Both treatments allowed for functional and pain improvements with respect to pre-operative levels lasting up to 100 months. MRI revealed consistent cartilage repair at 24 months in both groups. This study shows that AMIC and AMIC+ are effective treatments for focal chondral lesions with beneficial effect lasting up to 9 years. AMIC+ allows for faster recovery from injury, and is thus more indicated for patients requiring a prompt return to activity. Level of evidence: II, randomized controlled trial in an explorative cohort.The aims of the study were to evaluate long-term outcomes after autologous matrix-induced chondrogenesis (AMIC) in the treatment of focal chondral lesions and to assess the possible improvements given by the combination of this technique with bone marrow aspirate concentrate (BMAC). Twenty-four patients (age range 18–55 years) affected by focal knee chondral lesions were treated with standard AMIC or AMIC enhanced by BMAC (AMIC+). Pain ) and functional scores , Tegner, Knee injury and Osteoarthritis Outcome Score (KOOS)) were collected pre-operatively and then at 6, 12, 24, 60, and 100 months after treatment. Magnetic resonance imaging (MRI) evaluation was performed pre-operatively and at 6, 12, and 24 months follow-ups. Patients treated with AMIC+ showed higher Lysholm scores ( Articular cartilage injuries represent an issue in current orthopedics surgery, due to the high prevalence in the active population and to the low healing potential of hyaline cartilage . Bone maAMIC may benefit from the association with other biological procedures such as the addition of platelet-rich plasma (PRP) that can further stimulate the tissue repair ability of cells deriving from the microfracturing of the subchondral bone . HoweverBased on these observations, a prospective randomized study was performed to compare the clinical outcomes of standard AMIC technique versus the AMIC technique enhanced by the use of autologous BMAC harvested from the iliac crest (AMIC+) for the treatment of focal chondral defects. The hypothesis of the study is that the addition of BMAC would have a positive effect in improving the functional outcomes given the higher number of progenitor cells delivered to the injury site. 2, and normal surrounding cartilage . Patients with more than two chondral defects, immuno-mediated pathologies including osteoarthritis, knee infection, untreatable instability, malalignment or meniscal tears, serious cardiologic pathologies, or problematic general conditions were excluded from the study. All the patients were informed about the study and signed an informed consent. Immediately before surgery, the patients were randomly assigned either to the standard AMIC  or to the concentrated bone marrow aspirate (BMAC)-enhanced AMIC technique  . Between December 2007 and February 2010, 34 patients with clinical and radiological diagnosis of chondral defects received the indication to undergo an AMIC procedure at our institution . Twenty-four of them satisfied the following criteria and were included in the study: age between 18 and 55 years, one or two grade III or IV articular surface lesions of the knee according to the International Cartilage Repair Society’s chondral defect classification system (ICRS) [ n = 12) . The ranTM device  according to the manufacturer’s instructions. At the end of the centrifugation a phase enriched in mononuclear cells, including mesenchymal progenitors, was obtained. Meanwhile, a mini-arthrotomy  was performed to allow access to the defect. Using a template, the collagen type I/III bilayer matrix  was then cut according to the dimensions and the size of the defect. Microfracturing of the subchondral bone was performed following Steadman’s technique [TM, Arthrex, Naples, FL, USA). For the standard AMIC group the matrix was positioned on the defect and fixed with synthetic fibrin glue . For the AMIC+ group, before being placed on the lesion, the shaped matrix was dipped into the concentrated bone marrow aspirate for 10 min, according to a previously described technique [The surgical interventions were performed by two senior surgeons. Tourniquet was used for all patients; after checking the inclusion/exclusion criteria during the arthroscopy, the lesion was debrided, removing degenerated cartilage and creating a regular-shaped lesion. For each patient in the AMIC+ group, a sample of 24 mL of bone marrow was harvested from the anterior iliac crest and added to 6 mL of Anticoagulant Citrate Dextrose Solution A (ACD-A). As suggested by several authors, bone marrow was collected by multiple localizations (about 4–5 mL per sampling) to avoid the peripheral blood dilution effect ,23; the echnique , and theechnique .The immunophenotype of cells cultured for two passages in human mesenchymal stem cells growth medium  was analyzed by FACSCalibur™  and CellQuest™ analysis software, after staining with the following conjugated antibodies: anti human CD45-PE, CD34-PE, CD31-PE, CD90-PE, CD73-PE, CD166-PE, CD44-PE , and CD105-FITC . Cells were incubated with saturating concentrations of the appropriate antibodies for 30 min at 40 °C. The physician in charge of the clinical controls as well as the radiologist were blinded to the allocation of patients in the study groups. Pre-operatively and then at 6, 12, and 24 months from intervention, the Visual Analogue Scale (VAS) , LysholmPre-operatively, all patients underwent magnetic resonance imaging (MRI) with a 1.5 Tesla scanner to assess the location, size, and severity of the chondral defect, as well as any other soft tissue damage including the menisci or ligamentous structures by T1 weighted sequences. Structural regeneration of the cartilage defect was then assessed at 6, 12, and 24 months by an independent radiologist, with a focus on the extent, signal intensity, and surface of the defect filling, integration to adjacent cartilage, and bone marrow lesion (BML). This adapted scoring system takes into account a variety of features that are currently believed to be relevant to the integrity of cartilage repair tissue as used in the Magnetic resonance Observation of CArtilage Repair Tissue (MOCART) score  after ACBoth groups followed the same rehabilitation program. In case of condylar chondral defect, immediate full range of motion was allowed without any weight bearing for 3 weeks, then full bearing was allowed after the sixth week; in case of patellar defects, patients were allowed to progressively restore the full range of motion and bearing starting from the early post-operative days. All patients were advised to return to sports after 4–6 months from surgery, depending on the sport.2 test was used to analyze the categorical score (IKDC). Analyses were performed with Graphpad Prism v5.0 . Data are expressed as means ± standard deviations (SD). Values of p < 0.05 were considered statistically significant. At the time of project design, it was not possible to perform a reliable sample size calculation due to a complete lack of data about the outcome of AMIC. For this reason, the power of our statistical significances was calculated ex post with Gpower software v3.1.9.2 [All data were collected in an electronic database. Analysis of variance was performed on continuous data , whereas the χv3.1.9.2 . Differen = 12) received the intended treatment they were randomly assigned to and were analyzed for the primary outcome. Two patients for each group were lost within 5 years of follow-up, resulting in 10 patients per group at that time point. In the AMIC group, one of the patients had arthrosynovitis that provoked his exclusion from the study 1 year after surgery. We are not aware of any other failure or patients who underwent a successive surgical procedure. For each group, all the patients . In all concentrated samples, the nucleated cells demonstrated the typical MSC surface markers expression pattern, as reported in In vitro analysis of bone marrow samples revealed a difference in terms of MSC concentration among patients, with an average concentration factor of 3-fold in the concentrated bone marrow samples with respect to whole bone marrow  as well as pre-operative scores were homogeneous between the two groups . With thp < 0.05; −72%, p < 0.001 in the AMIC+ group). At the 12-month follow-up, the VAS was not significantly improved with respect to the 6-month assessment, but patients treated with AMIC+ demonstrated a significantly lower pain score with respect to the AMIC group . This difference was no longer present at the 24-month follow-up, when both groups showed the minimum level of pain recorded (p < 0.001 with respect to pre-operative levels in both groups). The VAS remained constant up to 100 months of follow-up, with only a slight increase for the AMIC group at the latest follow-up. However, it was always significantly lower than the pre-operative level (p < 0.05). The VAS  significp < 0.001). A similar situation was observed at the 12-month follow-up, where the difference between the two groups was significant in favor of the AMIC+ group . After 24 months from surgery both groups presented significant Lysholm score improvements with respect to baseline (p < 0.001). At the 60- and 100-month follow-ups a slight progressive reduction of this score was observed, although AMIC+ patient scores always remained significantly higher than baseline (p < 0.001 at both follow-ups). The Lysholm score  improvedThe evolution over time of the Tegner activity scale  demonstrp < 0.05). On the contrary, no significant difference was observed in the standard AMIC group at any time point. The objective IKCD scores improved in both groups 6 months after surgery . At lateIn both groups the KOOS score was satisfactory, in particular for pain and daily activities subsections up to the 100-month follow-up. A slight progressive decrease with time was observed for sport and quality of life (QOL). No differences were observed between the AMIC and AMIC+ patients . MRI analysis was affected by a poor patient compliance in the standard AMIC group, where just five and two patients participated at the 12- and 24-month follow-ups, respectively. On the contrary, in the AMIC+ group, just two dropouts were recorded between the 12- and 24-month follow-ups. Nevertheless, it was possible to observe improved surface appearance and MRI signal in the AMIC+ group .Graft integration was achieved in higher proportions of patients in the AMIC+ group at the 6-month follow-up, while it was comparable between the two groups 12 months after surgery. Defect size and defect filling were similar in the two groups at each follow-up. Reduction in bone marrow lesion was observed in just 30% of the whole cohort of patients (6/20) at 24 months ; given tThe main finding of this study is that both AMIC and AMIC+ allowed for stable significant clinical and functional improvements in patients with focal knee chondral defects up to approximately 9 years. The patients treated with the AMIC technique augmented with autologous concentrated bone marrow aspirate from iliac crest showed significantly better results in functional recovery with respect to the patients treated by the standard AMIC technique at the 12-month follow-up. MRI analysis confirmed this observation, since a higher proportion of patients showed improvements in the AMIC+ group at the 12-month follow-up with respect to patients treated with the standard AMIC technique, although the decreased number of patients analyzed does not allow for any statistically significant difference.At later time points the outcome of the two groups were comparable, thus allowing to hypothesize that a larger number of mesenchymal progenitor cells is beneficial in the initial phases of cartilage repair, providing an acceleration of the healing process. With the exception of one patient in the AMIC group who developed arthrosynovitis, no other adverse event or complication was recorded. According to the available records, none of the patients required additional interventions to the treated knee during the time of observation, although we cannot exclude this event for the patients for whom we were not able to complete the last follow-up.To our best knowledge, this is the longest follow-up reported in the literature for the treatment of chondral lesion of the knee with the AMIC technique, as well as the only randomized prospective study providing a direct comparison of the standard technique with that enhanced by BMAC. The very satisfactory clinical results recorded almost 9 years after surgery in both groups of patients showed that AMIC, independently from the addition of bone marrow concentrate, provides stable improvements in patients affected by isolated chondral lesions, even in a population mainly composed by patients practicing high-level sports activities. The AMIC technique consists in the use of a synthetic matrix aimed to retain the blood clot deriving from subchondral bone and to provide a 3D environment on which the progenitor cells can adhere and proliferate, favoring their differentiation process. In fact, it has been demonstrated that bone marrow mesenchymal cells are able to efficiently differentiate in vitro towards different cell types, including chondrocyte-like cells, under proper stimuli such as the 3D culture environment, which enhances the ability of cells to acquire the new phenotype ,34,35. HThe stable results observed in this study are consistent with other reports in the literature of long-term follow-ups after AMIC procedures, where Lysholm and IKDC scores remained significantly higher with respect to pre-operative levels , as well® collagen membrane, Bioteck S.p.A., Oviglia, Italy) enhanced by BMAC at a 2.5-year follow-up. This study, although suffering from the lack of a control group and a limited follow-up, showed a statistically significant improvement in the mean IKDC subjective score and Lysholm score. Similar to our results, the median Tegner activity scale was not significantly different between the pre-injury and post-operative levels, confirming the efficacy of this technique at least at short-term follow-up. Interestingly, the authors showed the histological analysis of four patients 1 year after the treatment. The biopsies scored a mean overall ICRS II score of 64 and in one case a hyaline-like matrix was found, though it lacked the typical columnar cell arrangement [A paper by Enea et al.  reportedangement . ® in a small number of patients; however, these improvements were not confirmed by MRI [Other biologics were associated with the use of matrix-assisted microfracture techniques, in particular platelet-rich plasma (PRP). In a cohort of 52 patients treated with PRP-augmented polyglycolic-acid (PGA)/hyaluronan scaffold, the authors reported a statistically significant clinical improvement at the 12-month follow-up . Other ad by MRI . The main limitations of the present study are the limited number of patients and the heterogeneity of the chondral defect sites. Nevertheless, the statistical power of our observation was above 60% for all the analyses. Moreover, due to the unavailability of objective IKDC scores at the later follow-ups, the long-term outcomes rely on subjective questionnaires, which suffer from high variability among individuals. Another limitation is the poor compliance of standard AMIC patients to the radiological observations at the 24-month follow-up. In addition, a more specific MRI protocol, such as the delayed gadolinium-enhanced MRI of cartilage (dGEMRIC), may have given better and more detailed information about the regeneration process of the cartilage. In conclusion, this study demonstrates that both AMIC and AMIC+ are effective treatments for knee chondral lesions, providing durable results over time. Indeed, the findings demonstrate that both standard and enhanced AMIC procedures assure functional and pain improvements up to 9 years from surgery. AMIC associated with BMAC had better short-term clinical outcome, which could be due to the higher number of progenitor cells in the beginning of the regeneration process, indicating that this augmentation could be considered for patients with higher functional requirements such as competitive athletes.


Diagnosis of lung cancer frequently occurs in its later stages. Low-dose computed tomography (LDCT) could detect lung cancer early.Our objective was to estimate the effect of LDCT lung cancer screening on mortality in high-risk populations. A systematic review of randomised controlled trials (RCTs) comparing LDCT screening programmes with usual care (no screening) or other imaging screening programme (such as chest X-ray (CXR)) was conducted. RCTs of CXR screening were additionally included in the network meta-analysis. Bibliographic sources including MEDLINE, Embase, Web of Science and the Cochrane Library were searched to January 2017. All key review steps were done by two persons. Quality assessment used the Cochrane Risk of Bias tool. Meta-analyses were performed.p = 0.62). There was a statistically non-significant increase in all-cause mortality. Given the considerable heterogeneity for both outcomes, the results should be treated with caution.Four RCTs were included. More will provide data in the future. Meta-analysis demonstrated that LDCT screening with up to 9.80 years of follow-up was associated with a statistically non-significant decrease in lung cancer mortality (pooled relative risk (RR) 0.94, 95% confidence interval (CI) 0.74 to 1.19; Network meta-analysis including the four original RCTs plus two further RCTs assessed the relative effectiveness of LDCT, CXR and usual care. The results showed that in terms of lung cancer mortality reduction LDCT was ranked as the best screening strategy, CXR screening as the worst strategy and usual care intermediate.LDCT screening may be effective in reducing lung cancer mortality but there is considerable uncertainty: the largest of the RCTs compared LDCT with CXR screening rather than no screening; there is imprecision of the estimates; and there is important heterogeneity between the included study results. The uncertainty about the effect on all-cause mortality is even greater. Maturing trials may resolve the uncertainty. Lung cancer is the most common cancer in the world with 1.8 million new cases diagnosed in 2012 . The maiHowever, prognosis is related to stage at diagnosis. The 1-year survival rate is over 80% when lung cancer is diagnosed in stage I, but under 20% when diagnosed in stage IV , this beOver several decades, a number of potential screening tests for lung cancer have been investigated including chest x-ray (CXR) and sputum cytology. Neither of these has been found to be effective . As compThe National Lung Screening Trial (NLST) has been particularly influential comparing LDCT screening with CXR . 53,454 In the UK, population lung cancer screening is currently not carried out by the NHS, based on the recommendation of the UK National Screening Committee in July 2006 when they last assessed whether lung cancer screening should be recommended for adult cigarette smokers. They concluded that it should not be recommended but should be reviewed in 2015/2016. The research we report was commissioned to update the systematic review which underpinned the last guidance , 11.Our objective was to evaluate the clinical effectiveness of screening programmes for lung cancers with LDCT in high-risk populations using a systematic review, meta-analysis and network meta-analysis of RCTs. The wider project also considered cost-effectiveness. The systematic review was registered (PROSPERO CRD42016048530). All aspects of the work were undertaken in accordance with a pre-specified protocol  with somWe searched MEDLINE, MEDLINE In-Process, Embase, PsycINFO , Web of Science (Thomson Reuters), CDSR and CENTRAL (via The Cochrane Library) and CINAHL (EBSCO) from 2004 to January 2017. . RCTs evaluating the effectiveness of CXR but not LDCT were also included in the network meta-analysis. The outcomes of interest for this analysis were lung cancer mortality and all-cause mortality.Two researchers independently screened the titles and abstracts of all reports identified by the search strategy. Full-text papers were subsequently obtained and screened in the same way. Data extraction and quality assessment were undertaken by one researcher and checked by a second. The risk of bias of included studies was assessed using the Cochrane Risk of Bias tool . We also2 statistic. Based on the advice in the Cochrane handbook, 30% to 50% was categorised as moderate heterogeneity; 50% upwards as substantial heterogeneity [All data were tabulated and primarily considered in a narrative review. DerSimonian and Laird random-effects models meta-analyses were performed to pool the estimates of effect . We restogeneity . We consNetwork meta-analysis was performed to assess the relative effectiveness of three screening strategies . A multivariate random-effects meta-analysis using restricted maximum likelihood approach was performed . We estiStatistical analyses were performed using the ‘metan’ ‘mvmeta’ and ‘network’ commands in Stata 14  , 21.From 9655 records identified in the searches, four RCTs were included in the meta-analysis of mortality data , 22–25 aThe characteristics of the included studies are shown in Table Looking at the 12 included RCTs in the qualitative review  , was howThe additional RCTs for the network meta-analysis were of slightly poorer methodological quality than most of the LDCT RCTs, with greater lack of clarity about loss to follow-up and absence of power calculations. A mitigating factor may be that standards for reporting RCTs were not well established in the 1970s when the studies were conducted with the first Consolidated Statement of Reporting of Trials version being published in 1996 . The PLCp = 0.62) with up to 9.80 years of follow-up when compared with controls , given which the results should be treated with caution. A range of potential sources for heterogeneity were investigated. When removing the poor quality trial (MILD) [p = 0.02) in favour of LDCT screening compared with controls .The direct meta-analysis showed that LDCT screening was associated with a statistically non-significant decrease in lung cancer mortality  , sensitip = 0.95) with up to 9.80 years of follow-up  detected between studies, the results from this pooled analysis should be treated with caution. We also investigated the potential sources of heterogeneity. When removing the low-quality trial (MILD) [p = 0.05) compared with controls , suggesting that variation in trial quality could be a potential source of heterogeneity between studies.The direct meta-analysis also showed that, compared with controls, LDCT screening demonstrated a statistically non-significant increase in all-cause mortality outcome  , sensitip value of 0.29, indicating no evidence of inconsistency.Network meta-analysis assessed the relative effectiveness of LDCT, usual care and CXR screening. The main network consisted of three RCTs comparing LDCT with usual care –24; one p = 0.18)   or other imaging screening programme (such as chest X-ray (CXR)) are a statistically non-significant decrease in lung cancer mortality  and a statistically non-significant increase in all-cause mortality outcome . The network meta-analysis agrees with the direct meta-analysis that LDCT is the most effective screening option between LDCT, CXR and usual care, but also raises the possibility that CXR screening may be less effective than usual care. This in turn has implications for the interpretation of the largest RCT which compares LDCT with CXR rather than usual care, the most relevant comparator in trying to decide whether LDCT screening should be introduced.There is thus considerable uncertainty about the effect of LDCT screening on mortality, particularly the size of the effect. Contributors to this uncertainty beyond the relevance of the main included RCT, are the width of the 95% CI (which include no effect) and the statistical heterogeneity between the included study results. Further, although a RR of 0.94 looks like a useful effect, the rarity of the outcomes in the trials needs to be taken into account (4.7 lung cancer deaths per 100 persons over an 8-year period was found in the Detection And screening of early lung cancer with Novel imaging Technology and molecular Essays (DANTE) RCT , which iThe research we report was undertaken by an experienced health technology assessment group, working to a pre-specified protocol, informed by comments from a steering group with wide expertise. The technology assessment as a whole was informed by a patient and public involvement exercise. No members of the research team had any connection with the research teams doing any of the included RCTs. Although likely to increase in the foreseeable future, our main limitation was the small number of included studies. This limited our ability to investigate the heterogeneity between study results and to explore other important phenomena like publication bias. We also did not have opportunity to systematically contact each of the original research teams which may have helped fill some of the gaps in details about the RCTs, particularly randomisation methods. We were conscious that selected enquiry of particular studies might in itself introduce bias.Our findings are not consistent with recent guidelines by the US Preventive Services Task Force  nor withOn balance, the evidence does not yet clearly support the case for population LDCT screening and any final decision should await the results of trials in progress. Our recommendation is that the evidence should be re-reviewed when the trials in progress are reported. This should include additional detailed investigation about study quality of all RCTs as we have provisionally identified this as a potential explanatory factor of the heterogeneity between the four RCTs published so far. Up-to-date reviews of other outcomes and cost-effectiveness modelling will also be important aspects of further research. These have already been conducted as part of this project and are reported in a recent publication .Additional file 1. Web resources. Web Table S1-Table S6, Figure S1-S2.


Plantar fasciitis is a common cause of foot pain presenting with morning stiffness and plantar area pain. This case study is to optimize the management in patient with plantar fasciitis accompanied by apparent high-arch foot.A 55-year-old women presented with plantar fasciitis accompanied by apparent high-arch foot. The pain presents for the heel and pelvic areas with long-distance walking.She was diagnosed with plantar fasciitis accompanied by apparent high-arch foot for physical examination and plain radiographs. In muscle performance and dynamic postural stability tests, indicated the muscle weakness and postural instability.The patient was treated with manual therapy such as joint and soft tissue mobilization interventions including plantar fascia and gastrocnemius stretching, but the heel and pelvic pain were aggravated during long-distance walking. After hip strengthening exercises, the heel and pelvic pain significantly improved.The 3-month follow-up revealed that the heel and pelvic pain did not occur in the long-distance walking, and there was no pain and discomfort at one year follow-up.To optimize the management in patient with plantar fasciitis accompanied by apparent high-arch deformity, clinicians should try to identify the hip abductor muscles weakness, and therapists should consider incorporating hip strengthening exercises. To date, physical therapy clinical practice guidelines have emphasized the importance of manual therapies such as joint mobilization and soft tissue mobilization to improve limited ankle range of motion (ROM). However, recent studies have reported that PF was not directly related to limited ankle ROM.,7Plantar fasciitis (PF), one of the most common causes of foot pain and presents with morning stiffness and plantar area foot pain.,3,8,9 strengthening exercises are often recommended to address excessive foot pronation and supination, which can increase the stress on the soft tissue structures and plantar fascia. Recent studies have reported that foot postures such as apparent low-arch foot (pronated) and high-arch foot (supinated) were associated with weakness of the hip abductor muscles,,11 since the hip muscles play an important role in lower limb biomechanics. Herein, we report a case of PF with apparent high-arch foot that was successfully treated by hip strengthening exercises. The patient was informed that data concerning the case would be submitted for publication of the case, and she provided consent.Since PF is associated with foot posture,2A 55-year-old women  presented with right foot pain that developed 1 year ago. The initial symptoms began in the forefoot and heel regions, and she had received a one-time steroid injection at another hospital. Thereafter, there was a slight decrease in forefoot pain. However, the pain rapidly increased 5 months after the steroid injection and was focused in the heel area. The heel pain was aggravated immediately after rest, such as while getting out of bed in the morning. She also complained of intermittent right pelvic pain without radiculopathy and low back pain. The pelvic pain was aggravated with prolonged standing or after long-distance walking and decreased with rest. Although there was only minor heel pain during weight-bearing, it was aggravated with single-leg weight-bearing. The heel area and the right-side pelvic pain showed a visual analogue scale (VAS) of 8 and 3 during single-leg weight-bearing, respectively. and a pedobarograph system were used to measure the foot arch and foot pressure , function (50 points), and alignment (10 points), and pain (50 points), disability (90 points), and activity limitation (30 points), respectively. FAAM ADL consisted of 21 questions . A lower score in the AOFAS-AHS and FAAM ADL is associated with poor functional activities, while it is the opposite in the FFI. The initial scores were 32 of 100 points in the AOFAS-AHS, 60 of 170 points in the FFI, and 31 of 84 points in the FAAM ADL.Foot function was measured using foot questionnaires such as the American Orthopedic Foot and Ankle Society Ankle-Hindfoot Scale (AOFAS-AHS), Foot Function Index (FFI), and activities of daily living (ADL) subscale of the Foot and Ankle Ability Measure (FAAM).Muscle performance testing was conducted using a quantified isokinetic device  and performed in a semi-sitting position with a slight knee flexion. The test consisted of muscle strength (5 at 30°/s) and endurance (15 at 120°/s) for dorsiflexion and plantarflexion and hip abductors, with a rest time of 30 s. The dynamic postural stability test was evaluated using the Biodex Stability System . BSS provides a 20° tilt and a 360° rotation of the platform, and the patient was instructed to maintain a single-leg posture while the stability level automatically declined from level 12 (most stable) to level 1 (most unstable). Each test consisted of 2 trials of 20 s each. The muscle performance and dynamic postural stability tests  were performed immediately after the evaluation of the Silfverskiold test. After 20 minutes of manual therapy intervention, heel pain was diminished (VAS 8 to 5). Accordingly, instructions for a self-mobilization intervention to improve dorsiflexion ROM were given to the patient to perform at home. The patient was treated 2 times per week for 2 weeks for 30 minutes, and she performed the home exercise program 3 times per day for 30 minutes. However, heel pain did not improve any further, and pedobarograph examination still showed foot pressure concentrated on the lateral forefoot and heel area .Initial manual therapy such as joint and soft tissue mobilization interventionsrea Fig. B.[20] FuThe patient experienced increased calf muscles spasms during muscle performance and dynamic postural stability tests. Therefore, the physiotherapist decided to change the treatment protocol to include strengthening exercises, such as plantarflexion muscle and hip and core muscles training, including the gluteal muscles, along with single-leg postural control reeducation. After the application of strengthening exercises, the symptoms of heel and pelvic pain were significantly diminished during walking and single-leg standing  and foot pressure on pedobarograph reexamination also showed a remarkable change Fig. C. In add2.2The patient was treated 2 times per a week during the 10 weeks with manual therapy and strengthening exercises. All parameters, including heel and pelvic pain , AOFAS-AHS, FFI, and FAAM ADL , dorsiflexion ROM , foot pressure (Fig. 3,21,22 Therefore, weakness of the plantarflexion muscles can increase the load on the plantar fascia.,23 In this patient, the muscle performance test showed plantarflexion muscle weakness, and increased calf muscle spasms were observed during walking, which indicated that plantarflexion muscle weakness was one cause of the aggravated heel pain.The patient in this case study had a previous history of PF treatment and presented with apparent high-arch foot with concurrent intermittent pelvic pain. The initial treatment strategy was manual therapy based on the physical therapy clinical practice guidelines for PF. Although the heel pain was slightly decreased after the initial manual therapy intervention, it still increased during walking and single-leg standing. We believe that the increasing heel pain during walking may have been caused by weakness of the plantarflexion muscles. The main roles of the plantarflexion muscles are to prevent excessive loading on the plantar fascia and to maintain the longitudinal arch.,25 including the function of decreasing forefoot plantar peak pressure during gait. In this patient, increased compensatory mechanisms of the hip joint to decrease forefoot plantar peak pressure from forefoot pain may have led to fatigue and weakness of the gluteal muscles, which resulted in aggravation of the right pelvic pain during walking. Conversely, increased compensatory mechanisms of the ankle joint caused by weakness of the hip abductor muscles may have caused the intractable heel pain. Weakness of the hip abductor muscles during walking may result in plantarflexion muscle overuse, which can increase the load on the plantar fascia. Hip abductor muscles contribute to postural stability in the frontal and sagittal planes of the lower extremities, and weakness can cause Trendelenburg gait, resulting in the lateral shift of concentrated pressure to the forefoot and heel areas. Our patient showed poor dynamic postural stability and Trendelenburg sign, suggesting that the heel and pelvic pain were likely caused by hip abductor muscle weakness. Based on the results of this study, we believe that strengthening exercises, especially for the hip joint, may be an effective method to improve various PF symptoms in patients with pelvic pain.The right pelvic pain in this patient had persistently increased during long-distance walking. We believe this may have been due to weakness of the hip abductor muscles. Previous studies reported that the hip abductor muscles play a significant role in the biomechanics of walking and ankle stability,4This case highlights the importance of hip muscle strengthening for the optimal management of PF patients with apparent high-arch foot and concurrent intermittent pelvic pain. To optimize the management in PF patient with apparent high-arch deformity, clinicians should try to identify the hip abductor muscles weakness, and therapists should consider incorporating hip strengthening exercises.Conceptualization: Jin Hyuck Lee, Jong-Hoon Park, Woo Young Jang.Data curation: Jin Hyuck Lee.Formal analysis: Jin Hyuck Lee.Supervision: Jong-Hoon Park.Writing – original draft: Jin Hyuck Lee, Jong-Hoon Park.Writing – review & editing: Woo Young Jang.Woo Young Jang orcid: 0000-0003-1775-7715.


Relapsing polychondritis (RPC) is a rare progressive autoimmune disease characterized by inflammation in the cartilage of multiple organs. Tracheobronchial involvement appears in nearly half of RPC patients during the course of their disease and represents the main cause of death. Localized tracheobronchial RPC is much rarer, and the pathogenesis remains unclear. Matrilin-1 is a non-collagenous cartilage matrix protein and has been suggested to be a potent autoantigen that induces the airway disease of RPC in animal models. However, the expression of matrilin-1 in tracheobronchial tissue in human remains unclear. Therefore, we examined the expression of matrilin-1 in the tracheal and auricular tissues in a localized tracheobronchial RPC patient.A 62-year-old man with systemic sclerosis presented with cough and dyspnea on exertion. The lung function test showed an expiratory flow limitation and chest computed tomography showed diffuse thickness from the trachea to the bronchiole. No other tests showed abnormal findings. To evaluate further, bronchoscopy was performed and endobronchial ultrasonography showed thickness in the fourth-marginal echo layer suggesting inflammation of the cartilage. However, the tracheal biopsy showed no specific findings. The subsequent surgical tracheal biopsies showed inflammatory cell infiltration with destruction of the cartilage. Neither auricular nor nasal deformity, except for a tracheobronchial lesion, was detected. Biopsy from the left auricular cartilage also did not show any inflammatory changes. Finally, we diagnosed the patient with localized tracheobronchial RPC. To address the hypothesis that autoimmunity against matrilin-1 is involved in the pathogenesis of localized tracheobronchial RPC, we evaluated the expression level of matrilin-1 in a tracheal and auricular specimen from this patient. Immunohistochemical staining with anti-matrilin-1 antibody showed matrilin-1 in the tracheal but not in the auricular cartilage.We first demonstrated the expression of matrilin-1 in tracheal but not in auricular cartilage in a localized tracheobronchial RPC patient. This result supports the possibility that matrilin-1 is involved in the pathogenesis of localized tracheobronchial RPC. However, this is only one case report and further observations will be needed to confirm this result. Relapsing polychondritis (RPC) is a rare progressive autoimmune disease affecting cartilaginous structures including ear, eye, nose, larynx, trachea, bronchus, joints and heart valves , 2. TracMatrilin-1, one of a four-member family of oligomeric extracellular adaptor proteins , has beeA 62-year-old man who had systemic sclerosis presented with cough and dyspnea on exertion. Lung function test showed an expiratory flow limitation Fig.  and chesTo clarify the hypothesis that autoimmunity against matrilin-1 is involved in the pathogenesis of localized tracheobronchial RPC, we evaluated the expression level of matrilin-1 in tracheal and auricular specimens from this patient. We performed immunohistochemical staining of the tracheal and auricular tissues using antibodies against matrilin-1  after antigen retrieval by autoclaving the slides. As we expected, immunohistochemical staining with anti-matrilin-1 antibody showed that matrilin-1 immunoreactivity was detected mainly in the boundary region of cartilage in the tracheal Fig. a, c, butRelapsing polychondritis (RPC) is a rare systemic inflammatory disease affecting the cartilaginous structures, and diagnosis of RPC is based on criteria suggested by Michet, et al , 3, 12. Occasionally, RPC has been known to coexist with other autoimmune diseases , 19. AntAt present, some types of cartilage antigens are presumed to be involved in the RPC pathogenesis. One of the candidates is type II collagen, which consists of 95% of the total collagen in cartilage, and anti-type II collagen antibody is detected in one third of RPC patients, the same as in rheumatoid arthritis , 20. In In conclusion, we first demonstrated the expression of matrilin-1 in tracheal but not auricular cartilage in a patient with localized tracheobronchial RPC. This result supports the possibility that matrilin-1 could be involved in the pathogenesis of localized tracheobronchial RPC. However, this is only one case report and further observations will be needed to confirm this result.


This review covers the current knowledge about plant stanol esters as a dietary treatment option for heterozygous familial hypercholesterolemia (he-FH) children. The current estimation of the prevalence of he-FH is about one out of 200–250 persons. In this autosomal dominant disease, the concentration of plasma low-density lipoprotein cholesterol (LDL-C) is strongly elevated since birth. Quantitative coronary angiography among he-FH patients has revealed that stenosing atherosclerotic plaques start to develop in he-FH males in their twenties and in he-FH females in their thirties, and that the magnitude of the plaque burden predicts future coronary events. The cumulative exposure of coronary arteries to the lifelong LDL-C elevation can be estimated by calculating the LDL-C burden (LDL-C level × years), and it can also be used to demonstrate the usefulness of dietary stanol ester treatment. Thus, when compared with untreated he-FH patients, the LDL-C burden of using statin from the age of 10 is 15% less, and if he-FH patients starts to use dietary stanol from six years onwards and a combination of statin and dietary stanol from 10 years onwards, the LDL-C burden is 21% less compared to non-treated he-FH patients. We consider dietary stanol treatment of he-FH children as a part of the LDL-C-lowering treatment package as safe and cost-effective, and particularly applicable for the family-centered care of the entire he-FH families. This review discusses the current knowledge about plant stanol esters as a dietary treatment option for heterozygous familial hypercholesterolemia (he-FH) children. Since the majority of reviews on dietary phytosterols (the common term for plant sterol and plant stanols) focus on plant sterols ,2, we soIt is estimated that as many as 4.5 million people in Europe suffer from the heterozygous form of familial hypercholesterolemia . The strLDLR) gene [(apoB) [(PCSK9) gene [LDLR gene, and indeed, so far, more than 1,700 mutations in this gene have been discovered, while much fewer mutations in the apoB gene or in the PCSK9 gene cause FH [FH is the most common inherited metabolic disease, and it is caused by a mutation in the low-density lipoprotein receptor (LR) gene , apolipo [(apoB) , or the K9) gene . Most cocause FH ,13,14,15cause FH ,17,18,19Although a healthy diet is an essential part of the treatment of he-FH children, according to current recommendations, medical treatment in he-FH is also necessary already in childhood starting from the age of 8 to 14 years ,24,25,26As stated already in the Introduction, the term phytosterols is used to cover both plant sterols and plant stanols. Phytosterols can be extracted from vegetable oils and are also present in nuts, fruits, and vegetables ,30. The In the Cochrane review including only controlled studies, in which cholesterol-lowering diets were compared with dietary interventions aimed at increasing the intake of plant stanols, only one study was found in which he-FH children were involved . This stIn a study of 19 he-FH families in the Finnish North Karelia, dietary treatment with stanol ester margarine was examined . A totalRegarding ho-FH children, it should be noted that in the study carried in an FH-family having two heterozygous parents and one homozygous child, dietary consumption of either plant stanol esters or plant sterol esters reduced the levels of serum cholesterol in the heterozygous parents by about 14% and in the homozygous child by about 9% . HoweverAccording to the recent guidelines provided by The Task Force for the Management of Dyslipidaemias of the European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS), functional foods with plant stanols  may be considered in children (from the age of six years) with he-FH . This reOnly very few studies exist in which hypercholesterolemic patients, and especially he-FH patients, are on a stanol diet on top of statin treatment ,40. One n = 21), and of these patients, four had consumed and six had not consumed plant stanol or plant sterol ester supplements regularly (duration not characterized) prior to the surgery, while no information on supplement consumption was available from 11 patients. It appeared that the higher the intestinal absorption of cholesterol, the higher the plan plant sterol contents in the stenotic aortic valves. Moreover, the sterol/cholesterol-ratios in serum closely correlated with those in the valves. However, no differences in plant sterol concentrations in either serum or aortic valves between individuals with (n = 4) and without (n = 6) using plant stanol or sterol ester supplements were detected. In the second study by Weingärtner et al., of 82 consecutive patients admitted to hospital for elective aortic valve replacement, a structural interview revealed that 10 patients had consumed plant sterol esters either irregularly or regularly for up to 4 years [n = 11) or with plant stanol ester (n = 12) or plant sterol ester (n = 13) supplementation until valve replacement surgery  [Interestingly, statin treatment induces increased plasma plant sterol levels, while plant stanols decrease such statin-induced elevation of circulating plant sterol levels . Accordi 4 years . Patientaverage) . In thisAnother issue has been the concern related to the adherence to statin therapy if the stanols are used as a part of the diet. Eussen et al.  showed tA study by computed tomography angiography has shown that in he-FH patients on non-optimal statin therapy, atherosclerotic plaques started to develop in males and females at a mean age of about 20 and 30 years, respectively . AdditioThe cumulative LDL-C burden can be demonstrated by LDL-C burden = LDL-C levels × years of age . We haveThere is a saying on the website of the FH Foundation: “We say we never find an individual with FH; we only find families with FH . Such a It has been shown that he-FH patients underestimate their risk of suffering from heart disease later on in life and they are also stressed by the uncertainty about the ability of the health behavior changes to reduce their risk of CHD . Thus, wPositive information from family members can be also potentially used to enforce the motivation for CHD prevention. Dietary stanol consumption in the whole family could be this positive non-stressful preventive measure. Furthermore, it is possible to proceed step-by-step with a young he-FH child in a family starting with dietary stanol treatment from six years age toward starting the statin treatment when the child is about 10 years old. The first step is that the whole family, including the non-FH members, starts stanol consumption as a part of their daily eating habits, as has been shown in previous study of FH families . As noteThis review discusses the current knowledge about plant stanol esters as a dietary treatment option for he-FH children. It is estimated that as many as 4.5 million people in Europe alone suffer from the heterozygous form of familial hypercholesterolemia, and accordingly, the numbers of he-FH children must also be very high. Quantitative computed tomography angiography studies among the he-FH patients have shown that atherosclerotic plaques start to develop in males and females in their twenties and thirties, respectively, and that such early plaque burden significantly predicts premature coronary events. The cumulative LDL-C burden can be calculated by multiplying the LDL-C level by the years of age, and it can be used to demonstrate the usefulness of dietary stanol treatment. We consider dietary stanol consumption as a component of the LDL-C-lowering package to be safe, cost-effective, and applicable not only for an he-FH child, but also for the whole he-FH family. Dietary stanol treatment serves, at its best, as a supportive measure for the early start of a lifelong treatment of the he-FH child.


Synthesis of a triporous nanographene with 102 sp2 carbon atoms by combining solution and surface chemistry. 2 carbon atoms by combining solution and surface chemistry. The carbon skeleton was obtained by Pd-catalyzed cyclotrimerization of arynes in solution, while planarization of the molecule was achieved through two hierarchically organized on-surface cyclodehydrogenation reactions, intra- and inter-blade. Remarkably, the three non-planar [14]annulene pores of this nanographene further evolved at higher temperatures showing interesting intra-porous on-surface reactivity.The synthesis of porous nanographenes is a challenging task for solution chemistry, and thus, on-surface synthesis provides an alternative approach. Here, we report the synthesis of a triporous nanographene with 102 sp Recently, nanographenes have attracted much attention in materials sciences due to their unique optoelectronics properties.72 carbon atoms by the on-surface cyclodehydrogenation of a starphene derivative through the formation of three C–C bonds 4 led to the isolation of dodecaphenyl[7]starphene 2 in 37% yield. Then, we attempted the preparation of porous nanographene 1 by DDQ-promoted cyclodehydrogenation of compound 2 in solution. Unfortunately, this approach was unsuccessful since we only detected the formation of nanographene 5 as the result of nine intra-blade cyclodehydrogenations. Based on our recent experience,1 by on-surface cyclodehydrogenation of 5 through inter-blade C–C bond formation. However, the deposition of compound 5 on a gold surface by sublimation from an effusion cell proved to be infeasible, most probably due to significant intermolecular interactions. Then, we decided to study sequential on-surface cyclodehydrogenations of trimer 2 as explained below.We started the synthetic route by preparing triflate 3 , which s2 on Au(111), we found that these molecules tend to gather into disordered π-stacked assemblies, whose structures are difficult to determine by STM. This is due to the high degree of conformational flexibility due to the rotation of the peripheral phenyl rings. This flexibility combined with the steric hindrance between neighboring phenyl rings leads to the molecules adopting a non-planar geometry. On a metal surface, which is known to catalyse cyclodehydrogenation reactions,2 into the polycyclic three-bladed propeller 5. Each of the wide propeller blades would be then composed of 9 rings. It is instructive to note that the transformed molecule would retain the non-planar character due to the steric interactions between the outermost rings of neighboring propeller blades within the necking of the conjoined bay regions.After thermal deposition of precursors 2 on Au1, we fou5 was formed as the major reaction product  as documented in 1. The above described observations suggested that at 340–380 °C inter-blade fusion between neighboring propeller blades of the intermediate compound 5 was initiated. The formation of a single C–C bond between peripheral benzene rings of neighboring propeller blades resulted in a partial planarization, closure of conjoint bay regions and disappearance of the bright lobe in STM images. The presence of molecules exhibiting two  were expected to exhibit a severe non-planar structure, we decided to inspect compound 7 with at greatest two maxima. Imaging at an elevated height confirmed that the bright lobes in STM images came from the significant non-planarity of the molecules . I/dV curves  complex with a formal negative charge on the gold atom (ESI Fig. S88 confirmed the stability of the proposed structure (Interestingly, further increase of the annealing temperature could be used for initiation of new transformations within the [14]annulene pores of nanographene tructure , hence pIn conclusion, by combining solution chemistry with sequential on-surface reactions, we have synthesized a non-planar three-fold porous nanographene with [14]annulene units embedded. The structure of the target nanographene and the corresponding intermediates has been analysed in detail by means of combined nc-AFM and STM experiments, and the porous nanographene electronic structure has been visualized by STM/STS measurements corroborated by theoretical modelling. The specifically designed precursor, consisting of three centrally fused branches with twelve peripheral phenyl rings, allowed on-surface intra-branch cyclodehydrogenation leading to the formation of a three-blade propeller. The following second-step cyclodehydrogenation processes finally led to the porous nanographene by closing fourteen-membered rings. Overall, twelve new C–C bonds accompanied by nine benzene rings and three [14]annulene pores were generated. By this we set a new path for the synthesis of large exotic nanographenes, where a single-step cyclodehydrogenation is not sufficient for their formation. Finally, we have explored the reactivity of these 14-membered ring pores on a gold surface by visualizing three different transformations within the same nanographene.There are no conflicts to declare.Supplementary informationClick here for additional data file.


The translational gap to treatments based on gene therapy has been reduced in recent years because of improvements in gene editing tools, such as the CRISPR/Cas9 system and its variations. This has allowed the development of more precise therapies for neurodegenerative diseases, where access is privileged. As a result, engineering of complexes that can access the central nervous system (CNS) with the least potential inconvenience is fundamental. In this review article, we describe current alternatives to generate systems based on CRISPR/Cas9 that can cross the blood–brain barrier (BBB) and may be used further clinically to improve treatment for neurodegeneration in Parkinson’s and Alzheimer’s disease (AD). These achievements have shown a huge reduction in the marked gap between basic medical research and translational medicine.The most common and prevalent worldwide neurodegenerative diseases are Alzheimer’s disease (AD) and Parkinson’s disease (PD). Thus far, there is no cure, nor is the etiology of either condition completely understood. Progression and ultimately death in patients with a very advanced degree of AD/PD may possibly be attributed to not having fully elucidated the molecular mechanisms that underlie these diseases . Previously, the guides were created without consideration for several parameters that have been identified as key factors to increase specificity over time. Several studies have shown that the off-target activity of Cas9 depends on the chemical composition of sgRNA and on the conditions in which the experiment is performed , by deletion of the deficient exon to produce the dystrophin protein, a direct cause of this pathology  repair mechanism and the union of nonhomologous ends. However, this efficiency decreases dramatically during the production of knock-in models due to the nature of repairing with the homologous recombination mechanism  transcript of genes, which has been adapted in embryo editing by microinjection , which has the function of isolating and protecting neural tissue, controlling the entry of molecules and therefore hindering delivery. About 7,000 drugs have been assessed in the Comprehensive Medical Chemistry database, with only 5% able to cross the BBB to enter the CNS  due to their inherent beneficial properties, such as the release of pro-regenerative trophic factors and the generation of bilipid vesicles. These can mediate the transport of small molecules, reported in cell–cell communication and signaling, arousing a great interest as a therapeutic option  gene to reduce exaggerated signaling in the striatum of a mouse model of fragile X syndrome (FXS) in a Fmr1 KO mouse. The team observed gene editing in neurons and non-neuronal cells, including microglia. Remarkably, they inhibited 40%–50% of the expression of the autism-causing gene, Grm5, in the striatum after intracranial injection, leading to rescue from autism-associated behavioral phenotypes .in vivo gene editing. Among the most outstanding recent achievements, Suzuki et al. (in vivo system consisted of HITI constructs and two AAV vectors and packaged both AAVs with serotype 8 or 9 (AAV-Cas9 and AAV-mTubb3). Afterward, they performed inducible Tubb3–GFP HITI targeting constructs, where Cre-dependent Cas9 expression was under the control of tamoxifen (TAM), by in utero electroporation of E15.5 fetal brain. The results revealed efficient GFP knock-in with the HITI donor and minimal knock-in with the HDR donor (Tubb3-HDR).In recent years, the manufacturing of new AAV serotypes is having an enormous effect on the field of gene therapy, especially in the neurosciences, and as a potential treatment of neurodegenerative diseases  indigenous to Sweden  who had increased Aβ levels . AAV-mediated delivery of SW1 gRNA was applied unilaterally into the hippocampus of adult mutant mice. After 6 weeks, the results confirmed the disruption of the mutation (indels) in primary cortical neurons and in the hippocampus in the APP allele , presenilin 1 (PSEN1), and presenilin 2 (PSEN2) triggers the production of beta-amyloid peptide. Therefore, the study of the familial component in AD can highlight tremendous opportunities to intervene through gene therapy. György et al.  harvestein vivo therapeutic approaches based on AAV-CRISPR correction constructs. Regardless, new variants of AAVs with greater penetration and efficiency are still needed.The study by György et al.  could leS. aureus (Sa)-Cas9  and a GMF-specific guide RNA (GMF-sgRNA). They found a few cells expressing SaCas9 while lacking GMF expression, confirming successful GMF gene editing. Nevertheless, they achieved low transduction efficiencies. Therefore, the authors suggest the possibility of exploring transduction efficiencies in embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), primary neurons, astrocytes, and microglia, and also the suitability of tropism-modified AAVs . They observed highly specific enrichment of the genes encoding vesicular glutamate transporter 1 (Vglut1) in pCaMKIIα::dCas9-KRAB+ neurons and glutamic acid decarboxylase 1 (Gad1) in pVGAT::dCas9-KRAB+ neurons. Remarkably, they found subtype-specific expression of the CRISPRi in the DG. Furthermore, Syt1 expression was selectively abrogated by conditional CRISPRi within the Syt1-targeting glutamatergic or GABAergic neurons but not in both, emphasizing the versatility of efficient disruption in a specific subtype of neurons in living mammals , fused to a Krueppel-associated box (KRAB) domain, could achieve precise and programmed transcription activation and repression, epigenetic remodulations of local histones, DNA modifications, labeling of the genomic locus, and single-base genome mutagenesis  of multiple genes involved in proteinopathy-induced neurodegeneration. They transfected HEK293T simultaneously with dCas9 sgRNAs targeting the alpha-synuclein, microtubule-associated protein tau, APP, and huntingtin . Notably, they performed precise transcriptional modulation through CRISPRa of neurodegenerative disease-related genes in human iPSC-derived neurons. TSS2-2 sgRNA and dCas9-VPR transcriptional activator mediated the activation of alpha-synuclein in normal alpha-synuclein levels (NAS) iPSC-derived neurons from healthy control patient and iPSCs derived from a patient with PD caused by alpha-synuclein triplication (AST). They achieved an eightfold activation of endogenous SNCA expression in the NAS iPSC-derived neurons and through dCas9-KRAB repression, a 40% reduction in alpha-synuclein mRNA levels in the AST iPSC-derived neurons  through CRISPR/Cas9-loaded nanocomplexes in post-mitotic neurons in vivo and demonstrated their therapeutic application in five familial AD (5XFAD) and APP knock-in AD mouse models. In the present study, despite not being a viral vector-based delivery, it was nonetheless shown to have a high editing efficiency, thus highlighting the emerging technology of nonviral NPs as an interesting therapeutic option for AD. Despite that CRISPR-based gene-editing and gene-therapy approaches have been seen as powerful tools to restore monogenic diseases, new approaches have provided a compelling basis to suggest the possibility of application to the sporadic component as well. Sun et al.  are caused by known mutations in the APP protein or gene products involved in processing APP to form beta-amyloid. Despite the small percentage of cases caused by these mutations, they all trigger the production of beta-amyloid peptide  in healthy human volunteers, presenting interesting data for the discussion of the implementation of gene therapies clinically. Nonetheless, both studies call on the international community of researchers to exercise a more rigorous assessment of the possible immunologic responses to the microbial origin of the system  resulted in the specific deletion of the same gene in the brain of knock-in mice, without detection of the vector in the liver  in the CA1/CA2 regions of the hippocampus (Long et al., TREM2 gene can cause a three- to four-fold increase in the risk of developing AD in a 5XFAD murine model (Wang et al., in situ.These studies agree that with the repeated administration of doses of AAV, concomitant disturbance caused by stereotactic surgery for effective infection and the proper distribution of the virus in the different regions of the brain are necessary. Despite the low risk present during surgery, an insult is consistently generated with the perfusion of the BBB, which could increase the release of inflammatory factors, such as IFN-γ and TLR4, coupled with the inherent reaction of the glia reactive inherent, observed in disorders such as PD and AD (Booth et al., in vivo. This was the first report of epigenomic manipulation performed in vivo based on CRISPR technology and should promote gene modification in living organisms, including modifications in the CNS.One of the great advantages that have been explored by some research groups is the performance of multiplex gene editing and the usefulness that CRISPR systems exhibit relative to other nucleases. Cong et al.  first devia nonviral and viral approaches throughout the large mammalian brain in vivo is achievable. Nevertheless, error-prone mechanisms remain dominant after CRISPR/Cas9 execution; in the adult brain, it is virtually impossible for post-mitotic neurons to utilize the HDR mechanism efficiently for ssODN replacement integration. Therefore, the refinement of new alternatives, such as HITI (Suzuki et al., The recent findings indicate that temporal and region-specific editing of gene expression AC-C made a substantial, direct, and intellectual contribution to the work. ND made an intellectual contribution and conducted manuscript review. EP-C made direct intellectual contribution and final approval to the manuscript. ED-M made a substantial intellectual contribution and performed the final revision of the 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.


The presence of aryl sulfides in biologically active compounds has resulted in the development of new methods to form carbon-sulfur bonds. The synthesis of aryl sulfides via metal catalysis has significantly increased in recent years. Historically, thiolates and sulfides have been thought to plague catalyst activity in the presence of transition metals. Indeed, strong coordination of thiolates and thioethers to transition metals can often hinder catalytic activity; however, various catalysts are able to withstand catalyst deactivation and form aryl carbon-sulfur bonds in high-yielding transformations. This review discusses the metal-catalyzed arylation of thiols and the use of disulfides as metal-thiolate precursors for the formation of C-S bonds. Over the last thirty years, significant strides have been made in organometallic processes that form carbon-sulfur bonds. Substantial growth in the transition metal-catalyzed formation of carbon-heteroatom bonds has been observed, however, the development of effective C-S bond formation reactions is underdeveloped with respect to the corresponding C-N and C-O coupling reactions. The necessity for the advancement of carbon-sulfur bond forming reactions is warranted by the prevalence of biarylsulfides in natural and unnatural products that exhibit activities against cancer, HIV, Alzheimer’s disease, inflammation, and asthma ,9,10,11.Methods to synthesize aryl sulfides without the use of transition metals are generally inefficient, require impractical reaction conditions, and have limited functional group tolerance. Some of these methods include nucleophilic attack on disulfides, aromatic substitution reactions, and metal-mediated disulfide reductions. The development of practical and efficient methods to create aryl sulfides has been realized through transition metal catalysis. Transition metal-thiolate interactions are strong and numerous stable complexes have been reported in the literature. This strong coordinating ability often leads to the belief that sulfur will hinder transition metal catalytic activity. Despite this notion, thioethers can be excellent ligands for metal-catalyzed processes and metal-thiolate complexes can undergo facile reductive elimination to form C-S bonds. Reviews discussing metal-catalyzed carbon-heteroatom bond forming reactions have been reported, including a recent excellent review on organometallic approaches to C-S bond formation . This re3]4 as a catalyst  was observed by 31P-NMR spectroscopy and it rapidly forms PhPd(SR)dppf upon the addition of Et3N. This reversible metallation of the thiol is proposed to facilitate the deprotonation step and would thus be applicable when weakly acidic thiols are employed. Further, the rate of reductive elimination to form the aryl sulfide was determined to be the slow step of the process. The final proposed catalytic cycle is depicted in Campagne and Jutand reported mechanistic studies on the palladium-mediated C-S bond forming reaction using a cysteine-derived thiol . PalladitBu) is an electon-rich alkylbisphosphine that creates a highly reactive palladium complex for the arylation of thiols. Through the isolation and reaction of each palladium complex of the catalytic cycle, it was observed that each step  proceeds within minutes at or below ambient temperature. However, the overall catalytic reaction requires temperatures of 110 °C. Based on these results, it is clear that the resting state of the reaction lies off the catalytic cycle. The resting state of the catalytic process was probed through analysis of the rate of reaction of stable complexes that preceed the catalytic cycle. Through a series of studies from isolated palladium complexes it was determined that the resting state of the reaction depended greatly on the source of palladium. A palladium-dithiolate complex represents the resting state of reactions using Pd(OAc)2 as the source of palladium. For reactions using Pd(dba)2 as the Pd-source, (LPd)2(dba) was determined to be the resting state. Lastly, for reactions catalyzed by the complex of the initial oxidative addition of aryl halide [Pd(L)(X)(Ar)], the resting state lies at a palladium hydridothiolate complex that arises from oxidative addition of Pd(0) to the S-H thiol bond. Furthermore, these results strongly correlate to the catalytic reactions of other electron-rich bisphosphines. The catalyst system developed by Buchwald using DiPPF was demonstrated to act similarly to the CyPF-tBu-ligated catalyst. The classical approach to improving a catalyst by accelerating the slow step of the catalytic cycle would fail for these reactions. For this system, acceleration of the rate of stable Pd-complexes to enter the catalytic cycle is necessary.More recently, Hartwig has performed intensive mechanistic studies of the Josiphos-ligated catalyst system . The JosShortly following Migita’s report on palladium-catalyzed arylation of thiols, Cristau and co-workers reported a nickel-catalyzed process to synthesize biaryl sulfides . Using aPercec reported that aryl mesylates are feasible coupling partners for nickel catalyzed C-S bond forming reactions . Using 1Most nickel-catalyzed protocols require high catalyst loadings and long reaction times ,63,64,65Nickel catalysts have also been shown to insert into disulfide bonds and undergo C-S bond formation. Recently, Taniguchi has reported the arylation of disulfides in the presence of bipyridyl nickel(II) bromide (Equation 10). Various aryl iodides are transformed into their corresponding aryl sulfide using 0.5 equivalents of alkyl or aryl disulfides. The reaction is postulated to proceed through an initial zinc mediated reduction of nickel(II) to the active nickel(0) complex . At thisNickel pincer complexes have also been shown to catalyze C-S bond forming reactions using disulfides and aryl iodides Equation (11) . Over the last decade, copper has emerged as a viable catalyst for the arylation of thiols. ,102,103 2CO3 can couple thiols with aryl iodides in good to excellent yields. The substrate scope is excellent with good functional group tolerance. Notably, the reaction proceeds in the presence of anilines and phenols. The ethylene glycol likely acts as a ligand to stabilize copper during the course of the reaction.Buchwald reported the first practical synthesis of aryl sulfides using a copper catalyst (Equation 13) . Under t2CO3 allows the coupling of iodoarenes with thiols to occur in the presense of aryl bromides. Switching the base to Cs2CO3 under the same conditions gives a chemoselective coupling of the aryl bromide with aryl thiols. The chemoselectivity is attributed to the ability of a stronger base (Cs2CO3) to polarize the aryl bromide bond and allow copper to undergo a more facile oxidative addition of the aryl bromide compared to the K2CO3 system. Notably, the aryl amine is also not coupled with aryl bromide under these conditions.Recently, a highly regioselective process for the thiation of aryl halides was reported by Ranu and co-workers (Equation 14) . Under tDisulfides and thioimides have been employed as thiolate surrogates in the copper-catalyzed thiation of arylboronic acids. This modified Chan-Evans-Lam cross-coupling reaction demonstrates the diverse utility of copper salts to construct carbon-heteroatom bonds. Guy first demonstrated the ability of arylboronic acids to react with thiols to construct the arylsulfide bond in the presence of stoichiometric copper. Based on this report, Liebeskind utilized thioimides as a copper-thiolate precursor (Equation 15).  A copperMechanistically, the reaction is presumed to proceed through a Cu(I)-Cu(III) catalytic cycle . InitialS-arylation of thiols (Equation 16) [Bolm reported the use of catalytic iron(III) chloride in the tion 16) . The rea3 makes for an attractive, cost-friendly process. Shortly after this report, experiments performed in the Buchwald laboratory determined that copper, as little as 10 parts per million, was essential for catalytic activity . AlthougCheng and co-workers disclosed a cobalt catalyzed process for the arylation of thiols (Equation 17) . This meThe mechanism of the cobalt catalyzed reaction is thought to occur through a cobalt(I)-(III) catalytic cycle . Zinc meExtensive work in metal-catalyzed C-S bond forming reactions has resulted in significant advances for late-metal catalyzed processes. It is evident that the palladium-catalyzed reaction creates systems with the highest yields, lowest catalyst loadings, and highest functional group tolerance. Despite this fact, expensive chiral ligands are required for high activity. Progress has been made to eliminate the requirement of these ligands through the use of zinc chloride and through the development of “ligand-free” catalysts. Other metals are growing in synthetic utility for C-S bond forming processes, such as copper and nickel. Mechanistic investigations are becoming more common in order to fully understand these catalytic processes and to ultimately advance the development of more efficient catalysts.


In 2014, the Ethiopian government passed a new smoking legislation that banned smoking in public and workplaces including health care facilities. However, data’s on level of non-compliance and associated factors with non-compliance towards smoke-free legislation in hospital settings of the country has not been studied yet.p-value of < 0.05 at 95% CI was considered to be statistically significant.Hospital-based Cross-sectional study design triangulated with observational study was conducted in five hospitals. Data were collected through direct observation and interviews using checklist, structured and pre-tested questionnaires for observational study and survey of hospital employee respectively. Nine data collectors and one supervisor were involved in data collection. Three hundred fifty (350) health care staffs were interviewed. Fifteen (15) buildings were purposively observed for observational non-compliance in the selected hospitals. Data were entered by Epi Info and analyzed using SPSS version 21 software. Logistic regression was used to compute the crude and adjusted odds ratios for the factors affecting employee non-compliance with the legislation. A p value = 0.001), having poor knowledge  and having Unfavorable attitudes .Anti-smoking signs were absent from a high proportion of hospital areas  although visible cigarette butts were generally not observed in most areas of the hospitals. Non-compliance level among health care staffs was 50(10.3%).Associated factors affecting to the non-compliance level of the staff were: being male (AOR = 5.89, Non-compliance level was high and needs careful implementation for 100% smoke-free legislation in addressing knowledge and attitudes of health care staffs. Tobacco consumption and exposure to second-hand smoke (SHS) are a leading cause of morbidity and mortality worldwide. Cigarette smoking is the main source for SHS exposure. Second-hand tobacco smoke is the combination of smoke emitted from the burning end of a cigarette or other tobacco products and smoke exhaled by the smoker. Second-hand smoke contains over 7000 chemicals of which at least 69 are known carcinogens or otherwise toxic . MoreoveGlobally, more than 7 million people die each year from tobacco-related illnesses  , and if As part of efforts to prevent this growing problem related to tobacco smoking, which can protect all peoples from the harmful effect of exposure to second-hand tobacco smoke, the World Health Organization’s Framework Convention on Tobacco Control (WHO FCTC) has called a comprehensive legislation to eliminate tobacco smoking in all indoor public places and workplaces and adapted the framework convention on Tobacco Control (FCTC) in 2003 , 7.Following this, many countries in the WHO Africa Region are enacting and implementing domestic legislation for tobacco control . EthiopiHealth care facilities are among the most influential setting for modeling abstinence from smoking and encouraging smoke-free environment. Health professionals have the trust of the population, the media and opinion leaders, and their voices are heard across a vast range of social, economic and political arenas. Hospitals should promote, implement and comply with tobacco control legislation, particularly smoke-free legislation. However, this may not be an issue in Ethiopia, where the legislation smoke-free workplace enacted after the FCTC has yet to be enforced broadly across the region of the country and within health care facilities particularly in the hospitals settings .In February17, 2014, the Ethiopian government passed a new smoking legislation through a house of parliament that banned smoking in public places and workplaces including health care facilities . HoweverThis assessment was conducted in five hospitals located in the city government of Addis Ababa from June 1 to December 30, 2017. These hospitals include Tikur Anbessa general specialized hospital, St. Paul’s hospital millennium medical college, Amanuel mental specialized hospital, Alert hospital, and St. Peter specialized hospital. We choose these hospitals because they have started implementation of the smoke-free legislation in their hospital premises.A hospital-based Cross-sectional study design triangulated with the observational study was employed in five selected hospitals located in the city government of Addis Ababa, Ethiopia.Five hospitals located in the city government of Addis Ababa which have started the implementation of smoke-free legislation in their hospital premises were purposively included for this study. For the observational study data were collected by observing the 15 buildings located in the hospitals premises of five purposively selected hospitals according to the compliance guide .Three hundred and fifty four(354) employees were engaged from five selected hospitals by assuming confidence interval of 95%, a power of 80%,OR of 4.6 and ratio of 1:1 . EmployeSecond-hand smoke (SHS) exposure: is secondary exposure of individuals to tobacco smoke as a result of others smoking tobacco.Smoke-free legislation: is the legislation or policies/law which makes all or almost all workplaces, including hospital settings, totally smoke-free; with no smoking at all allowed in any indoor area.Observational non-compliance to the legislation: Observation of at least one of the following anywhere in the hospital premises including; the presence of smoking, presence of cigarette butts, presence of ashtray and absence of anti-smoking sign.Employee non-compliance to the legislation: is a cigarette smoking in the hospital premises by the employee.Compliance: the degree to which a smoke-free legislation is being obeyed in the hospital premises.urrent smoker: is someone who had smoked cigarettes at least 6 months during their lifetime and was smoking tobacco products at the time of the survey  with the hospital smoke-free legislation during their working hours in the hospitals premises. Staff those self-identified as smokers was asked to indicate specific areas where they have been smoking during their working hours in the hospitals. Based on this employee non-compliance level was estimated by dividing the total number of employees who smoke in the hospital premises to the total number of surveyed employee.To assess the knowledge of hospital health care staff, eight specific questions related to the smoke-free legislation were requested. Those questions were including, “exposure to tobacco smoke in the workplace is not a significant cause of tobacco-related diseases”, “designated smoking areas in the same room are effective in protecting non-smokers and workers from the hazards of tobacco smoke”, “separate rooms with separate ventilation offer almost the same amount of protection as smoking bans and are a good alternative to bans”, “tobacco product advertising has no effect on consumption”, “because of all the publicity about how harmful tobacco use is, anyone who starts using tobacco these days is fully aware of the risks”, “warnings/messages on tobacco product packages, while attention getting, are not effective in motivating users to quit”, “only smokers have to worry about health risks from tobacco smoke” and Smoke free legislation in hospital protects your health and/or the health of other visitors and patients. We created a scale using these items where 1 point was given for each correct answer with a possible score ranging from 0 to 8.A possible answer listed for each question was true or false  with theTo assess attitudes of hospital health care staff, a series of 11-questions related to smoke-free legislation was requested with five-point scale from “Strongly disagree to strongly agree” . These qThe practice of health care staff was assessed by asking one question about their practice in enforcing the legislation when a violation was observed in their hospital premises as per compliance guide . Staffs Data collection tools were adapted from other literatures and prepared by a principal investigator. A well-structured questionnaire was translated into Amharic language and back-translated to the English language to maintain its consistency and corrective. Training was given for those data collectors and field supervisors on the objective of the study, contents of the questionnaire and how to maintain confidentiality and privacy of the participants. Finally, pre-test was performed at 5% of the sample size in AaBET hospital before the actual data collection. Data collection tools were revised, edited and modified according to the results of the pretest. A principal investigator and field supervisor had checked collected data for their completeness on the daily basis before the data were entered into the software. More orientation/guidance for those data collectors come up with incompleteness and inconsistency was provided in between data collecting period. In the case of the incompleteness and inconsistency, the collected data were discarded as incomplete.P-value less than 0.05 at 95% CI were considered as significant factors for non-compliance towards smoke-free legislation among hospital employees.Observational data were analyzed by Microsoft Excel and univariate analysis was performed to estimate observational non-compliance level. Interviewed data were cleaned and entered into EPI info7 version software and analyzed by SPSSwindow version 21 and univariate analysis was also conducted to estimate employee non-compliance level in the selected hospitals. Bivariate and Multivariable analysis was applied using binary logistic regression to identify predicting factors with employee non-compliance towards smoke-free legislation. Variables at Almost at all inspected places  in five selected hospitals presences of “anti-smoking sign” was nonexistent. Only 2.8% of the inspected places have “anti-smoking sign”. The places where such signs were most likely absent were the physicians’ room, Nurses’ room, OPD, Elevators, and toilets. However, the place where this sign was more existent was at entrances (23.8%) compared to other places  and toilets (14.8%) compared to other places. Site visits indicated the evidence of smoking on the hospital premises was observed in 3 (60%) of the selected hospitals. However, cigarette butts were observed in all selected hospitals, which indicate evidence of recent cigarette smoking in these hospitals  at a category of less than 30 years old. Of the 350 participants, 190(54.3%) were males. The majority of the study participants, 117 (30%), were from Tikur Anbessa General Specialized hospital. About 196(56%) of the respondent had a monthly income of less than 4708 birrs (mean monthly income). When considering the professional category of the study participants; the majority,  were health professionals,  were health-related staffs,  were administration staffs and  were support staffs. Among 350 respondents the majority,  and  of them were married and single respectively. One hundred eighty-two(52%) of the respondents worked less than or equal to 40 h per week (Table The median age of the respondents was 29 years (IQR 26 to 35 years) with the majority of the respondents  and 168(53.5%) of them have a respectively good knowledge and favorable attitude towards smoke-free legislation and exposure to SHS. However, the majority of non-compliant employee, 2569.4%) and 30(83.3%) have a respectively poor knowledge and unfavorable attitude towards smoke-free legislation and exposure to SHS  of study participants were aware of the hospital smoke free legislation, 119(34%) were not aware and 85(24.3%) were not sure of the existence of the smoke-free legislation that prohibits smoking in the hospital premises. Among those aware of the hospital smoke-free legislation,14(9.6%) of the participants were non-compliant to the legislation. However, among non-compliant employees, 13(36.1%) were not aware of the legislation and 9(25%) of them were not sure of the existence of the legislation in the hospitals (Table n = 50), only 10% of them were practicing the enforcement of the smoke-free legislation and 45 (90%) them were not practicing when a violation was observed. Even the majority of non-smokers, 69%, were also not practicing the enforcement of the legislation (data not shown).Only 98(28%) of the participants had been enforcing the smoke-free legislation. While the majority, 252 (72%), of them were not practicing the enforcement of the legislation Table . AccordiP-value less than or equal to 0.20 were entered to the model. Next multivariable logistic regression was performed to assess associated risk factors to the non-compliance level of employee within hospital settings and this logistic regression analysis depicted that being male , having poor knowledge  and having unfavorable attitude  were the three significant risk factors associated with non-compliance level of health care staffs towards the smoke-free legislation within the selected five Hospitals (Table Socio-demographic, Knowledge, attitude and practice of the study participants in relation to non-compliance were analyzed by bivariate analysis using simple binary logistic regression model. Binary logistic regression analysis was carried out to assess the relative effects of independent variables on the dependent variable. Those variables with The purpose of this study was to estimate observational non-compliance level, employee non-compliance level and associated factors towards the smoke-free legislation in hospital settings among hospital health care staff, by assessing the knowledge, attitudes, practice, and smoking habits of health care staff towards the smoke-free legislation. Observational non-compliance was also assessed by observing the hospital premises towards the legislation. This study showed non-compliance level among health care staffs was high Fig.  and affeNon-compliance level among health care staffs shown here was higher than non-compliance levels reported in the UK  and loweSite visits also indicated observational non-compliance level was high in the premises of the hospitals as per WHO FCTC guideline and indicator of success , 23. ThiLogistic regression analysis indicated sex, knowledge, and attitudes of health care staff were the three positively associated factors with non-compliance level of health care staff towards smoke-free legislation. Being a male is almost six times more likely to be non-compliant than being a female, having a unfavorable attitudes six times more likely to be non-compliant than having a favorable attitude while having poor knowledge is almost three times more likely to be non-compliant than having good knowledge towards the legislation.The significant association of health care staffs’ sex with their non-compliance level in the current study was not consistent with research done in the United Kingdom ; in whicDifferent studies –31 pointRegarding the significance of hospitals health care staffs’ attitude with their non-compliance level, different studies , 33 poinAlthough this study has important findings, it has some limitations related to its cross-sectional design like recall bias and the reliance on self-reported data. Especially, non-compliance status of hospital employee was based on what they self-reported during working hours which may make over or under report. We had also focused on the federal government hospitals due to budget limitations; which might not reflect non-compliance level in other health care facilities of the country. Being the first study in the hospital setting of our country is also another limitation which we are unable to compare and contrast current findings with others.Findings of this study showed that non-compliance level among health care staff was high, which implies the legislation has not been fully implemented as per WHO FCTC guideline. Especially, observational non-compliance level towards the legislation was high compared to the individual non-compliance level. The factors associated with the non-compliance level of health care staffs were; being male, having poor knowledge and unfavorable attitudes towards the smoke-free legislation. This study suggests careful implementation of the legislation was needed in addressing the knowledge and attitudes of health care staffs.


A comparative study of the electrical performance of triboelectric nanogenerators (TENGs) with plain- and 2/1 twill-woven cotton textiles was conducted. Furthermore, the microstructures of the cotton fiber surfaces were examined to understand the fundamental mechanical interaction among the cotton fibers in the TENGs. The TENG with 2/1 twill-woven cotton textiles exhibited higher output voltages compared to that with plain-woven cotton textiles. The difference in the output voltage between the two types of TENGs resulted from the difference in triboelectric charge generation between the constituent cotton textiles. The higher output voltage of the TENG with 2/1 twill-woven cotton textiles was attributed to the higher density in triboelectric interactions among the cotton fiber molecules. Our daily life is filled with diverse forms of ambient energies that are continuously generated and observable, but mostly wasted without being exploited. Various types of energy harvesters have been developed in order to harness such energies. For example, many promising technologies have been developed to convert solar and wind energies to electrical energy . FollowiIn this work, a comparative study of the output voltages of TENGs fabricated with plain- and 2/1 twill-woven cotton textiles was conducted. The microstructures of the cotton fibers were examined using atomic force microscopy (AFM) and scanning electron microscopy (SEM) analyses. The difference in the output voltages of the TENGs was elucidated on the basis of triboelectric charge generation in the cotton textiles.TENGs were fabricated by using cotton textiles as active triboelectric layers. The chemical composition of cotton fibers should be considered in order to understand triboelectric charge generation in cotton textiles. Cotton fibers contain various organic compounds, including cellulose, waxes, pectins, organic acids, and some inorganic substances; cellulose accounts for approximately 90% of the dry weight of cotton fibers . Figure The surface microstructures of cotton fibers were examined to understand the basic mechanical interaction among the cotton fiber yarns in the TENGs. Unlike film-type TENGs based on film-to-film interactions, examining the morphological properties of cotton fibers would be important and helpful in understanding the working mechanisms of fiber-type TENGs based on fiber-to-fiber interactions. 2 on the textiles at a frequency of 5.8 Hz for 5 s. The output voltages of the TENGs with plain- and 2/1 twill-woven cotton textiles were measured by applying a pressure of 0.47 N/cmTo explain the difference in output voltage between the TENGs, an understanding of the relationship between the mechanical properties of the cotton textiles and the triboelectric charge density in the cotton textiles is necessary. TENGs were fabricated using plain- and 2/1 twill-woven cotton textiles, and their output voltages were compared. The TENGs with plain- and 2/1 twill-woven cotton textiles exhibited average positive peak output voltages of 1.59 and 12.47 V, respectively. Their average negative peak output voltages were −0.86 and −4.13 V, respectively. The difference in the output voltages is explained on the basis of the difference in the mechanical properties of plain- and 2/1 twill-woven cotton textiles. The results of this study are expected to improve our understanding of triboelectric charge generation on cotton fiber surfaces and help in designing the structure of cotton textiles from the viewpoints of triboelectric engineering and wearable electronics.