However, its roles in human liver fibrosis and the underlying mechanism in HSC activation are not yet defined. In our current study, the expression of Lnc-MALAT1 in the fibrotic liver tissues and in the plasma extracelllar vesicles (EVs) of liver fibrosis patients was detected by FISH and qRT-PCR. The results revealed that enhanced expression of Lnc-MALAT1 was co-localized with increased expression of the fibrotic markers (collagen I and α-SMA) and the Wnt/β-catenin signaling proteins  in the fibrotic liver tissues. The level of Lnc-MALAT1 in the plasma EVs isolated from 60 liver fibrosis patients was significantly increased compared with that of the 46 control patients, and area under receiver operating curve (AUROC) analysis showed that plasma EVs-Lnc-MALAT1 was a potential diagnostic marker for liver fibrosis, especially for high liver fibrosis. Plasma EVs with highly expressed Lnc-MALAT1 derived from high liver fibrosis patients up-regulated the expression of the fibrotic markers and enhanced the Wnt/β-catenin signaling in human hepatic stellate cells LX-2, and the fibrogenic effects in LX-2 were inhibited by Lnc-MALAT1 knock-down. Interestingly, TGF-β1, a potent pro-fibrotic cytokine, promoted the expression of Lnc-MALAT1 in LX-2 and its pro-fibrotic effects were also abolished by siRNA for Lnc-MALAT1, suggesting that Lnc-MALAT1 probably functions as a common mediator in the activation and fibrogenesis of HSCs. Our results indicate that enhanced expression of Lnc-MALAT1 in the fibrotic liver stimulate the activation of HSCs and thus promote their fibrogenic activity. These results also provide evidences that Lnc-MALAT1 is a potential therapeutic target and plasma EVs-Lnc-MALAT1 is a potential diagnostic biomarker for liver fibrosis.Evidence shows that the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Lnc-MALAT1) is associated with activation of hepatic stellate cells (HSCs) and liver fibrosis in animal and  Liver fibrosis is a long-term pathological process characterized by increased extracellular matrix (ECM) around the liver parenchyma . It oftevia different signaling pathways (The mechanism of hepatic fibrogenesis is complex and HSC activation is one of the most important contributors . HSCs bepathways .4- and bile duct ligation-induced liver fibrosis mouse models. Lnc-LFAR1 promoted HSC activation and liver fibrosis through TGF-β and Notch pathways. Other lncRNAs including nuclear paraspeckle assembly transcript-1 (NEAT-1), Hox A distal transcript (HOTTIP), metastasis-associated lung adenocarcinoma transcript 1 (Lnc-MALAT1), highly upregulated in liver cancer (HULC), small cajal body specific RNA 10 (SCARNA10), small nuclear RNA host gene 7 (SNHG7) and plasmocytoma variant translocation 1 (PVT1) have been found to be up-regulated in fibrotic liver tissues to induce HSC activation and enhance accumulation of ECM through various mechanisms , RNA transcripts longer than 200 nucleotides with regulatory roles in many biological processes, participate in activation and fibrogenesis of HSCs. chanisms . Among tchanisms .in vitro and in human fibrotic liver tissues.In our current study, EVs-Lnc-MALAT1 in the plasma of liver fibrosis patients was detected to evaluate whether it had diagnostic values for liver fibrosis. Simultaneously, the role of EVs-Lnc-MALAT1 in liver fibrosis and the associated fibrogenic mechanism were investigated in the human hepatic stellate cells LX-2 Plasma and tissue samples from 60 informed patients with liver fibrosis and 46 informed controls were collected from the First Affiliated Hospital of Anhui Medical University  between August 2019 and June 2021 and accordance with the declaration of Helsinki. Inclusion criteria: patients in normal group are hepatitis B virus infected, diagnosed to be normal by liver biopsy and do not received treatment, while patients in fibrosis group are hepatitis B virus infected, diagnosed to be liver fibrosis by liver biopsy and do not received treatment. Exclusion criteria: alcoholic liver, non-alcoholic fatty liver disease, autoimmune hepatitis, other types of hepatitis, cirrhotic decompensation, liver cancer and other systemic fibrotic diseases. Liver fibrosis tissues were obtained from patients with liver fibrosis who undertook biopsy and hepatectomy, while the control liver tissues were collected from patients with hepatic hemangiomas or hepatic cysts who received hepatectomy. Signed informed consent was obtained from all patients before the specimen collection. Characteristics of all the patients are shown in g for 30 min to remove cellular debris and then centrifuged at 4°C, 100,000 × g for 90 min. The EVs pellet was resuspended in 4 ml PBS and filtered with 0.22 μm filter  centrifuged at 4°C, 100,000 × g for 90 min. The EVs pellet was resuspended in 50 ul PBS and stored at −80°C for later use. For observation of EVs structure, the EVs solution was loaded on a carbon-coated copper grid, washed and negatively stained with uranyl acetate, and then observed under Transmission Electron Microscopy . For analysis of the components of the isolated EVs, TSG101 and CD81 were detected by western blotting. For analysis of the size distribution, the EVs solution was diluted 1:20 in PBS and analysized using particle size analysis System . The protein concentration of the EVs was detected using Bicinchoninic Acid Assay .In total samples, 4 ml of plasma were centrifuged at 3000 × The immortalized human hepatic stellate cell line LX-2 was provided by Dr. Scott Friedman  and was detected to be free of mycoplasma. LX-2 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 U/mL streptomycin, and maintained at 37°C in a humidified environment containing 5% CO2. For EVs related experiments, Exosome-Free FBS  was used to exclude the EVs contamination from fetal bovine. The culture medium was changed every 1–2 days.6 LX-2 cells on a 6-well plate were transfected with 0.8ug/ml of Lnc-MALAT1-specific small interference RNA  or irrelevant small RNA  by lipo3000  for 4 h. After replacement of the culture medium with fresh medium, the cells were treated with 100 ug plasma EVs from liver fibrosis patients or from the control subjects and continued to culture for another 44 h. The cells were collected for quantitative real-time-PCR (qRT-PCR) and western blot analysis. Treatment with TGF-β1 (10 ng/ml in PBS) was used as a positive control and with equivalent PBS as a negative control.For assessment of the effect of EVs isolated from the plasma of the liver fibrosis patients on LX-2 fibrogenesis, 1 × 106 LX-2 cells on a 6-well plate were transfected with 0.8ug/ml of siRNA- Lnc-MALAT1) or siRNA-NC by lipo3000  for 4 h. After replacement of the culture medium with fresh medium, the cells were treated with PBS or 10ng/ml TGF-β1 for another 44 h. The cells were collected for qRT-PCR and western blot analyses.For assessment of the effect of endogenous Lnc-MALAT1 on TGF-β1-induced fibrogenesis, 1 × 10g for 30 min. The supernatants were collected and the protein concentration was determined by BCA protein assay kit. Total 20 mg proteins in the samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes . After blocking in 5% skim milk, the membranes were washed with Tween 20-buffered saline (TBS) and incubated overnight with the specific primary antibodies at 4°C. Rabbit polyclonal antibodies against α-SMA , Collagen I , β-catenin , β-actin , c-myc , Cyclin-D1 , TSG101 , CD81 , and Calnexin  were used, respectively. The membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing with TBS, protein bands were visualized with an ECL chemiluminescence kit  by a chemiluminescent western blotting detection system . The intensity of the bands was analyzed with Image J software.Tissues, cells and EVs were lysed with RIPA buffer  and centrifuged at 4°C, 12,000 × Total RNA was isolated in cell, tissue and EVs samples using Trizol reagent  according to the manufacturer’s instructions. Extracted total RNA was resuspended with RNase-free water and the concentration was measured and using Nano Drop 2,000 . In total RNAs, 500 ng was reverse transcribed and then quantitively analyzed using RT reagent Kit and SYBR-Green Master Mix . Relative primer sequences are listed in in situ hybridization (FISH) was performed using a probe specifically targeting Lnc-MALAT1  according to the manufacturer’s instructions. Briefly, paraffin sections were dewaxed with xylene and proteins were removed by proteinase K. After heat denaturation, the sections were incubated overnight at 37°C with the probe  target Lnc-MALAT1, then subjected to DAPI staining for the nucleus and finally observed under a fluorescent microscope .Fluorescence s = 3–4) and low fibrosis scores group (s = 1–2) by two pathologists independently according to the Ishak scoring (kappa = 0.8453) staining and Masson staining were performed according to a standard procedure and used to evaluate the changes in liver pathology and collagen deposition, respectively. Liver fibrosis patients were divided into high fibrosis score group (= 0.8453).For immunohistochemistry (IHC), the slides were treated with 0.3% hydrogen peroxide, blocked in 2% bovine serum albumin. Primary antibodies against α-SMA , Collagen I , β-catenin , c-myc  and Cyclin-D1  were used for incubation overnight at 4°C. The sections were then incubated with biotinylated secondary antibody at room temperature for 1 h. All the slides were observed under a light microscope  and assessed by using the software of Image-pro plus 6.0.t-test was used to compare differences between groups. When the data were not normally distributed, we used the Mann-Whitney U-test for data analysis. Associations between gender were analyzed using 2-tailed χ2 testing. P-values less than 0.05 were considered to be significantly different. All statistical analyses were performed with Graph pad Prism 8.0.Area under curve (AUC) and 95% confidence interval were used to assess the diagnostic efficiency of plasma EVs-Lnc-MALAT1 for detecting liver fibrosis. The Youden indices were used to confirm the optimal cutoff values and their best sensitivity and specificity combination in the training set. The level of Lnc-MALAT1 in EVs were expressed as medians with interquartile ranges, and the remaining data are expressed as mean ± SD (standard deviation). The student’s The liver tissues from 60 liver fibrosis patients, together with 46 patients with hepatic hemangiomas or hepatic cysts as the control liver tissues, were first subjected for HE staining and IHC analysis. Obvious fibrotic liver tissues were observed in the liver fibrosis patients . Also, eFor observation of Lnc-MALAT1 expression in the fibrotic liver tissues, the consecutive sections of the liver tissues were used for FISH and Masson staining. Lnc-MALAT1 was found to be enriched in the liver tissues of the liver fibrosis patients compared to in the liver tissues of the control patients, and mostly located within and surrounding the fibrotic lesions . QuantitTo find out whether Lnc-MALAT1 exists in EVs, we first isolated the EVs from the plasma samples of the patients and then determined Lnc-MALAT1 by qRT-PCR. The level of lnc-MALAT1 is 0.8589 (± 2.3145) in fibrosis group and 0.0887 (± 0.1299). Characterization of the isolated EVs, including TEM observation of shape and structure, western blotting identification of EVs-components, and Nanoparticle tracking Analysis (NTA) size distribution, was presented in p-values and critical values were shown in For analysis of correlation of the plasma EVs-Lnc-MALAT1 with liver fibrogenesis, the 60 liver fibrosis patients were further divided into a high fibrosis group  and a low fibrosis group  according to the pathological Ishak scoring. Detection by qRT-PCR showed that relative expression of plasma EVs-Lnc-MALAT1 was 0.089, 0.208, and 2.068 in the control, the low fibrosis and the high fibrosis patients, respectively, showing that relative expression of plasma EVs-Lnc-MALAT1 was correlated with liver fibrogenesis . TherefoDue to its etiological diversity, liver fibrogenesis has been reported to involve several signaling pathways . In the Activation of HSCs is an important contributor in liver fibrosis . For undFor elucidation of the role of EVs-Lnc-MALAT1 in liver fibrosis, LX-2 cells were incubated with the EVs isolated from the plasma of patients in the high fibrosis group or from the control plasma, with TGF-β1 as a positive control. Similar to TGF-β1 Lane 2, , the plavia up-regulation of endogenous Lnc-MALAT1 expression.The results describe above indicated that TGF-β1 induced the activation and fibrogenesis and unexpectedly, up-regulation of Lnc-MALAT1 expression in LX-2 cells. For further understanding whether the TGF-β1-induced activation and fibrogenesis depend on increased Lnc-MALAT1 expressions, we first transfected the si-RNA specific for Lnc-MALAT1 (si-Lnc-MALAT1) into LX-2 cells and then observed the fibrogenic effects of treatment with TGF-β1. Similar to the results of Liver fibrosis is a continuous wound-healing process, triggered and sustained by pro-fibrotic cytokines such as TGF-β1 and associated with increasing in Collagen I and α-SMA expression . HSCs arin vitro studies  as well as the Wnt/β-catenin signaling proteins  in the fibrotic liver tissues. Similar to TGF-β1, EVs with highly expressed Lnc-MALAT1 that were derived from the plasma of liver fibrosis patients up-regulated the expression of the fibrotic markers and enhanced the Wnt/β-catenin signaling in HSCs, and blocking of Lnc-MALAT1 expression by Lnc-MALAT1-specific siRNA abolished these effects in both the EVs-treated and TGF-β1-treated HSCs. As TGF-β1 stimulated the expression of Lnc-MALAT1 in HSCs, Lnc-MALAT1 probably contributes to the activation of HSCs. These results suggested that Lnc-MALAT1 is a potential therapeutic target for liver fibrosis. Another finding in our present study is that plasma EVs-Lnc-MALAT1 may be used as a diagnostic biomarker for liver fibrosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Faculty of Science, First Affiliated Hospital of Anhui Medical University. The patients/participants provided their written informed consent to participate in this study.ChaZ and ChoZ: study design. TW: experiment conduction. BZ: collection of clinical samples and data. SW and WY: statistical analysis. TW and XM: draft. JX and SZ: manuscript revision. All authors have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Social networks on the Internet have seen an enormous growth recently and play a crucial role in different aspects of today’s life. They have facilitated information dissemination in ways that have been beneficial for their users but they are often used strategically in order to spread information that only serves the objectives of particular users. These properties have inspired a revision of classical opinion formation models from sociology using game-theoretic notions and tools. We follow the same modeling approach, focusing on scenarios where the opinion expressed by each user is a compromise between her internal belief and the opinions of a small number of neighbors among her social acquaintances. We formulate simple games that capture this behavior and quantify the inefficiency of equilibria using the well-known notion of the price of anarchy. Our results indicate that compromise comes at a cost that strongly depends on the neighborhood size. Opinion formation has been the subject of much research in sociology, economics, physics, and epidemiology. Due to the widespread adoption of the Internet and the subsequent blossoming of social networks, it has recently attracted the interest of researchers in computer science at large .strategy that minimizes an implicit cost for the individual.An influential model that captures the adoption of opinions in a social context has been proposed by Friedkin and Johnsen . Accordineighbors. So, in their model, opinion formation co-evolves with the neighborhood for each individual: her neighborhood consists of those who have opinions similar to her belief. Then, the opinion expressed is assumed to minimize the same cost function used by Bindel et al.  such that r(i) and Case I:i (while player r(i) does so by definition), i belongs to the neighborhood of player Case I.1:ualities  and 15)Case I.1:ualities  yields \mentclasspt{minimaCase I.2:≥sℓ∗(i),  yields \mentclasspt{minimamentclasspt{minimamentclass2pt{minimCase II:i belongs to the neighborhood of player Case II.1:equality , equatioequality  becomes equality  yields \Case II.2:i does not belong to the neighborhood of player equation  becomes -zi2 and  yields \So, we have shown that in the pure Nash equilibrium i, we have that Recall that, for player equality  and the i) that minimizes the distance For the case of 1-COF games we can prove an even stronger statement following a similar proof roadmap as in the previous section, but using simpler (and shorter) arguments. We denote by Consider a 1-COF game with belief vector i with kangaroo player and associate the quantity covered by player i and define i and define We begin by classifying the players into groups and, subsequently, we show how the costs of different groups can be combined so that the lemma holds. We call a player L of large players such that, for any S that contains the remaining players who we call small. See also Fig. Let i.We proceed to prove five useful properties Claims –13.5; reLet i. Then, We assume that Let i is a small player. Hence, i would be covered. Let j be the player with the leftmost belief that is covered by player We assume that Let i is a small player. Hence, i would be covered. Then,i is small, we have We assume that N(S) denote the set of players j that are neighbors of players in S  does not contain small players.S. Without loss of generality i is covered nor Assume otherwise that for some player For every two players i or i or j. Now the fact that i and Assume otherwise and let S  is the neighbor of player entclass1pt{minimaCase IV:i.This completes the proof. In particular, we will show that k-COF games and we show lower bounds for pure and mixed Nash equilibria  opinion vector L and the R constitute local neighborhoods, that is, First, observe that the players in sets i consists of the remaining M as well as players r. The expected cost of player i in r is at distance y, her expected cost does not decrease. Clearly, if i is at least i when deviating to y isa and b.Next, let us focus on a player r; the case of player M always belong to the neighborhood r and it remains to argue about the identity of the last player in R belongs to r is r contribute r cannot decrease her expected cost by deviating to another opinion y. The expected cost of player r when deviating to y isa and b. Hence, we conclude that Now, let us examine player As in the proof of Theorem We have introduced the class of compromising opinion formation games by enriching coevolutionary opinion games with a cost function that urges players to “meet halfway”. Our findings indicate that the quality of their equilibria deteriorates as the neighborhood size increases; in particular, the price of anarchy and the price of stability are k-COF games for Another natural question is about the complexity of pure Nash equilibria in Finally, our modeling assumption that the number of neighbors is the same for all players is rather restrictive. Extending our results to the general case of different neighborhood size per player deserves investigation. One possible such generalization is to combine our approach to the Hegselmann-Krause model, i.e., each player’s neighborhood consists solely of those players with opinions sufficiently close to her belief.


Physical rehabilitation (PR) is recommended following spinal cord injury to help improve and maintain muscle elasticity, joint mobility, and nerve health. It can also be used to relieve pain and improve cardiopulmonary fitness in an immobile patient. There is evidence, in human medicine, that PR plays a critical role in mental health and the psychological state of the patient. As part of the assessment phase, human physical therapists often identify psychosocial symptoms and barriers at the start of PR that ultimately may affect improvement in human patients suffering from injury and the loss of mobility. Patient psychological state plays an integral role in healing and outcome during treatment and rehabilitation. Specific interventions set to address these symptoms can better outcome. Arguably, one of the most emotionally traumatizing injuries suffered by a canine patient can be immobility secondary to spinal cord injury. Poorly understood is the role the canine cognitive state plays in the success of rehabilitation following spinal cord injury. Should breed, age, sex, physical fitness, personality, previous experiences and history or home lifestyle, affect the PR assessment of these patients? Do these factors affect eventual outcome following PR? The purpose of this manuscript is to explore psychosocial barriers encountered during injury rehabilitation in humans and determine if the similar barriers need to be considered when assessing a canine patient for spinal cord injury rehabilitation. Physical rehabilitation (PR) is gaining more and more popularity in veterinary medicine. In the past 20 years, the number of clinics in the United States of America actively offering and performing PR continues to grow. In 2014, there were over 110 facilities in the United States offering physical rehabilitation services to their veterinary clients –8. The fIt is well-recognized in human medicine that treatment of injury is influenced by emotional, behavioral and situational factors –11. ThisThese biopsychosocial perspectives can act like barriers to healing and rehabilitation success. In 2010, a systematic review was published in the Journal of Manual Therapy, the purpose of which was to discuss barriers to physical therapy in patients undergoing musculoskeletal rehabilitation . The autIt is interesting and important to consider these biopsychosocial perspectives as they relate to our canine physical rehabilitation patients. When considering spinal cord injury specifically, the biological perspective in the canine patient is well-established. It is common knowledge that the presence or absence of limb deep pain sensation or incontinence following spinal cord injury are biological aspects of disease that directly affect both short and long term outcome and quality of life. Also of importance are previous injuries or medical co-morbidities that one patient may have that directly affects his or her ability to be treated. A paraplegic Corgi, who, for years has been clinical for elbow osteoarthritis, will have a different rehabilitation assessment and different rehabilitation plan than a dog who has no orthopedic disease pre-spinal cord injury, for example. As this patient rehabilitates and is forced to bear an increased percentage of weight on his thoracic limbs, his already diseased elbows may suffer as will his therapy success.Equally important to assessment and outcome should be the psychological state of the canine patient. Canine patients with spinal cord injury many times may require prolonged hospitalization during recovery from surgery and/or initial injury. They suffer a loss of independence as they may require assistance to walk and even urinate. This change in lifestyle results in a need to adapt; this acute loss of physical function causes stress , 18. Sinn = 8) compared with CKCSs without the condition (n = 13). Dogs with the condition had more a negative affective state  assessed by one of the cognitive tests  that was not correlated with clinical signs . Although more research into the behavioral and cognitive state in dogs with medical conditions is necessary, these previous studies suggest that the effect of disease  may directly alter behavior and cognition ultimately affecting treatment and prognosis. It is critical that the rehabilitation professional consider the patient's personality, behavior, cognitive state, and level of motivation when assessing the patient for PR as it directly affects outcome and expectation.Recently behavior and cognitive states in dogs with medical conditions has gained attention. It is reported that dogs with idiopathic epilepsy tend to have behavioral changes before and after the development of epilepsy , 21. In The social impact of injury is perhaps the hardest aspect of the biopsychosocial perspective to parlay over to the canine patient. That being said, dogs too, are social beings within a family unit. Though dogs do not have societal pressures weighing down on them, they do have a role within the household. Structured routine and predictable schedules help to reduce dog stress. In a household in which the dog holds a position equitable to the human members of the family it may be easier to obtain owner participation and PR buy-in than in a household in which the dog is simply an outdoor lawn ornament. As such, the role dogs play in the social environment within their home must be considered when assessing the patient for PR and predicting rehabilitation outcome and success.Because most dogs are very valued members of the family unit, attention should be paid to caregiver burden. The definition of caregiver burden in human medicine can apply to veterinary medicine; it is defined as the strain associated with caregiving. This strain comes in two varieties: subjective  response plus objective  demands of caregiving . For manIn an attempt to consider objectively these biopsychosocial barriers as they pertain to assessment and success in human physical therapy following injury, a flag system was created . Flags aThough it is clear that some of these “flags” cannot directly be applied to veterinary medicine, the idea that patient behavior and cognitive state are integral to the PR assessment, outcome and PR success traverses species. A “flag” system for canines suffering spinal cord injury could be adapted to assess objectively the injured canine patient  at the iThe use of a flag system would help give the rehabilitation professional a more objective way to assess his or her patients for PR. It would allow the professional a way to objectively incorporate the biological, but also behavioral, cognitive and social aspects of injury into his or her rehabilitation assessment. It would also force the rehabilitation professional to consider the cognitive state, behavior and also social barriers when formulating a rehab assessment and plan.Considering the biopsychosocial perspective has merit when assessing any canine patient for physical rehabilitation. These perspectives may even be of use when creating an in hospital and/or at home physical rehabilitation plan, and ultimately, can be used to predict overall patient outcome and success following injury and subsequent rehabilitation. As future studies exploring the use of physical rehabilitation in the veterinary spinal cord injury patient are designed and conducted, differences in the canine patient biopsychosocial perspective should be considered.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fomitiporia mediterranea (63–94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (−90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions  were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was  Plasmopara parasitica; powdery mildew, Erysiphe necator; gray mold, Botrytis cinerea), there is currently no efficient method against Grapevine Trunk Diseases (GTDs), a threat for viticulture worldwide ever since the ban of sodium arsenite in the 2000s [Phaeomoniella chlamydospora, Phaeoacremonium minimum, Eutypa lata, Fomitiporia mediterranea, and Botryosphaeriaceae species  [P. chlamydospora, for instance, is frequently isolated from black punctuations and sectorial necrotic tissues, whereas F. mediterranea is mainly isolated from sectorial and central white-rot necrotic tissues [Although various fungicides or biocontrol agents control the grapevine foliar diseases  ,7,8. The parvum) . P. chla tissues ,11.Phomopsis spp., responsible for cane and leaf spot diseases [P. chlamydospora and F. mediterranea in trunks from esca-diseased plants [F. mediterranea and Botryosphaeria spp., isolated from esca-diseased grapevine trunks [Arsenic compounds were used in the 18th century as insecticides to protect plants against pests ,13. In vdiseases ,16,17,18diseases ,22,23,24d plants . In addie trunks . These oe trunks  and subsTo improve our understanding of sodium arsenite curative effect on grapevine trunk diseases, we analyzed its impact on fungal and bacterial wood microbiota of grapevines displaying esca-foliar symptoms from three French vineyards . We also assayed the sensitivity of different fungal species isolates from grapevine wood tissues to sodium arsenite. Overall, this study highlights the strong impact of sodium arsenite on the fungal community colonizing woody tissues of grapevine trunks with esca-disease symptomsThe sampling sites were located in three French vineyards, each cultivated with a specific cultivar ‘Gewürztraminer’ with rootstock 33EM, in Alsace ; ‘Chardonnay’ with rootstock SO4 or Fercal, in Champagne , and ‘Merlot’ with rootstock SO4, in Languedoc . These established vineyards, aged 25 for Alsace, 27 for Champagne, and 40 for Languedoc, were examined for esca-foliar symptoms in both 2013 and 2014. In 2013 and 2014, before sodium arsenite treatment in 2014 and 2015, 10 plants expressing esca-foliar symptoms were selected at random in each vineyard. Five of the 10 selected plants were treated with sodium arsenite  at 1250 g/hL until streaming, at the end of winter after pruning, and before bud bursting, BBCH 00 [The DNA extraction protocol used here was similar to that of Bruez et al. ,28. SSCPhttps://submit.ncbi.nlm.nih.gov/subs/sra/SUB8307285/metadata) (accessed on 1 April 2021). In brief, sequences were selected in accordance with the following initial criteria: (i) ≥450 nucleotides in length; (ii) a perfect match to the primers and barcodes; with (iii) no ambiguous base allowed. Although the bacterial primers were designed to amplify bacterial DNA, we verified, using Metaxa software, that no sequences corresponding to chloroplast or mitochondria DNAs were present in the bacterial dataset [https://www.mycobank.org) (accessed on 1 April 2021) and UNITE database [https://www.ncbi.nlm.nih.gov/sra/PRJNA732838) (accessed on 1 June 2021).Fungal and bacterial data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (version 1.7.0)   indexes with the EstimateS software package . To compPhaeomoniella chlamydospora, Fomitiporia mediterranea, and Eutypa lata or (ii) saprobes such as Trichoderma spp. and Penicillium spp. [Sodium arsenite  was suspended in sterile water and added to molten malt-agar medium poured into Petri dishes  at a concentration ranging from 1 to 500 mg/L, with MA as control. Each plate was inoculated with a 6 mm diameter mycelial plug from the actively growing margins of each tested fungal strain grown on MA. The culture was left at room temperature in the dark for 7–14 days, depending on the growth rate of the fungus tested. When the margin of the colony growing on MA without sodium arsenite (control) approximately was at 1 or 2 cm from the edge of the Petri dish, mycelial growth was recorded by measuring colony diameter. The growth tests in this study were performed in 2014 on fungi isolated from the trunks of treated and untreated grapevines . The funium spp. ,39. All The vineyards studied were located in Alsace, Champagne, and Languedoc. The grapevine cultivars sampled in each region were ’Gewürztraminer’, ’Chardonnay’, and ’Merlot’, respectively. In 2014 and 2015, 10 plants expressing esca-foliar symptoms were selected at random in each vineyard. Half of the plants were treated with sodium arsenite at the end of winter. All 10 plants, treated or not, were uprooted in September 2014 and 2015, respectively. All untreated plants displayed both esca-foliar symptoms, and central white-rot in woody tissues WR, . WR tissp = 0.0492; p = 0.007; fungal p = 0.049, p = 0.049; fungal index p = 0.049, p = 0.502; bacterial communities p = 0.328; p = 0.062). A comparison of the fungal microbiota between treated and untreated grapevines indicated major differences (p = 0.001). When focus was placed on the different types of tissue , further differences became apparent (p = 0.001). Bacterial communities showed differences for all three regions (p = 0.029). These differences did not depend on the simple distinction between treated and untreated grapevine (p = 0.203), but on the types of tissue involved (p = 0.001).Thirty-six DNA samples  in all wp = 0.492; p = 0.001; p = 0.001) in fungal communities when different types of woody tissue from untreated grapevines were studied. The SSCP fingerprint data of microbiota from white-rot necrosis and sectorial necrosis tissues strongly differed from those of non-necrotic tissues according to the PCA axis 2  and phylogenetic analyses . Most OTUs could be assigned to a single fungal species without ambiguity including all GTD associated fungi detected in these samples . Five OTF. mediterranea was the most abundant fungal species in Alsace and Champagne grapevines , an unknown Agaricales genus (0.3–14%), Cladosporium spp. (0.1–11.4%), and Mucor circinelloides (0–9.8%). White-rot necrotic woody tissues of untreated Alsace and Champagne grapevines were almost exclusively colonized by F. mediterranea . In all these tissues, F. mediterranea was associated with P. chlamydospora as the second most abundant species . These two fungi dominated the necrotic tissues  or tissue (p = 0.218). An additional statistical analysis of each species highlighted significant differences among tissues in the relative abundances of Bradyrhizobium spp., Curtobacterium spp., Erwinia spp., Pantoea spp., Pseudomonas spp., Sphingomonas spp., and Yersina spp. . Differences in relative abundance were plotted for each species/tissue combination by region . The second fungal species displaying a significant reduction in abundance upon sodium arsenite treatment was S. vitis , and by 800-fold the abundance of Aureobasidium pullulans in white-rot necrotic tissues of Champagne grapevines . The taxa compositions of these communities were characterized by using metabarcoding, revealing 718 fungal and 3886 bacterial OTUs. Significant differences in fungal microbiota were observed between treated and untreated grapevines, independently of their region or tissue origin. Sodium arsenite, however, had no significant impact on bacterial microbiota.F. mediterranea, a GTD fungus, was the most abundant species colonizing all trunk woody tissues of diseased grapevines from two of the three vineyards studied  compared to healthy ones, reaching up to 35% of all woody tissues. It should be noted that as this study used drilling for wood sampling, it was not possible to split different types of tissue. Another recent study of young French Cabernet Sauvignon grapevines (Bordeaux vineyard) showed that esca was always associated with the presence of white-rot necrotic tissues in cordons [F. mediterranea (>90%), as observed in the Alsace and Champagne grapevine trunks  and vineyard . Analysis showed that reported ,41. A re cordons . These we trunks . Our res 38-55%, . Still, nes 23%, . This fuapevines ,45,47. Tapevines . HoweverP. chlamydospora was detected in high abundance (>15%), specifically in borders between white-rot necrotic tissues and non-necrotic tissues in all three vineyards. In these borders, P. chlamydospora was the only other fungal species associated with F. mediterranea. No other GTD fungi exceeded 5% abundance in any woody tissues, except for D. seriata. This fungus, D. seriata, was detected in non-necrotic tissues of Alsace and Languedoc grapevines at 5.2% and 7%, respectively, and in sectorial necrosis of Languedoc grapevines up to 18%  than in other tissues  [F. mediterranea was mostly located. We also tested whether in vitro sensitivity to sodium arsenite differed among fungi. F. mediterranea was the most sensitive species among those tested . Sodium arsenite, however, did not affect the bacterial microbiota, either non-pathogenic or the pathogenic including bacteria that could be involved in wood degradation. Based on our results, it would be interesting to develop products as efficient as sodium arsenite on F. mediterranea. One possible way is to design fungicides mimicking sodium arsenite effects on wood microbiota. This goal should include a good delivery of the fungicide to woody tissues including white-rot and a high toxicity toward F. mediterranea.Sodium arsenite was efficiently used to protect and cure grapevines from GTDs before its ban. The present metabarcoding study showed sodium arsenite induced major changes in the fungal microbiota colonizing grapevine woody tissues. Its major effect was to decrease massively the abundance of


ObjectiveIn this retrospective study, we aimed to investigate the impact of the coronavirus disease 2019 (COVID-19) pandemic on rehabilitation services including outpatient rehabilitation clinic visits, speech therapy, and occupational therapy and physiotherapy sessions.Materials and methodsThis was a retrospective, observational, single-center study, which included all patients who presented to the rehabilitation clinic at the King Fahad Specialist Hospital, Dammam (KFSHD) from January to August 2020 (study period), and they were matched with parallel groups from 2017 to 2019 (control period).ResultsA total number of 1,435 patient visits were recorded in the outpatient rehabilitation clinics during the study period as compared to the 1,496 patient visits during the control period. However, the number of patients seen by the speech therapists, physiotherapists, and occupational therapists significantly came down from 482, 963, and 171, respectively in the control period to 77, 218, and 130, respectively in the study period. The occupational therapy, physiotherapy, and speech therapy services were significantly affected during the COVID-19 pandemic.ConclusionsOur findings revealed the negative consequences of the COVID-19 pandemic on the outpatient rehabilitation services, which was reflected in the sharp decline in the number of patients who attended the rehabilitation clinics as well as in the reduced number of sessions. The rehabilitation facilities should be better prepared for such pandemics in the future to deliver appropriate and timely rehabilitation programs with a patient-centered approach. Rehabilitation is an essential component in healthcare and medical management, and it helps in enhancing functional capacity, promoting health and wellness, preventing disease, and achieving the best possible outcomes overall ,2. RehabThe world is currently experiencing a major healthcare crisis in the form of the ongoing coronavirus disease 2019 (COVID-19) pandemic, and it has heavily affected global healthcare; similarly, it has affected the rehabilitation of people with functional limitations ,7. The WThis was a retrospective observational study that included outpatients who attended rehabilitation clinics between 2017-2020 at the King Fahad Specialist Hospital, Dammam (KFSHD).Data sourceData were retrieved from the central computerized database [MedicaPlus ] of the sole tertiary care center at KFSHD in the Eastern Region of KSA. The data were reviewed and reported by rehabilitation consultants (AN and IA) and analyzed by another co-author (SB). The center provides inpatient and outpatient rehabilitation services. However, data were also included from outpatient clinics of rehabilitation physicians, speech therapists, and inpatient services of physiotherapists and occupational therapists. The study period was between January and August 2020, and the data were compared with that of the control period from January to August, from 2017 to 2019. All adult and pediatric patients who attended outpatient rehabilitation clinics were included in the study, while inpatient admissions were excluded.Data analysisThe SPSS Statistics version 22.0  was used for data analysis. Continuous variables were assessed for normality and were described using mean and standard deviation, and median with interquartile range (IQR), as appropriate. An independent sample t-test was used to compare the means of normally distributed data.The number of patients who visited the rehabilitation clinic is shown in Figure However, a significantly lower proportion of patients received occupational therapy during the study period in 2020 than during the control periods in 2017, 2018, and 2019  and the same period previously .This study aimed to investigate the impact of the COVID-19 pandemic on the provision of essential services involving multiple rehabilitation disciplines including speech therapy, occupational therapy, and physiotherapy.The findings of this study showed a marked decline in the number of patient visits and sessions provided at the outpatient rehabilitation clinics at KFSHD due to the sudden ending of treatment and changed working circumstances during the lockdown from January to August 2020. As a result of the abrupt closing down of all non-critical and non-urgent medical services in hospitals treating COVID-19 patients, the rehabilitation services in multiple disciplines were significantly affected. Outpatient visits attended by physicians were at their lowest in May 2020 and the same was the case with inpatient services. Similarly, physiotherapists and occupational therapists attended the least number of in-hospital patients visits during the month of April 2020. A 77% drop in the number of patients treated in the physiotherapy clinics was observed from January to August in 2020 when directly compared to the same period in 2019 (pre-COVID-19 period).With regard to the lockdown and reorganization of medical services that altered the accessibility to outpatient rehabilitation services, many reports from across the world have described the cessation or significant decline in outpatient services ,16,17,18It is well documented that COVID-19 increases rehabilitation needs not only for infected patients with novel coronavirus but also for those who suffer from long-term sequelae of their diseases or disabilities . The COVWe have clearly described the negative consequences of the COVID-19 pandemic on outpatient rehabilitation services in our study. Due to the pandemic, there was a sharp decline not only in the number of patients but also in the outpatient rehabilitation services such as physical and occupational therapy sessions. One of the limitations of our study was not taking into account the profiles of the patients, which would provide a better assessment. In addition, we took into account the number of patients and the number of outpatient rehabilitation services only, while other important parameters were not considered in this study. Further studies are warranted to analyze the impact of the COVID-19 pandemic on the functional status of patients as well as the ways in which delayed or obstructed rehabilitation services affect the patients' prognosis. Studies on the importance of timely rehabilitation are required to assess the consequences of such delays in the time of any pandemic. The major concern is that COVID-19 or any other similar pandemic in the future would obstruct rehabilitation patients’ healthcare services due to many factors. With a steeper rise in the number of patients with disabling conditions, the rehabilitation facilities may in the future confront the take-back effect, and the question is how we can be better prepared for such pandemics in the future and deliver appropriate and timely rehabilitation programs with a patient-oriented, tailored, and individualized type of rehabilitation design. Further large-scale and multiple-center studies are needed to understand the role of other factors including lifestyle and the scope of online rehab programs (telerehabilitation). Future emphasis might be placed on organizing virtual clinics with video facilities to reduce outpatient visits.


Augmented reality (AR) and head-mounted displays (HMD) in medical practice are current research topics. A commonly proposed use case of AR-HMDs is to display data in image-guided interventions. Although technical feasibility has been thoroughly shown, effects of AR-HMDs on interventions are not yet well researched, hampering clinical applicability. Therefore, the goal of this study is to better understand the benefits and limitations of this technology in ultrasound-guided interventions.Hololens) which overlays live ultrasound images spatially correctly at the location of the ultrasound transducer. We chose ultrasound-guided needle placements as a representative task for image-guided interventions. To examine the effects of the AR-HMD, we used mixed methods and conducted two studies in a lab setting: (1) In a randomized crossover study, we asked participants to place needles into a training model and evaluated task duration and accuracy with the AR-HMD as compared to the standard procedure without visual overlay and (2) in a qualitative study, we analyzed the user experience with AR-HMD using think-aloud protocols during ultrasound examinations and semi-structured interviews after the task.We used an AR-HMD system (based on the first-generation Microsoft n = 20) placed needles more accurately  but not significantly faster  with the AR-HMD. All participants in the qualitative study (n = 6) reported limitations of and unfamiliarity with the AR-HMD, yet all but one also clearly noted benefits and/or that they would like to test the technology in practice.Participants  contains supplementary material, which is available to authorized users. Augmented reality head-mounted displays (AR-HMDs) have been envisioned as tools for visualizing image data in medical interventions since the mid-1990s . Since tIntended benefits of AR-HMDs are, for example, reduced workload, fewer complications or shorter interventions .Despite the small sample size, there were strong contrasts between participants, particularly between participant 2  and the other was only mildly positive (P6). A similar difference was mentioned by P6, an ultrasound tutor, which they observed throughout the ultrasound courses they held: Some students found it difficult to look at and interpret the ultrasound image while simultaneously orienting the transducer (instead of looking at the transducer), whereas other do not. Their half-joking explanation for this observation was that those who struggle likely did not play video games when they were younger.Do relevant benefits exist and if so, on which mechanisms are they based? The AR-HMD provided quantifiable as well as subjective benefits; we could not verify all predicted benefits, though. Through qualitative data analysis, we were able to outline that improvements to spatial perception and understanding may be important intermediate mechanisms and be subject to individual differences. Even though the qualitative study design does not allow for causal inference, these insights may be foundations for hypotheses to be examined in future studies.Our results provide partial, though still incomplete, answers to the research question: p = 0.022). While we see this as further evidence for benefits of AR-HMDs, our results are not entirely conclusive: Task duration and perceived task load were both not significantly reduced. These inconclusive results may be due to unfamiliarity with the new technology, which has played an unexpectedly large role in the qualitative study. Unfamiliarity could lead to a higher task load  and slower execution time—and possibly explain why task load and duration were not significantly reduced. The influence of unfamiliarity on the needle placement experiment is also supported  by remarks of participants, recorded in field notes. Additionally, the strong effect of the order of conditions  possibly obscured the effect we hypothesized. But despite these mixed results, we argue that an underlying effect exists: In addition to improved accuracy, exploratory data analysis shows moderate correlations between differences in task load and differences in accuracy and task duration; participants who found the task easier with the AR-HMD also tended to perform better.Participants were significantly more accurate when using the AR-HMD, reducing the mean error by a third .Our data point to multiple potential causes for such benefits. Four out of six participants  mentioned an improvement of their spatial understanding of the ultrasound images. They stated that they could better locate the image in the patient anatomy and/or three-dimensionally imagine the anatomical structures they examined. Additionally, participants often mentioned use cases that require precise spatial navigation which implies benefits in such scenarios. Accordingly, we suggest Several participants stated they would rather use the AR-HMD in complex scenarios than for routine cases. On one hand, this could mean that they perceived the technology as a burden that should be used sparingly. On the other hand, they might feel that AR-HMDs augment their existing skills—which would only be relevant in cases they cannot handle proficiently already. If that is the case, AR-HMDs could help close the skill gap between novice and experienced surgeons or guide procedures that are only performed infrequently at a clinic. Accordingly, we suggest examining the relationship between the difficulty of a case and measurable benefits of the technology in future studies.Hedonistic aspects were frequently mentioned in the qualitative study; that is, the technology was perceived as novel and/or exciting. Though it may temporarily improve user experience, we doubt that novelty offers major clinical benefits. It may even skew perception and inflate expectations of HMDs—both for study participants and in scientific debate.did not seem to benefit more from using AR-HMDs. Instead, exploratory analysis shows that this number of ultrasound-guided tasks does not even correlate with placement performance when using the conventional ultrasound system. Accordingly, we consider it to be an ineffective predictor for beneficial effects of AR-HMD in this task and possibly for ultrasound-based needle placement performance in general.Our a priori assumption about underlying causes did not hold: Less experienced participants  Given the ambivalence in participants’ responses, we conclude that intermediate mechanisms seem to vary among users. The qualitative study suggests that some may not benefit from the AR-HMD technology, depending on, for instance, individual differences in 3D perception or visuomotorics. Additionally, benefits may only be noticeable during certain types of tasks and/or difficulty levels of interventions rather than in routine tasks. In conclusion, improved spatial understanding (through various pathways) seems to be a likely cause for the benefits of AR-HMDs. Based on these insights, we suggest that future research into the benefits of AR-HMD should investigate the intermediate mechanisms underlying the spatial understanding of ultrasound imaging and their interaction with person and task variables.Limitations of our study can be divided into three main categories: sampling, study design, and unfamiliarity. Both the quantitative experiment and the qualitative study have limitations related to the sampling of participants. Sample sizes were small because we were limited by the availability of the technical equipment. We also had to stop the needle placement experiment early because the training model had accumulated too much damage—previous needle cuts were clearly visible in the ultrasound image and distorted experimental conditions for later participants (Online Resource 3). Furthermore, our sampling was skewed in the following ways: (1) Participants were arguably inclined to be in favor of HMDs, considering they agreed to volunteer, (2) females were underrepresented, and (3) students were overrepresented in the quantitative experiment.In addition to sampling issues, generalizability of our results is limited due to study design. Though we would like to draw conclusions for image-guided procedures in general, only ultrasound was used for imaging and we examined just two (relatively easy) tasks. Furthermore, we suspect that the accuracy of error measurements was negatively impacted by inhomogeneity in the MRI dataset and by participants accidentally bending the needle during measurements. We tried to mitigate these issues by averaging two measurements per condition per participant. Additionally, it is possible that overlay inaccuracies may have negatively impacted needle placement accuracy when using the AR-HMD. Finally, the impact of the order of the conditions (on the differences in task durations at least) implies that participants should perform ‘test runs’ before the initial measurement. Though we did consider this, we decided against it as we suspected limited durability of the biopsy model.We initially considered no familiarity with HMDs to be beneficial for controlling variance. However, displaying ultrasound images in their ‘spatially correct’ position is not per se intuitive. Instead, participants generally found this way of displaying images to be unfamiliar or even strange. Though some seemed to adjust quickly during the experiments, multiple participants mentioned that lack of familiarity made the AR-HMD difficult to use in the qualitative study. We consider it likely that this effect was also present in the quantitative experiment. Generally, comparing familiar technology  against a novel system (AR-HMD) may be considered biased for similar reasons: Adapting to novel situations generally results in  cognitive load, making it harder to perform other tasks simultaneously. Furthermore, familiarity with a technology may have given users the opportunity to find strategies/techniques to better utilize benefits and mitigate limitations of the technology. As a result, we recommend effects of unfamiliarity to be sufficiently considered in the design of future studies.Our results add to the existing evidence that AR-HMDs can help improve performance in image-guided interventions. We additionally provide new evidence concerning potential intermediate mechanisms underlying these benefits. In particular, we suggest that the benefits of AR-HMDs are  mediated by improvements of the spatial understanding of (ultrasound) images. Potential mechanisms are that transferring 2D images into mental 3D representations of the anatomy and/or estimating the position of the image in 3D is facilitated.mixed methods  to be useful for interpreting results and providing a more differentiated perspective on study results, in particular at early stages of research. For instance, based on statements from the qualitative study, AR-HMDs may be particularly useful in difficult scenarios. Future studies could validate whether there is indeed a correlation between task difficulty and the magnitude of benefits; such a correlation would be important for identifying good clinical use cases.Based on our initial insights, we suggest various implications for future research, ideally to be performed in multicenter studies: Our participants consistently found the concept (and usage) of AR-HMDs to be unfamiliar, even strange. As a result, we recommend mitigating unfamiliarity by allowing participants to practice extensively before starting the actual experiment. Furthermore, we found intermediate mechanisms, rather than direct clinical benefits, should be the focus of upcoming studies in this field. Ultimately, we think that the current goal should be to determine the causal pathways underlying the clinical benefits of the technology—which skills and processes can it facilitate and how? By leveraging such knowledge, we believe that AR-HMDs can soon be applied and translated into clinical use cases with maximum impact.We also reaffirm that Online Resource 1: Video of needle placement, filmed through the AR-HMD (MP4 4827 kb)Online Resource 2: Video of ultrasound examination of a test patient, filmed through the AR-HMD (MP4 10760 kb)Online Resource 3: Example image of ‘used-up’ model (PNG 246 kb)Online Resource 4: Interview guideline (PDF 277 kb)Online Resource 5: Category definition  (PDF 109 kb)Online Resource 6: R code used for data analysis and plotting (R 9 kb)Online Resource 7: Quantitative Data (PDF 667 kb)Below is the link to the electronic supplementary material.


In addition, people with physical disabilities reported poor referral pathways, financial constraints, transport and road consideration and equipment unavailability as barriers to accessing rehabilitation services. Moreover, these barriers to access predisposed individuals to finance-, health- and person-related harm. Conclusion: Rehabilitation service availability is constrained by a lack of service capacity in rural South Africa. In addition, the rehabilitation services in district hospitals are not adequately accessible because of existing barriers to enable key populations to achieve optimised functioning.Introduction: Rehabilitation services aim to optimise individuals’ functioning and reduce disability. However, people with disabilities, who represent a key population of users of rehabilitation services, continue to have unmet needs for rehabilitation services that include the provision of assistive devices. This paper examines the availability and accessibility of rehabilitation services in a rural district of South Africa in order to explore why unmet needs for rehabilitation services persist. Methods: All nine district hospitals in a rural district of South Africa were included in the study. Rehabilitation services capacity was assessed by examining the available assistive devices, consumables and human resources at the level of the health facility. Data collection was conducted using the Global Co-operative Assistive Technology [GATE] Assistive Products List, AT2030’s ATScale priority list and the South African National Catalogue of Commodities for Primary Health Care Facilities. Descriptive statistics were then used for the analysis. For the qualitative component, semi-structured interviews were conducted with adults with physical disabilities at household level to explore barriers to accessing assistive device inclusive rehabilitation services and the consequences thereof in the same rural district. An interview guide based on the WHO health system building blocks was used. Thematic content analysis guided the analysis of the interview transcripts. Findings: The findings of the research demonstrate that rehabilitation service capacity in the district was constrained as a result of low availability of assistive devices [2–22%] and consumables [2–47%], as well as, possibly, a shortage of rehabilitation providers [ Rehabilitation services are health services whose chief aim is to optimise individuals’ functioning and reduce disability . These sTherefore, this paper aims to examine the availability of rehabilitation services in district hospitals of a rural district of South Africa and assess what factors may be influencing access to rehabilitation services amongst people with physical disabilities in the same district. The first objective is to determine the rehabilitation capacity in health facilities according to the elements of assistive devices, consumables and rehabilitation workforce. The second objective is to describe the barriers to access and their consequences for people with physical disabilities.OR Tambo District is a largely rural district in the province of the Eastern Cape in South Africa. The district has a population 1.4 million, making it the district with the highest population in the Eastern Cape . Most ofAccording to recent health estimates, the largest contribution to mortality in the district is attributable to non-communicable diseases (46%), followed by HIV and TB (32%) . In compRegarding rehabilitation services in the district at the primary health care level, services are offered in district hospitals, with outreach services intermittently provided at clinics and community health centres in each district hospital’s catchment area. Recent estimates report that the availability of rehabilitation providers may be low relative to national and global estimates. For instance, per 100,000 population, in 2019, there were 1.5 occupational therapists and 2.1 physiotherapists, which falls below the provincial  and national  estimates . Althougconversion factors include age, gender, co-morbidities, impairments, social factors, economic factors or political factors. Conversion factors are important because they affect how resources may be translated into a number of capabilities from which individuals may then choose  across low- and middle-income countries . HoweverA study in an urban and low-income area of South Africa found that unmet need for rehabilitation services, including assistive devices to be about one third of people with disabilities . AnotherRegarding human resources for rehabilitation, in the current study, there were 30 rehabilitation providers employed at district hospitals in OR Tambo district. However, they were not equally distributed across health facilities. Additionally, the distribution of rehabilitation providers according to professional experience was sub-optimal with most rehabilitation departments in OR Tambo district being led by professionals in their first year of work. While it is not known whether the quantity of the 30 providers is adequate to meet the needs, the findings suggest that their distribution in the district’s health facilities is sub-optimal.Gupta and colleagues’ global assessment of human resources for rehabilitation found that lower income countries had ratios of 0.5 rehabilitation providers per 10,000 population . In contThus, local and global evidence points to a shortage of rehabilitation providers in South Africa and other countries. Based on this literature, the number of available rehabilitation providers in the current study likely means that the number of rehabilitation providers in OR Tambo district is low relative to population-level rehabilitation needs, although this is not certain. This is because the full core rehabilitation team was available in only one of the nine health facilities, two health facilities had only one rehabilitation provider each and most of the rehabilitation workforce consisted of recently qualified providers, meaning that the availability of services was directly dependent on graduate output on a yearly basis. While comparisons may be made within and across countries about the numbers of rehabilitation providers, there is a paucity of literature that establishes thresholds for what an adequate number of rehabilitation providers per population should be . This isWith regards to access, the findings from this study demonstrate that adults with physical disabilities in OR Tambo district experience notable demand and supply-side barriers when accessing public health sector rehabilitation services. This aligns with other research in both HIC and LMIC which shows that, despite being more likely to have poorer health outcomes than those without disabilities, access to rehabilitation for people with disabilities is low ,57,58. SHowever, barriers were not simply inconveniences; they exposed individuals and their households to harm related to their finances, health and personal dignity. Since transport was a barrier to accessing rehabilitation services, some participants reported hiring a car from a member of the community, but this was very costly relative to their income. This has been reported in another South African study in which private car hire was reported to cost approximately R500 or more depending on the distance from the health facility . This fiMoreover, people with disabilities are more likely to be poorer than non-disabled individuals . FinanciDrawing from the findings of both the supply-side and demand-side, the Capability Framework presents an opportunity to understand why people with disabilities in OR Tambo district still report unmet needs for rehabilitation services despite these services seemingly being offered in health facilities. The Capability Framework also elucidates the mechanisms which may result in optimal functioning through rehabilitation not being achieved amongst people with physical disabilities in OR Tambo district. First, rehabilitation services are comprised of many components including at the least assistive devices, consumables and rehabilitation providers. What the current study’s findings demonstrate is that a lack of availability of assistive devices and consumables and a possible shortage in rehabilitation providers results in a resource (rehabilitation services) which is deficient and unable to result in desired health outcomes amongst individuals. We were unable to calculate staff to population ratios in OR Tambo district since the study examined only district hospitals and not regional and tertiary hospitals. Second, the presence of barriers [conversion factors] such as inaccessible transportation, lack of finances and absence of assistive devices prevents the ability for accessing rehabilitation services in a way that adequately results in its chief aims of optimising functioning and reducing disability. Lastly, the combination of deficient resources and barriers to access not only reduces opportunities for people with disabilities to achieve what is optimal functioning to them, but this combination also results in finance-, health- and personal-related harm to people with physical disabilities in OR Tambo district. In other words, the reduced access to rehabilitation services as a result of inadequate rehabilitation services accompanied by barriers to access is not only inconvenient but it actively reduces the potential for achieving the kinds of lives that individuals value. Although individuals exercised their agency by hiring private transportation to reach rehabilitation services, the alternative would result in forgoing rehabilitation services. Therefore, while the choices individuals could make were constrained by the barriers (conversion factors), the barriers did not altogether remove the agency of individuals.A major strength of this study is that all district hospitals in one district were included thus findings may be generalised to the district. A second strength is that both supply-side and demand-side aspects relevant to rehabilitation services were examined, thus enabling the interactions between these aspects to be demonstrated. However, this study also has limitations. First, the assessment of rehabilitation capacity was limited to a selection of readiness indicators to the exclusion of other relevant indicators such as equipment and process related factors such as observed provision of care and guidelines availability. Even so, this study represents an important first step in addressing the gap in rehabilitation capacity assessment in South Africa. Further work is required to develop a health-facility level assessment tool which examines rehabilitation service readiness and processes of care. Second, only people with physical disabilities were included as users of rehabilitation services to the exclusion of other types of disabilities. However, barriers such as finances and equipment are likely relevant for all types of disabilities in the district. Further research should include a diversity of disability types when examining barriers and their consequences. Third, only one investigator conducted the data analysis which may have reduced the rigor somewhat. To mitigate this, the codes and themes were discussed with the advisor as they developed, and the interpretation was discussed with the same advisor. Finally, further work should consider rehabilitation policies and their implementation in order to identify what factors contribute to the available rehabilitation capacity at the health facility and their accessibility to key populations.This study showed that the rehabilitation service capacity in a rural district of South Africa is constrained by a low availability of assistive devices, consumables and rehabilitation human resources in health facilities. In addition, people with physical disabilities are hindered by barriers when accessing rehabilitation services. These barriers result from the deficiencies in rehabilitation capacity but also from factors on the demand-side. Implications of this work point to a need for South Africa to develop its own national list of essential assistive products which spans all domains of functioning. Further, additional financing and improved procurement processes for assistive devices and consumables at the health facility level in OR Tambo district are required in order to increase the availability of these items. Finally, there is a need for greater collaboration between the stakeholders involved in social development, transport and roads and health in South Africa in order to address the existing demand-side barriers to rehabilitation services.


Transient receptor potential melastatin 4 (TRPM4) is a nonselective Ca2+-activated cation channel gene defined as a novel disease-causing gene of AVB. So far, 47 mutations of TRPM4 have been recorded in Human Gene Mutation Database. The aim of this study was to explore the relationship between TRPM4 mutation and pathogenesis of AVB. We investigated a Chinese family with AVB by whole-exome sequencing. An arrhythmia-related gene filtering strategy was used to analyze the disease-causing mutations. Three different bioinformatics programs were used to predict the effects of the mutation result. A novel mutation of TRPM4 was identified (c.2455C>T/p.R819C) and cosegregated in the affected family members. The three bioinformatics programs predicted that the novel mutation may lead to damage. Our study will contribute to expand the spectrum of TRPM4 mutations and supply accurate genetic testing information for further research and the clinical therapy of AVB.Atrioventricular block (AVB) is a leading cause of sudden cardiac death, and most of AVB cases are presented as autosomal dominant. The electrocardiogram of AVB patients presents an abnormal progressive cardiac conduction disorder between atria and ventricles.  Inherited progressive cardiac conduction disease (PCCD) is defined as an abnormal progressive cardiac conduction disorder , in whictransient receptor potential melastatin 4 (TRPM4) encodes a nonselective Ca2+-activated cation channel which is highly expressed in Purkinje fibers and nodal tissue . Ac. AcTRPM4disorder . Currentdisorder , 26.TRPM4 was identified, not only further explain the possible cause of AVB in the family but also expand the spectrum of TRPM4 mutations and contribute to genetic diagnosis and counseling for families with AVB.In conclusion, we identify a novel TRPM4 mutation (c.2455C>T/p.R819C) in a two-generation family with AVB by genetic sequencing. The present study, in which a novel mutation of


The aim of this study was to identify differences in serum metabolomics profiles of house-dust-mite (HDM)-induced allergic rhinitis (AR) patients compared to controls and to explore novel biomarkers reflecting disease severity. Serum samples were collected from 29 healthy controls and HDM-induced 72 AR patients, including 30 mild patients (MAR) and 42 moderate to severe AR patients (MSAR). Metabolomics detection was performed, and orthogonal partial least square discriminate analysis was applied to assess the differences between AR patients and controls and for subgroups based on disease severity. These analysis results successfully revealed distinct metabolite signatures which distinguished MAR patients and MSAR patients from controls. MSAR patients also could be discriminated from MAR patients based on their metabolic fingerprints. Most observed metabolite changes were related to glycine, serine, and threonine metabolism, pyrimidine metabolism, sphingolipid metabolism, arginine and proline metabolism, and fatty acid metabolism. Levels of sarcosine, sphingosine-1-phosphate, cytidine, and linoleic acid significantly correlated with the total nasal symptom score and visual analogue scale in AR patients. These results suggest that metabolomics profiling may provide novel insights into the pathophysiological mechanisms of HDM-induced AR and contribute to its evaluation of disease severity. Allergic rhinitis (AR) is an IgE-mediated immunologic disease characterized by mucus hypersecretion and airway hyperresponsiveness caused by common allergens such as house dust mite (HDM), pollen, and animal dander , 2. AmonPrevious publications reported that AR was a heterogeneous disease with a wide degree of severity; there was no available objective indicator or biological marker that is specific for its disease activity , 8. CurrMetabolomics, a branch of omics science that systematically analyzes the concentration profiles of low molecular weight endogenous metabolites generated by living systems, is a promising approach to identify new biomarkers and novel metabolic pathways for several diseases, simultaneously providing new insights into the underlying pathophysiological mechanisms , 12. RecTherefore, the primary aim of this study was to explore the metabolic profiling of HDM-induced AR patients and determine the relationship between the metabolite changes and clinical severity, thus to provide new insights into the complex pathophysiological mechanisms and monitor disease activity. In this study, ultrahigh-performance liquid chromatography mass spectrometry (UHPLC-MS) was performed to detect metabolites in serum samples of MSAR, in comparison with MAR and healthy controls. In addition, linear regression analysis was performed to evaluate the correlation between metabolites and disease severity.n = 30), MSAR group (n = 42), and control group (n = 29). The diagnosis of HDM-induced AR was done on the basis of medical history and allergic symptoms  for at least 2 years, positive skin test results , and positive specific IgE to HDM (>0.35 IU/mL). Patients with persistent AR were categorized into MAR and MSAR according to ARIA criteria [This is a prospective study with 101 participants recruited between June 2018 and January 2019. All participants were divided into three groups: the MAR group . After a 30-second vortex, the samples were sonicated for 10 minutes in ice-water bath. Then, the samples were incubated at -40°C for 1 hour and centrifuged at 12000 rpm for 15 minutes at 4°C. 100 μL of the supernatant was transferred to a fresh glass vial for UHPLC-MS analysis [Serum samples were collected from HDM-induced AR patients and healthy controls with serum separator tubes without anticoagulation or coagulant before breakfast, and serum samples were stored for 1 hour at room temperature. All blood samples were centrifuged at 4°C (3000 rpm for 10 minutes); then, we collected the supernatants and stored them at -80°C in equal aliquots for subsequent detection and analysis. The serum samples were prepared for UHPLC-MS analysis by mixing 100 analysis . The quaanalysis .μL of reconstituted sample was injected on a UPLC BEH Amide column . The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). Each sample was analyzed in the positive ion mode and negative ion mode. The Triple TOF 6600 mass spectrometry  was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software  continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on the preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at a collision energy (CE) of 30 eV. The cycle time was 0.56 second. Electrospray ionization (ESI) source conditions were set as follows: gas 1 as 60 psi, gas 2 as 60 psi, curtain gas as 35 psi, source temperature as 600°C, declustering potential as 60 V, and ion spray voltage floating (ISVF) as 5000 V or -4000 V in the positive or negative modes, respectively.Samples were analyzed on a 1290 Infinity series UHPLC System  as previous study described . BrieflyR2Y(cum) (goodness of fit) and Q2(cum) (goodness of prediction). Meanwhile, the 7-fold cross-validation and 200 permutation tests were conducted to reduce the risk of overfitting and the possibilities of false-positive findings. Metabolites contributing were selected according to the variable importance for project (VIP) values (VIP > 1.0) and P values (P < 0.05) [MS raw data (.wiff) files were converted to the mzXML format by Proteo Wizard and processed by R package XCMS V3.2. The process includes peak deconvolution, alignment, and integration. Minfrac and cut-off are set as 0.5 and 0.3, respectively, as before . In-hous < 0.05) . To detet test was utilized for comparison between two groups; nonnormally distributed data were described as median and interquartile range, and Kruskal–Wallis H test and Mann–Whitney U test were utilized for comparison among three groups and between two groups, respectively. Categorical variables are described as number (%) and compared utilizing Chi-square test. To explore the correlation between the levels of metabolites and the severity of AR, Spearman's correlation analysis was conducted. Differences were considered as significant when P < 0.05. All the above statistical analyses were carried out using SPSS statistics software version19.0 .Normally distributed variables were displayed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA) was performed for comparison among three groups, Student's P < 0.001).The main characteristics and clinical information of the participants are shown in P < 0.05, Figures R2s were below or around 0.6, and all permuted Q2s were below 0, which means that all R2s and Q2s are lower than the original on the right . However, linoleic acid levels were negatively correlated with TNSS and VAS (P < 0.05). In order to evaluate the prediction power of four significantly distinctive metabolites in reflecting the disease severity, we performed a single-composite ROC analysis. The result showed that the composite predictor exhibited good accuracy and utility  (The distinctive metabolites among the three groups with good predictability (AUC > 0.7) were included in Spearman's correlation analysis to evaluate their correlation with the severity of AR. As presented in < 0.001) .In the current prospective cohort study, we described a novel application of metabolomics in identifying the serum metabolic signatures and assessing the association between the distinctive metabolites and the severity of HDM-induced AR. The OPLS-DA model showed that obvious discriminators between patients with different disease severity and health controls. Thirty-five and 59 metabolites responsible for differentiating MAR and MSAR patients from health controls, respectively, were identified. In addition, 30 metabolites were found to be responsible for discriminating MSAR patients from MAR patients. After analyzing the relationships between the major discriminative metabolites and clinical parameters of patients, we observed that sarcosine, sphingosine-1-phosphate, cytidine, and linoleic acid levels were associated with the disease severity. These results showed that the identified potential serum metabolites might be useful for diagnosing HDM-induced AR and developing objective indicators for evaluating its severity. We will next discuss the most significant metabolites and related metabolic pathways, which may help us to better understand the underlying pathogenesis of HDM-induced AR and monitor its disease severity.Most importantly, arginine and proline metabolism pathway was significantly perturbed among the most affected pathways in HDM-induced AR patients. Arginine and proline metabolism is of particular importance in the nitric oxide synthesis and integrally links to cellular respiration, metabolism, and inflammation , 23. A rOur results also provide evidence that the sphingolipid metabolism alteration is involved in the occurrence and progression of HDM-induced AR. Sphingolipids are ubiquitous components of the cell membrane and play an important role in cell growth, inflammation, and tissue remodeling , 28. AmoWe firstly found that cytidine, identified from UHPLC-MS analysis, was associated with HDM-induced AR, and it might be a novel marker and potential therapeutic target. In our study, we observed that the levels of cytidine were elevated in the MAR and MSAR groups, and the concentrations of cytidine were positively correlated with TNSS and VAS. Cytidine, a pyrimidine molecule, is considered the precursor of the cytidine triphosphate (CTP), which is vital in the synthesis, interconversion, and degradation of DNA, RNA, and lipids , 33. Pretrans-vaccenic acid) were lower in the serum of MAR or MSAR patients in comparison with health controls, while the concentrations of palmitic acid were elevated. Moreover, the levels of linoleic acid were correlated negatively with TNSS and VAS. Our results were in line with most previous studies. However, further studies should be conducted to confirm these results and to clarify the underlying mechanism of AR subtypes.Interestingly, we also found that the fatty acid metabolism was dysregulated in all OPLS-DA models. In recent years, growing evidence suggested that the fatty acid metabolism played important roles in the modulation of immune responses . Most reWe acknowledge several limitations in our study which may affect the clinical applications of obtained results. First, the total sample sizes were relatively small and a validation cohort study was needed to confirm the conclusions. Second, the recruited participants were from a single center with the same ethnicity and region, which might limit the applicability of our findings. Third, only one biological sample (serum) was used in the present study; future studies should collect other biological samples, such as urine and nasal lavage fluid, to further verify whether the identified differential metabolites were associated with AR. Last, we did not compare serum metabolites between moderate AR and severe AR patients, but it does not mean that there are no differential metabolites. Future multicenter prospective clinical studies with larger sample sizes utilizing untargeted and targeted metabolomics will be important to support and extend our present findings.Our results suggest that serum metabolomics approaches can be successfully used to discriminate MSAR patients, from MAR patients and health controls, and establish a metabolite signature associated with the severity of HDM-induced AR. These results will be useful for diagnosing HDM-induced AR and developing objective indicators for evaluating the disease severity.