TM II H28 Detection assay . The H28 assay is a multiplex semi-quantitative real-time PCR test using the tagging of oligonucleotide cleavage and extension (TOCE) technology. The VL was semi-quantified as high , intermediate , or low . Out of 5940 HPV VL analyses, 356 assays (5.99%) were reported as low VL (1+) of HPV DNA. Matched cytology diagnoses were mostly negative findings , except for seven cases of atypical squamous cells of undetermined significance (1.9%) and two cases of atypical glandular cells (0.6%). During the follow-up periods, abnormal cytologic diagnoses were identified, including one case of high-grade squamous intraepithelial lesion (HSIL) and two low-grade squamous intraepithelial lesions (LSILs). The matched, confirmative histologic diagnosis of HSIL cytology was compatible with chronic inflammation, wherein the two LSILs had regular check-ups. None revealed clinically concerned outcomes associated with HPV-related squamous lesions. The cytology was most likely negative for malignancy when the VL of HPV DNA was low (1+). Additional strategic monitoring and management may thus be unnecessary.Infection with high-risk (HR) Human Papillomavirus (HPV) is associated with the development of precancerous lesions or invasive carcinoma of the uterine cervix. Thus, the high viral load (VL) of HR-HPV DNA currently serves as a representative quantitative marker for cervical cancer. However, the clinical significance of low HPV DNA VL remains undetermined. This study aimed to evaluate the clinical association between the low HPV DNA VL and cytology/histologic diagnosis of cervical samples. We searched the electronic medical databases for the resultant analyses of HPV genotyping among patients who underwent treatment for any cervical lesion or who had undergone gynecological examinations with any positive HPV results according to the national cancer screening service between 2015 and 2016. HPV testing with genotyping and semi-quantitative VL measurement was conducted using an Anyplex Worldwide, cervical cancer is the fourth most common cancer and the fourth leading cause of cancer death in women, with an estimated 604,000 new cases and 342,000 deaths in 2020 . Cervicawww.hpvcenter.se; accessed on 20 May 2022), and this number continues to expand [More than 200 different HPV types have been listed by the International HPV Reference Center  is due to HR-HPV . IntegraAs an effective screening test for cervical cancer, cervical cytology has so far played a key role in reducing the incidence of cervical cancer in countries with organized screening ,12,13,14To date, the viral load (VL) of HPV has been proposed as a potent discriminator of significance from insignificant HPV infections ,19. SeveThe VL of HPV can be measured by various methods, including semi-quantitative  or quantitative polymerase chain reaction (PCR) ,27. MostTM II HPV28 Detection assay  is a multiplex qRT-PCR test using the tagging of oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 19 high-risk (HR) HPV types , and 9 low-risk (LR) HPV types  in a single reaction . The H28 assay also estimates the VL through a repeated melting temperature analysis during the TOCE reaction and reports a semi-quantitative system as low , intermediate , or high  [In recent practice, a qRT-PCR method is commonly used to estimate the amount of HPV DNA. The Anyplex cycles) . Meanwhile, unlike the high VL of HPV, there has been little research on the clinicopathologic significance of low (+) VL of HPV based on H28 assay in cervical lesions. It is not surprising that the clinical association between low HPV VL and cytology/histologic diagnosis of cervical samples remains undetermined, mainly because of the over-sensitivity and inconsistent reproducibility of the test. Such a little-known characteristic may impede gynecologic physicians’ and patients’ best shared decision making for therapeutic plans and consequently bring about unexpected problems, such as excessive levels of patient-perceived anxiety or unnecessary waste of time and money. Therefore, a clear association between low HPV VL and cytology/histologic diagnosis of cervical samples using H28 assay needs to be investigated thoroughly.In this study, we aimed to evaluate the clinicopathologic significance of an HPV infection with a low (1+) viral DNA load on cervical/vaginal cytology samples obtained from patients with a clinical follow-up due to previous cervical lesions, such as SIL or invasive cervical carcinoma, and from patients who have shown a positive result for HPV in an earlier HPV examination.We searched the electronic medical database of the Department of Pathology of Kyungpook National University Chilgok Hospital in the Republic of Korea for HPV genotyping analyses among patients with any previous cervical lesions or who had undergone gynecological examinations according to the National Cancer Screening Services, between January 2015 and December 2016 at Kyungpook National University Chilgok Hospital.A total of 5940 HPV analysis results were obtained. Each of the matched clinical characteristics included age and HPV status at the initial cytology/histopathologic diagnosis plus the follow-up information for the subsequent cytology/histopathologic diagnosis and HPV status. A flowchart shows the sample selection and analysis process in this study .Briefly, liquid-based cytology (LBC) samples were collected using broom-like devices with detachable heads  according to the manufacturer’s instructions. The cervical broom was placed into the cervical canal and was rotated 360 degrees around the entire cervical canal. The detachable head was placed in a vial with preservative fluid. The LBC sample collection and transient storage temperature followed the manufacturer’s instructions.Subsequently, the HPV test with Anyplex HPV28 assay using LBC specimens was performed according to the manufacturers’ instructions. DNA from each LBC sample was extracted using STARMag 96 X4 Universal Cartridge Kit . HPV genotyping was conducted using a multiplex polymerase chain reaction (PCR) using a CFX96 PCR Thermal Cycler , according to manufacturers’ guidelines, using 5 μL of template DNA in a total volume of 20 μL for Anyplex™ II HPV28.TM II H28 Detection kit .At the initial examination and at least once during a follow-up visit, HPV testing with genotyping and semi-quantitative VL measurement were conducted according to the manufacturer’s instructions using the AnyplexTM II H28 assay. Descriptive statistical analysis using SPSS Statistics software for Windows, version 21.0  was only conducted for the demographic data, including age and follow-up.We performed a double-blind test for the cytology/histopathology diagnosis and the HPV status using the AnyplexAt a baseline examination for the cytology and HPV typing, the mean age was 48.1  years old. The mean follow-up period was 58.3 ± 12.2 months.Out of 5940 HPV analyses, 356 assays (5.99%) were reported as a low (1+) VL of HPV. Of 356 cases with low (1+) VL of HPV, 150 patients had a previous confirmative diagnosis of HPV-related epithelial lesion. The results are summarized in From the HPV test at the starting time (baseline) of this study, 233 (65.4%) out of the 356 cases were HR-HPV genotypes . HoweverDuring the follow-up periods, 263 (73.9%) of 356 patients with a low VL of HPV DNA underwent at least one repeated HPV test. Almost all (97.3%) showed a negative HPV result at the initial repeated test, while the remaining patients (n = 7) were finally negative for HPV at the subsequent tests . FurtherHR-HPV genomic integrations into the host genome are associated with a persistent infection . Either The recurrence risk of SIL or invasive carcinoma remains higher among women who undergo treatment for primary HPV-related epithelial lesions. For that reason, such patients need to be put under close surveillance and follow-up examination . Given tTM II HPV28 assay is a multiplex semi-quantitative real-time PCR assay using TOCE technology. The viral load is semi-quantified as high , intermediate , or low  [The Anyplex cycles) . TOCE te cycles) . The sen cycles) . The ass cycles) .TM II HPV28 and AnyplexTM II HPV HR assays had superior performances with higher sensitivity than HC2 (p < 0.0001) and were concordant with other commercial assays for HR-HPV detection despite presented semi-quantitative results [TM II HR assay, sharing the same TOCE technology as the AnyplexTM II HPV28 assay, compared to that of Cobas 4800 HPV and HC2. They assessed the precision of the assay by comparing the positive control results from 15 replicates using clinical specimens with low , intermediate (Cq < 32.2), and high (Cq < 26.7) concentrations of HPV DNA in triplicate per run a day on five different days [TM II HR assay and the Cobas 4800 [TM II tests, including the H28 assay, would be helpful in follow-up testing and patient management by providing genotyping information beyond HPV 16 and HPV 18 positivity [The H28 assay has been validated in recent HPV-related studies. Cornall et al. showed that both Anyplex results . Furtherent days . The prebas 4800 . Therefositivity .However, few studies have been conducted on the significance of low VL of HPV. Its value has been virtually ignored due to the difficulty in accurate quantification of low VL and (in some cases) the possibility of contamination by sexual partners or another viral status, which may lead to a false-positive reaction of viral DNA for a short term . Such a We initiated this study to contribute to medical science by addressing the clinical relevance between the low VL of HPV and the risk of cervical lesions. In this study, none of the patients who reported as low (1+) VL of HPV revealed clinically concerned outcomes associated with HPV-related epithelial lesions during follow-up. Therefore, in accordance with what the study found, we carefully propose that clinicians and patients would have a better shared decision making with simply the monitoring and management of HPV VL, instead of unnecessary additional tests or procedures, when the low (1+) VL of HPV status is detected in an H28 assay.In particular, there are two further significant implications obtained from this study. Although cervical cancer can develop as long as HPV infection persists, a quantitative contribution to cervical carcinogenesis remains unclear regarding the duration and viral load of HPV infection. Fortunately, our results could provide a practical reference for the clinical treatment of patients. Those with a low VL of HPV should be managed more conservatively and safely or in a step-by-step manner considering familial and social environmental factors, such as pregnancy, rather than immediate management.The other is that, even though the LR-HPVs are not subject to screening and follow-up for cervical cancer, it is impossible to know whether the viral subtype is from an LR- or HR-HPV group before performing the HPV subtyping test. Moreover, one should be aware of the possibility of cross-reaction or multiple HPV infections. This study demonstrated the clinical significance of the low VL of HPV in practical management. In other words, the cases with a lower VL of LR-HPV, as expected, did not have any precancerous or cancerous lesions during the follow-up; furthermore, in cases of HR-HPV groups, they did show identical findings. Therefore, we proposed that the low VL, regardless of LR- and HR-HPV subtyping, can emphasize the significance of conservative strategies for screening or follow-up of cervical cancer.It is also obvious that quality control of the laboratory performance is very important for either the primary screening test or a strategic assessment of the post-treatment testing utility for HPV infection. The laboratory performance should adhere to a well-established protocol of intra-laboratory quality assurance and be kept under close review, thereby ensuring a reliable data processing system within the institution. Once an individual laboratory is accredited for HPV molecular testing, intra-institutional strategic guidelines may also be proposed, which ultimately contribute to a standardized decision on “what to do” for the best follow-up care.This study had several inherent limitations regarding small sample size, sample type, and analytic methods. In detail, first, by selecting patients with HPV infection from one tertiary medical institute in the Republic of Korea, we might have biased our cohorts towards patients with HPV DNA persistence, thus undermining generalization. Second, we only assessed the HPV DNA load using a semi-quantitative measurement by a repeated melting temperature analysis of the HPV28 assay, which may damage validity to some extent. Further studies should propose a clinical cut-off for the viral load for the reliability of HPV testing. Third, the method of HPV28 detection may present inflated positive results due to its excessive sensitivity for the primary HPV screening test. By extension, there remain the possibilities of transient HPV infection or contamination, requiring further efforts to refine the sensitivity of the measurement and increase reliability in future studies. These limitations support a need to verify the study finding through large data obtained from rigorous replication of research in multi-institute settings and the general population. However, we believe that our results outweigh the limitations in that it is the first study to provide at least a minimum of medical evidence on the possibility of strategic utilization of low VL of HPV for monitoring cervical lesions.This study showed that none of the patients with a low VL of HPV DNA revealed clinically concerned outcomes associated with HPV-related squamous lesions, regardless of HR- or LR-HPV genotypes. Therefore, we could provide a practical guideline for the patient with a low VL of HPV DNA. When referring to the strategic monitoring and management of patients who undergo treatment for previous HPV-related epithelial lesions and patients who had an HR-HPV detection earlier, those with a low VL of HPV using the AnyplexTM II H28 assay should be managed in a conservative and safe or step-by-step manner considering familial and social environmental factors, such as pregnancy, rather than immediate management. In these particular situations, the accuracy and confidence of laboratory HPV testing need to be consistently guaranteed.


Background: Interstitial lung disease (ILD) is a common pulmonary disease often associated with significant morbidity and mortality in patients with connective tissue diseases (CTD). Currently, no gold-standard therapies are available for CTD-ILD. Recently, several studies have proposed that rituximab (RTX) may be effective for the treatment of CTD-ILD.Objectives: This study aimed to systematically evaluate the efficacy and safety of RTX for the treatment of CTD-ILD.Methods: Studies were selected from PubMed, Embase, and Cochrane Library, up to 20 July 2022. Improvement and stable rates were extracted as the main outcomes and pooled using the weighted mean proportion with fixed or random-effects models, in case of significant heterogeneity (I2 > 50%). Safety analysis was performed based on the adverse events reported in all of the studies.Results: Thirteen studies (312 patients) were included in the meta-analysis. The follow-up durations ranged from 6 to 36 months. The pooled improvement rate was 35.0% (95% CI: 0.277–0.442), while the pooled stable rate was 59.2% (95% CI: 0.534–0.656). Anti-synthetase syndrome associated with ILD  and idiopathic inflammatory myopathies associated with ILD  had higher improvement rates than the other types. A total of 106 adverse events associated with RTX or progressive ILD were reported among the 318 patients, 55.7% of which were mild. Among 19 deaths, 17 were due to ILD progression, one to severe pulmonary arterial hypertension, and one to Pneumocystis jirovecii infection.Conclusion: RTX, which exhibits a satisfactory safety profile, is an effective treatment option for CTD-ILD, even in patients who fail to respond to other therapies. Further randomized trials are needed to assess the efficacy of rituximab compared to other treatments for CTD-ILD.Systematic review registration: PROSPERO, identifier (CRD42022363403). Connective tissue diseases (CTDs) are a group of disorders characterized by diverse symptoms and autoantibodies that circulate throughout the body and damage internal organs . InterstThe pathogenesis of CTD-ILD is complex and not fully understood; however, it is generally accepted that underlying immune system dysfunction, immune-mediated pulmonary inflammation, and subsequent fibrosis are crucial steps. Therefore, corticosteroids and immunosuppressive drugs are considered crucial for the treatment of CTD-ILD . EvidencBiological treatments .The forest plot for the subgroup analyses is shown in n = 44), fever (n = 6), infusion reactions (n = 5), fatigue (n = 3), and cardiac involvement with arrhythmia (n = 1). Among grade 3–4 events, 28 adverse events occurred, including infection requiring hospitalization (n = 23), serum sickness (n = 2), gastrointestinal complications requiring surgery (n = 2), and anaphylaxis (n = 1). Nineteen deaths were reported in 318 patients: 17 due to respiratory failure secondary to ILD progression, one with severe pulmonary arterial hypertension, and one with Pneumocystis jirovecii infection.All patients treated with RTX were evaluated, some of whom were not included in the pooled analysis of lung response outcomes because lung function data and survival conditions were unavailable. Twelve  studies Sensitivity analysis was performed by removing individual studies one by one from the pooled results with high heterogeneity. The pooled analysis of improvement rate and stable rate did not change significantly when studies were omitted, indicating that our combined results are reliable .p = 0.17) (p = 0.21)   and stab = 0.21) . This waTo the best of our knowledge, this is the first meta-analysis of observational studies on the efficacy and safety of RTX in CTD-ILD. Considering that most of the involved patients were refractory to conventional treatments and had progressive ILD, improving or stabilizing lung function was regarded as an effective response. In our results, the improvement rate was estimated to be 35.0% in 312 patients with CTD-ILD, while the stable rate was 59.2%. This result indicates that RTX was an effective treatment option for patients with CTD-ILD based on the lung function test, even in those who failed to respond to other conventional therapies, such as CYC. A total of 106 adverse events associated with rituximab treatment or progressive ILD were reported among the 318 patients. Most were mild, indicating that RTX was tolerated in most patients.An optimal management for CTD-ILD has not been established because of the relative rarity of the disease and the high variability in disease presentation. Currently, corticosteroids are widely used to manage CTD-ILD . Other iNowadays the treatment of CTD-ILD has a more extended measure with the presence of antifibrotic treatments. The main antifibrotic drugs, pirfenidone  and nintt-test or Wilcoxon’s rank sum test. No consensus criteria for assessing the treatment efficacy of CTD-ILD are currently available. Some studies were excluded from our pool analysis because of different assessment methods that focused on the efficacy of RTX. These excluded studies assessed efficacy by comparing mean/median FVC and/or DLCO pre- and post-RTX treatment using the Student I2 = 54%) and stability rate (I2 = 43%). Subgroup analysis revealed that IIM-ILD (non-ASS)  and ASS-ILD  had higher improvement rates than the others. This adds to the weight of the evidence regarding the heterogeneity of CTD-ILD. However, the pathogenesis of CTD-ILD is not fully understood, and the mechanism by which IIM-CTD responds better to RTX therapy than the other types of CTD-ILD is uncertain. Further research that investigates on the pathogenic mechanisms of CTD-ILD is required.There was medium heterogeneity in the improvement rate  or ASS-ILD responded better to rituximab than those with other CTD-ILDs. Regarding side effects, most patients showed good tolerance to RTX. Considering these limitations, prospective randomized trials are needed to assess the efficacy of rituximab compared to other drugs in CTD-ILD. Consensual criteria based on PFT and HRCT for the assessment of CTD-ILD treatment efficacy should be established in the future.


Despite any post-stroke conditions, the evaluated total accuracy rate by the 10-fold cross-validation using SVM among four-, five-, six-, and seven-gesture models were 96.62%, 94.20%, 94.45%, and 95.57% for non-paretic and 90.37%, 88.48%, 88.60%, and 89.75% for paretic limbs, respectively. LDA had competitive results using PCA whereas k-NN was a less efficient classifier in gesture prediction. Thus, we demonstrate partial efficacy of the combination of sEMG and supervised learning for upper-limb rehabilitation procedures for early acute stroke motor recovery and various treatment applications.In clinical practice, acute post-stroke paresis of the extremities fundamentally complicates timely rehabilitation of motor functions; however, recently, residual and distorted musculoskeletal signals have been used to initiate feedback-driven solutions for establishing motor rehabilitation. Here, we investigate the possibilities of basic hand gesture recognition in acute stroke patients with hand paresis using a novel, acute stroke, four-component multidomain feature set (ASF-4) with feature vector weight additions  and supervised learning algorithms trained only by surface electromyography (sEMG). A total of 19  acute stroke survivors (12.4 ± 6.3 days since onset) with hand paresis  performed 10 repetitive hand movements reflecting basic activities of daily living (ADLs): rest, fist, pinch, wrist flexion, wrist extension, finger spread, and thumb up. Signals were recorded using an eight-channel, portable sEMG device with electrode placement on the forearms and thenar areas of both limbs (four sensors on each extremity). Using data preprocessing, semi-automatic segmentation, and a set of extracted feature vectors, support vector machine (SVM), linear discriminant analysis (LDA), and k-nearest neighbors (k-NN) classifiers for statistical comparison and validity (paired  Post-stroke motor deficits directly influence post-stroke recovery due to reduced self-care ability and participation in social life, leading to poor outcomes in one-third of survivors within 5 years after onset ,2,3. TheHealthcare fields are currently undergoing explosive growth in fields related to individualized and user-centric rehabilitation for neurological motor impairments that promote motor learning , such asParetic hand bioelectrical signal evaluation as a key domain for feedback-driven solutions can be partially reflected in the field of limb prosthetics in which the priority is artificial limb control by tracking the remaining atrophic muscle signals ,24,25. SRecently, valid feature selection for gesture recognition in paretic hands is being extensively reported due to the capacity for wide-ranging application to diverse methods ,38. AmonOverall, previous reports on sEMG-driven gesture recognition for stroke were narrowly focused on the chronic stage, although post-acute stage applications remain challenging ,34,35,41However, the acute stage of stroke requires specific attention since recovery plateaus and remains constant within 1 month after onset . AdditioWe conducted an observational study of 59 patients, among which 19 patients  had an acute stroke with brachial monoplegia. The candidate pool included hospitalized patients admitted to the University of Tsukuba Hospital within the post-acute stroke period  from December 2020 to June 2021.Written, informed consent was obtained for every participant. Eligible hospitalized patients who passed screening tests were assessed during routine procedures with an occupational therapist. Organizing and collecting data took 1 h, on average, and did not interfere with scheduled treatment procedures. Study regulations were approved by the University of Tsukuba Hospital Ethics Committee . Research protocols and design were implemented according to the Declaration of Helsinki  and the University Guidelines for Clinical Trials.Physical examinations were evaluated with the following stroke upper-limb motor impairment scales: Brunnstrom stage , which measures the state of post-stroke movement recovery; Fugl–Mayer assessment for upper extremity , which evaluates limb functioning; the single-task various function evaluation Stroke Impairment Assessment Set ; the Modified Ashworth Scale , where lower scores represent normal muscle behavior and higher scores indicate the development of post-stroke spasticity ,46,47.To establish the technical aspects of the procedure, we used removable, portable foam hydrogel Ag/AgCl sEMG sensors using a wireless eight-channel Biosignal Plux device  certified for biomedical research. The real-time recording mode was set to 16 bit analog-to-digital (ADC) converter resolution and 1000 Hz sampling rate.Participant hands and forearms were prepared with alcohol wipes to minimize possible artefacts and deviations during the sEMG recording. Before electrode attachment, forearm muscles were palpated by specialists. Five total sensors were placed according to the functional upper-limb anatomy, targeting muscle bellies and forearm anterior/posterior locations . Sensors were placed on the projection of the flexor carpi radialis muscle, the flexor carpi ulnaris muscle area, the abductor pollicis brevis muscle in the thenar area, and the extensor digitorum communis muscle, plus a grounding sensor that was placed on the lateral elbow epicondyle. We aimed to preserve 2 cm of distance between the electrodes ; howeverThe procedure involved a one-time sampling of surface electromyography from patients in a controlled environment to minimize possible biases and monitor participant conditions. In the research design , patientDuring the experiments, post-stroke neurological impairments caused some of the patients to have difficulty in performing hand movements on the first attempt. Hence, non-segmented data had a portion of artefacts and signal gaps that were unfit for statistical evaluation and required additional preparation.VCC is equal to 3 V, sensor gain ADC is equal to the sampled value from each channel, and n is the number of the channel bandwidths represented in bits.First, real-time bilateral electromyography data from both limbs were combined into two datasets with standardized dimensions. The initial raw signal was converted into a micro voltage (mV) format factor in accordance with the following transfer function that represents the device configuration  :EMGV=ADCA fourth-order Butterworth bandpass filter (BPF) ranging from 20 to 300 Hz;Hampel filtering for artefact reduction by identifying outliers deviating from an average of more than double the standard deviation in the neighboring 100 samples;Root-mean-square signal envelope and sEMG data normalization.Second, taking into consideration MAS scores and adjacent muscles on the forearm which could introduce movement artefacts, we applied high-pass (HPF) and low-pass frequency filters (LPF). According to related studies, filters defined by the International Society of Electrophysiology and Kinesiology (ISEK) standards of sEMG analog processing have initial ranges of 5 Hz cutoff for HPF and 500 Hz for LPF to ensure an optimal window signal range for upper-limb applications ,51. HoweThe maximum peak variable likelihood estimation was set to 8, cutting off the first and last gestures from each 10-gesture repeated set.Minimal temporal distances between the peaks were configured as 0.1 s.To avoid false variables, the estimation of peak selection was set at 0.25 percentile of the difference from neighboring signal peaks.For temporal standardization, signal boundaries  were determined by the window length of 30 ms for the inferior limit and 60 ms for the superior limit with regard to the peak.For our experiments, hand gesture recognition workflow included seven gesture labels . The segmentation started from a manual selection of active segments that contained one label of a single gesture. Furthermore, except for the ‘rest’ label, we used a centered, moving average function for automatic peak detection to process temporal analysis for four channels simultaneously with the following settings:To avoid peak misdetection, sEMG data were visualized and inspected ; the usuIn terms of biophysical definitions, electromyography of the upper extremities can be expressed as a set of time series of myoelectrical static events; the multichannel summation of which can store specific patterns of the hand gestures in the form of vector features ,30,31. FFirst, to emphasize the functionality of our ASF-4 set , we usedConsidering the peculiarity of the myoelectric signature of post-stroke patients, we defined a diagram  for the Initially, we applied our novel, stroke-oriented ASF-4 feature set as the base; however, in experiments, model performance was optimal for affected and non-affected extremities for three and four gestures, respectively. Further model enhancement met predictive pitfalls due to the fist (both flexor and extensor static muscle bursts) and thumb (included fist components posture) gestures.To manage that issue, we added noise-resistant functions  and our custom features to upscale multilabel class prediction (up to 6 gestures and rest state). Furthermore, we implemented nonlinear parameters to provide better accuracy for each gesture within the model ,60,61,62Keeping with the same approach, this tendency was also proposed for frequency-domain and time–frequency-domain features. In summary, aiming to control the level of model overfitting, we established two feature subsets of initial ASF-4 for non-paretic (ASF-14NP with 14 features) and paretic (ASF-24NP with 24 features) orientation.ASF-14NP  includesASF-24P  includesIn our framework, as features were concatenated, we converted each participant’s 4-ch sEMG data to Euclidean distances (ED) and standard deviation (SD) as a measure of distance for statistical evaluation. The mathematical definition of the ED is the root of the mean squared difference between the pair-channel signal’s sEMG data, while SD is the measure of the variance .As a separation index, the ratio of the ED to SD (RES) was used ,56, haviTo investigate optimal performance settings, we applied principal component analysis (PCA) to tune concatenated feature vectors. PCA establishes dimensional reduction by removing similar linear subspace within the feature vector , therebyGL) using the rest gesture label as the baseline for evaluation of all possible combinations of hand gestures, where GL4 has 20 combinations . To validate results, we applied 10-fold SVM cross-validation (with 100 iterations each) amid 19 participants , which resulted in a total accuracy rate of predicted and true gesture labels. This first classification was performed without PCA and subsequently with PCA to identify the impact of dimensional reduction on performance; we could, therefore, extend the usability of supervised learning in post-acute stroke paretic recognition.Acc) and F1-score (F1) which were derived from the confusing matrix output values of true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) [In general, to evaluate the multiclass supervised classification of biosignal pattern recognition in stroke victims, we deployed statistical measures of accuracy (ves (FN) . OverallF1-score, as a harmonic mean of precision  and recall (true positive rate), demonstrates the summarized accuracy of the method which is used in bioelectrical signal processing:The Acc, as the main performance metric obtained from SVM, LDA, and k-NN, was validated using canonical paired-sample t-tests  in each gesture label group (GL4–7). Thus, in the statistical evaluation, we tested Acc and F1 in pairs amid classifiers for the non-affected side and affected side to reveal tendencies in prediction between the identic and different algorithms. The same procedures were performed for the PCA-dependent performance using certain best principal components (PCs) of each classifier to study dimensional reduction and statistical significance. For this testing, the best PC values were derived from the best harmonic mean scores.MATLAB R2020a  software environment.Statistical evaluation was developed using custom scripts in the In 19 total patients, 11 cases of cerebral infarction  and eight cases of intracranial hemorrhage  were studied. Participating candidates  had the GLs from four to seven on a non-paretic dataset (NP19), whereas In both paretic and non-paretic extremities, we revealed that the use of a fist gesture label led to a decrease in the predictiveness of the model by an average of 7.7% ± 2.4% . GL4 and GL5 combinations on both extremities with an average accuracy rate above 80% in most scenarios. On the affected side, we observed a trend that flexor-related gestures had the most label misclassification. Moreover, the model complexity of a single-class dispersion (ability to recognize certain gestures) was varied and organized in a specific fashion in every classifier.Results indicate that supervised learners were capable of distinguishing GL with each component from 1 to 50 having a one-dimensional step . The dimensional impact on the CM charts is shown in To reveal the best settings in the PCA technique , we crosnal step . This prGLs. For k-NN, figures turned out to be distorted in terms of accuracy where the optimal parameters were obtained from only low PCs values. As for harmonic mean or F1-score, in general, those metrics were correlated with accuracy, turned out to be sensitive in dimensional investigation, and had larger deviation parameters across GLs.Here, we were able to visualize and conceptualize the PCA relationship with classification performance; for P19, SVM result metrics remained stable without the sharp accuracy spikes throughout the PCs, whereas the NP19 figure was heavily deformed when using small PCs numbers. LDA metrics had a similar shape but the P19 experienced an accuracy trend in which a smaller number of PCs had a greater recognition score for each To conclude, the optimal PC parameters above 80–90% accuracy rate for classifiers were the following: 23 (NP19) and 35 (P19) for SVM ; 8 (NP19) and 19 (P19) for LDA (this linear classifier tended to target one-third of the complete vector length both for ASF-14NP and ASF-24P); 5 (NP9) and 8 (P19) for k-NN .F1-scores.We also discovered that, according to the empirical results, a combination of LDA and PCA increased classifier performance by up to 12.2% in certain models . NotablyGLs in order to specify the best PCA settings and understand the optimal feature vector weights.In the first instance, we gradually inspected the statistical validity of supervised learners and the dimensional shift for each p < 0.05) was not obtained in every gesture model, whereas only GL6 demonstrated statistical validity among PC pairs.For this task, we evaluated the model performance between the same classifiers for paretic and non-paretic sides without  and withp-values between the classifiers ; black indicates a nonsignificant comparison between classifiers with a p-value of more than 0.05 ; light blue reveals an absence of statistical relationships since a paired t-test evaluation was not performed due to logical inconsistency .Furthermore, we summarized the cross-dimensional comparison of ssifiers . In thisAcc and F1 (SVM without and LDA with the PCA) were evaluated to validate pairs of highest interest for statistical validity  over an increasing number of gesture predictors. GL7, whereas the paretic side remained flat over the GLs. Using multidimensional layering of concatenated feature vectors, LDA upscaled predictive performance up to 9–10% for both datasets, targeting the paretic side as primary impact. F1 also increased for most of the gestures in each GL. As a consequence, the use of PCA elevated LDA performance and allowed for the outperformance of SVM for most myoelectric pattern recognition scenarios.For LDA, prediction capacities varied with a certain deviation between P19 (near to 80%) and NP19 (near to 85%). The linear parametric classifier had a tendency of increased accuracy rate related to the number of used gestures, with the best results at GLs, thereby having close prediction results amid all gesture clusters. Moreover, k-NN indicated close accuracy to SVM amid both datasets and flattened the total predictive capacities between each gesture whereas the standard deviation of Acc and F1 between paretic and non-paretic sides were the lowest  at the early acute stage (within a month) using only a simple, four-channel sEMG system.To start, according to the results , non-parDb4 for affected and Db8 for non-affected .Considering those limitations and other attempts in stroke hand gesture decoding ,31,32,33p < 0.05) between classifier groups. These results show that our method is suitable for practical application since we broke through the pre-determined 70% accuracy threshold for human–computer interfaces in rehabilitation [Over the course of the study, using supervised learning and stroke-oriented feature sets  extracted from simple sEMG signal input, we were able to discriminate static hand gestures with statistically viable results . For SVM, PCA downgraded the prediction models for each GL on average by less than 2% while having only one-third the length of a concatenated feature vector. Maintenance of such a shorter length of the extracted feature vector, coupled with predefined optimal PCs values, provided competitive model accuracy to the initial feature vector. For real-time use, even with lower accuracy scores, PCA application for SVM can be beneficial since dimensionally redacted ASF feature vectors will be processed faster and, at the same time, will serve as a primitive filter  [GL4 model), while the capabilities of the linear classifier increased with the number of gesture predictors. However, PCs increased mean accuracy for GL4 by 6.5% and 1–2% for the remaining GLs.In general, our results suggest that the behavior of the unaffected limb is specific but tends to be similar to those reported in healthy individuals’ gestures  ,36,38. Wl input) . In the As for the affected side, myoelectrical decoding without using PCs revealed that only SVM had outstanding performance metric scores usable across a set of gesture models. Adding the PCA method explicitly increased LDA performance by 12–13% in some scenarios, while PCs downgraded model performance both for k-NN and SVM. However, depending on the number of predicted labels used (4–7), certain algorithms have advantages with similar accuracy rate trends in both extremities.In contrast to our results, Zhang and Zhou  used higDuring the experiments, we discovered that the fist (mostly mismatched with wrist flexion and thumb) is the most complicated in terms of random gesture prediction , whereas rest state and wrist extension had the best prediction. Nonetheless, by referring to related studies conducted on stroke patients, we discovered at least several cases of similar tendencies in fist-like gesture recognition. As an example, Yang et al. (12 patients) obtained lower accuracy scores precisely in grip movements both for main and control groups among eight static hand gestures . TheoretOn the contrary, gesture models without the fist gesture in both datasets generally saw increases in total accuracy results by an average of 7–9% after model complexity reduction ,48. Withp < 0.05), we discovered an interesting observation that, within each GL, the statistically viable pairs in both datasets had precisely specific patterns in spite of the model complexity and number of PCs . Thus, additional insight related to paretic GL6 and GL7 combinations, in which significance between the groups was achieved only within a single inter-class of classifiers between the affected and non-affected sides, is required from future experiments on an expanded patient population. For this, we hypothesize that, for each stroke-oriented feature set, the classifier behavior has a specific pattern depending on dimensional reduction [During testing of the difference between classifiers , improve the prediction capabilities, generate advances in signal pre-processing, and optimize classifiers. These novel ASF feature sets require additional attention and further testing to validate their performance parameters .In a pilot study on 19 post-acute stroke patients, we were able to validate the practical possibility of recognizing simple hand gestures on both paretic and non-affected sides using only a four-channel sEMG and supervised learning. The evaluated total accuracy rates using SVM for four-, five-, six-, and seven-gesture models were 96.62%, 94.20%, 94.45%, and 95.57% for non-paretic and 90.37%, 88.48%, 88.60%, and 89.75% for paretic limbs, respectively. LDA had a competitive accuracy performance using PCA whereas k-NN turned out to be a less efficient classifier in gesture prediction.For post-acute stroke application, we did find several points of interest with our system. Among these: early-stage pattern recognition should include up to five predictive labels only for supervised learning , while, for the follow-up recovery process, applied hand and forearm gestures should be distinct according to upper limb biomechanics with an aim to trigger specific flexor/extensor ratio bursts on the forearm muscles . Lastly, myoelectrical prediction of fine gestures can be enhanced using dimensional reduction approaches.The research results are protected by international law in a patent. This original manuscript was created during the patent pending process.


FIB-4 was the best score to identify non-survivor patients (sensitivity of 80% and specificity of 63%) and predict the need for ICU or mortality (71% of sensitivity and 65% of specificity), with a cut-off value of 1.94. Therefore, we present the predictive power and the cut-off values of several liver fibrosis scores here for disease severity and mortality in SARS-CoV-2 in-patients and we proposed the use of the present scores to identify ab initio the clinical therapeutic and diagnostic protocols for high-risk patients.Discovering novel risk and prognostic factors for COVID-19 may help not only in reducing severity and mortality but also in creating targeted therapies considering patients’ individual features. Liver fibrosis is considered a complication in Non-alcoholic Fatty Liver Disease (NAFLD), it is a feature of steatohepatitis (NASH), and it has already been related to an increased risk for a wide range of diseases. Here, we aimed to define if any parameter assessing metabolic status has predictive power in identifying inpatients at risk for poorer prognosis and an increased mortality from COVID-19. This retrospective study was conducted at the Sub-Intensive Medicine Care Unit of the Presidio Maxi-Emergenze Fiera del Levante, Azienda Ospedaliero-Universitaria Policlinico di Bari, Italy. We evaluated 271 inpatients with moderate-to-severe SARS-CoV-2-related respiratory failure by comparing biochemical features and non-invasive liver fibrosis scores among discharged, transferred to Intensive Care Units (ICU) and non-survivor patients. Moreover, by performing ROC curves, we defined cut-off values to predict mortality and disease severity for each score. We found that non-invasive scores of liver fibrosis, obtained at day of admission, such as AAR ( SARS-CoV-2 enters the peripheral blood from the lungs and spreads into cells expressing angiotensin-converting enzyme 2 (ACE-2), which represents the cognate receptor of the envelope Spike protein of SARS-CoV-2. Then, the intracellular lifecycle of the virus starts. The infected host cells trigger an immune response with the recruitment of T lymphocytes, monocytes, and neutrophils . In seveSimilarly, the inflammation and release of a plethora of cytokines and adipocyte-specific hormones are linked to excessive fat accumulation, specifically visceral adiposopathy, representing a risk factor for cardiovascular, metabolic and chronic obesity-related diseases, including cancer .On the one hand, patients with increased abdominal obesity show a decreased diaphragmatic excursion that compromises the pulmonary function in a supine position, making ventilation more difficult . MoreoveAAR, the ratio of Aspartate Transaminase (AST) to Alanine Transaminase (ALT), which is typically less than 1, can rise to greater values as fibrosis and cirrhosis develop. According to a study conducted by Giboney et al., 87 percent of patients with an AAR of 1.3 or less had NASH . The severity of NASH as measured by the degree of fibrosis increased, as did the AAR. The mean ratio of 1.4 was found in patients with cirrhosis related to NASH. Wilson’s disease can cause the AAR to exceed 4. In short, certain AARs are suggestive of certain conditions. Consequently, since there is significant overlap between AAR in different conditions, and the exact mechanism of AAR alteration in the progression of liver disease is unclear, its accuracy in predicting the degree of fibrosis and the presence of cirrhosis is controversial and this ratio cannot be used alone to make a diagnosis .Fibrosis-4 index (FIB-4) is a non-invasive score used to assess liver fibrosis in outpatient settings. The index is considered to be accurate, non-invasive, and easily available, and may be useful in evaluating patients with Hepatitis-C Virus (HCV), NAFLD, and other liver complications , althougThe modified fibrosis-4 (mFIB-4) index was elaborated on as an instrument to assess the stage of liver fibrosis in patients with chronic hepatitis B (CHB) or C (CHC). However, it is used for the detection of advanced liver fibrosis in all patients with chronic liver disease .The FORNS index is based on the assessment of four routine parameters ). The most important study to evaluate its diagnostic accuracy considered a cohort of 250 patients with CHC. The FORNS index is able to exclude the presence of severe fibrosis with a Negative Predictive Value of 96% and to identify the presence of severe fibrosis with a Positive Predictive Value of only 66%. It is therefore a useful test in identifying patients with minimal fibrosis but it has limited value in identifying patients with more advanced liver disease .APRI score (AST to Platelet Ratio Index) is a non-invasive index for the assessment of liver fibrosis in patients with viral hepatitis, and represents an alternative to liver biopsy in the follow-up of patients with hepatitis C and liver cirrhosis in an outpatient setting. In a meta-analysis of 40 studies, it was concluded that an APRI score of above 1.0 presents a sensitivity of 76% and a specificity of 72% for predicting cirrhosis. An APRI score of above 0.7 presents a sensitivity of 77% and a specificity of 72% for predicting significant liver fibrosis . It is lAARPRI (AAR to Platelet Ratio Index) represents another important non-invasive score for the evaluation of hepatic fibrosis. In particular, it can be considered as one of the most reliable scores in the diagnosis of advanced stages of liver fibrosis. Moreover, AARPRI was also proposed as a predictor for chronic liver disease-associated complications ,19. All The aim of this study was to identify novel metabolic parameters such as liver fibrosis scores at admission with the putative ability to predict disease severity and mortality in COVID-19 patients.The initial phase of the study focused on analysing the bio-humoral and haemato-chemical parameters and liver fibrosis scores to determine metabolic abnormalities in the study population. In a second phase, attention was focused on identifying whether there were statistically significant differences between patients who died during hospitalization and patients discharged home or moved to Intensive Care Unit (ICU). We analysed and correlated the various markers, identifying the most representative ones in predicting mortality and prognosis, also studying which of these parameters showed a correlation with the length of hospitalization in our Unit.n = 13) and who admitted POTUS (n = 11) were excluded from the study. Acute heart failure and other acute diseases were excluded before admission to our unit.Patients’ recruitment, and clinical and biochemical analyses were registered consecutively in the electronic health register of the Medicina Sub-Intensiva Unit of Presidio Maxi-Emergenze (MSI-PME) at Teaching Hospital Policlinico di Bari, Italy, from April 2021 to April 2022. Admitted patients presented respiratory failure due to SARS-CoV-2 infection and required oxygen therapy up to noninvasive ventilation. A total of 443 patients with 18 or more years were initially enrolled in this study, among which 148 were excluded because biochemical data such as AST, ALT, GGT, or blood count were lacking on day 1, since the data were recorded before the admission to our ward, at the Emergency Department. Patients with previous viral hepatitis, cirrhosis, benign liver tumours (BLT), primary liver cancer (PLC) or hepatic metastasis at baseline . All participants underwent a detailed anamnesis and physical examination at admission. Unfortunately, precise anthropometric assessment at time 0 was not performed for all patients due to their critically ill status. Morning blood samples were obtained after 12 h of fasting from the antecubital veins of patients on the first day after admission to our unit. After blood clotting and centrifugation, serum was processed for an analysis of the biochemical markers of glucose and lipid metabolism. Liver, renal, thyroid and inflammatory markers were also studied following standardised biochemical procedures. All biochemical measurements were centralised and performed in the ISO 9001 certified laboratories of the University Hospital of Bari. Specifically, a complete blood count with a determination of the leukocyte subpopulation was performed. Measurements of total and High-Density Lipoprotein cholesterol (HDL-c), fasting plasma glucose (FPG), and triglycerides (TG) were obtained using an enzymatic colorimetric assay . CRP was performed via nephelometry . The low-density lipoprotein cholesterol (LDL-c) level was obtained using the Friedewald formula; the Neutrophil to Lymphocyte Ratio (NLR) and Monocyte to HDL-c ratio (MHR) were calculated manually.t-test (for continuous variables) and the Pearson χ2 test . Analyses between more than two groups were performed through one-way analysis of variance (ANOVA), which was followed, where required, by Bonferroni’s post hoc test. The correlation between continuous variables was also analysed and estimated using Pearson’s Correlation Coefficient (r).Descriptive statistical analyses of the study sample were performed, and their results are expressed as mean ± standard error of the mean (SEM) and frequencies (%), depending on the nature of variables. Comparisons of socio-demographic and clinical variables between two groups were conducted with the The receiver–operating characteristic (ROC) curves were used to determine the optimum cut-off levels of non-invasive liver scores in predicting mortality and severity of SARS-CoV-2 infection. Empirical ROC curves were plotted for these variables along with a calculation of the area under the curve (AUC) with 95% confidence intervals and one-sided upper p-values for the null hypothesis AUC = 0.5. The condition variable for severity was in-hospital death or ICU admission.p-values lower than 0.05 were considered significant. All analyses were performed using the NCSS 12 Statistical Software, version 12.0.2018  and GraphPad Prism, version 9.1.0 .The average age of our 271 patients was 70. Mean length of the stay in our Unit was 13 days. A total of 38 patients  died during hospitalization in our unit, while 233  were still alive when they moved from our unit. Of them, 208 patients  were discharged, while 25 patients  were transferred to ICU due to the worsening of their clinical conditions.p < 0.001) and exhibited increased lymphocytopenia (p < 0.05) and glycemia (p < 0.001) at admission. Surprisingly, no significant differences were found for inflammation markers  except for Procalcitonin (p < 0.05). With regard to lipid assessment, total cholesterol and LDL-cholesterol (LDL-c) were significantly lower in the non-survivor group (p < 0.05), while no statistical differences were found for AST, GGT, Total Bilirubin, and NT-proBNP. Myocitolysis parameters such as lactate dehydrogenase (LDH) (p < 0.001), creatine phosphokinase (CPK) (p = NS), myoglobin (p < 0.05), and troponin (p < 0.05) were all increased in the second group.As shown in p < 0.001), FIB-4, (p < 0.05), mFIB-4 (p < 0.05), FORNS (p < 0.05), and AARPRI (p < 0.05) scores, but no significant difference was observed for APRI , FIB-4 (p < 0.05), mFIB-4 (p < 0.05), FORNS (p < 0.01), and AARPRI (p < 0.05), but not for APRI.To better understand if these scores might predict not only the mortality but also the evolution of critical disease and prognosis, we then performed a one-way ANOVA test by dividing our population into three groups according to their final outcomes, i.e., if they were discharged after clinical resolution, moved to ICU because of the worsening of their conditions, or died during hospitalization. The comparison was significantly different for AAR (p < 0.05). Multiple comparisons also showed that only AAR (p < 0.001) or ICU-admitted (p < 0.05) patients, whereas other comparisons did not reveal any difference between these two groups.APRI only showed a statistically significant difference when compared by T-student test between ICU-admitted and survivor patients (only AAR a was sigp < 0.05), apart from APRI .Furthermore, we found that this association remained when we performed the same analysis only in patients who were discharged, excluding those who succumbed or worsened in condition  in predicting mortality, with a cut-off value of 1.94 a. The saIn this study, we demonstrated that non-invasive scores of liver fibrosis predict mortality and clinical outcomes in COVID-19 patients, and also correlate with the length of the hospitalization.Previous studies found that liver fibrosis was independently associated with mortality, regardless of the demographic characteristics of patients . SpecifiIn fact, in the setting of acute COVID-19 illness, Metabolic Syndrome and its hepatic features, namely NAFLD and NASH that represent an ongoing pro-inflammatory state, might exacerbate the virus-induced cytokine storm, possibly through the hepatic release of pro-inflammatory cytokines, which are responsible for worse prognosis. Moreover, in up to 20% of cirrhotic patients admitted to critical care units, respiratory viruses are usually detected and pneumonia is one of the most common infections in patients with advanced liver fibrosis . A more In the present study, we did not have the patients’ pre-admission metabolic status and previous anthropometric data. Thus, although we present data for time 0, we cannot exclude the idea that collateral liver damage from virally induced cytotoxic T-cells and the induction of a dysregulated innate immune response could also explain the association between the deranged liver markers and COVID-19 disease severity . AdditioFinally, elevated aminotransferases could also originate from myositis rather than liver injury, since myoglobin, hs-Troponin, and LDH values were higher in the non-survivors group. To exclude the influence of this circumstance on the predictive power of non-invasive fibrosis scores, we performed our analysis by considering clinical and biochemical assessment at admission; theoretically, this time period from infection was not sufficient to determine myositis. In addition, no patients reported previous myositis in their anamnesis.We found that non-invasive scores of liver fibrosis such as AAR, FIB-4 and mFIB-4, FORNS, and AARPRI strongly predict not only in-hospital mortality but also the length of hospitalization and eventual admission to intensive care units in SARS-CoV-2 infected patients. The strength of the present study is the identification of the cut-off values of FIB-4 and mFIB-4 at admission, which are able to predict disease severity and mortality in COVID-19 patients.


Low-frequency whole-body vibration , a low impact form of exercise, intervention for a month following moderate transient middle-cerebral artery occlusion (tMCAO) reduces infarct volume and improves motor function in reproductively senescent, middle-aged female rats. Since post-stroke cognitive decline remains a significant problem, the current study aims to investigate the efficacy of WBV in ameliorating post-tMCAO cognitive deficits and to determine the underlying putative mechanism(s) conferring benefits of WBV in middle-aged rats. Middle-aged rats of both sexes were randomly assigned to tMCAO (90 min) or sham surgery followed by exposure to either WBV (twice a day for 15 min each for 5 days a week over a month) or no WBV treatment groups. Following the last WBV treatment, rats were tested for hippocampus-dependent learning and memory using a water maze followed by harvesting brain and blood samples for histopathological and inflammatory marker analyses, respectively. Results show that post-tMCAO WBV significantly lessens cognitive deficits in rats of both sexes. Post-tMCAO WBV significantly decreased circulating pro-inflammatory cytokines and increased serum levels of irisin, a muscle-derived hormone that may play a role in brain metabolism and inflammation regulation, which suggests putative beneficial mechanisms of WBV. Stroke is the leading cause of disability worldwide, and most stroke survivors suffer functional and cognitive deficits . DeclineIn recent years, whole body vibration (WBV) emerged as a cognitive-enhancing therapy, especially in persons unable to perform active/aerobic forms of exercise . The aerIn several recent studies, physical activity of skeletal muscle resulted in the release of extracellular vesicles (EVs) into circulation . These eWhole body vibration is a method that amplifies baseline irisin levels in humans . Irisin Our published studies demonstrated that (1) low-frequency whole-body vibration (WBV) therapy after spinal cord injury improved selected biomarkers of bone turnover and gene expression and reduced osteoclastogenesis , and (2)in Vivo Experiments guidelines to the best of our knowledge.All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Animal Care and Use Committee of the University of Miami. Results are reported according to the Animal Research: Reporting n = 60) or age-matched male (n = 52) Sprague–Dawley rats were used to perform the experiments  a WBV intervention group or to (2) a No-WBV control group. Animals randomized to the WBV group underwent 30 days of treatment performed twice daily for 15 min (once in the morning between 8:00 and 10:00 a.m. and once in the afternoon between 2:00 and 4:00 p.m.) each session for 5 days each week. The vibration device  was programmed in order to achieve a frequency of vibration within a range of about 40 Hz similar to that used in clinical studies . The durHemodynamic measures were evaluated using the tail cuff method . For 5 consecutive weeks, 10 measurements were taken for each animal and the mean value was reported in Rat activity levels were tested in an open field (43.5 × 21.5 cm) with four white arenas using a video tracking system (EthoVision). Rats were placed in each arena, and 5-min recordings were taken prior to and after completion of WBV/No-WBV treatment. The total duration spent in the center of the open field was measured.Animals were evaluated for spontaneous forelimb placement in a transparent Plexiglas cylinder (20 cm diameter × 30 cm height) for 5 min. The cylinder geometry encouraged vertical exploration . AnimalsRats exposed to tMCAO and WBV treatment were monitored for memory and spatial learning using the Morris Water Maze . In thisSeparate cohorts of rats of both sexes exposed to tMCAO and tMCAO + WBV/No-WBV were sacrificed at 24 h and 1 month after tMCAO respectively, for brain tissue and blood/serum collection .The sera derived from middle-aged females that underwent tMCAO followed by WBV or No-WBV were used to isolate EV. Serum EV were isolated using the Total exosome isolation from serum kit (Invitrogen) as described in a previous publication . BrieflyThe protein content in homogenized cortical tissue and serum-derived extracellular vesicles (EV) was assessed and proteins were separated by 12% stain-free SDS-PAGE as described in previous publications . Proteinp < 0.05 was considered statistically significant.The data are shown as mean ± SD. All the data were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison tests for two or more groups. Data were normally distributed, using a D’Agostino–Pearson test for normality. A We hypothesized that post-tMCAO improves motor deficits, and in order to evaluate sensorimotor deficits, we utilized the cylinder test. This task quantifies spontaneous asymmetrical forelimb use and reliably detects deficits in models that produce substantial unilateral damage, which occurs after focal cerebral ischemia . For eacWe conducted open field activity tests to monitor alterations in animal activity following tMCAO and to study the impact of WBV in female rats. The results demonstrate that prior to tMCAO-induction, the time spent in the center of the open field is different amongst groups; however, the observed differences are not significant. Furthermore, the results demonstrated no significant difference between sham and post-tMCAO plus No-WBV activity measurements of distance traveled or frequency to visit the center of the field . Post-tMCognitive impairments following stroke are recapitulated in animal models, and emerging studies demonstrate that neurodegeneration occurs in the rat hippocampus and striatum after MCAO . Since, After a learning period of 4 days, a probe trial (30 s duration) was given with the platform removed and the time spent in the goal quadrant, where the platform used to be, is measured. The data presented in Our published study demonstrated that inflammasome activation and the pro-inflammatory cytokine IL-1β were significantly higher in the brain and serum of reproductively senescent females as compared to their younger counterparts and male rats . TherefoWe have demonstrated that WBV treatment reduced the expression of IL-1β in the brains of female rats after stroke . Others Vascular endothelial growth factor is known to increase after stroke. It is known to promote neuroprotection, neurogenesis, angiogenesis, and brain vessel repair . We obseSeveral recent studies have shown that following physical activity, skeletal muscle releases extracellular vesicles (EVs) into the circulation . These eThe most important outcome of the current study is that post-tMCAO low-frequency WBV therapy for a month significantly improves hippocampus-dependent cognition in middle-aged rats of both sexes. Using the spatial probe, the most sensitive test for post-stroke learning deficit , we obseStudies have demonstrated that physical exercise induces systemic inflammation, which improves cognition . The croThe data presented in the current study demonstrated that post-tMCAO WBV increases circulating serum-derived EVs containing irisin. At this juncture, we like to mention that circulating EVs released post-tMCAO WBV treatment likely contain more factors than only irisin, and future proteomic analyses are needed to characterize the contents of EVs. Further studies are also needed to determine the cellular origin and derivation of these EVs in order to confirm that are brain-derived. Given the fact that apart from the presence of irisin in EVs, post-tMCAO WBV also increases circulating irisin protein levels in the serum and cortical penumbra of middle-aged female rats. Overall, these data suggest a key role for irisin in WBV-conferred cognitive improvement. Although the mechanism by which irisin confers neuroprotection and improves cognition is not fully known, the hormone plays a crucial role in preserving mitochondrial function, mitigating oxidative stress, and elevating expression of BDNF, among other neuroprotective measures . Irisin Finally, the current study suggests that WBV improves post-stroke functional and cognitive deficits in middle-aged rats of both sexes and that irisin may be responsible for conferring the beneficial effects of WBV. While more studies are needed to characterize the role of irisin, based on data in the current study, WBV has a potential for clinical translation to improve post-stroke cognitive deficits. The most attractive aspect of low-frequency WBV therapy is that it could be implemented at an early stage as it is non-invasive and stress-free. In support of our approach, a study simulating helicopter-induced LFV/WBV immediately after tMCAO showed a significant reduction in infarct size and better neurological outcomes compared with control or actual helicopter-induced vibrations. Helicopter transportation is a common occurrence and will become routine with use of Tenecteplase, in which a “drip and ship” standard of care has been implemented (stroke patient receives tPA while transported by helicopter) . HelicopThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the IACUC University of Miami.AR, HB, and WD: conceptualization, resources, and writing—review and editing. NK, HB, and AR: methodology. NK and JS: formal analysis. NK, AR, and JS: investigation. NK, WM, and OF-A: data analysis. NK and AR: writing—original draft preparation. All authors contributed to the article and approved the submitted version.


The role of microparticles (MPs) and cold in high altitude pulmonary hypertension (HAPH) remains unexplored. We investigated the impact of long-term cold exposure on the pulmonary circulation in lowlanders and high-altitude natives and the role of MPs. Pulmonary hemodynamics were evaluated using Doppler echocardiography at the end of the colder and warmer seasons. We further examined the miRNA content of MPs isolated from the study participants and studied their effects on human pulmonary artery smooth muscle (hPASMCs) and endothelial cells (hPAECs). Long-term exposure to cold environment was associated with an enhanced pulmonary artery pressure in highlanders. Plasma levels of CD62E-positive and CD68-positive MPs increased in response to cold in lowlanders and HAPH highlanders. The miRNA-210 expression contained in MPs differentially changed in response to cold in lowlanders and highlanders. MPs isolated from lowlanders and highlanders increased proliferation and reduced apoptosis of hPASMCs. Further, MPs isolated from warm-exposed HAPH highlanders and cold-exposed highlanders exerted the most pronounced effects on VEGF expression in hPAECs. We demonstrated that prolonged exposure to cold is associated with elevated pulmonary artery pressures, which are most pronounced in high-altitude residents. Further, the numbers of circulating MPs are differentially increased in lowlanders and HAPH highlanders during the colder season. Prolonged living at high altitudes causes a sustained rise in pulmonary artery pressure (PAP). It is general knowledge that pulmonary vasoconstriction and vascular remodeling due to chronic hypoxia exposure underlie high altitude pulmonary hypertension (HAPH) . Yet, beIn the past, we have reviewed and analyzed the knowledge about the influence of low temperatures on pulmonary circulation and existence of the cold-induced PH . Based oMPs are shed cell membrane fragments released into the circulation to a certain extent in healthy individuals during different cellular processes . InteresOverall, we hypothesize that endothelial and inflammatory cell-derived MPs are actively involved in the pathology of HAPH. Also, we suggest that a long-term cold environment as a regularly present factor together with hypoxic conditions at high-altitude locations, may provide additional effects on patients with HAPH and may further dysregulate the MPs profile. The objectives of the current study were, therefore, to explore the impact of chronic cold exposure on the pulmonary circulation in high-altitude residents and to investigate the role of MPs in HAPH.During 2017 we performed two expeditions in Kyrgyzstan, the first one at the end of the colder season (February–March) and the second one at the end of the warmer season (September–October) . Both exg/15 min, followed by 10,000× g/5 min at room temperature), as we have described before [g (90 min), at 4 °C).Based on the living location and echocardiography-derived tricuspid regurgitant systolic pressure gradient , the study participants were divided into three groups: lowlanders (LA), highlanders controls (HA), and highlanders who developed pulmonary hypertension (HAPH) . For detd before . In ordeAs we have already stated in our previously published studies derived from the expedition in Kyrgyzstan, written informed consents were obtained from all human subjects enrolled in the study ,23. The Flow cytometry analysis of circulating MPs (CD68 (macrophages), CD3 (T-cells), and CD62E ) in PFP was done as described previously by our group with subtle modifications . Please For assessment of apoptosis, Kinetic Apoptosis Kit (Abcam) and IncuCyte live cell imaging system were used. The proliferation of human PASMCs was determined by Cell Proliferation ELISA, 5-bromo-2′-deoxyuridine (BrdU) assay , following the manufacturer’s instructions. Please find the details in the Isolated MPs were lyzed by the addition of 700 µL of QIAzol lysis buffer and total RNA was isolated using the miRNeasy Mini Kit (Qiagen), according to the manufacturer protocol. The amount of recovered RNA was quantified by Nanodrop . Expression levels of selected miRNAs: miRNA-210, miRNA-145, miRNA-17, and miRNA-133a-3p were assessed by qRT-PCR and TaqMan miRNA assays (Thermo Fisher Scientific). Please find the details in the Primary hPAECs (PromoCell) were seeded into 0.1% gelatin (Sigma-Aldrich)-coated 35 mm culture dishes at 37 °C and pre-incubated with 10 µg/mL of isolated MPs or NaCl control for 24 h prior to trypsinization. hPAECs were re-suspended in Endothelial Cell Basal Medium (PromoCell) containing 0.2% FBS and 10 µg/mL of isolated MPs or NaCl control. A total of 12,500 cells were seeded in a 96-well plate coated with polymerized Matrigel Matrix (Corning) and transferred to IncuCyte live cell microscopy imaging system. Images obtained after 2 h were analyzed by ImageJ software and Angiogenesis Analyzer tool using various parameters, such as number of nodes, total length, and total segments length.2 for 48 h prior to protein extraction. Western blot analyses were performed as described previously [Primary hPAECs (PromoCell) passage 7 were cultured in Endothelial Cell Growth Medium (PromoCell) in the presence of 10 µg/mL of isolated MPs and incubated at 37 °C in a humidified atmosphere of 5% COeviously . Anti-ent-test with Welch’s correction and two-way ANOVA with Sidak´s multiple comparisons test were used. p-value < 0.05 was considered statistically significant.All results are presented as mean ± SEM. ROUT test was used for the identification of outliers. For statistical analysis of data, First, we investigated the effects of cold exposure on pulmonary circulation in both Kyrgyz lowlanders (LA) and highlanders (HA). We compared intra-individually the changes in PAP, as estimated by TRG, between the colder and warmer seasons in 63 LA and 87 HA individuals. Interestingly, there was a trend toward higher TRG values in LA individuals during the cold season, as compared to those during the warm season . This efThe circulating levels of CD62E-positive MPs were comparable between the groups, except there was a significant increase in the number of these MPs in LA exposed to cold in comparison to the warm a. There We have further investigated the potential effects of MPs isolated from the blood of human LA, HA, and HAPH exposed to the warmer or colder seasons on relevant cellular processes, such as the proliferation and apoptosis of human PASMCs. Our data revealed that isolated MPs from all experimental groups increased the proliferation of PASMCs a. CorresWith regard to the miRNA-145 and miRNA-133-3p, there were no changes in the expression among the experimental groups a,b. FurtWe have also investigated the potential effects of isolated MPs on other relevant cell type, such as hPAECs. Initially, we analyzed whether there were effects of isolated MPs on angiogenesis. However, there were no changes among the experimental groups, as evident from different parameters of angiogenesis, such as number of nodes, total length, and total segments length .In addition, we have investigated the potential effects of MPs isolated from the blood of human LA, HA, and HAPH exposed to the warmer or colder season on expression profiles of VEGF and eNOS . Our resOverall, we have demonstrated that long-term exposure of humans to low temperatures is associated with PAP elevation. Interestingly, a recent study conducted on a large number of ambulatory patients in Israel showed that echocardiograms performed during Summer-Fall had a 19% lower chance to show systolic PAP > 40 mmHg compared with those done in Winter-Spring . In our MPs release is increased under stress conditions and disease. It has been suggested that a higher number of circulating MPs might be linked with different cardiovascular diseases . In addiHypoxia is known to activate endothelial cells  and CD62Several studies reported a positive correlation between endothelial cell-derived MPs and the severity of PAH ,29. HypoWe have recently demonstrated enhanced levels of circulating CD3 cell-derived MPs in different forms of PH . In contSimilar to our previous report on other forms of PH , levels Accumulating evidence has suggested a role for MPs as carriers of various bioactive mediators, including miRNAs ,41. InteGrowing evidence suggests that miRNAs are critically involved in responses to adverse environmental conditions such as low temperature ,54. We dMPs were suggested to affect the functions of PAECs and PASMCs. Circulating MPs isolated from hypoxia-induced PH rats have been demonstrated to affect the vascular tone and modulate vascular reactivity and influence neoangiogenesis. Cultured endothelial cells exposed to MPs isolated from rats with hypoxia-induced PH displayed decreased expression of eNOS and had lower nitric oxide production . AdditioVEGF is a potent endothelial cell-specific mitogen and permeability factor that is involved in angiogenesis . Recent Cold exposure has been shown to stimulate VEGF expression in various tissues ,70. IntePASMCs in remodeled pulmonary vessels are characterized by over-proliferation and apoptosis resistance. Remarkably, extracellular vesicles isolated from monocrotaline-treated mice induced PH with increased muscularization of peripheral pulmonary arteries and right ventricular hypertrophy in healthy animals . In accoTaken together, our study demonstrated that prolonged exposure to cold is associated with elevated PAPs, which is most pronounced in high-altitude residents. Further, the numbers of circulating MPs were differentially increased in lowlanders and HAPH highlanders during the colder season.


Background: The incidence of ischemic stroke (IS) is much higher among patients with chronic kidney disease (CKD) compared to the general population. Few studies have evaluated the association between the risk of IS and the use of Chinese herbal medicine (CHM) in patients with CKD. We aimed to investigate the risk of IS among patients with CKD using CHM as add-on therapy.Methods: We conducted a retrospective cohort study based on Taiwan’s National Health Insurance Research Database to assess 21,641 patients with newly diagnosed CKD between 2003 and 2012. Patients were classified as either the CHM  or the non-CHM group  based on whether they used CHM after first diagnosis of CKD. We used the proportional subdistribution hazards model of Fine and Gray to examine the hazard ratio (HR) of IS in propensity-score matched samples at a ratio of 1:1 for two groups.Results: The risk of IS was significantly reduced in the CHM group  compared with the non-CHM group. Those who used CHM for >180 days had an even lower risk of IS than those in the non-CHM group . Additionally, frequently prescribed formulae, such as Ji-Sheng-Shen-Qi-Wan, Liu-Wei-Di-Huang-Wan, and Zhen-Wu-Tang were associated with a 30%–50% reduced risk of IS.Conclusion: Our results suggest that patients with CKD who used CHM as add-on therapy had a lower hazard of IS than those in the non-CHM group, especially for patients taking CHM for >180 days. Further experimental studies are required to clarify the causal relationship. Chronic kidney disease (CKD) has recently been reported as an independent risk factor for stroke. The incidence of ischemic stroke (IS) was much higher among patients with CKD compared to the general population . A strokThe primary medications for preventing stroke among patients with CKD included antiplatelets, anticoagulants, statins, and antihypertensive agents . These cAbelmoschus manihot (L.) Medik  plus lorsatan for 24 weeks had less proteinuria than those treated with lorsatan alone . The main reason for CAM usage was based on the potential benefits of CAM . The potan alone . In addian alone . Moreovean alone . Furtheran alone . Additioan alone . Given tTaiwan launched a single-payer National Health Insurance (NHI) Program in 1995 that coveres almost the entire population. The annual coverage rate has ranged from 96.1% to 99.6% of the population, with more than 20 million Taiwan residents enrolled since 1997. The National Health Insurance Research Database (NHIRD) derived from the NHI program has been used for research purposes. The data we used in this study was from a longitudinal health insurance database from 2002 to 2013 comprised of medical claim data of one million beneficiaries randomly selected from the NHIRD, representing about 4.2% of all enrollees. Data in 2002 were used to identified patients’ medication history and comorbidities. Data during 2003–2012 were used to identify cases with newly diagnosed CKD. Data in 2013 were used for the subsequent follow-up. We extracted data regarding demographic characteristics, including encrypted identification numbers, gender, date of birth and death, and diagnostic information. The diagnostic data contained International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) as well as diagnostic and procedure codes. This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of the China Medical University (CMUH104-REC2–115 [CR-6]).The study cohort was composed of the patients with a diagnosis of CKD (ICD-9-CM codes 585 and 586) and having at least three outpatient visits or one inpatient visit within 1 year from 2003 to 2012. The cohort was divided into two groups: those using CHM because of CKD diagnosis  during the study period were allocated to the CHM group; those who did not receive any CHM treatment during the study period were allocated to the non-CHM group. The index date for the CHM group was the first date of taking CHM because of the CKD diagnosis, and the index date of the non-CHM group was randomly assigned a date between CKD diagnosis date and 31 December 2013. We excluded patients who were under 18 years of age, experienced a stroke (ischemic stroke or hemorrhage stroke) before the index date, had dialysis history before the index date (possible history of CKD), and had IS occurring during the first 6 months after CKD diagnosis date. Additionally, we conducted propensity score matching at a ratio of 1:1 cohort sample by age (per 5 years-groups), gender, index year, comorbidities and medication to balance the basic characteristics of two groups.The main outcome of the study was IS (ICD-9-CM codes 433–437). We followed patients until IS occurred, death, loss to follow-up or 31 December 2013. Death was considered as the competing event; loss to follow-up and the end of this study were considered as censored events.Baseline comorbidity was defined as comorbid disease with at least three outpatient visits or at least 1 inpatient visit before the index date within 1 year. According to the recommendation of the previous study, hypertension (ICD-9-CM codes 401–405), diabetes mellitus (ICD-9-CM code 250), hyperlipidemia (ICD-9-CM code 272), and atrial fibrillation (ICD-9-CM code 427.31) are thought to be the traditional risk factors for patients with CKD . In addip < 0.05 and the SAS 9.4  was used for the statistical analysis.Continuous variables are reported as mean and standard deviation, whereas categorical variables are reported as frequencies and percentage. We used standardized mean difference to compare balance of basic characteristics and potential confounders between the two groups, and a value of <0.1 indicates a negligible difference between the two groups. Considering the presence of competing risk, we used the proportional subdistribution hazards model of Fine and Gray to examine the effect of CHM on the risk of IS, reporting as hazard ratio (HR) and 95% confidence interval (CI). A multivariable model was performed by controlling for potential confounding variables, such as gender, age, comorbidities of hypertension, diabetes mellitus, hyperlipidemia, atrial fibrillation, COPD, chronic liver disease and cirrhosis, chronic artery disease, cancer, and the use of diabetes medications, antihypertensive medications, anti-lipid medications, steroid, NSAIDs, and antiplatelet or anticoagulation agents. We applied cumulative incidence function to assess the cumulative incidence rate of IS between the two groups. The Gray’s test  was usedWe identified 21,641 patients diagnosed with CKD during the years 2003–2012. According to the exclusion criteria, 11,816 patients were excluded. A total of 6,299 patients received CHM because of CKD diagnosis, and 3,529 patients received conventional treatment only. After matching with propensity score at a ratio of 1:1 cohort sample by age (per 5 years-groups), gender, index year, comorbidities and medication, there were 3,149 patients in either the CHM group or the non-CHM group. The sampling procedure is presented in p = 0.02). Additionally, the risk of IS was significantly lower in the CHM group than that in the non-CHM group . After multivariate adjustment, the adjusted HR (aHR) for the risk of IS among patients with CKD taking CHM as add-on therapy remained significantly lower . After stratification by gender, both male and female patients with CKD treated with CHM were significantly associated with a reduced risk of IS, and the risk of IS was much lower in female patients  than that in male patients . Furthermore, patients with CKD aged ≥65 years who received CHM had significantly lower IS risk compared with patients of the same age not receiving CHM . Regarding comorbidities, CKD patients with the traditional IS risk factors, such as hypertension, diabetes mellitus, and hyperlipidemia receiving CHM treatment were associated with a 45%–52% reduced risk of IS compared to those not treated with CHM . Moreover, in the stratification analysis of the medication, CKD patients receiving the CHM in combination with conventional medications showed a trend of reduced risk of IS compared with those receiving conventional medications only ], Rheum palmatum L  and Astragalus membranaceus Bunge  and the three most commonly prescribed formulae were Ji-Sheng-Shen-Qi-Wan, Liu-Wei-Di-Huang-Wan, and Zhen-Wu-Tang (The information about composition and processing were shown in The top three single herbs prescribed for CKD patients were p = 0.02).In the sensitivity analysis of extending the period of case exclusion from 6 to 12 months, there were 2,808 patients in both the CHM group and non-CHM group after matching with propensity score at a ratio of 1:1 cohort sample by age (per 5 years-groups), gender, index year, comorbidities and medication. The basic characteristics in each group were similar with all standardized mean difference values < 0.1 . The incThis nationwide cohort study using real-world data revealed that the use of CHM as add-on therapy was associated with a lower risk of IS among patients with CKD. In the subgroup analysis of different cumulative CHM usage days, we identified an association between longer CHM usage (>180 days) and a reduced risk of IS. To date, few studies have examined conventional medicine along with CHM as adjunctive therapy in relation to the risk of IS among patients with CKD. This study used a large-scale nationwide cohort to address this knowledge gap.Astragalus membranaceus Bunge  and Salvia miltiorrhiza Bunge , the most commonly prescribed single herbs, have also been reported to attenuate kidney injury via anti-inflammatory effects by suppressing the activation of NF-κB signaling pathways in the animal model ] has been shown to have effects of attenuating renal inflammatory injury through decreasing the level of certain proinflammatory cytokines, including prostaglandin E, tumor necrosis factor-α, and TGF-β1 ], was shown to alleviate indoxyl sulfate-induced kidney injury through suppressing oxidative stress response ] could significantly enhance the dialysis removal of protein-bound indoxyl sulfate ] has also been found to inhibit the elevation of indoxyl sulfate, which is thought to accelerate atherogenesis in patients with CKD ] and Astragalus membranaceus Bunge  have been shown to confer benefits in ameliorating these comorbidities -induced endothelial dysfunction. For examples, previous investigators  have demresponse . Salvian sulfate . In addiwith CKD . Furtherwith CKD . Certainbidities  thus conAbelmoschus manihot (L.) Medik  combined with losartan for 24 weeks showed a more reduction in 24-hour proteinuria than those taking losartan alone ], Rheum palmatum L  and Astragalus membranaceus Bunge  have the potential to enrich Qi and remove blood stasis to invigorate these patients. Furthermore, the clinical signs of kidney Yin deficiency in CKD patients are dizziness, tinnitus, and oliguria. The second most commonly prescribed formula, Liu-Wei-Di-Huang-Wan, could nourish kidney Yin to ameliorate the associated signs. Our findings provide evidence suggesting that those herbs may lower the IS risk in CKD patients, a benefit that is beyond the relief of clinical symptoms.The Yin-Yang theory of traditional Chinese medicine may explain why clinical doctors choose those herbs as CKD treatments. CKD is characterized by three syndromes based on the theory of Chinese medicine, syndrome of Yang deficincy and water overflowing, syndrome of Qi deficiency and blood stasis, and syndrome of kidney Yin deficincy . The cliThe strength of our study was using large-scale real-world data with longitudinal follow-up. Compared to randomized clinical trials, real world studies had broader generalizability and give evidence of effectiveness in daily practice settings . The stuIn this real-world, nationwide cohort study, the use of CHM as add-on therapy for patients with CKD was associated with a decreased risk of IS compared to using conventional treatment alone. In particular, CKD patients taking CHM for >180 days had a significantly lower risk of IS. Additionally, Ji-Sheng-Shen-Qi-Wan, Liu-Wei-Di-Huang-Wan, and Zhen-Wu-Tang might be associated with a 30%–50% reduced risk of IS. Although adjunctive CHM may have beneficial effects for IS prevention in patients with CKD, further randomized clinical trials are required to clarify the causal relationship of these results. Also, patients with other chronic inflammatory diseases such as rheumatoid arthritis  also hav