Interpenetrating Polymer Network Capturing FRET-Sensitive Polymers Available for an Enzyme Assay.

Fluorogenic glycomonomers have been used for biological evaluations, and water-soluble and Förster resonance energy transfer (FRET)-sensitive glycopolymers have also been reported. A FRET-sensitive polymer was conveniently prepared from a fluorogenic donor monomer and a fluorogenic acceptor monomer by means of simple radical polymerization in high yield. Continuous fluorospectroscopic monitoring of the polymer in the presence of an enzyme was performed, and the results showed the possible application of the FRET-sensitive glycopolymer for practical use. In addition to the use of aqueous solution phase, the water-soluble and FRET-sensitive glycopolymer was completely captured into an interpenetrating polymer network (IPN) by means of radical polymerization with a combination of acrylamide and bis-acrylamide as used for the cross-linking reagent system. The IPN including the FRET-sensitive glycopolymer was allowed to react with amylases in an aqueous buffer solution at 37 °C, and the enzymatic reaction was continuously and conveniently monitored by means of fluorometric spectroscopy.

Timing of Initiation of Acute Stroke Rehabilitation and Management Corresponding to Complications at Primary Stroke Centers in Japan: A Nationwide Cross-Sectional Web-Based Questionnaire Survey.

INTRODUCTION: Many guidelines now recommend early rehabilitation for acute stroke patients. However, evidence remains lacking regarding the specific timings for initiation of various rehabilitation steps and management when complications are encountered in acute stroke rehabilitation. This survey aimed to investigate actual clinical situations in acute stroke rehabilitation in Japan and to improve the medical systems for rehabilitation and plan further studies. METHODS: This nationwide, cross-sectional, web-based questionnaire survey was administered between February 7, 2022, and April 21, 2022, targeting all primary stroke centers (PSCs) in Japan. Among several components of the survey, this paper focused on the timing of the initiation of three rehabilitation steps (passive bed exercise; head elevation; and out-of-bed mobilization), along with the management of rehabilitation (continued or suspended) in the event of complications during acute stroke rehabilitation. We also investigated the influence of facility features on these contents. RESULTS: Responses were obtained from 639 of the 959 PSCs surveyed (response rate: 66.6%). In cases of ischemic stroke and intracerebral hemorrhage, most PSCs initiated passive bed exercise on day 1, head elevation on day 1, and out-of-bed mobilization on day 2 (with day of admission defined as day 1). In cases with subarachnoid hemorrhage, rehabilitation steps were delayed compared to other stroke subtypes or showed wide variation depending on the facility. Passive bed exercise was accelerated by the presence of protocols for rehabilitation and weekend rehabilitation. Out-of-bed mobilization was accelerated by the presence of a stroke care unit. Facilities with board-certified rehabilitation doctors were cautious regarding the initiation of head elevation. Most PSCs suspended rehabilitation training in the event of symptomatic systemic/neurological complications. CONCLUSION: Our survey revealed the actual situation of acute stroke rehabilitation in Japan and indicated that some facility features appear to influence early increases in physical activity levels and early mobilization. Our survey provides fundamental data to improve the medical systems for acute stroke rehabilitation in the future.

Synthetic assembly of α-O-linked-type GlcNAc using polymer chemistry affords sugar clusters, which effectively bind to lectins.

Fischer's glycoside synthesis was applied to linker precursor alcohols of two different lengths having appropriate alkane chains to obtain the corresponding α-glycoside and it was found to be applicable with moderate yields. Water-soluble glycomonomers were systematically prepared from N-acetyl-d-glucosamine (GlcNAc) by introducing two kinds of alcohols having different methylene lengths. Typical radical polymerizations of the glycomonomers with acrylamide as a modulator for control of the distance between carbohydrate residues in water in the presence of ammonium persulfate (APS)-N,N,N',N'-tetramethylethylenediamine (TEMED) gave a series of glycopolymers with various α-glycoside-type GlcNAc residue densities. Fluorometric analysis of the interaction of wheat germ agglutinin (WGA) with the glycopolymers was performed and the results showed unique binding specificities based on structural differences.

Impact of the COVID-19 Pandemic on Acute Stroke Rehabilitation in Japanese Primary Stroke Centers: A Nationwide Cross-Sectional Study Using a Web-Based Questionnaire Survey.

INTRODUCTION: Acute stroke rehabilitation is crucial for achieving good functional recovery, even during the coronavirus disease 2019 (COVID-19) pandemic. This study aimed to clarify the impact of the COVID-19 pandemic on acute stroke rehabilitation and identify which components in the acute stroke rehabilitation provision system were susceptible to the pandemic. METHODS: A web-based questionnaire survey was conducted in all primary stroke centers (PSCs) in Japan between February 7 and April 21, 2022. The survey included questions about the current status of acute stroke rehabilitation and the influence of the pandemic. We classified the influences and investigated the relationship between the influence of the pandemic and the provision of rehabilitation. Additionally, we investigated a group of prefectures divided according to the grade of the pandemic. RESULTS: The survey response rate was 67% (639 of 959 PSCs). Among them, 387 PSCs (61%) reported that the COVID-19 pandemic affected acute stroke rehabilitation. In PSCs affected by the pandemic, more rehabilitation-related staff were working, and the proportion of weekend/holiday rehabilitation implementation was higher compared to those unaffected. In PSCs from highly infected prefectures, no significant relationship was observed between the influence of the pandemic and the rehabilitation provision system. CONCLUSIONS: PSCs that provide denser acute stroke rehabilitation may be affected to a greater extent by the pandemic. We conclude that stroke hospitals should formulate infection control procedures for acute stroke rehabilitation in advance, depending on the conditions of the region and facility.

A nationwide survey for the provision of acute stroke rehabilitation in Japan: initial dose and weekend/holiday rehabilitation.

OBJECTIVES: The early initiation of acute stroke rehabilitation with a sufficient dose, including at weekends/holidays, is important to improve functional outcome. We investigated the status of acute stroke rehabilitation in Japan by using a nationwide survey. MATERIALS AND METHODS: Facility features, rehabilitation dose provided in the first week in each stroke subtype, and weekend/holiday rehabilitation were investigated by using the results of a web-based survey among primary stroke centers. The relationships between facility features and weekend/holiday rehabilitation were also analyzed. RESULTS: A total of 639 stroke centers (66.6%) completed the questionnaire. The overall median dose was 2.0 (interquartile range, 1.7-3.0) U/day (1U = 20 min). After 7 days, the overall median dose increased to 4.0 (2.0-5.4) U/day. Almost 50% of facilities replied that they could not provide a sufficient dose of rehabilitation; the main reason was a lack of therapists (31%). For rehabilitation on long weekends, no rehabilitation was provided on 3-day weekends in 19% of facilities, and in 5% of facilities on ≥4-day weekends. The mean number of therapists was almost 50% less in the facilities that provided no rehabilitation on 3-day weekends compared to those that provided daily rehabilitation (19.4 vs. 36.2 therapists, respectively, p < 0.001). CONCLUSIONS: In this survey, the provision of acute stroke rehabilitation, including non-working days, was clarified. According to the results, prospective interventional or observational studies are needed to design more effective rehabilitation programs to improve outcome. In particular, it is important to determine the optimal dose and intensity of acute stroke rehabilitation.

Clinical applicability of automated tractography for stroke rehabilitation: Z-score conversion of fractional anisotropy.

[Purpose] To expand the applicability of diffusion-tensor tractography fractional anisotropy for stroke rehabilitation, this study aimed to provide references for representative neural tracts from non-lesioned hemispheres. Therefore, we applied the assessment of neural integrity to representative stroke patients using Z-score conversion. [Participants and Methods] Fractional anisotropy values were assessed in neural tracts, including the corticospinal tract, inferior fronto-occipital fasciculus, uncinate fasciculus, and anterior thalamic radiation, of stroke patients receiving acute care. [Results] Data were collected from 60 patients for the non-lesioned right hemisphere and 68 patients for the non-lesioned left hemisphere. Mean fractional anisotropy values in the corticospinal tract and inferior fronto-occipital fasciculus were notably elevated, reaching approximately 0.6 and 0.5, respectively. The mean fractional anisotropy values for other neural tracts were approximately 0.4, and, the overall standard deviations were approximately 0.04. In two typical stroke patients assessed using Z-scores, the scores in the corticospinal tract corresponded to the severity of the hemiparesis. The scores in the anterior thalamic radiation and inferior fronto-occipital fasciculus were associated with more significant brain dysfunction, including inattention and aphasia. [Conclusion] In this study, the Z-score findings related to stroke symptoms align with those reported in the literature, indicating the appropriateness of the methodology used and its potential in future applications.

Proteolytic polymer: polyacrylamides functionalized with amino acids cleave bovine and human serum albumins.

Polyacrylamides with various compositions of serine, aspartic acid, and histidine, which are the amino acids involved in the catalytic triad of natural serine protease chymotrypsin, were synthesized and their protein cleavage activity was investigated. SDS-PAGE analysis showed that some of the synthesized ternary copolymers showed cleavage activity against bovine and human serum albumins. Polyacrylamides incorporating a single type of amino acid were also able to cleave the protein substrates. These homopolymers exhibited unique cleavage profiles and pH and temperature sensitivities that differed from those of α-chymotrypsin. The results indicate the potential of polymers functionalized with amino acids as proteolytic artificial enzymes.

Synthetic assembly of a series of glycopolymers having sialyl α2-3 lactose moieties connected with longer spacer arms.

Glycopolymers having sialyl α2-3 lactose moieties via longer spacer arms were systematically prepared from the corresponding glycomonomers. Radical polymerization of glycomonomers gave a series of glycopolymers displaying various sugar densities. Fluorometric analyses of wheat germ agglutinin (WGA) against the glycopolymers were conducted and the results showed unique binding specificities on the basis of sugar densities.

Brain regions associated with Brunnstrom and functional independence measure scores in patients after a stroke: a tract-based spatial statistics study.

[Purpose] We aimed to assess diffusion tensor fractional anisotropy to outline the brain regions associated with the long-term motor and cognitive functional outcomes of patients with stroke. [Participants and Methods] Eighty patients from our previous study were enrolled. Fractional anisotropy maps were acquired on days 14-21 after stroke onset, and tract-based spatial statistics were applied. Outcomes were scored using the Brunnstrom recovery stage and Functional Independence Measure motor and cognition components. Fractional anisotropy images were assessed in relation to outcome scores using the general linear model. [Results] For both the right (n=37) and left (n=43) hemisphere lesion groups, the corticospinal tract and the anterior thalamic radiation were most strongly associated with the Brunnstrom recovery stage. In contrast, the cognition component involved large regions encompassing the anterior thalamic radiation, superior longitudinal fasciculus, inferior longitudinal fasciculus, uncinate fasciculus, cingulum bundle, forceps major, and forceps minor. The results for the motor component were intermediate between those for the Brunnstrom recovery stage and those for the cognition component. [Conclusion] Motor-related outcomes were associated with fractional anisotropy decreases in the corticospinal tract, whereas cognitive outcomes were related to broad regions of association and commissural fibers. This knowledge will help scheduling appropriate rehabilitative treatments.

Applicability of fractional anisotropy from standardized automated tractography for outcome prediction of patients after stroke.

[Purpose] Diffusion-tensor fractional anisotropy has been used for outcome prediction in stroke patients. We assessed the clinical applicability of the two major fractional anisotropy methodologies-fractional anisotropy derived from segmentation maps in the standard brain (region of interest) and fractional anisotropy derived from standardized automated tractography-in relation to outcomes. [Participants and Methods] The study design was a retrospective survey of medical records collected from October 2021 to September 2022. Diffusion-tensor imaging was conducted in the second week after stroke onset. Outcomes were assessed using the total score of the motor component of the Stroke Impairment Assessment Set (null to full, 0 to 25). Correlations between fractional anisotropy and the outcomes were then assessed. [Results] Fourteen patients with hemorrhagic stroke were sampled. The fractional anisotropy from standardized automated tractography of the corticospinal tract on the lesion side (mean ± standard deviation, 0.403 ± 0.070) was significantly and tightly correlated (r=0.813) with the outcomes (13.4 ± 9.2), whereas the fractional anisotropy from a region of interest set in the cerebral peduncle on the lesion side (0.548 ± 0.064) was not significantly correlated with the outcomes (r=0.507). [Conclusion] The findings suggest that fractional anisotropy derived from standardized automated tractography can be more applicable to outcome prediction than that derived from a region of interest defined in the standard brain.

Applicability of automated tractography during acute care stroke rehabilitation.

[Purpose] To assess the clinical applicability of a novel automated tractography tool named XTRACT during acute stroke rehabilitation. [Participants and Methods] Three patients with left hemisphere stroke were sampled. Diffusion tensor images were acquired on the second week, and automated tractography was then applied. Tractography images and fractional anisotropy (FA) values in the corticospinal tract (CST) and arcuate fasciculus (AF) were assessed in relation to hemiparesis and aphasia. [Results] Patient 1 was nearly asymptomatic; FA in the left CST was 0.610 and that in the AF was 0.509. Patient 2 had severe hemiparesis and mild motor aphasia. Tractography images of the CST and AF were blurred; FA in the left CST was 0.295 and that in the AF was 0.304. Patient 3 showed no hemiparesis or aphasia at initial assessment. Tractography image of the CST was intact but that of the AF was less clear; FA in the left CST was 0.586 and that in the AF was 0.338. Considering the less clear images of the AF and lower FA value in Patients 2 and 3, further examinations for aphasia were performed, which revealed agraphia. [Conclusion] Visualization and quantification of neural fibers using automated tractography promoted planning acute care rehabilitative treatment in patients with stroke.

Dendritic maleimide-thiol adducts carrying pendant glycosides as high-affinity ligands.

We synthesized N-acetylglucosamine-terminated hexavalent carbosilane dendrimers and investigated their binding to wheat germ agglutinin (WGA). The glycodendrimers were prepared by the conjugation of 3-mercaptopropyl, 4-mercaptobutyl, or 5­mercaptopentyl glycosides to maleimide-terminated hexavalent carbosilane dendrimers. Titration of WGA with the glycodendrimers yielded quenching of tryptophan fluorescence. All of the glycodendrimers exhibited high affinity with nanomolar dissociation constants (KD values). The best dendrimers were 1a and 1b with KD values of 6.5 ± 1.7 and 5.3 ± 1.7 nM, respectively. The magnitude of fluorescence quenching increased with decrease in the length of the thioalkyl spacer. Maleimide-pendant carbosilane dendrimers provide ready access to multivalent ligands with high-affinity potential.

Relationship between the independence level of individual motor-related functional independence measure items and its total score in patients after hip fracture: an ordinal logistic modelling study.

[Purpose] The independence level of activities of daily living during the recovery period should be predicted to plan a rehabilitation program. This study aimed to assess the relationship between the independence levels of individual motor-related Functional Independence Measure (FIM-motor) items and total FIM-motor score in patients after hip fracture. [Participants and Methods] This study retrospectively analyzed 40 patients who had stayed in a convalescent rehabilitation hospital after hip fracture. The FIM-motor score was assessed for each patient on admission, after 2 and 4 weeks of hospitalization, and at discharge. [Results] The median FIM-motor scores were 52.5 on admission and 83.0 at discharge. The results of ordinal logistic modeling were statistically significant for all 13 FIM-motor items. The independence levels for transfer to toilet and bed/chair/wheelchair were proportionally distributed across the entire range of total FIM-motor scores. However, a weak relationship was observed between the scores for bladder and bowel management and total FIM-motor scores. Although eating and grooming were relatively easy items, stair-climbing and locomotion were difficult. [Conclusion] The relationship between the independence level of individual FIM-motor items and the total FIM-motor score varied widely. This knowledge might be useful while scheduling rehabilitative treatments for patients after hip fracture.

Preparation of glycopolymers having sialyl α2 → 3 lactose moieties as the potent inhibitors for mumps virus.

A water-soluble glycomonomer having a sialyl α2 â†’ 3 lactose (SLac) moiety was prepared from a known imidate derivative of the SLac and an acrylamide alcohol by means of Schmidt's protocol followed by transesterification. Polymerization of the monomer proceeded in water as the solvent in the presence of ammonium persulfate (APS)-tetramethylethylenediamine (TEMED). Since acryl amide (AAm) was used as a regulator for the arrangement of sugar density, three kinds of glycopolymers having different sugar densities were obtained. Infection inhibition assays of mumps virus (MuV) for Vero cells using the glycopolymers were performed, and the results showed that a glycopolymer having a low sugar density has the highest inhibitory potency. In comparison to sialyl Lewis X (SLeX) as the strongest inhibitor in a previous study, SLac polymer with the low sugar density showed ten-times stronger inhibitory potency than that of SLex. This finding suggested that multivalent conversion of the monomeric SLac with appropriate spatial arrangement are able to effectively inhibit the interaction between the attachment glycoprotein of MuV and glycan receptors on Vero cells.

Outcome prediction in patients with putaminal hemorrhage at admission to a convalescent rehabilitation ward based on hemorrhage volume assessed with computed tomography during acute care.

[Purpose] The aim of this study was to assess the usefulness of computed tomography for outcome prediction in patients with putaminal hemorrhage at admission to a convalescent rehabilitation ward. [Participants and Methods] Patients admitted to our convalescent rehabilitation ward after transfer from acute care hospitals were included in this study. Multiple regression analyses were performed using the score in the motor component of the Functional Independence Measure at discharge as the target value. Hemorrhage volume assessed with computed tomography during acute care and age were set as the explanatory variables. The motor component of the Functional Independence Measure score at admission and the time (days) from onset were also recorded. Correlation analyses between all the possible pairs of explanatory variables were then performed. [Results] Hemorrhage volume and age were both significant contributors to the motor component of the Functional Independence Measure score at discharge. However, the contribution of hemorrhage volume disappeared when the time from onset and motor component of the Functional Independence Measure score at admission were added. Hemorrhage volume significantly correlated with the time from onset and motor component of the Functional Independence Measure score at admission. [Conclusion] The present findings suggest that computed tomography may be useful for outcome prediction from the acute stage in stroke patients with putaminal hemorrhage. However, because of multicollinearity, its predictive power was reduced when the patients were transferred to a convalescent rehabilitation ward.

Fluorogenic glycopolymers available for determining the affinity of lectins by intermolecular FRET.

A convenient assembly of fluorogenic glycopolymers having various polymer compositions was accomplished from the corresponding glycomonomer and dansyl monomer by means of radical polymerization, and the water-soluble glycopolymers gave typical fluorescence spectroscopic profiles due to the dansyl moieties on the glycopolymer in aqueous media. Biological evaluation of the polymer against wheat germ agglutinin (WGA) was accomplished on the basis of fluorescence changes due to tryptophan residues on WGA, and the affinities between the glycopolymers and WGA were estimated to be 4.7 × 105 to 9.3 × 105 M-1. In order to apply the fluorogenic glycopolymers for further biological measurements, efficient resonance energy transfer from tryptophan moieties on WGA to dansyl moieties on the fluorogenic glycopolymers was examined. FRET profiles of both fluorophores were similar compared to the binding profiles on the basis of fluorescence changes of tryptophan residues. This approach is applicable for the determination of an affinity constant between a carbohydrate and a lectin in which no fluorophore exists near the binding site.

Neuraminidase-triggered activation of prodrug-type substrate of 4-nitroaniline.

Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator.

Comparison of Fractional Anisotropy from Tract-Based Spatial Statistics with and without Lesion Masking in Patients with Intracerebral Hemorrhage: A Technical Note.

BACKGROUND: Diffusion-tensor fractional anisotropy (FA) is an index of neural-fiber damage in patients following stroke. To better characterize FA, tract-based spatial statistics (TBSS) is frequently used, which involves spatial transformation into the standard brain space. Despite its utility, this technique is susceptible to space-occupying hematoma in patients with intracerebral hemorrhage. To correct this, "lesion making" has been proposed. Here, FA values from TBSS without lesion masking and TBSS with lesion masking were compared, and the clinical utility was evaluated. METHODS: Forty patients from our previously published work were entered into the study. Diffusion-tensor imagings were acquired 14-21 days after onset and FA maps were generated. Lesion masks were produced in reference with nondiffusion (b = 0) brain images. Two types (with or without lesion masking) of TBSS were then performed. For both types, using individual data we extracted mean FA values within for the corticospinal tract (CST) and the superior longitudinal fasciculus (SLF). FA ratio (rFA) between the lesioned hemisphere and the unaffected hemisphere was then calculated. The two sets of the data were then compared by assessing residuals of mean root sum square error (RMSE). RESULTS: Although rFA obtained from TBSS with lesion masking tended to be slightly smaller, the estimated RMSE was .025 for both the CST and the SLF. CONCLUSIONS: The estimated FA differences between the two sets of TBSS were very small. Considering the time for manual labor for producing lesion masks, regular TBSS without lesion masking may be sufficient in terms of clinical utility.

A constraint scaffold enhances affinity of a bivalent N-acetylglucosamine ligand against wheat germ agglutinin.

Bivalent glycoconjugates have a minimal valence with avidity potential on protein-carbohydrate interactions as well as simplicity of chemical structures enabling simple synthesis with low cost. Understanding the way to maximize the affinities of bivalent glycoconjugates is important for the development of cost-effective tools for therapeutic and diagnostic research. However, there has been little discussion about the effects of constraints imposed from ligand scaffolds on the binding abilities. We synthesized three kinds of biantennary N-acetylglucosamine glycosides with different scaffolds using isobutenyl bis(propargyl)ether as a common scaffold precursor. Decoration of the scaffold branches with GlcNAc moieties through copper-catalyzed azide-alkyne cycloaddition and grafting of the alkenyl focal point to another bivalent biotin dendron through thiol-ene and nucleophilic substitution reactions were successfully carried out in an orthogonal manner. The association constants of the ligands against wheat germ agglutinin were determined by a fluorometric titration assay. A bivalent biotin counterpart provided higher affinity than an isobutyl scaffold, whereas an isobutenyl scaffold yielded more enhancement than a bivalent biotin counterpart. The present work suggested that the constraint and steric bulk of ligand scaffolds are possible factors for improving binding properties of glycoconjugates against lectins or proteins.

Synthetic construction of sugar-amino acid hybrid polymers involving globotriaose or lactose and evaluation of their biological activities against Shiga toxins produced by Escherichia coli O157:H7.

Synthetic assembly of sugar moieties and amino acids in order to create "sugar-amino acid hybrid polymers" was accomplished by means of simple radical polymerization of carbohydrate monomers having an amino acid-modified polymerizable aglycon. Amines derived from globotriaoside and lactoside as glycoepitopes were condensed with known carbobenzyloxy derivatives, including Z-Gly, Z-l-Ala and Z-ß-Ala, which had appropriate spacer ability and a chiral center to afford fully protected sugar-amino acid hybrid compounds in good yields. After deprotection followed by acryloylation, the water-soluble glycomonomers were polymerized with or without acrylamide in the presence of a radical initiator in water to give corresponding copolymers and homopolymers, which were shown by SEC analysis to have high molecular weights. Evaluation of the biological activities of the glycopolymers against Shiga toxins (Stxs) was carried out, and the results suggested that glycopolymers having highly clustered globotriaosyl residues had high affinity against Stx2 (KD = 2.7∼4.0 µM) even though other glycopolymers did not show any affinity or showed very weak binding affinity. When Stx1 was used for the same assay, all of the glycopolymers having globotriaosyl residues showed high affinity (KD = 0.30∼1.74 µM). Interestingly, couple of glycopolymers having lactosyl moieties had weaker binding affinity against Stx1. In addition, when cytotoxicity assays were carried out for both Stxs, glycopolymers having highly clustered globotriaosyl residues showed higher affinity than that of the copolymers, and only highly clustered-type glycopolymers displayed neutralization potency against Stx2.