Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease.Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs.Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-β-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-β-dependent pathway, and validated in a saphenous vein into carotid interposition pig model.Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors. China Coronary artery bypass grafting is used in cardiac surgery for more than 50 years to combat the consequences of coronary artery disease in vivoEx vivo vessel culture systems (EVCSs) and bioreactors to stimulate cells mechanically offer the unique possibility to investigate the effects of isolated or combined mechanical stimuli under well-controlled and reproducible biomechanical and/or metabolic conditions in human large vessels. In this framework, the aim of the present investigation was to characterize the effect of coronary mechanical conditions on molecular programming of vein graft disease using an integrated tissue/cell biomechanical approach.The role of mechanical forces in the progression of graft failure has been recognized, although the nature of cell-based mechanosensing in the vascular tissue remains unclear due the difficulty of decoupling distinct components Extended Methods are provided in the Methods section in the online only Data Supplement.The experimental investigation on human-derived tissues and cells was approved by the local ethical Committee at Centro Cardiologico Monzino, IRCCS. Patients were required to sign an informed consent. The use of human material was done in compliance with the Declaration of Helsinki. The main patient characteristics are shown in Mechanical stimulations of SV grafts were performed using a custom-made bioreactor After mechanical stimulation, tissues, cells and culture supernatants were prepared and appropriately processed for histological, immunohistochemical, immunofluorescence, protein/secretome and RNA analyses, as already published in vitro experiments was performed as previously described Isolation of cells for Tissue/cells RNA and protein content was analysed by Q-RT-PCR and Western blotting performed with protocols already published P value < 0.05 was considered significant. The type of statistical test employed for data comparison is specified in figure legends. As a general rule, comparisons between two independent samples were performed by unpaired/paired t-test (two-tails), while for comparisons between 3 or more groups we adopted one-way ANOVA with post-hoc comparisons tests. The specification of the number of independent samples included in the analyses and the type of statistical tests used to compare data are specified in the legends to figures. Further details about data processing are provided in the online methods section.In all graphs throughout the manuscript, data were plotted as mean ± standard error using GraphPad Prism 7. A Our previous contributions showed that application of an 80-120 mmHg pulsatile pressure regimen determined a change in the SV structure at 7 days, consisting of a significant thinning of the vessel wall, and elevation in cell death by apoptosis and enhanced proliferation ex vivo cultured SVs exhibited a similarly elevated cellular apoptosis , and later in the media, as detected by expression of PCNA marker.Recruitment of cells from the vascular adventitia has been identified as a key early event in VGD setting in animal vein arterialization models  vasorum , this sul Figure A-C, whicin silico the level of cell deformation associated to the circumferential strain of the two major SV layers occurring in the presence of coronary flow mechanics. We were interested in this strain component considering the orientation of the circumferentially arranged SMCs bundles, which appeared the most affected structures in the media of CABG-stimulated SV conduits  TSP-1 as mechano-responsive gene. Finally, we performed immunohistochemistry and Western blotting analyses on tissue sections and protein extracts of samples treated with VP or CABG flow. This clearly showed a specific elevation of TSP-1 in the intima and media compartments of CABG-stimulated vs. VP and control SVs  s Figure A. Results Figure B. To subs Figure C or the s Figure D; in bot+/SM22α+ cells growing in SV conduits exposed to coronary mechanics are actively recruited from the adventitia where they initially reside; conversion from contractile to synthetic SMCs due to circumferential strain component attracts these cells in the medial layer through secretion of TSP-1 and CD47-mediated chemotaxis. To get further insights in this process, we reasoned that transitioning from the adventitia to the media could expose cells to a combination of stimuli resulting from TSP-1 and classical vascular pro-remodeling factors, such as TGF-β +/CD44+ cells  a substantial rearrangement of the intima and the media layers structure, ii) a major change in cell composition resulting from an early induction of apoptotic death in a significant portion of the SMCs, accompanied by a transition from a contractile to a synthetic phenotype of the surviving SMCs associated with TSP-1 release in the media, and iii) proliferation of a vessel-resident cell subset arising from the SV adventitia, characterized by expression of CD44 and SM22α, leading to a fibrotic-like response associated to TSP-1 release.We and others have hypothesized an important role of hemodynamic forces in molecular programming of the SV graft disease vs. the venous flow/pressure pattern.The first questions that we aimed to resolve was to define the structural rearrangement of the vessel wall subjected to CABG conditions and the cellular dynamics occurring in consequence of the coronary stimulation post mortem showing narrowing of the vein wall and absence of intima hyperplasia at early times after implantation As shown in Figures + cells occurred at day 14 of CABG stimulation. Thus, the shift in SMCs phenotype in the media was likely an effect of the adaptation of the cells surviving the early apoptotic peak to the new mechanical conditions. This conclusion is in line with reported differences in biological responses of synthetic SMCs to mechanical strain compared to their contractile counterparts IF data indicated that coronary mechanics induced synthetic SMCs phenotype, with a decrease in αSMA and Calponin and a parallel increase in Vimentin in the media layer Figure . This is+/SM22α+ SMCs or of progenitors with pericyte characteristics associated to vasa vasorum+/CD44+ cells were found almost uniquely in the adventitia in the vasa vasorum region, from where they appeared to invade the media at the later time point, especially in CABG condition   Figure . It was s Figure , or in is Figure . TogetheTAGLN1, TGFBR1 and Col1A genes expression, SM22 protein expression and proliferation (Figure vasa vasorum and in modulating the latent TGF-β signaling, to promote maturation and proliferation of these cells in the media + in the adventitial region, and this correlated with neointima accumulation.Inspired by the vision of a mechano-paracrine mechanism involving TSP-1 in SV remodeling process, we finally established a mechanism for activation of adventitial cells in SV graft pathophysiologic process. Even if these cells have been extensively characterized for their vascular regeneration potential n Figure -S11. TakIn conclusion, our study unravels for the first time a molecular mechanism linking mechanical injury occurring in coronary SV grafts with programming of intima hyperplasia by a mechano-paracrine effect. While this evidence demonstrates the relevance of cell-based mechanosensing in fibrotic diseases of the cardiovascular system, it calls for more focused studies addressing the potential reduction of intima thickening, e.g. by treatments with peptides inhibiting the TSP-1 function to resolve the timely issue of venous CABGs occlusion Supplementary methods, figures, and tables.Click here for additional data file.


While this approach has not led to such success in other patients and is not applicable to HIV+ individuals without cancer, the encouraging results may point toward a breakthrough in developing a cure strategy for HIV. Adoptive transfer of virus-specific T cells (VSTs) post HSCT has been effectively used to treat and prevent reactivation of latent viral infections such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), making VSTs an attractive therapeutic to control HIV rebound. Here we will discuss the potential of using adoptive T cell therapies in combination with other treatments such as HSCT and latency reversing agents (LRAs) to achieve a functional cure for HIV.Human immunodeficiency virus (HIV) has caused millions of deaths and continues to threaten the health of millions of people worldwide. Despite anti-retroviral therapy (ART) substantially alleviating severity and limiting transmission, HIV has not been eradicated and its persistence can lead to other health concerns such as cancer. The only two cases of HIV cure to date are HIV A combination of drugs targeting different stages of HIV life cycle in anti-retroviral therapy (ART) has been used to treat HIV infection, rendering the virus undetectable and preventing transmission  in immunocompromised patients  Hutter, . The onlex vivo and endowed with antigen-specificity and improved persistence and function within the tissue microenvironment. While ACT had little success with HIV treatment in the past  is an artificial receptor with an extracellular antigen-binding domain connected to an intracellular signaling domain by a hinge region and a transmembrane domain. When expressed on the surface of a T cell, CAR can induce T-cell activation upon ligand binding independently of T cell receptor (TCR)-major histocompatibility complex (MHC) interactions, thereby bypassing MHC restrictions and MHC downregulation mechanisms often used by cancer cells or viruses, including HIV  was derived from a naturally-occurring TCR isolated from an HIV-infected individual and demonstrated a higher affinity to SL9 , but the results have not been reported. Engineering T cell specificity with TCRs may yield a highly efficient T cell response, but its use is limited by MHC restriction, antigen loss, and off-target toxicities including death as reported for cancer patients infused with MAGE-A3 TCR T cells  and did not reach statistical significance , a mixture of CD8+ and CD4+ T cells, were able to lyse peptide-pulsed targets and suppress the viral spread of an HIV lab strain in vitro. Moreover, HXTCs were also capable of controlling the outgrowth of autologous reservoir-derived HIV and, more importantly, clearing resting CD4+ T cell reservoir post LRA treatment in vitro . When infused to HIV-infected participants on ART, HXTCs were safe and well-tolerated, but showed little impact on the frequency of cells harboring replication competent HIV, measured by the quantitative viral outgrowth assay  to delete the CCR5 gene was largely safe, and that the gene-modified cells engrafted and persisted . Moreover, autologous CD4+ T cells transduced with a lentiviral vector expressing an antisense targeting HIV env were infused to 13 ART-treated patients followed by treatment interruption, attempting to block viral replication upon infection -enriched cell population lacking CD62L and CCR7 expression  or central memory (TCM) cells , but the infused T cells may be at disadvantages for three reasons: (1) the latent reservoir is expected to be substantially bigger without lymphodepletion, (2) the lymphoreplete host environment is suboptimal for the expansion of infused T cells, and (3) the infused T cells may be vulnerable to HIV infection upon viral reactivation. The occurrence of “elite controller” individuals who can keep the virus in check without ART by maintaining strong HIV-specific T cell responses (Saez-Cirion et al., P-HL wrote the manuscript. MK, PH, and CB reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.MK serves on an advisor board for Gilead Sciences. PH is a co-founder and serves on the board of directors of Mana Therapeutics. CB serves on an advisory board for Cellectis, serves on the board of directors for Cabaletta Bio and is a co-founder of Mana Therapeutics and Catamaran Bio and serves on their scientific advisory board. In addition she has stock ownership in Neximmune and Torque Therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lymphoma in HIV-infected patients is AIDS defining. This is the second most common AIDS defining malignancy after Kaposi’s sarcoma. Development of lymphoma in HIV patients is related to immunosuppression and high viral load. Co-infection with other lymphotrophic viruses especially EBV is also strongly associated with development of lymphoma in HIV patients. Despite advances in HAART therapy, incidence of diffuse large B cell lymphoma in HIV-infected patients remains significantly higher than in the general population.Early diagnosis is challenging due to presence of opportunistic infections and atypical presentation of the lymphoma in this subset of patients. Atypical imaging findings are not unusual, and the diagnosis of lymphoma on imaging is on many occasions unexpected as the patient would ideally be initially investigated for presumed opportunistic infection.Lymphoma treatment approaches in HIV patients are complicated by comorbidity with opportunistic infections and performance status of the patients. Treatment failure and early relapse are also common in AIDS-related lymphoma. This review article highlights the common and unusual multimodality imaging findings in HIV-associated lymphoma. Non-Hodgkin lymphoma is the second most common malignancy in HIV patients.AIDS-related NHL is an aggressive multisystem disease with a poor prognosis.CT and FDG PET CT imaging are crucial in initial diagnosis, staging, and follow-up of AIDS-related lymphoma.Knowledge of imaging features of diffuse features of HIV-associated lymphoma can help to differentiate lymphoma from infectious comorbidities which are common in HIV/AIDS patients.HIV remains a critical health problem; as of 2018, 37.9 million people globally were living with HIV, of which 24.5 million had access to highly active antiretroviral therapy (HAART) . Although rituximab has revolutionized the treatment of CD20-positive lymphomas in the HIV-negative population, its role in HIV-related lymphoma is not as clearly defined, given the profound cellular and humoral immunodeficiency that this agent can cause [24]. Many authors suggest the addition of rituximab to the treatment regimen if the CD4 count is over 50/mL and individualized consideration for patients with CD4 less than 50 [25]. Imaging is crucial in assessing response to treatment. An interim PET/CT is useful in assessment of treatment response.Current evidence shows no clinical benefit of surveillance imaging , 21, altImaging is valuable for diagnosis and differential diagnosis of AIDS-related lymphomas and facilitates staging and monitoring of therapeutic effects. However, there can be atypical imaging features of AIDS-related DLBCL compared to findings in HIV-negative patients. Knowledge of these atypical imaging features may facilitate the diagnosis of lymphoma over infectious disease in the setting of HIV/AIDS.


Adrenomedullin (ADM) is a peptide hormone with vasodilatory effects and involved in the regulation of the endothelial barrier function. Previous research found increased ADM plasma levels in patients with sepsis and ADM levels correlated with disease severity and mortality in sepsis . Althougn = 5).We present here data regarding ADM in patients with COVID-19. Starting from March 2020, we included 45 adult patients presenting with signs of respiratory infection (cough and/or fever) to the Emergency Department in this ongoing study. The study was approved by the ethics committee of the University of Regensburg. Each individual provided written informed consent prior to inclusion. SARS-CoV-2 infection status was evaluated by PCR analysis mainly using throat rinse water . Patients were classified as COVID-19 positive (PCR positive for SARS-CoV-2 and signs of respiratory infection) or control . Whole blood was drawn by venipuncture and lysed in Trifast (Ambion) buffer solution. Further, RNA expression analysis of ADM in whole blood was performed using qPCR and normalized to GAPDH as housekeeper gene. The final diagnosis after patient discharge was reviewed by a consultant physician and patients without evidence of respiratory infection were excluded from analysis (Baseline characteristics of the study population are described in Table p = n.s.). According to ROC-analysis, ADM was able to differentiate severe from non-severe COVID-19 cases with an AUC of 0.82 .ADM expression was significantly elevated in patients with COVID-19 than other respiratory infections (Fig. To strengthen our hypothesis, we analyzed ADM expression in the left ventricular myocardial tissue of patients who were deceased from COVID-19. Infection with SARS-CoV-2 had been verified by PCR in all of these patients. As control, we used a combination of left ventricular myocardial tissue of patients who died from other respiratory infections or from patients destined for organ donation, which could ultimately not be performed. We found a significantly elevated expression of ADM in patients who died from COVID-19 in contrast to controls (Fig. Our findings suggest a potential role for ADM in severe COVID-19. While ADM might be a therapeutic target in sepsis and septic shock, further research is needed regarding ADM in COVID-19. Further, the diagnostic potential of ADM as a marker for progression to severe COVID-19 at first medical contact should be evaluated.Limitations of this study are the small number of patients included and RNA expression analysis in contrast to direct measurement of ADM levels. This study, however, is the first to show an association between ADM and severity of COVID-19.


Drosophila melanogaster, approximately 150 so-called clock neurons express the molecular components of the circadian clock mechanism. The activity of these neurons enables the fly to anticipate and track cyclic changes in their environment and to respond to them accordingly. The core of the circadian clock is built by two interlocked transcriptional feedback loops. In the first loop, the transcription factors CLOCK (CLK) and CYCLE (CYC) initiate the expression of the core clock proteins PERIOD (PER) and TIMELESS (TIM), which are in turn repressing the activity of CLK and CYC as transcriptional activators, resulting in a cyclic accumulation of PER and TIM . In light:dark cycles, the intracellular photoreceptor Cryptochrome (CRY) mediates light information to the core clock and resets the molecular mechanism, thereby synchronizing the clock every day at sunrise to the 24h oscillation . However, in constant darkness, the oscillation of the clock proteins PER and TIM cycles with the endogenous period, which is close to, but not exactly 24hrs.In Drosophila chronobiology takes at least 80 flies per genotype, when the sampling occurs every 3hrs .A commonly used read-out for the molecular oscillations are immunohistochemical assays to quantify the clock protein abundance over time. For this endeavor, the researcher has to dissect and analyze a large number of animals, which is very labor-intensive and time-consuming. A standard timeseries in Drosophila by imaging PER-Luciferase (PER-LUC) activity . Even though this technique has the potential to overcome the labour-intensive timeseries sampling for immunohistochemical experiments, only one more study utilized the single-cell luciferase imaging approach resulting in two publications . We believe that this is on one hand partially caused by the complicated cultivation protocols available and on the other hand that the two mentioned studies have been performed on custom-built microscope setups, which might deter potential users. ).In 2011, the lab of Herman Wijnen was the first to describe a procedure which enables to assess the clock protein cycling of single clock neurons in individual brains of Drosophila brains, which is sufficient to keep the brains alive and healthy for more than a week, even without partial substitution of the culture medium on a regular basis. We further used a commercial Olympus Luminoview (LV200) luminescence microscope equipped with an EMCCD camera  to monitor the PER-LUC expression of single clock neurons over several circadian cycles to show the viability of the explanted brains in an independent assay.In this article, we briefly describe a culturing protocol for explanted 2+ free Ringer’s solution (see reagents), we placed the brains in a Poly-L-Lysine coated glass bottom petri dish (Greiner Bio-One) and added the culture medium. The culture medium contains 20% heat-inactivated fetal bovine serum, 1% Penicillin-Streptomycin and 0.75 µM Luciferin. Subsequently, the dish with the brain explants was transferred either to a culture incubator or to the luminescence microscope for imaging. For avoiding movement artifacts during long-term recording, we decided to culture the brain explants without partially substituting the culturing medium. To test the viability of these explants, we assessed pan-glial EGFP expression over the course of several days, since glia are essential for neuronal survival and suppression of cell death . We could not observe any dramatic loss of glial cells after eight days in vitro (DIV8) without adding fresh medium . The majority of the targeted cells express EGFP on DIV8, and we therefore concluded that the explants are viable for at least eight days in culture. In the next step we assayed brains from the y w; Pdf::RFP1; BG-luc line  under the LV200 luminescence microscope. The resolution of the setup is sufficient to identify single clock neurons by their PER-LUC expression by using either 30-fold or 60-fold super apochromat objectives and 300 s exposure time . The specificity of the y w; Pdf::RFP1; BG-luc line was confirmed by analyzing the native Pdf::mRFP1 expression and a separate staining against the firefly luciferase, which was colocalized in the Pigment Dispersing Factor expressing (PDF+) ventrolateral clock neurons . We could further show that the staining for the circadian output factor PDF also worked after the brain had been imaged and kept in culture for eight consecutive days, showing that the tissue is indeed viable throughout the complete recording time . Even though it was not possible to record z-stacks of the cultivated brains with the LV200 due to the long exposure time (300 s for one plane), the simultaneous recording of up to six synchronized brains is an option to increase the probability of recording several circadian clock clusters of different focal planes and/ or to increase sample size .The flies for this experiment were reared under 12:12 hr light:dark-cycles on standard corn agar medium at 25°C with 60% relative air humidity. After dissection in ice cold Caet al., 2002) identified a protein cycling with a circadian period of approximately 24hrs in one of the neurons . The period length estimated by MESA is shorter than the one obtained with autocorrelation analysis . In the other neuron both methods failed to detect rhythmicity, even though the oscillations appeared rhythmic . We estimated the period length by manually measuring the peak-to-peak intervals. Although the underlying rhythm is not significant, the manually calculated period length of approximately 23.6hrs falls into the circadian range. It is reported that the l-LNv would not cycle in constant darkness in the intact fly , but the aforementioned studies observe a PER cycling in those cells as well . This is most likely due to the loss of inhibitory signals from the periphery .The analysis of the recorded large ventrolateral clock neurons’ (l-LNv) PER-LUC rhythms via a MATLAB toolbox ; expresses mRFP1 under the control of the Pdf promotor and a PERIOD-LUCIFERASE fusion protein in all clock neurons. See Ruben et al. (2012) for information on the Pdf::mRFP1 construct and Stanewsky et al. (1997) for information on the BG-luc line.Repo-Gal4 ; expresses GAL4 in glia cells. See Sepp et al. (2001) for further information.UAS-S65T-GFP ; expresses GFP.S65T under the control of UAS.Primary antibodies:Anti-LUC ; raised in mouse, binds to the Luciferase (LUC) enzyme. Used in a final concentration of 1:2000.Anti-mCherry ; raised in rat, binds to red fluorescent protein (RFP) and its derivates. Used in a final concentration of 1:2000.Anti-PDF-C7 ; raised in mouse, binds to the pigment dispersing factor (PDF) neuropeptide expressed by the lateral clock neurons. Used in a final concentration of 1:4000.Secondary antibodies:AlexaFluor488 anti mouse ; used in a final concentration of 1:200.AlexaFluor568 anti rat ; used in a final concentration of 1:200.AlexaFluor647 anti mouse; used in a final concentration of 1:200.Buffers and solutions:2+ free Ringer’s solution CaCulture medium Fetal bovine serum Luciferin (Biosynth #L-8220)Paraformaldehyde 4% Penicillin/ Streptomycin Phosphate buffered Saline Poly-L-lysine (Sigma-Aldrich #P4707)Schneider’s insect medium Glass bottom culture dish Greiner Bio-One #627861


Malignant Pleural mesothelioma (MPM) is a rare disease which is associated with a poor prognosis. Front line chemotherapy represents the cornerstone in the management of MPM, and the place of radical surgery is controversial and reserve in early-stage disease. However prolonged survival (more than 24 months) can be observed in rare cases and only in the context of multimodal treatment including surgical management. We report the case of a patient suffering from an epithelial MPM with a 14-years progression-free survival after trimodal treatment including extrapleural pneumonectomy followed by chemotherapy and radiotherapy. This case illustrates that despite being an aggressive disease, multimodal management including radical surgery may allow a prolonged response in MPM but requires a whole-life surveillance. •Malignant pleural mesothelioma (MPM) is a rare disease.•Prognosis of MPM is very poor despite new therapeutic agents.•The place of radical surgery still remains controversial.•Prolonged survival can be obtained by multimodal therapy including radical surgery.•Complete remission after treatment requires a whole-life surveillance. Among them radical surgery remains controversial even in the setting of multimodal approach. However, the patients with prolonged survival are those managed with multimodal approach including extrapleural pneumonectomy (EPP) or pleurectomy/decortication (P/D). Usually these patients are macroscopically considered with complete tumoral resection (R0) and the challenge is the design of post therapeutic follow-up in terms of frequency and duration. Here we present, after his informed consent and according to SCARE criteria , the cas22) and oxaliplatin (130 mg/m2), was administered followed three weeks after by radiotherapy with 65 Gy of the involved hemithorax for 6 weeks. Three other cycles of chemotherapy was administered starting four weeks after the completion of radiotherapy with mild hematologic toxicity and no alopecia [18F-fluorodeoxyglucose (FDG) PET showed a metabolic nodular thickening of the left costo-diaphragmatic junction . Second, a recurrence can happened after a prolonged period leading to the necessity of whole-life surveillance. To our knowledge, it is the first case concerning a 15-year prolonged progression-free survival in patient suffering from a stage II malignant pleural mesothelioma.None.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not applicable.JG and PA collected the clinical data and wrote the paper.PA followed the patient for 14 years.BC, SL, HD approved the draft and contribute to this report.Not commissioned, externally peer-reviewed.Philippe ASTOUL is the guarantor of this work.None.


Traumatic lesions of articular cartilage represent a crucial risk factor for osteoarthritis. Even if several strategies exist to treat such damages, the optimal solution has not yet been found. A new strategy represents the scaffold-free spheroid-based autologous chondrocyte transplantation. In this method, spheroids of chondrocytes are synthesized after chondrocyte isolation and expansion, followed by the implantation in a second intervention.Fine Jamshidi-needle biopsies from five patients  treated with a spheroid-based autologous chondrocyte implantation (ACI) after traumatic lesions of the articular cartilage of the knee were analysed histologically and immunohistologically for collagen II, collagen X and aggrecan expression. The indication for a second look arthroscopy was given by arthrofibrosis or meniscus-lesions, respectively. The time between ACI and second-look arthroscopy ranged between 6 and 16 months.In all patients, the histological examinations revealed an avascular cartilage tissue with a homogenic extracellular matrix. The subchondral bone neither showed bleeding, necrosis nor hypertrophy. A homogenous alcian blue staining indicated high amounts of mucopolysaccharides and glycosaminoglycans. Collagen II staining was highly positive, whereas collagen X staining was negative in every patient, ruling out hypertrophic chondrocyte differentiation. In addition, intense aggrecan staining indicated a strong expression of this extracellular matrix component.The present case series represents the first histological and immunohistological analyses of spheroid-based ACI in humans. Spheroid-based ACI revealed excellent histological results regarding the regeneration of hyaline articular cartilage. These results indicate that spheroid based ACI is a promising strategy for treating traumatic lesions of the articular cartilage of the knee. Hyaline cartilage has a unique capacity to answer pressure transformations and is thus critical for the proper function of the musculoskeletal system . HoweverDifferent strategies have been developed to treat cartilage lesions, such as mosaic arthroplasty, microfracture, autologous matrix-induced chondrogenesis (AMIC) and autologous chondrocyte implantation (ACI) , 12. TheCurrently, several Matrix-based ACI (MACI) are in clinical use , 18, 19.The spheroid-based ACI represents a relatively new technique. It requires two surgical interventions, one for harvesting chondrocytes and one for the implantation of the generated chondrocyte spheroids , 14. TheEthical approval for the re-evaluation was given by the Institutional Review Board of the University Regensburg (ID-number: 19-1558-104).Samples were collected from five patients (one biopsy from each patient) treated with spheroid-based ACI  due to traumatic lesions followed by re-arthroscopy. All defects were localized on the medial femur condyle. Implantation was performed via mini-arthrotomy Fig. . Re-arthStandardized and automated methods were used in all procedures. The specimens were decalcified, dehydrated and embedded in paraffin . Sections of 5 µm thickness were deparaffinized and histological staining was performed with haematoxylin/eosin and alcian blue. Immunohistological detection was performed for collagen II, collagen X and aggrecan Table  in a fulSpecimens were histologically analysed. The existence of hyaline cartilage-like tissue, fibrous tissue and potential vascularization were examined. Furthermore, the potential hypertrophy of the subchondral bone was described.The evaluation was performed by two independent double-blinded investigators (D.G. and C.B.) using a visual-based scheme in which the stained area and the intensity of the staining were scored semi-quantitatively using a wide-spread scheme in histopathology. The staining intensity was evaluated as zero (negative/no staining), + (slight positive), +  + (moderate) and +  +  + (strongly positive). A positive control (+ +  + /strongly positive) and a negative control (0/negative) were used as references. No test of significance was possible due to the very low number of patients. Therefore, the focus was placed on the exact description of the tissue. The findings were documented by whole slide imaging using a Digital Microscope and Scanner Device .Histologically, an avascular cartilage tissue with an intact cartilage-bone border interface was observed in the regenerated tissue in all patients. Cells within the regenerated tissue showed a round and chondrocytic phenotype with cluster-formation. In three patients the typical layer-based architecture with flattened chondrocytes in the apical layer was detectable. The alcian blue staining revealed intensive and homogenous staining of the extracellular matrix in all patients, which represents an indicator for the presence of glycosaminoglycans and mucopolysaccharides. In three of five patients, the staining was intensive in the basal half of the biopsy and decreased in the apical direction, corresponding to the three patients with the typical layer-based structure. The other two patients showed a homogeneous alcian blue staining. There were no signs for degenerative changes, such as demarked collagen fibres in the tissue. Hypertrophy of the subchondral bone was not detected in any of the five analysed patients.Immunohistologically, one biopsy showed an intensive and homogenous expression of collagen II within the extracellular matrix. Two biopsies showed an intensive expression of collagen II in the basal and middle zone of the biopsy with a slight decrease of intensity in the apical half of the specimen. In one patient, higher expression in the apical zone could be detected than in the middle and basal zone. One patient was not evaluable due to loss of the cartilage from the biopsy after performing the histological staining. The immunohistological staining for aggrecan revealed a homogenous strong expression in all patients. No spatial differences of the aggrecan positivity could be detected in the basal, middle or apical zone of the biopsies. The immunohistological staining for collagen X antigen was negative in four patients. In one patient no more cartilage tissue was seen in the biopsy. The results of the immunohistological evaluation are summarized in Table The present study demonstrates for the first time that the regenerated cartilage tissue after spheroid-based ACI is histologically, histochemically and immunohistochemically very similar to hyaline articular cartilage. The typical layer-based architecture with a deep, middle and apical zone was identified in three patients. In addition, these three patients demonstrated a typical apical zone with two to three layers of flattened chondrocytes and a decrease of alcian blue-positive glycosaminoglycans, which is well known from initial articular cartilage. Specimens with high amounts of glycosaminoglycans, high expression of collagen type II and cells with chondroid phenotype, but without the typical layer-based architecture was defined as “very similar to hyaline articular cartilage”, which was present already 5 ½ months after ACI and almost of similar structure in the specimen which was taken 16 months after ACI. These findings implicate a good regenerative potential of the spheroids, making them an attractive alternative to other methods where changes in the regenerative tissue and long-term failures have been described , 6. LongIn this study biopsies taken between 5 ½ and 16 months after ACI clearly demonstrated the regeneration of predominantly hyaline cartilage-tissue. Nevertheless, the limited number of specimens without a homogenous study cohort is a major limitation of the present case-study. Further clinical trials with a larger number of patients have to be performed to consolidate these results and to generate statistically significant data . The indThe present study represents the first histological and immunohistological analysis of spheroid-based ACI. It shows that spheroid-based ACI yields excellent histological results in terms of regeneration with predominantly hyaline articular cartilage.


My brother and I are writing a letter in response to the paper “Follow-Up Investigation on the Promotional Practices of Electric Scooter Companies: Content Analysis of Posts on Instagram and Twitter” by Dormanesh et al .The paper discusses the promotion of proper safety equipment for riding electric scooters (e-scooters) online. The authors found that the social media accounts of prominent e-scooter companies Bird and Tier Mobility rarely, if ever, included photos of people wearing equipment such as helmets, knee pads, or wrist guards. To expand on this subject, Dormanesh and colleagues  could coThe importance of educating teenagers on appropriate safety gear is supported by recent research done reporting e-scooter injuries in the pediatric population. A previous study of 990 patients who sustained craniofacial injuries due to motorized scooter use shows that almost 50% of the injuries were to children under the age of 18 years, including 33% who were under the age of 12 . All of E-scooter accidents are currently difficult to accurately track in injury databases. A recent paper examined e-scooters using the “hoverboard” category from the National Electronic Injury Surveillance System, which includes electrically powered skateboards . The lac


Advances in spectral-domain optical coherence tomography (SD-OCT) technology have enhanced the understanding of external limiting membrane (ELM) and ellipsoid zone (EZ) in diabetic macular edema. An increase in VEGF has been demonstrated to be associated with sequential ELM and EZ disruption on SD-OCT. An intact ELM is a prerequisite for an intact EZ in DME. Anti-VEGF therapy leads to restoration of barrier effect of ELM. The ELM restores first followed by EZ restoration. Diabetic retinopathy (DR) is a major complication of diabetes mellitus . DiabetiAdvances in spectral-domain optical coherence tomography (SD-OCT) technology have enhanced the understanding of morphological alterations in individual layers of retina and their association with various molecular mechanisms . The extThe ELM separates the layers of rods and cones from the overlying outer nuclear layer and is a linear confluence of junctional complexes between Muller cells and photoreceptors , 10. It The EZ clinically defines the photoreceptor integrity. The biological EZ consists mainly of mitochondria. This enables higher levels of energy consumption within the photoreceptors. Focal or global absence of the EZ on SD-OCT corresponds to the reduced reflectivity or anatomic absence of the EZ. Dysfunction of mitochondria in the foveal photoreceptors results in reduced VA in DME .The ELM and EZ integrity is essential for the maintenance of normal vision . DME is Omri et al.  demonstrVEGF alters TJs and promotes vascular permeability in many retinal as well as brain diseases. The molecular mechanisms of this barrier regulation is however, not very well understood. Murukami et al.  highlighAnti-VEGFs are considered as the first-line treatment for DME. Administration of intravitreal anti-VEGF agents has been found to be associated with reduction in CST and improvement in visual acuity (VA) –29. RestDe et al. discovered the mechanism of ELM and EZ restoration after anti-VEGF therapy in DME. Anti-VEGF therapy led to restoration of barrier effect of ELM. The ELM was established as a retinal structural barrier and was found to restore first followed by EZ restoration. Decrease in logMAR VA was more pronounced in patients associated with restoration of ELM and EZ .An increase in VEGF results in sequential ELM and EZ disruption on SD-OCT. An intact ELM is a prerequisite for an intact EZ in DME. Anti-VEGF therapy leads to restoration of barrier effect of ELM. The ELM restores first followed by EZ restoration.


An efficient solution to increase the sustainability of building materials is to replace Portland cement with alkali-activated materials (AAM). Precursors for those systems are often based on water-cooled ground granulated blast furnace slags (GGBFS). Quenching of blast furnace slag can be done also by air but in that case, the final product is crystalline and with a very low reactivity. The present study aimed to evaluate the cementitious properties of a mechanically activated (MCA) air-cooled blast furnace slag (ACBFS) used as a precursor in sodium silicate alkali-activated systems. The unreactive ACBFS was processed in a planetary ball mill and its cementing performances were compared with an alkali-activated water-cooled GGBFS. Mixes based on mechanically activated ACBFS reached the 7-days compressive strength of 35 MPa and the 28-days compressive strength 45 MPa. The GGBFS-based samples showed generally higher compressive strength values. Th. Th33]. The MCA-ACBFS based mortars activated with the SS having the alkali modulus of 1 and containing 10 wt.% of the SS alkali activator showed comparable or higher 28-day compressive strength values than the GGBFS . MortarsThe compressive strength values tended to increase at lower w/b ratios for both types of the used slags. Usually the w/b ratio had a smaller effect on mechanical properties in the case of the AAM and the chemical composition of the precursor was the dominant factor . In thisThe workability of the GGBFS-based concrete was not affected by a higher alkali modulus . On the Both effects were observed also by others and were related to a higher pH values which accelerated dissolution of the slag ,35,36,37SEM-EDS analysis was performed on alkali-activated slag samples having w/b 0.45, alkali modulus of 1.5 and 10% of solid solution . The alkThe chemical composition of the solidified binder matrix was characterized by the SEM-EDX spot analysis. The calculated Ca/Al and Ca/Si ratios are shown in The ternary diagram  based onInert and crystalline air-cooled slag can be amorphized and have enhanced reactivity by application of an optimized mechanical activation process.MCA-ACBFS can be considered as precursor for alkali activated systems.Workability of ACBFS is strongly influenced by the used mechanical treatment and sodium silicate activator.The highest compressive strength of mortars based on MCA-ACBFS was achieved for mixes having the w/b = 0.45 and activated with 10% of SS. The AM was 1.The highest compressive strength of mortars based on GGBFS was achieved for mixes having the w/b = 0.45 and activated with 10% of SS. The AM was 1.5.Decreased intensities and increased area of the XRD peaks indicated an increased amorphization of the treated samples after application of the optimized mechanical activation process. SEM-EDX analysis showed a quite similar composition of the solidified binder matrixes in both tested slags. The main conclusions can be summarized as follow:Further tests are needed in order to evaluate the suitability of using MCA-ACBFS as a precursor for AAM. Standardized test methods should be used for full scale comparison with traditional materials.


Angioleiomyoma of the pulmonary artery is rare in the literature and few studies have been reported. Here we present a rare case of angioleiomyoma arising from the pulmonary artery in a young patient.A 27-year-old male patient presented to our clinic due to the incidental finding of a nodule in the right lower lobe of the lung, which was unchanged from the prior year. Preoperative CT scans showed a well-demarcated nodule of soft tissue density penetrated by the basal branch of the right anterior basilar artery (RA8b). Single-port video-assisted RS8 segmentectomy was performed under the guidance of preoperative 3-dimensional reconstruction for histologic confirmation of the tumour. The tumour appeared as a solid tumour of a tube-like structure with vascular endothelium, composed of spindle-shaped smooth muscle cells lacking nuclear atypia and homogenous red-dye substances. The spindle cells were positive for immunostaining for smooth muscle actin (SMA), desmin and Ki-67 and were negative for immunostaining for Dog-1, HMB45, and Melan-A. A pathological diagnosis of primary angioleiomyoma of the pulmonary artery was finally made.This report is a reminder for thoracic surgeons that angioleiomyoma should be included in the differential diagnosis of lung neoplasms, especially for the mass of soft tissue density penetrated by pulmonary blood vessels shown by CT. Awareness of this rare entity should potentially prevent underdiagnosis and improper surgical treatment. Angioleiomyoma is a benign soft tissue tumour comprising mature smooth muscle cells with a prominent vascular component . It typiA 27-year-old male patient complained of the incidental finding of a nodule in the right lower lobe of the lung on a routine health examination in October 2019, which was unchanged on repeat examinations from the prior year. No related subjective symptoms were noted and the physical examination showed no abnormalities. The results of routine blood biochemistry, oncological biomarkers and flexible bronchoscopy were normal. Contrast-enhanced computerized tomography of the chest showed a well-demarcated mass with a maximum diameter of 1.7 cm in the right anterior basilar segment of the lung (RS8). The lesion, which displayed a soft tissue density in the periphery, was penetrated by the basal branch of the right anterior basilar artery (RA8b)  in 2002, but reclassified under ‘pericytic tumours’ in the updated classification because of morphological features shared with myopericytoma, that is, showing a perivascular concentric arrangement of smooth muscle cells . AdditioClinically, angioleiomyoma usually occurs in the lower extremities and appears as a solitary, slow-growing, mobile, firm and occasionally painful cutaneous mass . The paiPreoperative diagnosis of angioleiomyoma is difficult before histopathology . CompletTo the best of our knowledge, only two cases of angioleiomyoma of the pulmonary artery have been reported so far , 7. The We believe this report emphasizes the necessary awareness of thoracic surgeons that angioleiomyoma should be included in the differential diagnosis of lung neoplasms, Although an accurate preoperative diagnosis can be challenging, in our opinion, CT imaging findings presenting as a soft-tissue density nodule penetrated by the pulmonary blood vessel may indicate the possibility of angioleiomyoma. Due to the benign nature of this tumour, sublobar resection is appropriate for the treatment of angioleiomyoma of the pulmonary artery.Additional file 1. Video 1 Legend. Single-port RS8 segmentectomy was performed through the fifth intercostal space between anterior axillary line and midaxillary line. Insufflation technique was utilized to establish intersegmental border (Asian Cardiovasc Thorac Ann 2019 Nov; 27 [


People with chronic kidney disease (CKD) have high levels of co-morbidity and polypharmacy placing them at increased risk of prescribing-related harm. Tools for assessing prescribing safety in the general population using prescribing safety indicators (PSIs) have been established. However, people with CKD pose different prescribing challenges to people without kidney disease. Therefore, PSIs designed for use in the general population may not include all PSIs relevant to a CKD population.The aim of this study was to systematically collate a library of PSIs relevant to people with CKD.A systematic literature search identified papers reporting PSIs. CKD-specific PSIs were extracted and categorised by Anatomical Therapeutic Chemical (ATC) classification codes. Duplicate PSIs were removed to create a final list of CKD-specific PSIs.Nine thousand, eight hundred fifty-two papers were identified by the systematic literature search, of which 511 proceeded to full text screening and 196 papers were identified as reporting PSIs. Following categorisation by ATC code and duplicate removal, 841 unique PSIs formed the final set of CKD-specific PSIs. The five ATC drug classes containing the largest proportion of CKD-specific PSIs were: Cardiovascular system (26%); Nervous system (13.4%); Blood and blood forming organs (12.4%); Alimentary and metabolism (12%); and Anti-infectives for systemic use (11.3%).CKD-specific PSIs could be used alone or alongside general PSIs to assess the safety and quality of prescribing within a CKD population.The online version contains supplementary material available at 10.1186/s12882-020-02158-0. People with chronic kidney disease (CKD) stand to benefit from careful prescribing of medications to prevent disease progression, manage comorbidities and relieve symptoms . MeanwhiPrescribing safety indicators (PSIs) distinguish prescribing events that put a patient at risk of harm . PSI libThe aim of this study was to use a systematic search of the published literature to produce a library of PSIs specific to outpatient prescribing for people with CKD. This library could be used alone or alongside general PSIs to assess the safety and quality of prescribing within a CKD population.A protocol was written to collate PSIs with specific relevance to people with CKD from the published literature (PROSPERO CRD42018109113). A systematic literature search to identify papers reporting PSIs was followed by extraction of CKD-specific PSIs. Pre-defined criteria classified PSIs as CKD-specific if they were of exclusive relevance to adults (age ≥ 18) with CKD and referring to medications prescribed within the outpatient setting.A sensitive search strategy was designed to capture all potentially relevant papers reporting PSIs. To capture PSIs from the general primary care population the search strategy used by Spencer et al. was updated to include literature published from 2012 until 2018 . Search Results from the two searches were combined and duplicate results were removed. Titles and abstracts were independently screened by two of the authors . An inclusive approach was used, excluding only those records from non-human or paediatric work (age < 18 years) or those referring solely to prescribing in an inpatient setting. There was no selection for CKD-specific PSIs made at this stage. If a record was recommended for inclusion by one of the reviewers it progressed to full text screening.Full-text screening was conducted by FS. Publications reporting one or more PSI in the main text, abstract or tables of the publication were included. The details of all PSIs from the literature search were entered into a data extraction form by FS. The elements extracted included details relating to the publication itself – title; authors; journal; date of publication; study design; setting; target population; methodology and method of indicator development – in addition to exact replication of each PSI from the original publication.A two-stage process followed to identify CKD-specific PSIs. Firstly, an automated filter was developed to shortlist possible CKD-specific PSIs. The automated CKD-specific filter terms  codes of each medication listed in the prescribing statement . DuplicaIn total 9852 papers were identified by the two searches . Following title and abstract screening 511 papers proceeded to full text screening . Of the 511 articles, 315 did not contain any PSIs. PSI extraction was performed from the remaining 196 articles, with a total of 3464 PSIs identified. [Fig. The CKD-specific PSIs were classified by ATC code. Three hundred seventy-one duplicates were identified and excluded. A further 19 were deemed not to fit the pre-specified criteria after discussion between reviewing authors (6 related to inpatient prescribing and 13 were deemed not to be CKD-specific). The remaining 841 unique PSIs became the final set of CKD-specific PSIs. Examples of PSIs are demonstrated in Table PSIs were found to take multiple different forms, including those that referred to single drugs  were found, with the majority considering cardiovascular, nervous and haematological prescribing. Drugs acting on the renin-angiotensin system, non-vitamin K oral anticoagulants, anti-diabetic medications, lipid-modifying agents, and antidepressants were particularly frequent. This library of CKD-specific PSIs is a first step towards generation of prescribing safety assessments and interventions for populations with CKD.CKD, defined by persistently abnormal glomerular filtration rate (GFR), and/or other evidence of damage or structural abnormality, has a global prevalence approximating 13% , 7. The Prescribing for people with CKD is further complicated by comorbid illness. CKD is both a cause and consequence of comorbidity, most notably microvascular, macrovascular and cardiac disease. Since the prevalence of CKD increases with age, acquisition of concurrent illness is frequent. People living with CKD may thus be recommended medications to manage causal conditions, coincidental co-morbidity, and complications of low-renal function – alongside agents which are preventative against cardiovascular disease and end stage kidney disease. Unsurprisingly, this can lead to the prescription of numerous drugs , 9. The An additional factor influencing renal prescribing is that conventional estimates of GFR use serum creatinine, which is affected by muscle mass and protein intake. Prescribers must be alert that age-related changes in body composition may result in lower accuracy of eGFR. KDIGO recommends prescribers use the most accurate method for GFR estimation when drug dosing . EstimatClinical support systems such as computerised alerts, manual-review and eGFR-prompts have been used to reduce inappropriate prescribing in CKD populations, and can be effective in both inpatient and outpatient settings . NeverthThe main strength of this work is the use of an inclusive systematic approach before focussing on CKD-specific PSIs. This process reduces the risk of missing CKD-specific PSIs that are reported in the general literature. The main weakness is an output that requires further work before application in research or clinical settings. Removal of near-duplicate PSIs, selection of uniform dosing and GFR-estimation criteria for individual agents, appraisal in light of present evidence and supplementation with PSIs for prescribing events that did not appear is needed. Such a process will involve expert appraisal and formal approaches to consensus development beyond the scope of this work. Chronic kidney disease-specific PSIs relating to a wide range of agents were found, but the frequency of PSIs relating to particular agents should not be seen as indicative of the clinical importance of individual agents, and may simply reflect the frequency with which these agents are used, and/or general familiarity with their CKD-specific risks .This work focused on outpatient prescribing for individuals with CKD, so some agents well-known to present risks in kidney disease are not captured (e.g. intravenous aminoglycosides). Inpatient prescribing differs substantially from ambulatory settings, with acute illness, acute kidney injury and daily prescription review. A separate approach for collating and categorising PSIs relevant to this setting is required. A further limitation in this study is that a single author completed full text screening of articles recommended for inclusion thus posing a risk of PSIs being missed from the literature.This study has collated a library of safety indicators relating to outpatient prescribing for people with CKD – a unique group with a higher proportion of prescribing challenges. The library generated could be used alone or alongside general PSIs to generate approaches for assessment of prescribing safety and quality for individuals and populations with CKD.Additional file 1: Appendix 1 - Systematic literature search terms. Appendix 2 - CKD-specific PSI selection.Additional file 2: Appendix 3 - 841 CKD-specific PSIs.


Pulmonary fibrosis has been identified as a main factor leading to pulmonary dysfunction and poor quality of life in post-recovery Severe Acute Respiratory Syndrome (SARS) survivor’s consequent to SARS-Cov-2 infection. Thus there is an urgent medical need for identification of readily available biomarkers that in patients with SARS-Cov-2 infection are able to; (1) identify patients in most need of medical care prior to admittance to an intensive care unit (ICU), and; (2) identify patients post-infection at risk of developing persistent fibrosis of lungs with subsequent impaired quality of life and increased morbidity and mortality. An intense amount of research have focused on wound healing and Extracellular Matrix (ECM) remodelling of the lungs related to lung function decline in pulmonary fibrosis (PF). A range of non-invasive serological biomarkers, reflecting tissue remodelling, and fibrosis have been shown to predict risk of acute exacerbations, lung function decline and mortality in PF and other interstitial lung diseases . We suggest that lessons learned from such PF studies of the pathological processes leading to lung function decline could be used to better identify patients infected with SARS-Co-V2 at most risk of acute deterioration or persistent fibrotic damage of the lung and could consequently be used to guide treatment decisions. The COVID-19 pandemic, caused by the novel virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has reached pandemic status, causing severe impacts on health, major reductions in life quality due to widespread quarantines and social distancing and huge economic impacts, all of which affecting many millions of people worldwide. Updated information on COVID-19 disease is continuing to emerge, including from Wuhan in China from where the virus was first reported . Risk faThe SARS-CoV-2 virus has been reported to use the angiotensin converting enzyme 2 (ACE2) receptor and the viral entry-associated protease TMPRSS2 for cellular entry in the upper airway. ACE2 and TMPRSS2 have been detected in nasal and bronchial epithelium by immunohistochemistry. Gene expression occurs in approximately 2% of type II alveolar epithelial cells (AECs) –11, althAECs are squamous cells with long cytoplasmic extensions that are adapted to carry out gas exchange –14. TheyPF has been identified as one of the main factors leading to pulmonary dysfunction and poor quality of life in post-recovery Severe Acute Respiratory Syndrome (SARS) survivors –20. ExisA growing body of research has been undertaken to understand changes which occur in ECM dynamics related physiological impairment and lung function decline in e.g. idiopathic pulmonary fibrosis (IPF) –28.The lungs have a highly specialized ECM Fig. . In healThe long-term effects of SARS-CoV-2 infection on the pulmonary system must be understood to better support future COVID-19 patients. An important research area to prevent the development of fibrotic consequences of COVID-19 is to understand the effects of the SARS-CoV-2 infection in to the lung ECM . Data deKey data on neoepitope biomarkers within PF are summarized in Table Neoepitope markers of wound healing should also be considered as valuable markers in COVID19 patients and are under investigation in PF at the moment. As eluted to earlier, it has been found that D-Dimer is elevated in ARDS patients compared to non-ARDS patients and indicating increased woundhealing during ARDS. The relationship between cell damage and the resulting wound healing and ECM remodelling is schematically represented in Fig. Several groups have investigated the use of routine clinical parameters assessed in patients infected by SARS-CoV-2. Interestingly, a machine learning approach was used upon 398 patients by Booth et al.  with theIn conclusion, there are important lessons learned from diseases leading to PF in which key ECM remodelling and wound healing biomarkers are predicting exacerbations of disease and death.These important data and key learnings have the potential to be repositioned for COVID-19 research. Potentially, systemic markers of ECM turnover, inflammation and wound healing, the latter based on data from previous publications, may provide value in the clinical evaluation of COVID-19 patients, with the aim of assisting in the prediction of prognosis during the acute phase of disease and could potentially identify COVID-19 survivors at risk of developing permanent pulmonary damage and fibrosis.


Weight stigma internalization among adolescents across weight categories leads to adverse psychological consequences. This study aims to adapt and validate a Chinese version of the Weight Bias Internalization Scale for Mainland Chinese children and adolescents(C-WBIS). A total of 464 individuals aged 9 to 15 years participated in the present study. Based on item response theory (IRT) and classical test theory (CTT), we selected the items for the C-WBIS and evaluated its reliability and validity. The item response theory yields support for the one-dimensional factor mode. All item parameters fit the IRT model , eight items were adopted. No evidence of significant differential item functioning (DIF) was found for gender and age groups. The C-WBIS was correlated with the Core Self-Evaluation Scale (CSES) and two subscales of the Social Anxiety Scale for Children (SAS), which indicated an acceptable criterion-related validity. The C-WBIS is a reliable and valid measure that can be used as a psychometrically sound and informative tool to assess weight bias internalization among children and adolescents. There has been a considerable increase in research on the stigma toward individuals of varying weight statuses since Goffman’s pioneering work . ExperiePrior research on self-reported measures for assessing self-stigma suggests that studies predominantly use the Weight Self-Stigma Questionnaire(WSSQ) for overweight and obese individuals and the Weight Bias Internalization Scale(WBIS) across weight categories among children and adults. The WSSQ consists of two underlying psychological dimensions using (a) self-devaluation and (b) fear of enacted stigma . The WBIContemporary literature indicates that the WBIS has been validated as a psychometrically sound and informative tool to assess weight bias internalization and has supported a unidimensional interpretation of the WBIS. More specifically, the original validation of the WBIS has analyzed the 11-item WBIS to measure weight bias internalization, and included a sample of 198 participants aged >18 years and self-identified as overweight. Cronbach’s alpha was 0.90. One-dimensional factors would be retained when the component extraction was set to one. The scale has significant partial correlations with the dislike subscale of the Antifat Attitudes Questionnaire(AAQ) and the drive for thinness in the Eating Disorders Inventory(EDI),suggesting that anti-fat attitudes, drive for thinness and weight bias internalization are related but different from each other .The revised German version of the WBIS contains 11 items in a large sample of 1092 participants aged between 14 and 89 to conduct the latent variable structure of the WBIS. Cronbach’s alpha was 0.91 after items one was deleted. The goodness-of-fit criteria supported the single-structure of the 10-item WBIS . To be bFurthermore, the 11 item Weight Bias Internalization Scale for Youth (WBIS-Y) provides a valid and reliable assessment tool in a sample of 191 German adolescents aged between 13 and 19. Items were modified to be adjusted for adolescents. Cronbach’s alpha was 0.87. The first eigenvalue (4.92) explained 44.70% of the variance, and the ratio of the first to the second eigenvalue was 4.0,which supported the single-factor structure of the WBIS . A recenAlthough researchers have made great efforts on the operational definition and empirical measurement, it remains somewhat contested, and raises questions about whether to measure the latent trait that it is associated with weight bias internalization. In addition, studies on the WBIS have been conducted in Western countries. Such studies on school-aged adolescents across weight categories remain under investigation, especially in East Asian regions.China is the most populous country in East Asia. Evidently, only a few studies conducted with children or adults have focused on assessing degree of self-stigma, such as perception of incorrect beliefs . In a HoPrevious studies have reported the psychometric properties of the WBIS. However, additional psychometric evaluations are necessary as most of the studies evaluating the psychometric properties of the WBIS merely use classical statistical methods, such as exploratory factor analysis, internal consistency, and test-retest reliability. These methods essentially use the correlation coefficient that falls within “classical test theory” as it has the same underlying weakness as the other methods analyzing correlations among items . These wtrans-cultural utilization of foreign scales significantly differs from its utilization in the Chinese mainland on school-aged children and adolescents across weight categories. It is unwise to assess weight bias internalization if the scale is introduced merely based on previous studies without rewording sensitive items to mitigate its controversial aspects. Thus, this study aims to evaluate the reliability and validity of a Chinese version of the Weight Bias Internalization Scale (C-WBIS), and to select and validate C-WBIS items based on different psychometric analyses. According to Anna and Roberto’s findings  have been used numerous times for other scales , they hafindings ,the hypo(1)The C-WBIS items would indicate an acceptable unidimensionality and yield reliable and valid scores for male and female children and in various age groups.(2)The total score of the C-WBIS would be associated with higher levels of core self-evaluation and social anxiety.M = 11.14 years; SD = 2.01 years) across weight categories. These participants were given a brief introduction and completed surveys under quiet conditions within the given 15-min period. There were 236 participants younger than 12 and 185 participants over 12. The second subsample included 43 students who were recruited for a student number to allow the matching of test–retest samples. Notably, the participation was voluntary. Consequently, the subjects’ decision to participate was made upon reading the informed consent document.The sample of this work comprised two subsamples and used the school class as a sampling unit. The first subsample comprised 200 female and 221 male primary and middle school students aged between 9 and 15  for treatment-seeking obese adolescents ,the WBISAfter minor revision, the C-WBIS was finalized. Specifically, we used the words “my weight” instead of the word “overweight” in evaluating the participants across different body weight statuses, and reworded controversial items that would cause additional distress to students. Thus, the item “My weight is a major way that I judge my value as a person.” was replaced with the item “My weight strongly influences what I think of myself confidence and worth as a person”see . A totalCore self-evaluation was assessed using the Chinese version of the Core Self-Evaluation Scale (CSES) ,which adRegarding the Social Anxiety Scale for Children (SAS) , we emplDemographic information. Participants were asked about their gender and age using a demographic questionnaire.Based on the principles of item response theory (IRT) and classical test theory (CTT), the items of the C-WBIS were selected and validated.n = 43) using Pearson’s correlation coefficients. Test–retest intervals were set at 2 weeks.An item was considered for deletion if its standard deviation was ≤1, which evaluated the sensitivity of the items. Items were removed from the analysis if the corrected item-total correlation (CITC) was ≤0.40 . To estin = 421). If the ratio between the first and second eigenvalues exceeded three, a single latent construct was reflected   to assess construct validity using AMOS 20.0. The goodness-of-fit criteria used in this study including the Comparative Fit Index(CFI) (good fit is ≥0.95), Tucker–Lewis Index(TLI) (good fit is ≥0.95), and root mean square error of approximation(RMSEA) (good fit is <0.06) were examined . The cri0 −0.50) .X-axis indicates latent weight bias internalization in a standardized unit, and the Y-axis indicates the probability of choosing the number of response categories  to evaluate the following item parameters : discrimtegories .Furthermore, Rasch analysis based on the Andrich rating scale model (joint maximum likelihood estimation) was used to detect differential item functioning (DIF), a method for studying measurement equivalence across groups which assesses the equivalence of both item discrimination and item difficulty parameters . The sigA combination of the results led to the removal of three items from the C-WBIS. In Basic definitions and criteria were provided for each item parameter based on IRT and the principles of classical test theory (CTT), such as discrimination (a), difficulty parameters (b), and CITC. At this stage, it was decided to delete three items but to retain eight items. Thus, the analysis was run with the remaining eight items. As a prerequisite for conducting analysis based on IRT, a one-dimensional factor model needs to be confirmed. Principal components analysis (PCA) presented the C-WBIS as a single factor measure. The first eigenvalue (3.604) explained 45% of the variance, and the ratio of the first to the second eigenvalue was 3.6. A comparison of the original dataset eigenvalues and randomly generated eigenvalues suggested that the one-dimensional factor should be retained , which could improve measurement precision. The table of DIF identification toward the overall items is displayed in the r = −0.33, p < 0.01). Small but significant correlations were observed between C-WBIS and FNE  and SAD .Associations were observed between weight bias internalization, core self-evaluation, and social anxiety. There were significant and moderate inverse correlations between core self-evaluation and weight bias internalization .In a nutshell, three items were removed based on the item response theory. The final eight-item C-WBIS showed acceptable internal consistency , and the test-retest reliability was satisfactory over 2 weeks  using IRT and CTT as a framework for investigating the item parameters. Additionally, the results were consistent with the aforementioned studies on adolescents and adults . These fn = 538) of the participants wanted to become slimmer. Thus, we categorized the participants according to their age characteristics into two levels: younger than 12 and over 12. Our results showed that the measurement invariance of the single-factor structure was supported for C-WBIS across age (younger than 12 vs. over 12). These results expanded upon Roberto’s work and suggested that the C-WBIS would be culturally competent for children and adolescents in the Chinese mainland.Another aspect highlights measurement invariance as a key concept that had drawn wide attention. Our findings support the measurement invariance of the unidimensional factor model of gender groups. Thus, females and males participants in this study have equivalent meaning, and are consistent with a previous report examining German children, which contributes to the global use of this tool . Weight The findings of this study highlighted important issues related to the psychometric properties and acceptability of the C-WBIS. These findings suggest that analyses should be conducted to ensure that the environment, time, and process of the sample survey mitigated the disturbance to the survey as much as possible. Further, they indicate that the C-WBIS should be used regardless of whether adolescents’ weight status was categorized based on relative body mass index or self-reported, in that a high correlation pointed to a strong relationship between reported and measured Body Mass Index (BMI) . LessonsAlthough the C-WBIS has shortcomings, every WBIS is technically deficient . Our resThe C-WBIS might be beneficial to interventions targeting weight stigma. With this tool, the comprehensive impact of treatment on weight stigma and its consequences  could be adapted to adolescents who might benefit from information on coping with weight stigma. School psychologists or social workers could help students adopt more reasonable coping strategies toward the consequences of weight stigma in their lives by advocating preventive efforts targeting children of different weights.In conjunction with existing research, this study finds that the C-WBIS, which has both psychometric and practical implications, has been adapted to assess weight bias internalization for Mainland Chinese children and adolescents across weight categories. This study supported a one-factor structure of the C-WBIS with eight items being defined more parsimoniously than in its original version. This study is novel in that it combines modern measurement theory, item response theory (IRT), with classical test theory (CTT) to assess the WBIS, which assesses weight bias internalization in the general population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Fujian Normal University Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HC and Y-dY conceptualized the study. HC analyzed the data and wrote the manuscript. Y-dY supervised the whole study. Both authors contributed to the article and approved the submitted version.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.


Compared with disease biomarkers in blood and urine, biomarkers in saliva have distinct advantages in clinical tests, as they can be conveniently examined through noninvasive sample collection. Therefore, identifying human saliva-secretory proteins and further detecting protein biomarkers in saliva have significant value in clinical medicine. There are only a few methods for predicting saliva-secretory proteins based on conventional machine learning algorithms, and all are highly dependent on annotated protein features. Unlike conventional machine learning algorithms, deep learning algorithms can automatically learn feature representations from input data and thus hold promise for predicting saliva-secretory proteins.We present a novel end-to-end deep learning model based on multilane capsule network (CapsNet) with differently sized convolution kernels to identify saliva-secretory proteins only from sequence information. The proposed model CapsNet-SSP outperforms existing methods based on conventional machine learning algorithms. Furthermore, the model performs better than other state-of-the-art deep learning architectures mostly used to analyze biological sequences. In addition, we further validate the effectiveness of CapsNet-SSP by comparison with human saliva-secretory proteins from existing studies and known salivary protein biomarkers of cancer.http://www.csbg-jlu.info/CapsNet-SSP/. We believe that our model and web server will be useful for biomedical researchers who are interested in finding salivary protein biomarkers, especially when they have identified candidate proteins for analyzing diseased tissues near or distal to salivary glands using transcriptome or proteomics.The main contributions of this study are as follows: (1) an end-to-end model based on CapsNet is proposed to identify saliva-secretory proteins from the sequence information; (2) the proposed model achieves better performance and outperforms existing models; and (3) the saliva-secretory proteins predicted by our model are statistically significant compared with existing cancer biomarkers in saliva. In addition, a web server of CapsNet-SSP is developed for saliva-secretory protein identification, and it can be accessed at the following URL:  Saliva comes mainly from three pairs of salivary glands  and numerous minor salivary glands spread throughout the oral cavity . Similaromics technology, researchers have acquired vast amounts of disease-related data. Therefore, it is desirable to use bioinformatics methods to identify highly sensitive and specific biomarkers from big data. Because there are many signals for various physiological and pathophysiological conditions in blood, most studies on body fluid biomarkers focus on blood biomarkers  is a 16D vector in the PrimaryCaps layer, Wi,j is the weight matrix of an affine transformation, and Vj is a 16D vector obtained by the weighted sum sj on all the outputs μj ∣ i of the PrimaryCaps layer. There are eight capsules  in the HiddenCaps layer of each group that receive inputs from the outputs of the corresponding PrimaryCaps layer. The squashing activation function shown in Eq. ci,j is the coupling coefficient determined by the iterative dynamic routing process, and these coefficients sum to 1 for the eight capsules of HiddenCaps in each group. To train the model by dynamic routing, five routing iterations are applied in this study. The complete dynamic routing algorithm can be found in the original CapsNet paper [The HiddenCaps layer in each lane consists of eight 16D capsules that map the input proteins to different states. The computation between the PrimaryCaps and HiddenCaps layers is shown in Fig. et paper . The outDuring model training, all the deep learning models are trained using identical training strategies. The parameters of these deep learning models are optimized using the Adam stochastic optimization method  with theThe dropout, early stopping and regularization strategies are used to prevent overfitting of the complex deep-learning model. The dropout strategy adds multiple dropout layers in the prediction model. During training, samples are taken from different sparse networks of index numbers. During testing, an unthinned network is used with a smaller weight to easily approximate the average of all the thinning network predictions . AnotherThe bagging ensemble learning method  is appliS+ contains 350 saliva-secretory proteins, and S− contains 1750 non-saliva-secretory proteins. The number of iterations T is 10, and the size of the random selection n is 350.Here, Additional file 1. Thirty-Seven human saliva-secretory proteins that do not overlap with training set. These proteins are collected using the LC-MS/MS analyses reported in the literature and databases of SPD, LOCATE and UniProt. Then, the proteins in the training set are removed.Additional file 2. Function enrichment analysis of top 4000 proteins. The function enrichment analysis is implemented by treating the entire set of human proteins as the background among the top 4000 proteins ranked by S-value, using DAVID against the GO and KEGG pathway.


Higher-order functions have become a staple of modern programming languages. However, such values stymie concolic testers, as the SMT solvers at their hearts are inherently first-order.This paper lays a formal foundations for concolic testing higher-order functional programs. Three ideas enable our results: (i) our tester considers only program inputs in a canonical form; (ii) it collects novel constraints from the evaluation of the canonical inputs to search the space of inputs with partial help from an SMT solver and (iii) it collects constraints from canonical inputs even when they are arguments to concretized calls. We prove that (i) concolic evaluation is sound with respect to concrete evaluation; (ii) modulo concretization and SMT solver incompleteness, the search for a counter-example succeeds if a user program has a bug and (iii) this search amounts to directed evolution of inputs targeting hard-to-reach corners of the program.


Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of “marker” SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons. In recent years, the study of the genetic causes of monogenic diseases has evolved from a basic science research area into widely accepted clinical testing protocols with substantial impacts on diagnostics and clinical decision-making  is a common phenomenon that reduces the efficiency of PCR-based targeted sequencing. It was first described in 1991 as a “partial amplification failure,” causing a potential source of misdiagnosis for both dominant and recessive diseases  according to the manufacturer's instructions.Mutational screening was performed using three custom targeted gene panels with two sets of oligoprimers designed automatically using Ion AmpliSeq Designer®  containing 1,049 primer pairs for 37 genes, with a total length of 153 kb. More detailed characteristics of each target genes panels are presented in Rare genetic variants detected by NGS were verified via bi-directional capillary Sanger sequencing on an ABI 3730XL DNA Analyzer according to the manufacturer's instructions (Thermo Fisher Scientific). Alternative pairs of oligoprimers flanking the coding and adjacent intronic regions of the 37 genes were designed for PCR using open-source PerlPrimer  . List of variants with accession numbers is summarized in All genetic variants newly detected in this study were registered in public database ClinVar  in the ll reads . This caquencing .SCN1B gene). This amplicon was sequenced separately by capillary Sanger sequencing and the loss of the allele containing the c.641G>A (p.R214Q) variant was reproduced  PCR, we performed a single control PCR with two oligoprimers designed by AmpliSeq and flanking genomic region chr19:35524839-35525003  allowed us to identify a case of allelic dropout in the syndrome . Heteroz proband  and prob proband . TheoretManual analysis of oligoprimer binding sites using gnomAD revealed the presence of a common genetic variant c.4542+89C>T [total minor allele frequency (MAF) 0.098] in the 3′-end of the R-primer which caused ADO. This heterozygous SNV was detected in DNA samples of patient II-4 and his mother by PCR-RFLP analysis using an additional pair of oligoprimers flanking this region. Absence of genetic variant c.4516C>T (p.P1506S) in the mother's DNA sample was confirmed by capillary Sanger sequencing with two independent oligoprimers and PCR-RFLP analysis. Control re-sequencing on the IonTorrent platform showed that family member II-4 is a carrier of heterozygous c.4516C>T (p.P1506S) .LDB3 gene. A variant of unknown significance—c.1051A>G (p.T351A)—in the LDB3 gene in sample ARVD19 was detected only by panel I    but not oteins”) . This SNoteins”) .PKP2 gene was located close to the 5′-end and led to ADO in nine samples studied  and two negative results (complete loss of the c.2091A>G variant). This ADO event was caused by the intronic variant c.1904-49T>A located at the 3′-end of the primer-binding site.All amplicons with identified ADO events were carefully re-sequenced using alternative oligoprimer pairs and selective allele amplification was confirmed in all cases .Currently, ADO is a known limitation of PCR-based molecular diagnostic approaches. Caused by different mechanisms, the single allele amplifies exclusively or pre-dominantly, leading to overrepresentation of homozygosity  Guidelines recommend that assay development and quality control should include measures aimed at both detecting allelic dropout and minimizing its occurrence CLSI, .In this study, six ADO events were identified in both PCR-based sequencing platforms. The causes of these events were also revealed in all cases. On the IonTorrent platform, ADO events were caused by rare or unique genetic variants in the oligoprimer binding sites. On the capillary sequencing platform, ADO events were caused by common SNVs in the oligoprimer binding sites . As a reThere are 2 types of causes of the ADO phenomenon described in literature  “Sample-specific” causes due to the quality of the DNA sample or the low DNA concentration. Such ADO cases are found in forensic diagnostics, where only fragmented or degraded DNA is available, as well as in preimplantation diagnostics, where genotyping is performed on DNA extracted from one blastomere;(2) “Locus-specific” causes due to the characteristics of the locus under investigation. The presence of single nucleotide polymorphisms (SNPs) in oligoprimer binding sites of forward and/or reverse primers disturbs the specificity of the complementary interaction between the oligonucleotide and the target DNA sequences, leading to the lack of oligoprimer hybridization, and elongation of the amplicon.All ADO events revealed in this study involved locus-specific causes due to the characteristics of individual loci in normal concentrations of DNA.PAH gene to evaluate the probability of ADO depending on SNV location in oligoprimer binding sites. Four alternative variants of forward oligoprimers were designed containing the SNV in the 3rd, 5th, and 7th positions of the 3′-end of the oligoprimer. Sanger sequencing was performed for patients carrying the heterozygous genetic variant rs2247836 and mutation p.Arg158Gln in exon 5 of the PAH gene. Loss of heterozygosity was detected for all positions of the ADO-causing SNV. The authors recommended careful consideration during primer design of the rare/common SNVs in areas within 7 nucleotides of the 3′-end of oligoprimers. We found that the presence of SNVs close to the 5′-end of oligoprimers may also cause ADO events. Convincing data suggest that any polymorphic position within the oligoprimer sequence potentially reduces the accuracy of DNA diagnostics , inborn metabolic disease genotyping (FAH testing), hematology research etc  were found in 4 of 521 amplicons in panel 1 (0.77%). There are increasing numbers of studies that discuss the importance of ADO in DNA diagnostic procedures . The patients/participants provided their written informed consent to participate in this study.AS, AB, and SS performed wet genetic investigation. AS performed data analysis and drafting the manuscript. EZ–management of the project, editing, and final approval of the manuscript. All authors read, discussed, and approved the manuscript as submitted.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.