BCNP1) or Family member of 129C (FAM129C) was identified as a B‐cell‐specific plasma‐membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein‐protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA , MAPK1 , KRAS  and AKT2 . We also found that PI3K (Phoshoinositide 3‐kinase) inhibition and p38 MAPK (p38 mitogen‐activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome‐mediated pathway.B‐cell novel protein‐1 ( Many components of the PI3K signalling pathways are frequently altered in a broad spectrum of human cancers et al. found that Niban is involved in the endoplasmic reticulum stress response by decreasing phosphorylation of p70 ribosomal S6 subunit kinase (S6K) 1 and eukaryotic initiation factor 4E‐binding protein1 (4E‐BP1) The structure and normal function of BCNP1 is not known. In addition, the relationship between the BCNP1 and cancer is also not very well understood. However, it is well‐known that some members of the FAM129 proteins (to which BCNP1 or FAM129C belongs) such as Niban (FAM129A) are indeed involved in cancer. For example, Niban was found to be up‐regulated in renal carcinoma The proline rich region of FAM129B at the carboxyl end is phosphorylated at six serine residues et al. showed that overexpression of a zinc‐finger protein (ZNF777) inhibited proliferation of cells at low density through down‐regulation of FAM129A (Niban) To understand the collective behaviour of cancer cells, we are studying the Ph+ Acute Lymphoblastic Leukaemia cell line (ALL3) that has excellent growth at high starting cell densities (HD) and no growth at low starting cell densities (LD) In the current study, we employed bioinformatics and biochemical approaches to investigate the functions of BCNP1, its relationship with various types of cancer and its biochemical relationship with PI3K and p38 MAPK pathways. Bioinformatics analysis predicted that BCNP1 contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions, and Leucine Zipper domain (LZ) suggesting that it may be involved in protein‐protein interactions. Additional bioinformatics analysis also suggested that BCNP1 is phosphorylated. When we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types using TCGA data sets, we concluded that BCNP1 is highly altered in some of the cancer types and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53, PIK3CA, MAPK1, KRAS and AKT2. In addition, when we investigated the function of BCNP1 in the PI3K‐dependent signalling pathway, we found that PI3K inhibition and p38 MAPK activation leads to reduction in serine‐phosphorylation of BCNP1 suggesting that BCNP1 phosphorylation is dependent on PI3K and p38 MAPK and it might be involved in cancer. The biomedical significance of our findings is discussed.2 in incubator at desired times depending on type of experiments. HEK293 cells were used in this study because they are amenable system for transfection.HEK293 cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat‐inactivated foetal bovine serum (FBS) and penicillin and streptomycin. Cells were kept at 37°C, 5% COAnti‐FLAG M2 monoclonal antibody (Sigma F1804) and Anti‐phosphoserine (Sigma P3430) antibody were purchased from Sigma‐Aldrich, St. Louis, MO, USA. Anti‐phospho Akt (Ser473) (CST#9271) and anti‐Akt (CST#9272) were from Cell Signaling Technology, Boston, MA, USA. Anti‐phospho p38 MAPK (Thr180/Tyr182) and anti‐MAPK were from Cell Signaling Technology, USA. All secondary antibodies and anti‐βactin antibody were from Abcam, Cambridge, MA, USA. Polyethyleneimine (PEI) was purchased from Polyscience Inc, Warrington, PA, USA. Protein A/G Plus Agarose beads (sc2003) for immunoprecipitation (IP) were from Santa Cruz Biotechnology, USA. The compounds wortmannin and LY294002 were from Selleck chemicals, Houston, TX, USA. Cycloheximide, sorbitol and MG132 were from Sigma‐Aldrich.The BCNP1 (FAM129C) (NM_173544.4) cDNA construct (HsCD00295103) cloned into pENTR223.1 was obtained from Harvard Medical School Plasmid DNA core facility, Boston, MA, USA. The pcDNA3 Flag HA empty vector [from William Sellers (Addgene plasmid # 10792)] was obtained from Addgene, Cambridge, MA, USA. The FAM129C cDNA was amplified by PCR. The PCR product contained EcoRI and NotI restriction sites at 5′‐ and 3′‐end, respectively. The Empty vector and PCR product were digested using EcoRI and NotI  enzymes, ran on the 1% agarose gel and purified using gel extraction kit (#28704) from Qiagen, Valencia, CA, USA. After purification, the PCR product was ligated into pcDNA3 Flag HA and transformed into 5‐α highly competent E. Coli as described in a protocol from NEB. A few colonies were selected to confirm the presence of the desired cDNA construct. The sequence fidelity was confirmed using Sanger‐sequencing method at DNA Sequencing Core Facility at Memorial Sloan‐Kettering Cancer Center, NY‐USA (MSKCC).Transient transfection of pcDNA3 Flag HA‐FAM129C was carried out using PEI. The HEK293 cells were plated 24 hrs prior to transfection. For transfection into 60 mm plate 3 μg of plasmid DNA was diluted in 100 μl high glucose serum‐free Dulbecco's modified eagle medium (DMEM). To dilute PEI, 0.75 μl of 1 mg/ml stock solution was added in 100 μl high glucose serum‐free DMEM. Diluted PEI solution was added to diluted DNA and incubated at room temperature (RT) for 30 min. After 30 min., DNA/PEI mixture was added drop‐wise to HEK293 cells and incubated at 37°C for 24–48 hrs in tissue culture incubator and processed further.Transiently transfected cells were washed twice with ice cold phosphate buffered saline (PBS) and scraped with 1–2 ml of ice cold PBS and centrifuged at ~525 g for 8–10 min. at 4°C using eppendorf 5810 high speed centrifuge. The pelleted cells were lysed with 0.2–0.4 ml of RIPA lysis buffer [radioimmunoprecipitation assay buffer  with protease and phosphatase inhibitors cocktails for 30 min. on ice and centrifuged at 4°C for 10 min. at 13,500 g eppendorf 5810 high speed centrifuge. The lysates containing supernatants were collected carefully without disturbing the pellets. Protein concentration of the supernatant was measured using a Pierce Protein Assay Kit . For IP, 500 μg of proteins were incubated with anti‐flag antibody at 4°C overnight. The protein–antibody immunoprecipitates were collected by protein A/G plus‐agarose . Following the final wash, the samples were boiled and centrifuged to pellet the agarose beads. To check protein levels from whole cell lysates (WCL), 50 μg of proteins were used for analysing for western blotting. All the protein samples were prepared in 2× Laemmali sample buffer (Biorad#1610737). WCL and immunoprecipitated (IP) proteins were separated by Sodium Dodecyl Sulphate Poly‐acrylamide Gel Electrophoresis (SDS‐PAGE) at RT and transferred to polyvinylidene fluoride (PVDF) membrane using BioRad transfer apparatus at 4°C. The membranes were blocked with 3% bovine serum albumin (BSA) or 5% non‐fat milk in TBS  with 0.1% Tween‐20 (TBST) for 60–90 min. Membranes were probed with a variety of primary antibodies at 4°C overnight. The membranes were washed three times with TBST, and developed with horseradish peroxidase‐conjugated (HRP) anti‐mouse or anti‐rabbit antibody   for 1 hr at RT. Membranes were then washed three times with TBST and developed with a Western Lightning Chemiluminescence reagent  for 1–5 min. at RT, and the signals were developed on film. The films were scanned and intensity was measured and bands were transformed using Image J  software available from National Institute of Health, USA (NIH).www.cbioportal.org); the data were collected on July 2015. We used OncoPrint tool to visualize the data We used comprehensive data available for cancer samples as part of the TCGA (The Cancer Genome Atlas) project (tcga.cancer.gov). All TCGA data were from the cBio Cancer Genomics Portal  as shown in Figure BCNP1 contains a PH domain , adenoid cystic carcinoma , uterine corpus endometrial carcinoma  and breast cancer  Fig. A and B.We also collected data to determine the percentage of cases wherein BCNP1 is mutated. The data shows the average of studies if there are of similar types. Tumour types having a higher percentage of BCNP1 mutations included stomach adenocarcinoma SC; 3.5%), oesophageal carcinoma , pancreatic adenocarcinoma  and colorectal adenocarcinoma    activation, KRAS amplification and TP53 amplification and mutation simultaneously. To generate an OncoPrint profile, we used data from various studies as follow: OSC , OSC , breast cancer (BC), adenoid cystic carcinoma (ACyC) and brain lower grade glioma (GBM). As shown in the Figure P = 0.0139 and P = 0.0137, respectively , OSC TCGA,  and GBM ely Fig. B. In othely Fig. A and C. ely Fig. C. Taken Protein phosphorylation acts as an essential posttranslational modification regulating signalling pathways and various cellular functions like differentiation, cell movement, apoptosis, intercellular communication and cell survival Next, we investigated whether p38 MAPK activation leads to change in BCNP1 phosphorylation at serine residues. Treatment of transiently transfected HEK293 cells with 0.5 M sorbitol, a well‐established osmotic shock inducer and selective p38 MAPK activator, led to a ~10–30% reduction of BCNP1 protein level Fig. . As expeWe also determined whether BCNP1 is degraded in transiently transfected HEK293 cells using the protein synthesis inhibitor cycloheximide at 20 μg/μl. Treatment led to decrease in BCNP1 protein levels with a half‐life of ~45 min. in transiently transfected HEK293 cells Fig. A.Next, treatment of transiently transfected HEK293 cells with proteasome inhibitors such as MG132 at 20 μM led to increased BCNP1 level doubled after 30 min. Fig. B. We conBCNP1 is a not very well studied protein with unknown function(s). BCNP1 is more highly expressed in cancer samples than normal tissues The p38 MAPK pathway plays an essential role in regulating inflammation, cell differentiation, cell growth and apoptosis. It is strongly activated in response to stress stimuli such as osmotic shock, UV and ionizing radiation, and inflammatory cytokines. It might have different roles depending on cellular context and can play an important role in cancer. Our work defines a functional connection between p38 MAPK and BCNP1 Fig. . Using KGenomic efforts to categorize human tumour types have provided further understanding into the molecular contribution of heretofore understudied gene products of BCNP1. Our findings revealed that BCNP1 is overexpressed in many types of human cancer cells. Undoubtedly, cell type‐specificity and even type of genetic alterations in tumours are critical determinants of the cell signalling pathways mediated by BCNP1. Given the relative frequency of BCNP1 alterations in many cancer types warrant further investigation of its function providing answers to question when BCNP1 is amplified, deleted or mutated. Our recent observation that BCNP1 (FAM129C) was the most highly up‐regulated gene when non‐growing low density Ph+ ALL3 cells were stimulated to proliferate by cell‐free supernate from the same cells growing rapidly at a high cell density, and Yuki's report that overexpression of ZNF777 inhibited proliferation of cells at low density by down‐regulating FAM129A, suggests that BCNP1 probably has a very important role in cell‐cell communication and corresponding intracellular signalling pathways We declare that we have no conflict of interest.Fig. S1. An OncoPrint showing the relationship of BCNP1 genetic alterations with TP53, KRAS, MAPK1 (ERK), PIK3CA (PI3K) and AKT2 mutational events.Fig. S2. Effect of PI3K inhibition on BCNP1 phosphorylation at serine residues.Click here for additional data file.


Intracerebral hemorrhage (ICH), a form of brain bleeding and minor subtype of stroke, leads to significant mortality and long-term disability. There are currently no validated approaches to promote functional recovery after ICH. Research in stroke recovery and rehabilitation has largely focused on ischemic stroke, but given the stark differences in the pathophysiology between ischemic and hemorrhagic stroke, it is possible that strategies to rehabilitate the brain in distinct stroke subtypes will be different. Here, we review our current understanding of recovery after primary intracerebral hemorrhage with the intent to provide a framework to promote novel, stroke-subtype specific approaches. In humans, primary ICH occurs because of a rupture of an intracerebral vessel, most often due to hypertension or cerebral amyloid angiopathy. Vessel rupture leads to mechanical tissue disruption (secondary to the jet of blood) and hematoma formation, followed by secondary injury processes, including disruption of the blood-brain barrier, expansion of perihematomal edema, inflammation, neuronal cell death, and chemical or cellular barriers to repair3. These secondary injury processes occur over hours to days after the primary injury and are attributable to toxins from lysed blood, including hemoglobin (Hgb), hemin (oxidized form of heme), and thrombi8. These toxins could affect cell survival or repair via interactions with a host of cell types, not only neurons.Intracerebral hemorrhage (ICH) accounts for about 15% of all strokes in the USA and Europe and leads to significant mortality and long-term disability despite advances in acute medical and surgical treatmentAlthough research in the field of stroke recovery and rehabilitation has grown, there have been very few studies specifically focusing on rehabilitation in ICH. In this review, we summarize the recent advances in our knowledge of the pathophysiology of primary ICH, how rehabilitation affects functional outcomes after ICH, and the mechanisms that may underlie the gains seen with rehabilitation in preclinical models and patients.9. The two most commonly used rodent models of ICH are autologous blood injection and bacterial collagenase infusion10. In the whole-blood injection model, lysed blood is injected directly into the brain parenchyma, with almost no spontaneous bleeding11. The bacterial collagenase model involves intraparenchymal infusion of bacterial collagenase, an enzyme that damages the basal lamina and causes bleeding that evolves and grows over 24 hours12. Models of hypertensive rodents in which ICH occurs spontaneously, which may more closely mimic what occurs in patients, have been less widely used. In reviewing the literature, one must consider that these preclinical models have different pathophysiological processes which may affect the course of recovery and response to rehabilitation; thus, the use of multiple models has been recommended to better understand post-ICH recovery in preclinical studies13.Preclinical models have provided important insights into the pathophysiological processes that occur after ICH, which differ from those seen after ischemic stroke14. In the collagenase model, there is markedly greater delayed cell death, leading to worse functional deficits even when the hematoma size is matched14. Historically, it was believed that Hgb or hemin induced toxicity following ICH by loading neurons with redox-active free iron leading to the formation of hydroxyl radicals via Fenton chemistry9. Indirect support for this model came from studies, not replicated by all groups, that iron chelators such as deferoxamine (DFO) improve functional recovery in pig15 and rodent16 models. More recent studies suggest that DFO and a more selective metal chelator, adaptaquin, act not by inhibiting Fenton chemistry but by inhibiting a specific family of iron-, 2-oxoglutarate-, and oxygen-dependent dioxygenases, the hypoxia-inducible factor proly hydroxylases (HIF PHDs)17. HIF PHDs are oxygen sensors and accordingly, with drugs such as adaptaquin, the brain can be fooled into thinking it is hypoxic when it is not. The consequence of turning on hypoxic adaptation in ICH with adaptaquin is to inhibit the synthesis of a cassette of genes  that are involved in mediating neuronal death. Accordingly iron, likely via its ability to act as a cofactor for the HIF PHDs, promotes the synthesis of ATF4-dependent genes that mediate neuronal death.After ICH, there is both immediate and ongoing cell death in both whole-blood and collagenase modelsOf note, ATF4 regulates the synthesis of a pseudokinase inhibitor of AKT, called tribbles homolog 3 (Trib3). Studies in non-neural cells have shown that Trib3 and ATF4 can dimerize to inhibit transcription by the pro-plasticity transcription factor, CREB (cAMP-responsive element-binding protein). Activators of CREB have been shown to improve learning and memory as well as functional recovery after stroke. Accordingly, iron’s ability to load the HIF PHDs could lead to the transcriptional induction of genes  that constrain neuronal survival and plasticity following hemorrhagic stroke. The model is consistent with data showing that adaptaquin, a small-molecule inhibitor of the HIF PHDs, improves functional recovery out to a month in collagenase and blood infusion models in mice and rats, respectively. Future studies will clarify the role of ATF4 in regenerative failure following ICH.Parthanatos19. By contrast, recent data suggest that cell death following ICH occurs via a ferroptotic pathway that also involves necroptosis20. Of note, inhibitors ofParthanatos have no effect on ICH-induced deathin vitro7, suggesting that the pathways of cell damage and, by extension, regenerative failure might be different.How do cells die following ICH in cellular and rodent models? Recent studies from two groups suggest that cell death in ICH is completely distinct from that found following ischemic stroke. In ischemic stroke, cell death occurs via a poly(ADP-ribose) polymerase-1 (PARP)-dependent pathway leading to AIF release from the mitochondria and21. Interestingly, activated microglia can take on pro-inflammatory (via classic activation) or anti-inflammatory  phenotypes, the latter associated with wound healing and repair23. A recent study demonstrated that, after ICH, microglia have dynamic changes in gene expression that result in a transition from an early, pro-inflammatory phenotype to an anti-inflammatory phenotype during the resolution phase of ICH24. Transforming growth factor-beta 1 (TGF-β1) was identified as the most likely mediator of this effect, and treatment with TGF-β1 improved functional outcomes in a murine model. Furthermore, early increases in TGF-β1 levels were also shown to be highly associated with improved 90-day outcomes in patients with ICH.Other studies have focused on the role of the immune system in secondary injury and repair. After ICH, there is a marked inflammatory reaction with activation of microglia and recruitment of leukocytes to the perihematomal region, leading to the production of inflammatory mediators, including cytokines, chemokines, and matrix metalloproteinases3. In a preclinical study, treatment with a PPARγ agonist, rosiglitazone, promoted hematoma resolution, reduced neuronal damage, downregulated the expression of pro-inflammatory genes , reduced oxidative stress, and was associated with improved neurological function25.Another area of research involves the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a significant role in phagocytosis and modulating inflammation and neuroprotective mechanisms29. Given the differences in pathophysiology between ischemic stroke and ICH, one could assume that the recovery outcomes or mechanisms would be dissimilar between the two stroke subtypes. Comparisons between recovery outcomes in patients with ischemic stroke and ICH have yielded mixed results; some have found comparable activity limitation and recovery30, whereas others have found greater recovery after ICH31.Post-stroke recovery has been widely studied, but the majority of research has focused only on ischemic stroke32. A recent longitudinal study of patients with ICH characterized the time course of recovery of motor and sensory impairment and ambulation in 11 patients up to six months post-stroke33. Sensory, truncal, and lower limb motor impairment reached a plateau after three months, whereas there was continued improvement of upper limb movement and ambulation to six months. The course of motor recovery has been shown to depend on the integrity of the corticospinal tract (CST), as measured by transcranial magnetic stimulation34 or diffusion tensor tractography32. In a study of 36 patients with putaminal hemorrhage, motor function recovered in all patients up to four months post-stroke, but patients who had preserved CST integrity at baseline had greater recovery of motor function32. More studies are needed to better elucidate the natural history of recovery after ICH, as has been done for ischemic stroke.The majority of recovery after ICH occurs early, within the first few months post-stroke36. Different types of rehabilitation have been tested in preclinical models, including environmental enrichment38, skilled reach training37, constraint-induced movement therapy40, and aerobic training41. Enriched rehabilitation (ER) combines environmental enrichment  and task-specific training (most commonly skilled reach training) and was first used as a successful rehabilitative intervention in preclinical models of ischemic stroke43. Rehabilitation has been shown to be associated with improved behavioral recovery and enhanced neuroplasticity, including dendritic reorganization44, astrocytic plasticity45, and synaptogenesis in ipsilesional motor cortex and striatum46.Rehabilitation has been shown to lead to functional improvements in rodent models using both collagenase-induced and whole blood-induced ICH36. ER also significantly reduced the amount of perihematomal neuronal death even after one week of treatment, suggesting a possible neuroprotective effect of rehabilitation. This study also examined whether rehabilitation influenced iron toxicity and inflammation post-ICH; rehabilitation did not have an effect on levels of iron-binding proteins (ferritin and transferrin) or number of inflammatory cells in perihematomal tissue. The results of a follow-up study also support a neuroprotective role of rehabilitation; ER beginning one week after ICH was demonstrated to augment the clearance of toxic blood components, Hgb and iron, and reduce oxidative stress at the hematoma/perihematomal interface35. The authors speculate that rehabilitation upregulates pathways for Hgb clearance  and downregulates anticlearance pathways  to expedite hematoma resolution and limit secondary injury. Further investigation is needed into a possible neuroprotective role of rehabilitation in preclinical models, as well as the extent to which secondary injury processes contribute to the functional deficits after ICH in humans, and the ability to reduce secondary injury with rehabilitation in humans.In a collagenase-induced rodent model, rats that participated in a two-week course of enriched rehabilitation beginning one week after ICH improved their skilled reaching and walking ability13. Interestingly, the behavioral improvements in the whole-blood model, unlike those in the collagenase model, were not accompanied by a decrease in lesion volume or increase in dendritic length13. The authors propose that the lack of delayed injury in the whole-blood model compared with the collagenase model may account for these differences. It is still unknown which of these models better exemplifies what occurs after ICH in humans.In a whole-blood injection model, ER beginning one week after ICH also led to significant improvements in reaching ability49.Taken together, evidence from preclinical studies shows a benefit of early rehabilitation after ICH, but the mechanisms underlying the behavioral gains are incompletely understood and vary according to model. Further refinement of neurorehabilitative interventions in preclinical models, which focuses on factors such as the timing, intensity, schedule, and total dose of therapy, is also needed to guide the development of optimal rehabilitation paradigms for patients50. Similar results were seen in ICH patients who received the same type and dose of inpatient rehabilitation and had greater recovery of neurological impairment and reduced activity limitation by time of discharge, relative to patients with ischemic stroke31.Rehabilitation may be more effective after hemorrhagic stroke than after ischemic stroke, when matched for baseline clinical severity. In a preclinical study in size- and location-matched ischemic or hemorrhagic stroke, rodents with ICH were shown to have greater recovery of skilled walking ability than those with ischemic stroke52 or excluded ICH patients altogether from study53. In animal models of ischemic stroke, there is an early ‘sensitive period’ after stroke, during which rehabilitation leads to larger behavioral improvements than when rehabilitation is delayed, even by one week54. However, owing to the paucity of clinical trials, evidence is lacking for a ‘sensitive period’ in humans for ischemic or hemorrhagic stroke.There have been very few rehabilitation studies undertaken in patients with ICH. Many of the larger neurorehabilitation trials have included both hemorrhagic and ischemic stroke patients55. Patients with moderate motor impairment were randomly assigned to either very early rehabilitation (within 48 hours) or standard care (rehabilitation starting after seven days). The very early rehabilitation group had shorter lengths of stay, improved quality of life, greater independence in activities of daily living, and lower six-month mortality when compared with the standard-care group. Because this study was conducted in China, where rehabilitation is delivered by family members and is less standardized, it is unclear whether the results are generalizable to the general ICH population. Furthermore, information on known predictors of outcome, such as hematoma volume, was not available, and the outcomes were self-reported and subject to responder bias. Nevertheless, it is a promising result in favor of very early rehabilitation, which has been a topic of recent debate56.The effects of early rehabilitation after ICH were recently investigated in a multicenter, prospective, randomized controlled study of 243 patients with ICH in China57, and multiple studies have shown that early therapy is safe and feasible59. However, the results of the recently published A Very Early Rehabilitation Trial (AVERT) indicate that very early, intensive mobilization may be detrimental after stroke52. In this single-blinded, randomized controlled trial of 2,104 patients with ischemic or hemorrhagic stroke, patients who received very early mobilization (< 24 hours post-stroke with more frequent sessions) were more likely to have an unfavorable outcome than patients who received usual care52. In a pre-specified subgroup analysis, this effect was stronger in patients with severe stroke and ICH. Furthermore, providing early rehabilitation for patients with ICH is particularly challenging because these patients are often admitted to intensive care units and require close neurological and hemodynamic monitoring. One must ensure that rehabilitation interventions do not cause detrimental fluctuations in blood pressure and intracranial pressure that can lead to hematoma expansion, which is associated with worsened functional outcomes after ICH60.Most clinical guidelines support early initiation of rehabilitation after strokeReducing impairment and disability after ICH requires a multifaceted approach with advances in the acute medical and surgical treatment and rehabilitation of patients with ICH. Rehabilitation leads to significant behavioral improvements in animal models of ICH and is associated with enhanced neuroplasticity and reduced neuronal degeneration, although the mechanisms contributing to behavioral recovery are not yet understood. Clinical rehabilitation studies in patients with ICH have been limited; more studies are needed to characterize the natural history of recovery after ICH and to examine the effect of rehabilitative interventions specifically in this patient population. Therapies to enhance neuroprotection and neuroplasticity are also being developed and may be used in conjunction with rehabilitation to promote recovery.


Rehabilitation plays an important role in the physical health of patients with hemophilia. However, comprehensive information regarding the utilization of rehabilitation for such patients remains scarce.This population-based study aimed to examine the characteristics, trends, and most important factors affecting rehabilitation usage in patients with hemophilia A using a nationwide database in Taiwan.Data from 777 patients with hemophilia A who were registered in the National Health Insurance Research Database between 1998 and 2008 were analyzed using SAS 9.0.Musculoskeletal or nervous system-related surgical procedures and clotting factor VIII concentrate costs were identified as factors affecting rehabilitation usage; musculoskeletal or nervous system-related surgical procedures  were the most important predictor of whether a patient with hemophilia A would use rehabilitation services. Joint disorders, arthropathies, bone and cartilage disorders, intracranial hemorrhage, and brain trauma were common diagnoses during rehabilitation use. The costs of physical therapy (physiotherapy) comprised the majority (71.2%) of rehabilitation therapy categories. Increasingly, rehabilitation therapy was performed at physician clinics. The total rehabilitation costs were <0.1% of the total annual medical costs.Musculoskeletal or nervous system-related surgical procedures and increased use of clotting factor VIII concentrate affect the rehabilitation utilization of patients with hemophilia A the most. The findings in this study could help clinicians comprehensively understand the rehabilitation utilization of patients with hemophilia A. The National Health Insurance (NHI) program was launched in Taiwan in 1995. This universal program covers all insured individuals and has enrolled more than 99% of all Taiwanese citizens and legal residents, with premiums generally ranging from 2% to 5% of the total household income [Before the NHI programs were implemented, patients with hemophilia A received insufficient treatment with clotting factors. Since its initiation, the NHI has classified hemophilia as a catastrophic illness. This classification exempts patients with hemophilia from copayment for clinic visits or hospitalization associated with hemophilia-associated diseases or comorbidities.Although research regarding the treatment of hemophilia has progressed to cell  and geneTwo previous studies , 13 mentth edition, Clinical Modification (ICD-9-CM) diagnostic codes and procedure codes, catastrophic illness and medical service registries, prescription orders, details, and expenditures at contracted pharmacies, and inpatient and outpatient claims and copayments. The NHIRD is among the largest administrative health care databases worldwide, and related studies have increased rapidly in both quality and quantity [In Taiwan, the National Health Insurance Research Database (NHIRD) comprises de-identified personal data available for research purposes. This database includes important information such as beneficiaries, International Classification of Diseases, 9quantity  since thThe study protocol was approved by the Institutional Review Board for Human Studies at Chang Gung Memorial Hospital . We included 777 patients registered for catastrophic illness between 1998 and 2008 whose medical records listed ICD-9: 286.0 and concomitant clotting factor concentrate (CFC) usage . CFCs inIn previous articles, factors such as gender , 16, ageThe monthly insured amount was divided as < United States dollar (USD) 528 (New Taiwan dollar (NTD) 15840) and ≥USD 528 (NTD 15840) (conversion rate of NTD:USD = 30:1). Values of NTD 15840 represented monthly salaries at tier 1 according to the 38-tier insured amount designed by the NHI.2), the proportions of people with college or higher educational levels, those older than 65 years, agriculture workers, and the number of physicians per 100,000 people according to Taiwan census data from the year 2000. This classification method, which has been used in other studies, provides the most accurate urbanization levels of all 359 townships in Taiwan [Places of residence (township level) for each year from 1998 to 2008 were estimated according to Lin [n Taiwan , 22.Because musculoskeletal and nervous system-related procedures are frequently associated with rehabilitation utilization, the procedures involving ICD-9-CM procedure codes 01–059 (nervous system) and 76–8499  were recorded.The total costs for clotting factor VIII (including both human plasma and recombinant sources) were also recorded. Inhibitor status was considered the total cost of bypass agents, including activated prothrombin complex concentrate  and activated recombinant factor VII .In Taiwan, rehabilitation therapy is categorized as physical, occupational, or speech/swallowing therapy. The most common diagnosis (ICD-9-CM) associated with inpatient and outpatient rehabilitation prescriptions, the total costs in each rehabilitation therapy category and the number of rehabilitation users in terms of the number of rehabilitation therapy sessions received in both inpatient and outpatient services during 1998–2008, and the total number of rehabilitation therapy sessions at different medical institution levels were recorded. The total annual costs of inpatient and outpatient rehabilitation from 1998 to 2008 and the annual percentages relative to total medical costs were also surveyed. Recorded costs included all claims and copayments both with and without the catastrophic illness card used at NHI-contracted medical institutions. The number of patients who visited the same medical institution for outpatient rehabilitation and CFC prescriptions was also surveyed.t test and chi-square test, respectively. Variables were also used for the analyses with Pearson’s and Spearman’s correlation to check the collinearity. A logistic regression analysis was used to identify the most important factors for rehabilitation usage. A P-value <0.05 was considered statistically significant.SAS 9.0 for Windows  was used for data management and analysis. For factors affecting rehabilitation usage from 1998 to 2008, continuous and categorical variables were analyzed with the independent Joint disorders, arthropathies, bone and cartilage disorders, intracranial hemorrhage, and brain trauma were common diagnoses among the top 20 ICD-9-CM codes associated with rehabilitation prescriptions of outpatients and inpatients with hemophilia A shown in  Tables. In this study, musculoskeletal or nervous system surgery and clotting factor VIII concentrates costs were identified as factors predictive of rehabilitation usage in patients with hemophilia A. Musculoskeletal or nervous system surgeries comprised the most important factor affecting the use of rehabilitation among patients with hemophilia A. This suggests that a patient’s present need determines their decision regarding rehabilitation. Joint disorders, arthropathies, and bone and cartilage disorders were all listed among the top 20 diagnoses associated with rehabilitation. Preoperative physical therapy assessments could predict and establish the limits of postoperative rehabilitation for hemophilic patients aiming to receive elective orthopedic procedures . The impThe costs associated with clotting factor VIII might result from the practice of clotting factor VIII use in patients with hemophilia. Some patients may use clotting factor VIII prophylactically to avoid bleeding during rehabilitation. Also, increased usage of clotting factor VIII might reflect more severe hemophilia, which is associated with greater destruction of the major joints. Patients with greater joint destruction may require more rehabilitation resources to maintain an appropriate musculoskeletal condition.Our regression model did not identify urbanization level as a factor. Lin et al.  used theIn Taiwan, increasing numbers of physician clinics have been established to provide rehabilitation services. According to our data, although CFC prescriptions were all provided by hospital-level medical services, approximately 40% of the patients with hemophilia sought rehabilitation and CFC prescriptions at the same hospitals. In other words, patients chose to receive rehabilitation both at hospital-level medical services and at physician clinics providing rehabilitation therapy. Although increasing numbers of Hemophilia Treatment Centers, which provide a wide variety of multidisciplinary interventions, have been established at Taiwanese hospital-level medical institutions, it remains questionable whether these centers can induce patients to undergo rehabilitation at hospitals.Physical therapy accounted for the majority of costs among the 3 rehabilitation therapy categories. Physical therapy not only played a role in the conditions of patients before and after musculoskeletal surgery , but alsAlthough the rehabilitation costs have increased since 2004, these values have fluctuated without additional year-over-year increases. However, the proportion of rehabilitation costs among the total medical costs has decreased annually since 2005; this is likely due to the dramatic and rapid increase in the costs associated with CFC use, as these comprised the majority (>85%) of total medical costs among patients with hemophilia A in our study. However, rehabilitation costs, unlike CFC costs, did not exhibit persistent year-over-year growth since 2005. Since 2005, the government of Taiwan has allocated an independent budget for NHI-covered rare diseases (including hemophilia) to counteract deficiencies in medical expenditures for these diseases. If the annual costs for rare diseases exceed the initial independent budget, the overage would be diverted from other budgets. We believe that this change in policy had a stronger influence on CFC usage than on rehabilitation resource usage.In our study, rehabilitation costs accounted for <0.1% of the total annual medical costs. To our knowledge, few articles have reported the cost of rehabilitation usage relative to the total medical costs for patients with hemophilia. In Belgium, Henrard et al. reported that the costs of physiotherapist visits  accounted for 2.2% of the total direct costs for 1153 patients with hemophilia in 2011 . Ballal Our study shares the limitations common to retrospective database analyses. We were unable to obtain additional information regarding factors that influence rehabilitation usage. In addition, we were also unable to acquire the database after the year 2008 owing to the limited grant. Furthermore, the diagnoses were sourced from NHIRD according to ICD-9-CM codes. These diagnoses might have been less accurate than diagnoses collected prospectively according to standard procedures. Despite these limitations, our study provides a clear profile of rehabilitation usage among Taiwanese hemophiliacs.This is the first comprehensive study to describe nationwide rehabilitation utilization in patients with hemophilia A. Musculoskeletal or nervous system surgeries and clotting factor VIII concentrates costs were all factors predictive of rehabilitation usage. Joint disorders, arthropathies, bone and cartilage disorders, intracranial hemorrhage, and brain trauma were common diagnoses among those using rehabilitation services. Only nearly 40% of the patients, however, sought outpatient rehabilitation services and CFC prescriptions at the same hospitals. Rehabilitation therapy has been increasingly performed at physician clinics. It is essential to promote the use of rehabilitation resources to achieve better muscle and joint condition among this population. It is also imperative to enhance the professional abilities of therapists to care for hemophilic patients.S1 TableICD-9-CM, The International Classification of Diseases, Ninth Revision, Clinical Modification.(DOC)Click here for additional data file.S2 TableICD-9-CM, The International Classification of Diseases, Ninth Revision, Clinical Modification.(DOC)Click here for additional data file.


Now we studied the direct effect of physiological dose of metformin on human umbilical vein endothelial cells (HUVEC) under conditions mimicking hypoxia-hyperglycemia. HUVEC migration and apoptosis were studied after induction with euglycemia or hyperglycemia and/or CoCl2 induced hypoxia in the presence or absence of metformin. HUVEC mRNA was assayed by whole transcript microarrays. Genes were confirmed by qRT-PCR, proteins by western blot, ELISA or flow cytometry. Metformin promoted HUVEC migration and inhibited apoptosis via upregulation of vascular endothelial growth factor (VEGF) receptors (VEGFR1/R2), fatty acid binding protein 4 (FABP4), ERK/mitogen-activated protein kinase signaling, chemokine ligand 8, lymphocyte antigen 96, Rho kinase 1 (ROCK1), matrix metalloproteinase 16 (MMP16) and tissue factor inhibitor-2 under hyperglycemia-chemical hypoxia. Therefore, metformin’s dual effect in hyperglycemia-chemical hypoxia is mediated by direct effect on VEGFR1/R2 leading to activation of cell migration through MMP16 and ROCK1 upregulation, and inhibition of apoptosis by increase in phospho-ERK1/2 and FABP4, components of VEGF signaling cascades.Cardiovascular disease is the leading cause of morbidity/mortality worldwide. Metformin is the first therapy offering cardioprotection in type 2 diabetes and non-diabetic animals with unknown mechanism. We have shown that metformin improves angiogenesis via affecting expression of growth factors/angiogenic inhibitors in CD34 Cardiovascular disease (CVD) remains the leading cause of death worldwide according to the World Health Organization 2016 mortality database. The outcome of CVD management is affected by diabetes mellitus (DM), which results in a two- to four-fold increased risk of CVD ,4.Diabetes associated endothelial dysfunction is a known early step in the adverse sequence of events leading to the development of micro- and macrovascular complications, which results in mortality linked to diabetes in 75% of cases ,7. Furth+ cells under hyperglycemia-hypoxia [Pre-treatment of diabetic patients with metformin was found to be associated with reduced myocardial infarction (MI) size compared to non-metformin treated . Additio-hypoxia . HoweverIn this paper, we hypothesized that metformin at physiological dose enhances endothelial cell (EC) survival and migration under the conditions of hyperglycemia and hypoxia. As it is important for the research to be of translational value, we specifically applied clinically relevant physiological doses of metformin. Although cell culture systems have certain limitations in mimicking and simulating CVD; they are indispensable tools to unravel critical underlying molecular mechanisms of the disease. Therefore, we employed freshly isolated HUVEC from different donors as an in vitro model to study the effect of physiological concentration of metformin on cell survival, migration and gene expression under chemical hypoxia and hyperglycemia.2 at 18 (p < 0.01) and 24 h , 12 (p < 0.001), and 18 h , 12 (p < 0.001), 18 (p < 0.001), and 24 h of CoCl2  under hyperglycemia-CoCl2 at 24 h of CoCl2  in HUVEC treated with hyperglycemia for 48 h  in the apoptotic cells under euglycemic conditions (p = 0.018) under hyperglycemia-CoCl2  assay revealed that metformin led to a significant downregulation of HIF-1α protein levels at 1 h of CoCl2 under euglycemia-CoCl2  but exhibited no effect under hyperglycemia-CoCl2  and hyperglycemia . WB revealed a significant increase in HIF-1α protein at 1 h of CoCl2 under euglycemia-CoCl2  but no significant increase under hyperglycemia-CoCl2 , whereas the level decreased at 12 h of CoCl2  versus parallel conditions without metformin  under euglycemia  and hyperglycemia . qRT-PCR validation showed upregulation of VEGFA upon exposure to CoCl2 at 3 and 12 h under euglycemia-CoCl2 , and hyperglycemia-CoCl2  as illustrated in Transcriptome analysis showed a time dependent increase in 2 but was significantly upregulated at 12 h of CoCl2 under hyperglycemia-CoCl2  but not at 1 or 3 h (In WB VEGF 165A protein expression was unchanged under euglycemia-CoCl1 or 3 h B.p = 7.36 × 10−3) under euglycemia-CoCl2 at 12 h of CoCl2 but not under hyperglycemia-CoCl2. A number of VEGFR1 downstream genes was significantly differentially expressed under euglycemia and hyperglycemia following 12 h of CoCl2 exposure. Among these genes was the mitogen-activated protein kinase kinase 1 (MAP2K1), a key molecule in the ERK1/2 MAP kinase pathway, with 1.7-fold (p = 5.37 × 10−9) upregulated under euglycemia-CoCl2, and 1.6-fold (p = 6.8 × 10−8) upregulated under hyperglycemia-CoCl2. Moreover, gene expression of phosphoinositide-3-kinase, regulatory subunit 1 (PI3KR1) which is a PI3K/Akt signaling molecule, was increased at 12 h CoCl2 with euglycemia to 1.9-fold (p = 2.25 × 10−7) and hyperglycemia to 1.7-fold (p = 7.37 × 10−6). In contrast, PI3KR3 was detected to be downregulated by −2.5-fold (p = 2.09 × 10−9) under euglycemia-CoCl2 at 12 h of CoCl2.Several key signaling cascades, including ERK/MAPK and PI3K/Akt pathways are known to be implicated in the modulation of VEGF induced angiogenic and proliferation effects mediated via VEGFR1 . Microar2 at 3 h of CoCl2 .2 at 1, 3, 12 h of CoCl2 exposure  compared to hyperglycemia alone  and hyperglycemia  compared to euglycemia. However, hyperglycemia-CoCl2 led to decrease in VEGFR2 at 12 h of CoCl2  compared to hyperglycemia  under euglycemia-normoxia (p = 0.024) was observed under hyperglycemia-CoCl2 at 12 h of CoCl2  at 12 h of CoCl2 under euglycemia, to 33.8% (p = 0.05) under hyperglycemia alone, and to 7.5% (p = 0.001) at 12 h of CoCl2 under hyperglycemia-CoCl2  ligand 8 (CXCL8) to 1.9-fold (p = 2.84 × 10−2), lymphocyte antigen 96 (LY96) to 1.6-fold (p = 2.01 × 10−3), MMP16 to 1.7-fold (p = 2.34 × 10−2), and Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) to 2.7-fold (p = 1.15 × 10−2) that are known to enhance cell migration. Furthermore, metformin led to a significant overexpression of tissue factor pathway inhibitor 2 (TFPI2), a regulator of EC migration [p = 2.07 × 10−4) under hyperglycemia-CoCl2 at 12 h of CoCl2.Whilst, this effect was not observed without metformin leading to downregulation of LY96 to −1.7-fold (p = 5.4 × 10−4), and TFPI2 to −1.7-fold (p = 1.4 × 10−5). The most differentially expressed genes affected by metformin under hyperglycemia-CoCl2 are summarized in Microarray data showed that metformin, under hyperglycemia-CoClFABP4) mRNA levels were downregulated at 12 h of CoCl2 in euglycemia  and hyperglycemia . Conversely, the expression of FABP4 was significantly induced by adding metformin to HUVEC cultured under hyperglycemia-CoCl2 at 12 h of CoCl2 .Fatty acid binding protein 4  compared to control. There was a decrease in MMP16 expression under hyperglycemia-CoCl2 at 12 h of CoCl2  compared to hyperglycemia alone  and 24 h (p < 0.001) as shown in 2, marimastat had no effect on cell migration of ECs treated with metformin under euglycemia-CoCl2 versus parallel conditions without metformin  concentrations [Impaired angiogenesis in response to ischemia is elaborated as major macrovascular complications in DM . Additioeviously . Furthertrations .2 (1 h), and this may be beneficial as a protective mechanism against prolonged angiogenesis. For instance, 1 h of chemical hypoxia is a critical time defined in clinical practice when patients benefit from cardiovascular intervention following acute myocardial infarction.Vascular remodeling by cell migration and angiogenesis is regulated mainly by VEGF signaling . It has MMP16, FABP4, ROCK1, TFPI2, CXCL-8, and LY96 under hyperglycemia-CoCl2, which play a part in cell migration. The role of metformin on migration was confirmed in our experiments using VEGF and MMPs inhibitors. MMP16 is a membrane-type MT3-MMP that contributes to angiogenesis by degrading extracellular matrix components, thus promoting cell migration and bioavailability of growth factors [2 treatment was inhibited by marimastat, an MMP inhibitor.Our microarray experiments demonstrated that metformin upregulated downstream targets in VEGFA pathway;  factors ,27. Park factors . The int factors ,30. The  factors . A prior factors . This isFABP4 [2 at 12 h of CoCl2 enhancing cell survival and migration. FABP4 knockdown in HUVEC has been shown to decrease cell migration and increase apoptosis [ROCK1 under hyperglycemia-CoCl2, another moderator of cell migration. RhoA/ROCK signaling had previously shown to promote endothelial migration in response to VEGFA through the modulation of the actin cytoskeleton organization [ROCK activation appears to be a key event in the initiation of angiogenic process by mediating an increase in endothelial permeability and migration, whilst ROCK1 inhibitor suppressed cell migration as shown recently [CXCL8. This chemokine revealed pro-angiogenic properties by preserving VEGFA expression and secretion [A downstream mediator of VEGF/VEGFR2 in our study, FABP4  was founpoptosis , whilst poptosis ,35. Addinization . Moreoverecently . We alsoecretion , whereasecretion .A Kunitz-type serine proteinase inhibitor TFPI-2 , upregul2 through VEGF and activation of VEGF downstream signaling pathways. This was confirmed by using VEGF inhibitor (sunitinib) in the apoptosis assay. It is known that VEGF activates the phosphorylation of ERK1/ERK2 leading to activation of MAPK pathway, which is an essential step in determining the survival of EC [2, hyperglycemia, and hyperglycemia combined with CoCl2 at 12 h which is measured by MAPK activation dual detection assay. A previous study demonstrated that the level of phospho-ERK1/2 in astrocytes was elevated after 1 h ischemia and reached a maximal level after 4 h ischemia, before decreasing at 5 h resulting in enhanced ischemia-induced cell death [2, augmenting cell survival. Therefore, metformin may have properties to enhance cardioprotection from ischemia-reperfusion; however, we have not studied the reperfusion phase in our study.Our results demonstrate that metformin inhibited apoptosis under combined hyperglycemia-CoClal of EC . We founll death . In anotll death . In our 2. Previous studies have shown that VEGFA expression was significantly elevated in diabetic patients with chronic coronary heart disease, and the cellular response to VEGFA was attenuated due to defect in VEGF receptors and downstream signal transduction ,48. Our 2. In line with these findings, clinical studies confirm that metformin offers no benefit in cardiovascular outcome in non-diabetic individuals [Thus, the pivotal effect of metformin as documented by us was to restore the VEGFA pathway to non-diabetic state by augmenting the expression of VEGFR1 and VEGFR2 that transduce the vasoprotective downstream signalings of VEGF ; a mechaividuals ,51. Our 2 for 1, 3, or 12 h using a final concentration of 150 µmol/L CoCl2  as per protocol [2 exposure was utilized in functional assays . Metformin concentration was selected according to the peak plasma concentration suggested by FDA fact sheet [The study was approved by the Biomedical Ethics Unit, Faculty of Medicine, King Abdulaziz University  and NRES Committee North East-Sunderland, UK . HUVEC were harvested from three independent umbilical cords as previously described . All expprotocol  in the sct sheet .5 cells/well). Cells were incubated under euglycemia or hyperglycemia in the presence/absence of metformin for 24 h. Scratch lines were created on confluent HUVEC monolayers using 1000 µL pipette tip. The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor  to a final concentration of 10 µmol/L in DMSO [2 chamber (OkoLab) for 24 h that was connected to the camera . Cell migration was examined by phase-contrast microscopy  using software-based autofocus . Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area in each image was measured using NIS Elements software. Cell migration is presented as a percentage of gap closure and has been calculated by the following equation: Area (0) is the area of scratch pre-migration, Area (t) is an area of the scratch after migration at time t.HUVEC were subcultured onto 24 well plate  was performed according to the manufacturer’s recommendations and acquiring 10,000 events by FACSAria III flow cytometer . Total RNA from HUVEC was extracted using the RNeasy Mini Kit  and on-column DNase digestion with RNase-Free DNase set (QIAGEN) was performed according to the manufacturer’s instructions. The integrity of RNA samples was assessed by using Agilent 2100 Bioanalyzer  yielding high RNA Integrity Numbers (RIN) between 9.1 and 10. Microarray experiments were performed using Affymetrix  Human Gene 1.0 ST arrays according to manufacturer’s instructions with minor modifications ,56. ThreAffymetrix CEL files were imported into Partek Genomic Suite version 6.6  and then data were normalized and analyzed as described previously . The com® VILO™ cDNA Synthesis Kit  in a final volume of 20 μL. cDNA were quantified by hydrolysis probe real-time PCR performed with TaqMan® Universal Master Mix II (Life Technologies) using StepOne Plus Real-time PCR system (Life Technologies) according to the manufacturer’s recommendations. Expression of the selected genes VEGFA and MMP16 was validated using TaqMan gene expression assays . Three independent biological replicates were tested for each gene, and all samples were run in triplicates. The Comparative Ct (ΔΔCt) method was applied to the mean value of each triplicate to quantify expression of the target genes, which were normalized to reference control gene for the large ribosomal protein P0 [Total RNA (100 ng) from each sample was reversed transcribed to cDNA using SuperScriptotein P0   anti-mouse IgG, 0.5:5000; Li-COR) was applied. β-actin  was used as a loading control. Anti-VEGF showed many extra protein non-specific bands; therefore, we studied VEGF165A in detail. Bands were detected by infrared imager Odyssey (Li-COR) and quantified by Image Studio Lite version 3.1 . For stripping, bound antibodies were removed by incubation in RestoreTM Plus Western Blot Stripping Buffer . Protein was extracted using the mirVana PARIS  and the concentration was measured using Pierce Microplate BCA protein assay  according to the manufacturer’s protocol. The concentration was scanned using ELISA plate reader . Western blots were performed using commercially available kits (Life Technologies) XCell SureLock (Life Technologies) for protein fractionation, and Mini Protean II  for blot transfer. Nitrocellulose membranes (Life Technologies) were blocked with blocking buffer from Li-COR  and blocking buffer was diluted 1:2 with 1× TBS-T for incubation with antibodies. Transferred protein were probed for hypoxia-inducible factor-1α , VEGFA , and VEGF165A  and subsequently a secondary antibody , VEGFR-2 and phospho-VEGFR-2  were assessed by ELISA according to the manufacturers’ instructions. ® 647 conjugated antibody. HUVEC were treated with 5.5 mmol/L) or 16.5 mmol/L glucose concentrations in the presence/absence of metformin (0.01 mmol/L) for 48 h and then were exposed to CoCl2 for 12 h. All conditions were assayed in 2 or 3 independent replicates. Culture media and metformin were replaced every 12 h. For VEGF inhibitor assays, cells were incubated with EBM-2 medium containing VEGF as a positive control or with EBM-2 medium containing 7 µmol/L sunitinib malate as a negative control. Subsequently, 2 × 105 cells were fixed by an equal volume of ice-cold fixation buffer for 20 min and processed according to the manufacturer’s instructions by acquiring 14,000 events using FACSAria III flow cytometer.The activation of mitogen-activated protein kinase (MAPK) signaling pathway was measured by phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) using the FlowCellect™ MAPK Activation Dual Detection kit  which includes two antibodies, a phospho-specific anti-phospho-ERK1/2 -PE and, for quantification of total levels of ERK, an anti-ERK1/2-Alexa Fluort-test between any two experimental groups was applied for the MAPK activation dual detection assay and Annexin V Apoptosis assay. Calculations were performed using IBM SPSS software version 21.0 . A p-value < 0.05 was considered statistically significant.Results are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by post-hoc analysis Fisher’s least significant difference (LSD) test for qRT-PCR, ELISA, western blot, and in vitro scratch assays. The Student’s MMP16 and ROCK1 upregulation, in addition to inhibition of apoptosis by increase in phospho-ERK1/2 and FABP4, components of VEGF signaling cascades.In conclusion, our in vitro study demonstrates that the effect of metformin in acute hyperglycemia-chemical hypoxia model is mediated through restoring VEGFA pathway. This occurs through upregulation of VEGFR1 and VEGFR2, leading to dual effect on activation of cell migration through


Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7.We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7.Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments.LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted.The online version of this article (10.1186/s12935-018-0568-1) contains supplementary material, which is available to authorized users. Over 40,000 men are diagnosed with prostate cancer, and over 10,000 die from advanced metastatic disease, annually in the UK. Initial treatment of metastatic disease is reliant on inhibiting androgen receptor (AR) signalling by systemic androgen deprivation therapy (ADT). Although almost all patients respond to ADT, disease progression, to a clinical phenotype termed castration resistant prostate cancer (CRPC), occurs in virtually all patients. Metastatic CRPC is almost uniformly lethal within a median of 2–3 years and is commonly accompanied by significant symptomatic and healthcare burden [Treatment options after transition to CRPC have developed significantly in the last few years. Approaches that are established to extend survival include further hormonal intervention with either the new generation AR antagonist enzalutamide or the CYP17A1 androgen synthesis inhibitor abiraterone acetate –5. TheseAs a conventional nuclear steroid hormone receptor transcription factor, AR activation involves ligand binding , phosphorylation and homo-dimerisation, nuclear translocation, formation of co-regulator complex interactions with chromatin modifiers, androgen response element (ARE) sequence binding within target gene promoters and AR target gene expression and suppression. Non-genomic/non-ARE mediated effects also occur through AR activation .Androgen deprivation therapy failure and transition to CRPC occurs through multiple mechanisms including expression of constitutively active AR point mutants and splice variants, AR co-regulator expression changes, AR expression change, altered AR ligand sensitivity/promiscuity and signalling pathway crosstalk . Critically we have no current routinely available means within the clinic to detect resistance mechanisms to either initial ADT or subsequent therapies –12.Intense interest currently surrounds the V7 splice variant of the AR which lacks a ligand  binding domain leaving a constitutively active N-terminal. AR-V7 expression represents a relatively common cause of transition to a CRPC phenotype and a cause of resistance to enzalutamide or abiraterone. Recent data found that AR-V7 detection in circulating tumour cells was possible in 19–39% of CRPC patients who were naive to new generation hormonal agents. AR-V7 expression was associated with almost complete loss of biochemical prostate specific antigen (PSA) response to enzalutamide or abiraterone and reduced median survival . SubsequCo-regulators of AR transcriptional activation include epigenetic mediators that induce chromatin remodelling at AR response element sites , 15. A kLSD1 expression is increased in prostate cancer compared with benign prostate and its expression correlates with higher Gleason score, risk of relapse and distant metastases, and reduced survival post prostatectomy , 21. HisWe and others have shown that chemical inhibition of LSD1 inhibits cell proliferation and AR signalling in prostate cancer cells , 23–25. l-glutamine and 1 mM pyruvate. For experiments, LNCaP cells were incubated for 24 h and then media was changed to RPMI-1640  supplemented, for the remainder of the experiment, with 10% charcoal stripped FBS (Life Technologies). Dihydrotestosterone (DHT) and 5α-Androstan-17β-ol-3-one were from Sigma-Aldrich; LSD1 inhibitors LSD1-C76 and HCI-2509 were from Xcess Biosciences; enzalutamide and apalutamide were from Selleckchem. Chemical inhibitors of LSD1 were synthesised, and a separate publication will describe full details of the preparation.Cell lines were maintained in Dulbecco’s Modified Eagle’s Medium  or Roswell Park Memorial Institute (RPMI) medium  supplemented with 10% fetal bovine serum (FBS) with 4 mM (HEK293) or 2 mM (LNCaP) 50 values for LSD1 activity after chemical inhibition were measured as previously described [2O2 using AmplexRed® (Invitrogen).ICescribed . Briefly® 2.0 Assay (Promega) according to the manufacturer instructions. Luminescence was measured using a Varioskan Flash Multimode Reader (Thermo Scientific).Cell proliferation assays were performed by incubating cells in 96 well plates in RPMI complete medium. After 24 h, different drugs or a DMSO control were added and analysis of proliferation was determined after 72 h using the CellTiter-GloCells were harvested and lysed by resuspension in protein sample buffer. DNA from samples was fragmented by sonication. Total protein from each sample was separated using the Laemmli method . After bKDM1A (LSD1). For the selection of the guide sequence oligonucleotide we used an online tool for CRISPR design (http://crispr.mit.edu/). Guide sequence oligonucleotides were, top 5′ caccgTGTGTTTTGATCGGGTGTTC 3′ and bottom 5′ aaacGAACACCCGATCAAAACACAc 3′.LSD1 knockout was undertaken in HEK293 cells according to the protocol from Zhang et al. . Briefly® Reporter Assay System (Promega) in a Varioskan™ Flash Multimode Reader (Thermo Scientific). All results show firefly luciferase activity normalised to renilla luciferase activity to control for transfection efficiency.Luciferase assays were performed using a vector (pARE-Luc) with the firefly luciferase gene under the control of the androgen receptor response element (ARE) in HEK293 cells co-transfected with peAR-Wt and peAR-V7 plasmids and with a renilla luciferase vector used as a transfection efficiency control as previously described . Briefly® High Sensitivity kit (Active Motif) according to the manufacturer’s instructions. Immunoprecipitation of the chromatin bound DNA was performed using the following antibodies from Active Motif: Histone H3 K9me3, Histone H3, Histone H3 K9me2, Histone H3K9me1. The ChIP-IT® Control Kit, Human was used with these assays as a control.CHIP assays were performed using the ChIP-IT® Universal PCR Master Mix, No AmpErase® UNG (ThermoFischer Scientific) and TaqMan® Gene Expression Assays probes for KLK3 (PSA) and for the reference gene GAPDH. Assays were performed in an Applied Biosystems 7500 Real Time PCR System. Quantification of mRNA expression levels was performed using the 2−∆∆C(T) method as described [Total mRNA extraction was performed using the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. Contaminating DNA was digested using RQ1 DNAse (Promega). Synthesis of cDNA was performed using M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s instructions. For qRT-PCR we used the TaqManescribed .1, Additional file http://www.clinicaltrials.gov) and a set of five second-generation tranylcypromine analogues  or endogenous expression (22Rv1 cells). We therefore propose that the impact of LSD1 on the androgen receptor is likely not lost by transition to a castration resistant phenotype where this arises through AR-V7 activity and regardless of the WT/V7 co-expression status. This would imply that AR-V7 retains a requirement for complex formation with LSD1, for functional activation. Having established that AR-V7 driven prostate cancer remains susceptible to targeting of LSD1, at least in the model systems used here, it will be important to dissect the molecular interaction between chemical LSD1 inhibition and an LSD1:AR interaction. Proper understanding of the molecular basis for a therapeutic effect will be critical to facilitate exploitation of this strategy. In addition, we fully acknowledge that LSD1 inhibition might function as a therapeutic strategy through other mechanisms beyond targeting of AR-WT and AR-V7.Our results would suggest broadly that depletion of LSD1 activity was able to reduce AR-V7 activation by about 50% in most of the experimental models that we utilised. This was seen in LSD1 knockdown models and so implies that this may be more than a simple ‘drug potency’ issue. It is likely that other epigenetic influences on AR activation remain relevant and thus combinatorial approaches may be required for optimal suppression of AR function . To whatAR-V7 splice variant models of CRPC are amenable to therapeutic targeting through LSD1 inhibition. This raises the opportunity to develop this therapeutic strategy further.Additional file 1: FIgure S1. Figure S1 Molecular structure of the LSD1 inhibitors used in this work. Figure S2. In vitro LSD1 inhibition assay for the indicated compounds. Data points show percentage of LSD1 activity when compared with the DMSO treated sample for three separate experiments. Figure S3. Cell proliferation assays for the indicated compounds in LnCAP prostate cancer cells. Data points show percentage luminescence compared to DMSO solvent exposed control samples after 72 h for a minimum of three separate experiments in each case. Figure S4. Androgen response element (ARE) luciferase reporter assay, in response to androgen receptor (AR) antagonists, for the AR wild type (WT) or V7 splice variant forms. (A) Luciferase reporter assay of ARE promotor activation in HEK293 cells co-transfected, as indicated, with either AR WT or V7 expression vectors, and incubated as indicated with dihydrotestosterone , enzalutamide (10uM) or apalutamide (10uM). Data are mean values ± standard deviation from four replicate determinations. P values were derived using a two-way ANOVA for comparison followed by Tukey's multiple comparison post hoc tests. (B) Western blot to confirm AR expression following transfection of either the WT or V7 or Q640X splice variant forms or an untransfected sample, as indicated, in HEK293 cells. ****, P < 0.0001; ns, non-significant; NT, non-transfected. Figure S5. Western blot analysis of LSD1 expression for different clones obtained after the CRISPR experiment. (A) immunoblot for the N-terminus of human LSD1. (B) immunoblot for the C-terminus of human LSD1. Actin expression is shown as a protein loading control.


Rehabilitation medicine plays an integral part in attainment of optimal functioning after injury or disease. The National Rehabilitation Policy of South Africa (NRP) (2000) highlights the need for access to professional health care services, redistribution and optimal utilisation of resources and research in the field of disability and rehabilitation. The government further ratified the Convention on the Rights of Persons with Disabilities (CRPD) (2007), which validate the urgency in advancing the agenda of persons with disabilities. This paper outlines the methodological plan for evaluating rehabilitation services in the Western Cape, South Africa against the aims and objectives of the NRP as well as its principles and concepts. The evaluation process further focused on specific articles in the CRPD that were aligned with disability, health and rehabilitation.A mixed-method design was used to evaluate the alignment of rehabilitation services with the NRP in the Western Cape. Four rehabilitation study settings were selected to ensure that both inpatient and outpatient rehabilitation levels of care were covered at different contexts . The sites were checked for the most prevalent rehabilitation-related conditions to ensure the identification of suitable instruments for measuring rehabilitation outcomes. Each study setting was linked to two researchers with one exploring the rehabilitation organizational structure of the sites and the other exploring the client outcomes after receiving rehabilitation services. Patients were evaluated at baseline and discharge, within seven days after admission and seven days prior to discharge. The evaluation was based on the rehabilitation organizational capacity to provide patient-oriented rehabilitation and the measurement of rehabilitation outcomes. Kaplan’s framework of organisational capacity was used in the context of each study setting. For the measurement of service users’ outcomes, the International Classification of Functioning, Disability and Health was used (ICF). Standardised outcome measures were adopted for the domains of impairment, activity and participation. The World Health Organisation Community-Based Rehabilitation guidelines were used as guiding principles and concepts as suggested in the NRP.This is a groundbreaking methodological exploration that offers both study methods and instruments to measure rehabilitation services at both in-patient and out-patient rehabilitation services. The aim of this paper is to present the protocol for the process of developing methodological steps for the realistic evaluation  of instiThe evaluation of service delivery systems is listed by Mitchel  as a focThe reforms of 1994 in South Africa necessitated adjustments of existing health policies and the development of new ones. The overarching aim was to improve access to health services especially to the poor. Primary health care was identified as the vehicle to deliver health services. Rehabilitation is the third pillar of the primary health care service model .To improve accessibility of rehabilitation servicesTo establish mechanisms for intersectoral collaboration in order to implement a comprehensive rehabilitation programme.To facilitate appropriate allocation of resources, and encourage their optimal utilisation.To facilitate human resource development that takes into account the needs of both the service providers and the consumers.To encourage the development and implementation of monitoring and evaluation strategies for rehabilitation programmes.To ensure participation of persons with disabilities in planning, implementation and monitoring of rehabilitation programmes.To encourage research initiatives in rehabilitation and related areas .According to the World Health Organisation, rehabilitation aims to enable persons with disabilities to reach and maintain their optimal functional levels by providing them with the tools they need to attain independence and self-determination . To achiThere is a need to evaluate the effects of these seven objectives on clients who have attended institution-based rehabilitation services in the Western Cape. Service delivery models that use the medical model of rehabilitation are rejected by disabled people’s organizations because of the emphasis on adjusting the person to the existing systems and norms through – mainly – medical and therapeutic interventions . In spitAs a practical means of adopting a rights-based approach to rehabilitation services and equalization of opportunities for disabled people, the NRP has adopted Community Based Rehabilitation (CBR) strategy and describes it as an approach to delivering services and not the service itself . At the The recently ratified United Nations Convention on the Rights of Persons with Disability (UNCRPD) is the latest instrument that raises issues of equity for persons with disability. It calls for governments to mainstream disability in policies of their countries. South Africa is the first African country that ratified the UNCRPD. Rehabilitation features strongly within the UN Convention with articles 9, 19, 20, 25 & 26 giving clear indicators regarding how to respond to the rehabilitation needs of the disabled people. These articles resonate in the objectives of the NRP . This evA small team of three researchers started planning an evaluation of one site in the Western Cape. The approach was to demonstrate the effect of rehabilitation with the aim of convincing the provincial government of the need for allocation of resources for rehabilitation service in this site. It was during this period of proposal writing for this one site that a wider call came from SANPAD inviting people to send proposals for a collaborative research study. The Centre for Rehabilitation Studies offers advanced interdisciplinary postgraduate rehabilitation studies. It is a platform for the disability and rehabilitation debate and is in partnership with the Provincial Government and Stellenbosch University to ensure evidence-based input into the programs and policies of the Province. Hence, it was in excellent position to coordinate this collaborative research. Initially, a comprehensive team of approximately 20 people joined the research team and developed a reference group for the project. The team was a mixture of young and more mature rehabilitation professionals from public institution-based rehabilitation services and academic departments . The proposal was accepted and funded by SANPAD that allowed the reference group the opportunity to implement the evaluation plan of the four sites.Firstly, we wanted to evaluate whether the NRP with its seven objectives is aligned with the rehabilitation services at the four sites in the Western Cape.Secondly, we wanted to measure whether rehabilitation services as an organizational structure in the four sites can deliver comprehensive rehabilitation services.Thirdly, we wanted to measure, using rehabilitation outcome measures, whether the patients that enter the four study settings do receive comprehensive rehabilitation.Lastly we wanted to align our findings with the 7 objectives of the NRP and do a gap analysis with regard to whether the NRP has lived to its promises.Rehabilitation services in the Western Cape are subject to several national and provincial policies. The rehabilitation of physically disabled persons in the Western Cape takes place at various levels of health care and at a variety of institutions. This realistic evaluation research will critically analyze services rendered at four sites within the public sector. The context, mechanisms and outcomes (CMO) for each site were investigated. The sites were purposively selected as each site represents a different level of intervention and serves a different population. The Western Cape Rehabilitation Centre (WCRC) is a specialized in-patient rehabilitation centre that offers both in and out-patient services. In patient referrals come from the Western, Eastern and Northern Cape, while out-patient therapy is limited to patients in the direct vicinity of the Centre. For inpatient rehabilitation, the WCRC admits annually approximately 1160 clients with mainly neurological health conditions. The five most prevalent health conditions constitute about 94% of all clients admitted to the centre Table . The ComA series of steps were developed using aspects of the realistic approach to evaluation research and taking into account the concept of the context of the rehabilitation services and the rehabilitation outcomes of the clients receiving these services , 16. MixThe capacity of the four sites as organizational structures to deliver rehabilitation servicesAnd the rehabilitation outcomes of the patients receiving the services as well as client satisfaction.Continuous discussions were held by the research group to develop a unified conceptual framework for the evaluation study. Discussions concentrated on:It has already been specified in the discussion of the NRP that the NRP has seven objectives and links to CBR and the UNCRPD. We concluded that these will be used as yardsticks to evaluate the ability of the rehabilitation services in the four sites to deliver rehabilitation services. On one hand we decided to develop indicators based on the seven objectives of the NRP, aspects from the CBR matrix and 5 health and rehabilitation articles from the UNCRPD. We saw that the fulfillment of these indicators by the four sites would give us the capacity to deliver rehabilitation services based on the organizational framework by Kaplan . ValidatWhen looking at the ability of the four sites to deliver rehabilitation services to clients, we decided to use Kaplan’s model of evaluating organizational capacity . Kaplan At the bottom of the hierarchy are quantifiable and measurable elements of capacity which can be worked with. The elements nearer the top of the hierarchy are not easily assessed and are observable only through the effects they have and they largely determine the organizational capacity. The framework describes the elements of capacity but it cannot predict or determine change processes. When tensions arise between the needs of the health care institution and those of the team and the client, a conceptual framework can help to retain the client focus . In thisThe NRP was seen as having promised certain deliverables through its objectives and principles, hence key indicators were drawn from the objectives of the National Rehabilitation Policy, the 5 relevant articles of the UNCRPD and the Community Based rehabilitation model, and we finally determined how these indicators were aligned with the 6 elements of organizational capacity named by Kaplan. Questionnaires were developed from these indicators for clients, service providers and managers to evaluate the service delivery of the four sites. A description of the methodological steps used to evaluate the alignment of the service provision of the four sites in the western Province with the NRP.The research team searched for instruments to be used in this study; however, no suitable questionnaires were found which could be used to ask relevant questions to the service providers and the managers. Various questionnaires were found which addressed some of the issues that needed to be interrogated but there were none that were able to interrogate all the objectives of the National Rehabilitation Policy, the selected articles of the UNCRPD, the CBR principles and the ICF. Also during the questionnaire design, we utilized some of the primary health care criteria for the delivery of comprehensive primary health care, for example accessibility, acceptability, affordability and equity. Therefore the research team designed three questionnaires, one for service providers, one for the manager of the centre and one for the manager of the non-governmental organization.The research team of the SANPAD funded study was tasked to do an in depth analysis of the objectives of the National Rehabilitation Policy, the relevant five articles of the United Nations Convention on the rights of Persons with disabilities, the Kaplan Organizational Model, Community Based Rehabilitation, the International Classification of Function and Disability framework and key Human Rights Documents. All documentation was analysed by the group and all possible questions drawn up to interrogate each of the seven objectives of the National Rehabilitation Policy. Next, the five relevant articles of the UNCPRD were analysed and questions drawn up relating to these five articles. These questions were then added to the original questions. The group checked that the objectives of Community Based Rehabilitation were also included as well as Human Rights issues.A spreadsheet was drawn up with indicators and the questions relating to each indicator. The relationship between each question and the five elements of the Kaplan Model was then determined. Three key informant groups were then decided upon. These four groups were the service providers, the manager of the Community Health Centre and a Non-governmental organization manager and persons with disabilities who were the consumers of the services. Questions relating to catchment area as well as the rehabilitation human resource provision were answered by the facility managers. Persons with disabilities participated in responding to questions related to participation of persons with disabilities and health service-related questions. We also explored issues on the structures and procedures and those related to guidelines on clinical treatment, availability and training of staff, procurement of resources as well as administration-related questions. For some questions, there were more than one key informant. Draft questionnaires were then drawn for the key informant groups. The fourth group of key informants was the rehabilitation service clients and standardised questionnaires were used that answered the relevant questions. The standardised questionnaire was mainly based on the ICF checklist for environmental factors. Once the questionnaires had been piloted and the adjustments made, they were sent to an expert in managing rehabilitation services and an expert in rehabilitation research for validation purposes. Minor changes were indicated, and suggestions were integrated to reflect the intended content.Moving from capacity of institutions rendering rehabilitation services to outcomes, the ICF was used to assist in the understanding of the complex interaction between features of the biological, psychological, cultural and social factors of disability . In thisAs well as recognising the impact of individual bodies and social factors in understanding disability, the main principle of the biopsychosocial model is to understand disability as a continuum. In analysing this notion of rehabilitation critically, it can be seen that using the ICF incorporates both the medical and social models in rehabilitation. For example, rehabilitation inputs can be focused directly on activity and participation, or might target body function issues  , 20. ThiThe process started with the identification of the five most prevalent conditions at each site Table . To be iFollowing the review, it was found that no suitable instrument was available to measure impairments and participations restrictions related to each of the health conditions. A condition-specific instrument was therefore developed to measure impairments, standardized measures were used to collect data concerning activity limitations of the five conditions at each site, and a previously used generic instrument was adapted and validated to collect participation data. In addition three questionnaires were developed to collect contextual information. The development of these instruments is presented below. The study on the rationale, design and pilot of the impairment outcome measure is in progress.Following a focus group discussion of therapists, researchers and academics involved in this project, a condition-specific impairment measure for each health condition was developed. For the development process, all primary impairments related to the health condition, given that it is routinely assessed in practice, were identified. The impairment variables were identified via the aforementioned study group consisting of therapists, researchers and academics, the ICF framework on body structures and functions , the ICFA data extraction sheet was developed and distributed to experts in the field of rehabilitation with the aim of ensuring the face and content validity. All questions and recommendations from the expert panel were considered and integrated, with the final version approved by them. In addition, the inter-rater reliability of the impairment extraction sheet was conducted on the medical records of 20 patients. Two reviewers independently captured the data from the 20 patient records. The data were captured and analysed using Statistical Package for Social Sciences version 18. The Intraclass Correlation Coefficient (ICC) of all the items averaged together for both raters ranged between 0.471 and 1, which indicates moderate to perfect agreement .A number of suitable outcome measures were available to measure activity limitations of all health conditions covered in this study. All the standardised measures decided on presented with good psychometric properties and translated versions were available for most. In cases where translated versions were not readily available, we initiated the process and we are currently busy with its validation. The Barthel Index was used to assess activity limitations in persons with stroke, amputations and traumatic brain injury, whereas the spinal cord independence measure III was used among those living with a spinal cord injury, and the Screening Activity Limitation and Safety Awareness outcome measure was used among persons with peripheral neuropathy. For back pain, we used both the Oswestry ODI version 2.0 and the Clinical Mobility Scale. For persons with arthritis and hands/upper limb injuries, we used the Arthritis Impact Measuring Scale and the Disability of the Arm Shoulder and Hand, respectively –31.Since limited participation measures are available, especially generic outcome measures, the Zambian survey on living conditions that was utilised to study the influence of physical disabilities on activity and participation of patients in Zambia  was adapConcerning health-related quality of life, the EQ-5D was used across all participating centres and health conditions. This measure consists of five items that are thought to best represent an individual’s quality of life in the presence of a disabling health condition. The EQ-5D has been widely used in South Africa for the valuation of quality of life, and the measure presents with adequate psychometric properties, as tested in the local context  Table 4.The first questionnaire, to be completed on admission, comprised five sections. Section one covered personal demographic factors  and medical information such as diagnosis of health condition, cause/nature of disability, and presence of co-morbidities. Section two described the socio-economic status of the participants, i.e. income, the participants’ monthly income, number of members in household, source of income and highest educational level. Section three described information related to the participants’ primary residence and the availability of basic needs that include running water, electricity, telephone and ablution facilities, while section four described information related to availability of transport for attending rehabilitation services. The last section related to involvement in prior research.The questionnaire completed at discharge comprised three sections. The first section covered participants’ awareness about their rights to rehabilitation and their participation in social roles and community life activities. Section two described information about the assistive devices received and the participants’ level of satisfaction with these appliances, and section 3 described the services and interventions patients received during rehabilitation.This study was largely descriptive in nature, with the focus on determining organisational capacity to deliver relevant services and the measurement of outcomes following rehabilitation. Since we selected commonly used standardised measures – specific to the selected health conditions –in the study, our sample size was determined by using the formula specific to a one-group confidence interval for means. Therefore, with confidence limits set at 95% and a desired total width of 5 for the confidence limits based on the mean score, and a standard deviation of 10 for the outcome variables , it was necessary to recruit at least 61 participants per site in order to detect a sufficiently powered mean value for the respective populations per site.This is a groundbreaking methodological exploration as it has managed to combine instruments that are pointing to both evidence and human rights. To classify rehabilitation as an organizational structure by using Kaplan’s conceptual framework of an organizational capacity, it managed to offer the evaluation process and measurement of clients' outcomes. It is important to clarify that thought the ICF is an important outcome measure to evaluate rehabilitation services, the implementation of this tool often requires the use of other sensitive outcome measures in evaluating activity limitation and participation restrictions in clients with impairments. Having designed both the study methods, accompanied by instruments, we believe that we now have a methodology to measure rehabilitation services at both in-patient and out-patient levels of rehabilitation services. This approach using a realistic evaluation philosophy also make this methodology adaptable to any setting thereby allowing researchers the ability to use this approach in numerous studies.At the time of manuscript submission the trial is in an ongoing phase of data management and analysis.