activation of Suprahyoid Muscles in Healthy Adults: A Preliminary Research.

The suprahyoid muscles play a major role in safe swallowing in the pharyngeal phase. Therefore, it is clinically important to design a therapeutic approach for strengthening the suprahyoid muscles for safe and normal swallowing. This study aimed to investigate the activation of suprahyoid muscles by resistance training using kinesiology taping (KT). We enrolled 23 healthy adults. All participants performed saliva swallowing five times at 5 s intervals in three conditions (without KT, 50% stretch with KT, and 80% stretch with KT). KT in the I and reverse V shapes was pulled vertically from the hyolaryngeal complex to the sternum and medially from the superior surface of the clavicle, respectively. Another KT horizontally covered the hyolaryngeal complex to enhance the movement restriction of the hyolaryngeal complex during swallowing. Activation of the suprahyoid muscles during swallowing in the two conditions was measured using surface electromyography. In addition, a 0-10 numerical rating self-report scale was used to evaluate the required effort and the resistance felt during swallowing. Both KT 50% and 80% were significantly higher in surface electromyography (sEMG) mean value, peak value, required effort, and resistance felt during swallowing compared to normal swallowing (p < 0.05). In addition, KT 80% was significantly higher in sEMG value, peak value, required effort, and resistance felt during swallowing than KT 50% (p < 0.05). This study demonstrated that KT applied to the area under the hyolaryngeal complex improves activation of the suprahyoid muscle during swallowing. Therefore, KT applied as resistance during swallowing is considered to have therapeutic potential in dysphagia rehabilitation.

Effects of chewing exercises on the occlusal force and masseter muscle thickness in community-dwelling Koreans aged 65 years and older: A randomised assessor-blind trial.

Chewing exercises have been applied in clinical settings to improve the occlusal force and function of the masseter muscle in elderly individuals. However, the clinical relevance and effects of chewing exercises are unclear. This study aimed to investigate the effects of bilateral chewing exercises on the occlusal force and masseter muscle thickness in community-dwelling Koreans aged 65 years. Forty community-dwelling healthy elderly individuals were enrolled in this study. They were assigned to the experimental or the control group. The experimental group performed chewing exercises using medical equipment developed to facilitate such exercises. The chewing exercises were divided into isometric and isotonic types and were performed for 20 min/d, 5 days/wk, for 6 weeks. The control group did not perform any chewing exercises. The outcome measures were occlusal force and masseter muscle thickness, which were evaluated using an occlusometer and ultrasound device, respectively. A paired t test and an independent t test were used to evaluate the training effects. Within-group comparisons showed that occlusal force and masseter muscle thickness improved significantly in the experimental group (P < .001 for both), while the control group showed no significant improvements (P = .098 and .130). Between-group comparisons showed that the experimental group had a greater increase in occlusal force and masseter muscle thickness (P < .05 for both) compared to the control group. These results suggest that chewing exercises are effective in improving occlusal force and masseter muscle thickness in healthy elderly individuals.

Inclusion complexes of cysteinyl ß-cyclodextrin with baicalein restore collagen synthesis in fibroblast cells following ultraviolet exposure.

Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a wide range of biological applications. In the present study, we used inclusion complexes of cysteinyl ß-cyclodextrin (ß-CD) with baicalein to enhance the stability and solubility of baicalein in aqueous solution. We examined the effects of inclusion complexes of cysteinyl ß-CD on collagen synthesis following ultraviolet (UV) irradiation, as well as the mechanisms underlying its effects. Our findings demonstrated that baicalein significantly restored collagen synthesis in the UV-exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized ß-CDs were found to promote baicalein-induced collagen synthesis. Inclusion complexes of cysteinyl ß-CDs with baicalein significantly upregulated the protein expression of type I collagen and activated the transcription of type I, II, and III collagen. Inclusion complexes of cysteinyl ß-CDs with baicalein also downregulated matrix metalloproteinase -1 and -3, and α-smooth muscle actin expression. In addition, inclusion complexes of cysteinyl ß-CDs with baicalein attenuated the expression of caveolin-1, but this treatment enhanced the UV-induced phosphorylation of Smad in the transforming growth factor-ß pathway. These results suggested that the newly synthesized derivative of CD can be used as a complexing agent to enhance the bioavailability of flavonoids such as baicalein, especially in restoring collagen synthesis.

Efficacy of modified chin tuck against resistance exercise using hand-free device for dysphagia in stroke survivors: A randomised controlled trial.

BACKGROUND: Chin tuck against resistance exercise was recently reported as a novel method for improving the swallowing function of patients with post-stroke dysphagia. However, as this exercise involves holding and fixing the device using the hand, physically weak patients may find it difficult to perform it. OBJECTIVES: This study aimed to investigate the effect of modified chin tuck against resistance (mCTAR) exercise on patients with post-stroke dysphagia. METHODS: In total, 30 patients with dysphagia were recruited in this study. They were randomly assigned to either the experimental or control group. The experimental group performed mCTAR exercise and received traditional dysphagia treatment, whereas the control group received only traditional dysphagia treatment. mCTAR exercise involved isometric and isotonic exercises. Aspiration and oral diet were evaluated using penetration-aspiration scale (PAS) and functional oral intake scale (FOIS), respectively. Moreover, the rate of nasogastric tube removal was analysed. RESULTS: Compared with the control group, the experimental group showed statistically significant improvement in PAS and FOIS (P < 0.001, both). The rates of nasogastric tube removal were 25% and 15% in the experimental and control groups, respectively. CONCLUSION: This study demonstrated that mCTAR exercise can reduce aspiration and improve dietary levels in patients with post-stroke dysphagia. Therefore, mCTAR exercise is expected to be beneficial for physically vulnerable patients with dysphagia who have limited hand strength and range of motion.

Effects of lingual strength training on oropharyngeal muscles in South Korean adults.

BACKGROUND: Lingual strength training (LST) has been reported to positively affect the activation of submental muscles, as well as to increase lingual muscle strength. However, there is little evidence to support its effectiveness. OBJECTIVES: This study aimed to investigate the effect of LST on the strength and thickness of oropharyngeal muscles in healthy adults. METHODS: The study included 30 subjects who were assigned to the experimental (n = 15) and the control groups (n = 15). The experimental group performed LST based on tongue-to-palate resistance exercise method. LST was categorised as isometric and isotonic exercise. The intervention was performed five times a week for 6 weeks. The control group did not receive any intervention. The lingual strength was measured using the Iowa Oral Performance Instrument. Changes in the thickness of the mylohyoid and the digastric muscles and the lingual were assessed ultrasonographically. RESULTS: After the intervention, the thickness of the mylohyoid and the digastric muscles in the experimental group was significantly greater than that in the control group (P = 0.037 and 0.042). CONCLUSION: This study demonstrated that LST increases the thickness and the strength of oropharyngeal muscles. Therefore, LST is a useful option in patients with dysphagia or in elderly patients prone to swallowing dysfunction.

The effect of exercise combined with a cognitive-enhancement group training program on cognition and depression in the community-dwelling elderly.

[Purpose] In this study, we investigated the effects of combining exercise with a cognitive-enhancement group program on cognition and depression in a group of community-dwelling elderly people. [Subjects and Methods] The study's subjects consisted of 30 community-dwelling elderly people of both genders, whose average age was 78 years. They participated in a program of physical exercise combined with a cognitive-enhancement group training program. This consisted of sessions lasting 60 minutes that took place once a week over 3 months. To assess the participants' levels of cognition and depression, we conducted batteries of tests using, respectively, the Korean versions of the Consortium to Establish a Registry for Alzheimer's Disease assessment packet (CERAD-K) and the Geriatric Depression Scale-Short Form (GDS-SF). [Results] The Verbal Fluency test, Word List Memory test, Modified Boston Naming test, Mini Mental Status Examination (Korean Version) (MMSE-KC), Constructional Praxis task and Constructional Recall task showed significant improvement, but improvement in the Word List Recall and Word List Recognition tests did not achieve significant levels. Meanwhile, the symptoms of depression were shown to decrease significantly. [Conclusion] Physical exercise combined with a cognitive-enhancement group training program was effective in improving, some of the components of cognition, as well as alleviating depression. This program should be used for the prevention of dementia in community-dwelling elderly, through the intervention should be complemented in order to improve more of the components of cognition.

Effects of observation of hand movements reflected in a mirror on cortical activation in patients with stroke.

[Purpose] The purpose of this study was to examine what changes occur in brain waves when patients with stroke receive mirror therapy intervention. [Subjects and Methods] The subjects of this study were 14 patients with stroke (6 females and 8 males). The subjects were assessed by measuring the alpha and beta waves of the EEG (QEEG-32 system CANS 3000). The mirror therapy intervention was delivered over the course of four weeks (a total of 20 sessions). [Results] Relative alpha power showed statistically significant differences in the F3, F4, O1, and O2 channels in the situation comparison and higher for hand observation than for mirror observation. Relative beta power showed statistically significant differences in the F3, F4, C3, and C4 channels. [Conclusion] This study analyzed activity of the brain in each area when patients with stroke observed movements reflected in a mirror, and future research on diverse tasks and stimuli to heighten activity of the brain should be carried out.

Characterization and Enhanced Antioxidant Activity of the Cysteinyl ß-Cyclodextrin-Baicalein Inclusion Complex.

Baicalein is a type of flavonoid isolated from the roots of a medicinal plant, Scutellaria baicalensis. Although it has attracted considerable attention due to its antiviral, anti-tumor, and anti-inflammatory activities, its limited aqueous solubility inhibits the clinical application of this flavonoid. The present study aimed to prepare and characterize a host-guest complex in an effort to improve the solubility and antioxidant activity of baicalein. The host molecule is a macrocyclic ß-cyclodextrin (ß-CD) functionalized with cysteine for a synergetic effect. The structure of the synthesized cysteinyl ß-CD was analyzed using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The inclusion complex with baicalein was studied by UV-vis, NMR spectroscopy, scanning electron microscopy, and X-ray powder diffractometry. The formed cysteinyl ß-CD/baicalein inclusion complex efficiently improved the solubility and antioxidant ability of baicalein. Therefore, we suggest that the present cysteinyl ß-CD is a potential host molecule for inclusion complexation and for bioavailability augmentation.

Synthesis, Characterization, and Retinol Stabilization of Fatty Amide-ß-cyclodextrin Conjugates.

Amphiphilic cyclodextrin (CD) has been the object of growing scientific attention because of its two recognition sites, the cavity and the apolar heart, formed by self-assembly. In the present study, mono[6-deoxy-6-(octadecanamido)]-ß-CD and mono[6-deoxy-6-(octadecenamido)]-ß-CD were successfully synthesized by reacting mono-6-amino-6-deoxy-ß-CD with N-hydroxysuccinimide esters of corresponding fatty acids in DMF. The structures were analyzed using nuclear magnetic resonance spectroscopy and mass spectrometry. The amphiphilic ß-CDs were able to form self-assembled nano-vesicles in water, and the supramolecular architectures were characterized using fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy. Using the cavity-type nano-vesicles, all-trans-retinol was efficiently encapsulated; it was then stabilized against the photo-degradation. Therefore, the present fatty amide-ß-CD conjugate will be a potential molecule for carrier systems in cosmetic and pharmaceutical applications.

Effects of experience-based group therapy on cognitive and physical functions and psychological symptoms of elderly people with mild dementia.

[Purpose] The purpose of this study was to investigate the effect of experience-based group therapy consisting of cooking and physical activities for elderly people with mild dementia on their cognitive and physical function, as well as on their psychological symptoms. [Subjects] The subjects of this study were 12 older adults with mild dementia (3 males, 9 females; 76.75 ± 3.61 years) who voluntarily consented to participate in the study. [Methods] In total, 12 subjects received experience-based group therapy for 2 hours per session once per week, totaling 10 sessions. Cognitive function was evaluated using the Mini Mental State Examination-Korean (MMSE-K), and physical function was evaluated using the Geriatric Physical health condition measurement Tool (GPT). The Geriatric Depression Scale Korean Version (GDS-K) and Geriatric Quality of Life-Dementia (GQOL-D) were used to measure psychological symptoms. [Results] There were significant differences between the MMSE-K, GPT, GDS-K, and GQOL-D scores of before and after group therapy. [Conclusion] In conclusion, it is regarded that cognitive function, physical function, and psychological health improved through experience-based group therapy.

Investigation of the effects of mirror therapy on the upper extremity functions of stroke patients using the manual function test.

[Purpose] The purpose of this study was to investigate the effects of mirror therapy on the upper extremity functions of stroke patients. [Subjects] The subjects of this study were 14 hemiplegia patients (8 males, 6 females; 9 infarction, 5 hemorrhage; 8 right hemiplegia, 6 left hemiplegia) who voluntarily consented to participate in the study. [Methods] The Korean version of the manual function test (MFT) was used in this study. The test was performed in the following order: arm movement (4 items), grasp and pinch (2 items), and manipulation (2 items). The experiment was conducted with the subjects sitting in a chair. The mirror was vertically placed in the sagittal plane on the desk. The paretic hand was placed behind the mirror, and the non-paretic hand was placed in front of the mirror so that it was reflected in the mirror. In this position, the subjects completed activities repetitively according to the mirror therapy program over the course of four weeks. [Results] There were significant increases in the grasp-and-pinch score and manipulation score. [Conclusion] In conclusion, the grasp-and-pinch and manipulation functions were improved through mirror therapy.

Comparison of gait before and after superficial trunk muscle exercise and deep trunk muscle exercise.

[Purpose] The purpose of this study was to examine the effects of superficial trunk muscle and deep trunk muscle exercise on gait. [Subjects] The subjects were 45 young adults who voluntarily consented to participate. The subjects were divided into a control group, a superficial muscle exercise group, and a deep muscle exercise group with 15 participants in each group. [Methods] Each group performed the exercises 5 times a week for 4 weeks. A Gait Analyzer was used to measure the subjects' gait. A one-way ANOVA was conducted for analysis between each group. [Results] After 4 weeks, the values from right heel contact to foot flat, left foot flat to heel off, right foot flat to heel off, and left heel off to toe off significantly differed among the groups. [Conclusion] The superficial trunk muscle exercise improved stability, such as the period of mid stance during gait. The deep trunk muscle exercise improved mobility, such as heel contact to foot flat and heel off to toe off during gait.

Muscle activation of drivers with hemiplegia caused by stroke while driving using a steering wheel or knob.

[Purpose] The aim of this study was to investigate three muscle activities of drivers with post-stoke hemiplegia while they were driving using a steering wheel or a spinner knob, and to compare them with those of non-disabled drivers. [Subjects and Methods] The subjects were four non-disabled drivers and five drivers with left hemiplegia. The subjects drove forward in a straight line for 5 m and then turned right or left using the steering wheel or spinner knob with only their right hand. EMG electrodes were placed over the anterior deltoid, biceps and triceps brachii on the right-side. [Results] While differences in muscle activation between the spinner knob and the steering wheel in the control group were not significant, those of the experimental group were significant. Activation of the biceps brachii while the control group turned the vehicle to the right using the spinner knob was significantly lower than when using the steering wheel. Activation of the biceps brachii while the experimental group turned the vehicle to the right using the spinner knob was significantly lower than that of the control group. [Conclusion] The results of this study indicate that a spinner knob requires less activation of the main muscle than a steering wheel, especially in drivers who have had a stroke. The results could be used as basic data when driver rehabilitation specialists prescribe the spinner knob for patients.

The effect of superficial trunk muscle exercise and deep trunk muscle exercise on the foot pressure of healthy adults.

[Purpose] The purpose of this study was to analyze the effect of superficial trunk muscle exercise and deep trunk muscle exercise on the foot pressure of healthy adults. [Subjects] The subjects were 30 healthy females and males who agreed to participate in this study. There were two groups, a superficial trunk muscle exercise group and a deep trunk muscle exercise group, with 15 participants in each. [Methods] The exercises were conducted 5 times a week for 4 weeks for both groups. A gait analyzer was used to measure foot plantar pressure while walking on a plate. Participants were measured before starting the exercise and after 4 weeks. The paired t-test was used to analyze the pre-and post-test results. [Results] There were no significant differences in foot pressure in any region in the superficial trunk muscle exercise group. In the deep trunk muscle exercise group, there were statistically significant increase in F1, F4, F5, R1 and R3. In addition, there were significant decreases in R2 and R4. [Conclusion] After the 4-week deep trunk muscle exercise group decreases in foot pressure on the inner foot and increases on the outside of the feet indicate normal and overall even distribution of body weight on the feet.