The tumour microenvironment (TME) plays a crucial role in the regulation of cell survival and growth by providing inhibitory or stimulatory signals. Extracellular vesicles (EV) represent one of the most relevant cell-to-cell communication mechanism among cells within the TME. Moreover, EV contribute to the crosstalk among cancerous, immune, endothelial, and stromal cells to establish TME diversity. EV contain proteins, mRNAs and miRNAs, which can be locally delivered in the TME and/or transferred to remote sites to dictate tumour behaviour. EV in the TME impact on cancer cell proliferation, invasion, metastasis, immune-escape, pre-metastatic niche formation and the stimulation of angiogenesis. Moreover, EV can boost or inhibit tumours depending on the TME conditions and their cell of origin. Therefore, to move towards the identification of new targets and the development of a novel generation of EV-based targeting approaches to gain insight into EV mechanism of action in the TME would be of particular relevance. The aim here is to provide an overview of the current knowledge of EV released from different TME cellular components and their role in driving TME diversity. Moreover, recent proposed engineering approaches to targeting cells in the TME via EV are discussed. Extracellular vesicles (EV) include 100–5000 nm vesicles released by almost all cell types. Exosomes are a subclass of extracellular vesicles ranging from 40 to 150 nm derived from multivesicular bodies, and are distinct for their biogenesis from ectosomes and pre-apoptotic vesicles, as the latter originate from the budding of the cell plasma membrane .EV activate signalling pathways in cells they fuse or interact with by transferring specific genetic and non-genetic components ,4. EV arDuring the last two decades, several studies have explored EV function in tumour from different origins, however, so far, the precise EV role remains uncertain. In cancer, EV from different cells of origin have been largely described as tumour promoters, however, available data also suggest that EV retain anti-tumour properties and can also act to restrain disease progression .The tumour microenvironment (TME) contains cancer cells, displaying different phenotypes and genetic features and a number of different cell types, including stromal cells, mesenchymal cells, endothelial cells (EC), and cancer associated fibroblasts (CAF) ,10. DiveNovel EV engineering approaches have offered the opportunity to control primary tumours and metastatic diseases ,13. TherThe TME drives pro-tumourigenic effects by boosting tumour expansion and metastatic spread. A number of studies have suggested that EV within the TME act as central mediators of angiogenesis, immune modulation, and metastatic spread . This imTumour lesions comprise multiple subpopulations of cells, including those endowed with “stemness” properties and named cancer stem cells (CSC). CSC are responsible for tumour initiation, metastasis formation, resistance to conventional therapies and disease recurrence . These rRecent studies have shed light on the role of CSC-derived EV as tumour promoters by regulating the TME remodelling. It has been found that colon cancer stem cells expressing the extracellular matrix metalloproteinase inducer (EMMPRIN), also named CD147, are able to release CD147+ EV, which activate a signalling cascade in recipient cells boosting their migratory phenotype. Moreover, the expression of CD147 was also found to regulate EV shedding. Consistently, EV release and their downstream effects on target cells were hampered by knocking-down CD147 or by using the anti-CD147 blocking antibody .EV conveying CSC signature have been recently proposed as tumour biomarkers and potential CSC targets . It has It has also been reported that EV enriched in Wnt3a are released from high grade lymphoma stem cells and can reprogram the TME by activating the Wnt pathway. This translates in the expansion of the tumour bulk . It has Wnt signalling plays an important role in many biological processes such as growth, development, metabolism, and stem cell maintenance . The abnMoreover, it has been found that signals driving CSC differentiation into non-stem tumour cells (non-CSC) is a bidirectional and dynamic process. EV released in the TME by CSC concur to the dynamic shift between the differentiated and undifferentiated state of cancer cells, the so-called tumour plasticity .It has also been reported that CSC can evade the immune surveillance and maintain their quiescent and dormant state through their immunomodulatory properties . They drThis suggests that further efforts to dissect the mechanisms regulating the immunological profile of CSC and their released, EV as well as their crosstalk with immune cells within the TME, would be useful to rationally design immunotherapeutic interventions to eradicate malignancy.Particular attention has been devoted to EV derived from stem cells, since they are generally involved in the network of inputs enabling cells to proliferate and migrate to sites of tissue injury ,33. StemThe formation of the tumour vascular network is crucial to supply nutrients and oxygen, to remove waste products, and to provide the soil for the homing of immune cells . AngiogeThe pro-tumourigenic action of MSC-EV has also been reported to contribute to MCF7 cell migration. The up-regulation of several cancer-related signalling pathways was found to be associated to the MCF7 cell migratory phenotype. The Wnt signalling cascade was found to be among the most relevant pathways activated in response to MSC-EV challenge .It has also been reported that MSC-EV control the expansion of nasopharyngeal carcinoma (NPC) cells. It has been noticed that MSC-EV taken up by NPC cells drive proliferative and migratory cues. After MSC-EV administration, tumour cells showed significant changes in the expression of EMT markers . In thisMSC-EV also play a role in metastasis formation by contributing to tumour cell homing at distant sites. It has been reported that a more efficient tumour growth and metastasis formation occurs when myeloma cells are administered together with MSC-EV . FurtherOf interest, and similar to MSC, the human liver stem cells (HLSC), a mesenchymal stromal cell-like population resident in the adult liver, display immunomodulatory properties by a mechanism dependent on prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase activity .The crosstalk between MSC-EV and cancer cells in the haematopoietic niche has also been investigated. It has been reported that in vivo, the growth of multiple myeloma (MM) cells is supported by the release of MSC-EV . MoreoveThe list of MSC-derived “messengers” is expanding, and different molecules have been reported to act as drivers of cancer progression in a paracrine fashion ,58. A reAs one of the most abundant TME components, CAF, and particularly their released EV, play key roles during tumour expansion and metastasis formation . MultiplCAF-EV have also been involved in the regulation of intercellular communication mechanisms leading to secondary organ localization in salivary adenoid cystic carcinoma (SACC). The authors have shown that CAF-EV are crucial for lung pre-metastatic niche formation and hence for the increased number of lung metastases . The rolCAF-EV can also promote the progression of breast cancers through the transfer of their miRNA cargo. It has been demonstrated that, upon the transfer of miR-21, miR-378e, and miR-143 via CAF-EV, breast cancer cells  acquire an aggressive phenotype. The authors have shown that breast cancer cells treated with CAF-EV are connoted by the increased expression of stem cell and EMT markers, and acquire an anchorage-independent phenotype .The tumour-promoting actions of CAF-EV, as well as the molecular mechanisms involved in these processes, have also been investigated in CRC. Depth biotype characterization of non-coding RNA (ncRNA) was analysed by next generation sequencing and bioinformatics, and revealed significant differences in CAF-EV (ncRNA) content when compared to CAF and EV derived from normal fibroblasts. In addition, since the ncRNA regulatory elements were found to be specifically packaged in CAF-EV, it has been proposed that CAF and CRC cells and/or stromal cells specific cross-talk may take place thanks to EV . StudiesAs supported by these data, CAF via EV contribute to many aspects of cancer development and progression by orchestrating a cell-to-cell signalling network involving different TME components . TherefoLikewise, CAF and EC are essential TME components ,74. SeveSeveral miRNAs enriched in EV are thought to be specifically involved in tumour angiogenesis reviewed in . For exaOf note, it has also been shown that, in response to chemotherapy, EC promote tumour development through the secretion of circulating miRNA-503. Using an exogenous miRNA, the authors demonstrated that EC via EV drive breast cancer cells toward a proliferative and invasive phenotype by transferring their genetic content . MoreoveAdditional studies revealed that HeLa cell-derived EV promote metastasis by triggering the endoplasmic reticulum (ER) stress and by breaking down the integrity of the endothelial layer. Indeed, the expression of genes involved in the ER stress was found significantly increased in EC after treatment with HeLa cell-derived EV. Knock down of the protein kinase RNA-like endoplasmic reticulum kinase prevented the down-regulation of Zonula occludens-1(ZO-1) and Claudin-5 and inhibited the ER stress in cells treated with HeLa cell-derived EV .As supported by these data, tumour angiogenesis can be modulated not only by soluble angiogenic factors, but also by EV derived from different cell types in the TME. Therefore, to face cancer angiogenesis and interfere with cell-to-cell communication in the TME, EV targeting should be considered a novel frontline.The anti-tumour activity of stem cells-derived EV, as well as the immunomodulatory properties of tumour-derived EV (TDE) and EV derived from immune cells, have gained attention as novel tools for EV-based anti-cancer therapies. In fact, the most promising results on EV application as anti-tumour delivery system rely on their immunomodulatory action ,91. HereIn cancer, stem cell-EV (SC-EV) are the most attractive therapeutic options, due to their natural tumour homing capability, and to the presence of chemokine and cytokine receptors on their surface, allowing their interaction with tumour cells . SeveralSuppression of tumour growth by SC-EV can be accomplished by prompting apoptosis, cell cycle arrest, inhibition of angiogenesis, or by interfering with the mechanisms involved in the metastatic spread . RecentlIn recent years, the impact of EV in regulating the adaptive and innate immunity has been extensively investigated . The modHence, EV can be considered attractive immune tools to fight cancer. EV derived from B-cells and DC have the ability to induce antigen-specific T- and B-cell responses . It has It has been shown that, besides activating CD4+ T and CD8+ T in patients with melanoma, DC-EV can be safe and feasible for clinical application . It has Among immune cells displaying anti-tumour activity, NK cells have deserved attention. NK cells can induce the immune response impairing dissemination of solid cancers and haematological malignancies ,111. HowLymphocyte-derived EV are not the sole immune modulators in the TME, since TDE are also considered a valuable tool for anti-cancer immunotherapeutic approaches . TDE canEngineered EV are a new frontier for anti-cancer-based therapies, particularly for tailored treatments in patients at different stages of disease . Among tRecent studies have been focused on the chimeric antigen receptor (CAR)-engineered T-cell (CAR-T) approach for cancer immunotherapy ,119,120.However, concerns related to their infusion in patients are emerging. The so-called cytokine release syndrome (CRS) is one of the most adverse event associated with CAR-T cell infusion . EV haveUnlike in lymphoid malignancies, in solid tumours, the CAR-T approach unveiled disappointing therapeutic effects . Two corThese data support the possibility that CAR EV may become an effective anti-cancer targeting approach overwhelming the limitations of current treatment modalities . ApproprInnovative technologies have been developed in recent years, exploiting EV-miRNA-loading . Since mA recent study investigated the use of synthetic particles to vehicle miRNAs as therapeutic drugs to suppress HepG2 growth . miRNA-3In addition, miRNA-146b, which is lost in most glioma tumours, was found to reduce invasion and motility of glioma cells by silencing the EGFR . KatakowRecent evidence showed that synthetic miRNA-143 introduced into MSC cells is released by EV, and is efficiently transferred to osteosarcoma cells. The delivery of MSC-EV enriched in miRNA-143 significantly reduced migration of osteosarcoma cells .Of interest, it has been reported that EV protonation can be useful to generate a pH gradient across the EV membranes to enhance miRNA loading without impairing EV cellular uptake nor inducing toxicity in vivo. The biological effect was verified by loading HEK293T-derived EV with different miRNAs and evaluating their cellular targets .In order to enhance the efficacy of EV-mediated miRNA delivery, a novel system has been developed . The autRecent in vivo studies have also reported that SC-derived EV loaded with exogenous anti-tumour molecules and activated with a pro-drug successfully kill cancer cells . Hence, Engineered EV have also been used to directly and specifically target the mutated form of the GTPase KRAS, a key driver of pancreatic cancers . EV deriEV have been increasingly investigated as novel intercellular communication mediators in the TME ,142. RecDue to the relevant role of the TME in tumour development, progression, and metastasis formation, targeting different cell types supporting tumour growth by EV engineering might represent a conceivable anti-tumour strategy to track . Moreovehttps://clinicaltrials.gov/ct2/results?cond=Cancer&term=Exosomes&cntry=&state=&city=&dist=). Biological fluids such as urine and serum are the most common proposed EV sources. Data on their impact as diagnostic or prognostic tools could be available in a few years and, should validated, their possible application in cancer patients will become part of the real world. Finally, even though only a few data on the effectiveness of radiotherapy and MSC-EV as combo approach [As EV cargo includes numerous proteins, lipids, and nucleic acids, valuable information on their origin and function can be garnered by a deeper cargo analysis. Particular efforts have been devoted to characterizing EV, specifically TDE collected and isolated from liquid biopsy . TDE havapproach  are so fHowever, to move towards EV, anti-cancer-based therapy several hurdles should be solved . They in


In the past few years, there has been increasing focus on the use of messenger RNA (mRNA) as a new therapeutic modality. Current clinical efforts encompassing mRNA-based drugs are directed toward infectious disease vaccines, cancer immunotherapies, therapeutic protein replacement therapies, and treatment of genetic diseases. However, challenges that impede the successful translation of these molecules into drugs are that (i) mRNA is a very large molecule, (ii) it is intrinsically unstable and prone to degradation by nucleases, and (iii) it activates the immune system. Although some of these challenges have been partially solved by means of chemical modification of the mRNA, intracellular delivery of mRNA still represents a major hurdle. The clinical translation of mRNA-based therapeutics requires delivery technologies that can ensure stabilization of mRNA under physiological conditions. Here, we (i) review opportunities and challenges in the delivery of mRNA-based therapeutics with a focus on non-viral delivery systems, (ii) present the clinical status of mRNA vaccines, and (iii) highlight perspectives on the future of this promising new type of medicine. Vaccination has had a tremendous impact on global health and the quality of human life by reducing the mortality and morbidity caused by infectious diseases. The development of vaccines is predicated on the classical 3I’s paradigm of “isolating, inactivating and injecting” the causative microorganism, coined by Louis Pasteur . VaccineNucleic acid-based vaccines, i.e., DNA (as plasmids) and RNA (as messenger RNA (mRNA)) vaccines, pave the way for safe and efficacious biologics to mimic inoculation with live organism-based vaccines, particularly for stimulation of cell-mediated immunity . This teThe central dogma of molecular biology states that DNA is transcribed into mRNA, which is subsequently translated into protein . The floTwo classes of mRNAs, i.e., non-replicating and self-amplifying mRNA, are commonly used as vaccine vectors. Non-replicating mRNA encodes only the protein antigen(s) of interest, while self-amplifying mRNA also encodes proteins enabling RNA replication . VaccineIn vitro transcribed (IVT) mRNA is employed therapeutically as it mimics fully mature native mRNA present in the eukaryotic cytosol . This maThe pharmacodynamic activity of both native and IVT mRNA takes place in the cytosol . HoweverThe pharmacokinetics of IVT mRNA is determined by the half-life of the mRNA and the resulting mature protein after post-translational modification. The two major factors influencing the bioavailability of exogenous mRNA in the cytosol are (i) rapid RNase-mediated degradation and (ii) lack of passive diffusion across the plasma membrane owing to high molecular weight and electrostatic repulsion between the negative charges of the proteoglycan-coated cell membrane and the negatively-charged mRNA molecules . Naked mThe development of mRNA-based drugs dates back to 1990 with the successful expression of a number of different proteins upon injecting mRNA encoding these proteins directly into the muscles of mice . This leIn a cell-free system, mRNA can be synthesized by IVT of a DNA template , which encodes all the structural elements of a functional mRNA. To perform an IVT reaction, all the elements of the natural transcription process are required, i.e., a DNA template, an RNA polymerase, and nucleotide building blocks. During the subsequent purification of the mRNA, the DNA template is often degraded by the addition of DNases, followed by purification by means of other conventional methods for isolating mRNA, e.g., precipitation and chromatography. This process results in highly pure mRNA products ready for use ,30,31. SStrategies including engineering of sequences and/or structure to enhance mRNA stability  and translation are often instrumental in increasing the protein expression levels. Techniques employed to achieve this includes elongation of the poly(A) tail, modification of the 5’ cap, engineering of the UTRs and the sequence patterns in the ORF, and/or incorporation of modified nucleotides .7G) cap [A synthetic cap analogue can readily be added to the mRNA because the 5’ end cap is not encoded by the DNA template. Natural eukaryotic mRNA has a 7-methylguanosine (m7G) cap coupled to the mRNA during the transcription process via a 5’-5’-triphosphate bridge (ppp) . The m7G7G) cap . Alterna7G) cap . A major7G) cap . The fra7G) cap . 27,3’−OGpppG) has been introduced to prevent incorporation in the opposite orientation [Three different classes of m7GpppG cap analogs are used : (i) antentation . ARCAs jentation . In addientation . Recentlentation , imidiphentation , locked entation , boranopentation  and otheentation . Conventionally, the synthetic 5’-Cap 0-capped RNA is transcribed by performing an in vitro transcription, where more than 80% of the GTP added to the reaction is substituted with a dinucleotide cap analog , resulting in initiation of transcription with the cap analog . However) regulation of mRNA export from the nucleus, (ii) regulation of translation efficiency [The importance of incorporation of 5’- and 3’-UTRs has been noted during in vitro post-transcriptional regulation of gene expression . The numficiency , (iii) oficiency , and (ivficiency . Introduficiency . The optficiency  and intrficiency . Moreoveficiency . In someficiency .The poly(A) tail plays a significant role in mRNA translation as well as for the enzymatic stability of mRNA. The poly(A) tail binds to several polyadenosyl binding proteins (PABPs) while working synergistically with 5’m7Gcap sequences to regulate translational efficiency . EukaryoIt has been shown that a gradual increase in the poly(A) tail length of IVT mRNA to 120 bases commensurately increases the protein expression level, while an increase in the number of bases beyond 120 does not further enhance protein expression . Poly(A)5–106 Da) [Despite the promising potential of mRNA-based vaccines, efficient intracellular delivery of mRNA to the cytosol continues to pose a major hurdle, especially for mRNA administered systemically. The large molecular weight (10–106 Da)  and high–106 Da) . Moreove–106 Da) . Consequ–106 Da) ,74. Diff–106 Da) , RNA pat–106 Da) , gene gu–106 Da) , protami–106 Da) , RNA adj–106 Da) , and enc–106 Da) . General–106 Da) , which c–106 Da) ,83,84,85The simplest administration strategy comprises intramuscular (i.m.) injection of naked mRNA, and proof of concept was originally demonstrated by in vivo reporter gene expression in mice . Since tDelivery of mRNA can be mediated by viral and non-viral vectors. Non-viral vectors can further be categorized into lipid-based delivery systems, polymer-based delivery systems, and lipid-polymer hybrid systems . For virgag. Gag-specific, IFN-γ secreting T cells were measured in the spleen and lymph nodes of mice immunized with gag mRNA complexed with cationic lipids but not in mice immunized with naked and polymer-complexed mRNA. PEI and its derivatives are among the most commonly employed cationic polymers [co-glycolic acid) (PLGA), are well suited for incorporation of hydrophobic and positively-charged molecules. They provide good colloidal stability, low toxicity, and the possibility of sustained release. However, due to the anionic nature of PLGA at physiological pH [Diethylaminoethyl (DEAE) dextran was the first polymer to be tested as a delivery reagent for IVT mRNA . Later, polymers . They arpolymers . Howeverpolymers . Variouspolymers . Polymergical pH , the mRNgical pH . A seriegical pH . While dgical pH . Despitegical pH .Vectors based on lipids or lipid-like compounds (lipidoids) represent the most commonly used non-viral gene carriers . VariousLiposomes are closed membrane structures, which are formed by self-assembly when phospholipids are dispersed in aqueous systems . They coAlternatively, new gene delivery vectors containing ionizable lipids  and lipiLipid-polymer hybrid nanoparticles (LPNs) have been demonstrated previously to exhibit effective functional delivery of siRNA in vitro  and therPeptide-based systems for mRNA delivery are gaining momentum due to the versatility peptides can offer. Peptide-based delivery systems, both alone and in combination with other materials such as polymers, have been reported in the literature. In a study concerning ovarian cancer therapy, the commercially available cell-penetrating peptide (CPP) PepFect14 was complexed with eGFP mRNA via attractive electrostatic interactions . This naRecently, a novel polymer-peptide hybrid mRNA delivery nanoplatform was introduced  combininCell-specific delivery of mRNA would be beneficial for the development of mRNA-based therapeutics. This can enhance the delivery of mRNA molecules to the targeted cells and hence reduce the required mRNA dose, as well as reducing potential off-target effects. It has been reported that lymphoid organs can be targeted by adjusting the net charge of the formulation . This isThe field of mRNA-based therapeutics spans from protein replacement therapy and gene editing to vaccination. With the dozens of mRNA-based vaccine candidates currently in pre-clinical and clinical phases of development, it is evident that the mRNA-based vaccine technology is a promising tool for the development of novel therapeutic and prophylactic vaccines against infectious diseases and cancer. However, the multifarious obstacles associated with mRNA’s extremely large size, charge, intrinsic instability, and high susceptibility to enzymatic degradation hamper the translation of mRNA-based therapeutics from the bench to the bedside. Therefore, the wider application of mRNA-based therapeutics is still limited by the need for improved vectors or drug delivery systems. Advanced delivery systems can be applied to overcome the poor stability, cell targeting, and translational efficiency of naked mRNA. However, many clinically tested mRNA vaccine candidates are formulated without any delivery system, which suggests a need for further improvement of delivery systems for mRNA vaccines. Presently, lipoplexes and lipid-based nanoparticles are mostly used for delivering mRNA. Additionally, polymers and lipid-polymer hybrid nanoparticles offer great promise in terms of safety, stability, high transfection efficiency, and low price. Continued advancement in mRNA formulation and delivery using different nanomaterials can improve the wider use of mRNA for the treatment and prevention of infectious diseases and cancers.


Adhesive capsulitis (AC) is a glenohumeral (GH) joint condition, characterized by decreased GH joint range of motion (ROM) and compensatory ROM in the elbow and scapulothoracic (ST) joint. To evaluate AC progression in clinical settings, objective movement analysis by available systems would be valuable. This study aimed to assess within-session and intra- and inter-operator reliability/agreement of such a motion capture system. The MVN-Awinda® system from Xsens Technologies  was used to assess ST, GH, and elbow ROM during four tasks  in 10 AC patients (mean age = 54 (±6), 7 females), on two test occasions (accompanied by different operators on second occasion). Standard error of measurements (SEMs) were below 1.5° for ST pro-retraction and 4.6° for GH in-external rotation during GH external rotation; below 6.6° for ST tilt, 6.4° for GH flexion-extension, 7.1° for elbow flexion-extension during combing hair; below 4.4° for GH ab-adduction, 13° for GH in-external rotation, 6.8° for elbow flexion-extension during grasping the seatbelt; below 11° for all ST and GH joint rotations during placing a cup on a shelf. Therefore, to evaluate AC progression, inertial sensors systems can be applied during the execution of functional tasks. Adhesive capsulitis (AC), or frozen shoulder, is a pathological glenohumeral (GH) joint condition, characterized by adhesions across the GH joint capsule and surrounding ligaments, which negatively affect active and passive GH mobility ,2. MovemThe evaluation of active and passive shoulder movement in current clinical practice of AC patients is generally done by visual observation or goniometry ,9. DespiHowever, before such a system is implemented in clinical settings, the assessment of the system’s reliability and agreement in calculating joint angles within and between assessors/clinicians should be assessed . Knowledge of a system’s measurement error makes interpretation of recorded motion data straightforward  . A recenApart from the assessment of GH and ST range of motion during analytical arm movement tasks, the assessment of GH and ST range of motion during functional movement tasks , is essential in the evaluation of AC progression and in the evaluation of the effect of AC treatment . Given tTen individuals with primary AC were recruited via the orthopedic department and rehabilitation ward of Jessa Hospital Hasselt . Participants were included if they received a diagnosis of adhesive capsulitis by a medical doctor in the past six months before inclusion. This diagnosis was based on the criteria described by The American Physical Therapy Association : (a) 50%Kinematic data from the GH, ST, and elbow joint were collected from the affected arm of each person with AC using the commercially available inertial sensor system ‘MVN Awinda® motion capture system’ . Data collection was done via wireless inertial motion sensors (sampling at 60 Hz), consisting of a tri-axial accelerometer, gyroscope, and magnetometer. The signals of the accelerometer, gyroscope, and magnetometer provided the orientation of the technical coordinate system of the sensor relative to a global, earth-based coordinate system. Although only interested in movement data from the affected upper extremity, the system’s upper limb configuration without hands was chosen. Within this configuration, sensors must be placed in the middle of the forehead, on the pelvis, on the sternum and on both scapulae, upper arms, and lower arms. Only the data collected by the sensors on the sternum, and on the scapula, humerus, and lower arm of the affected arm were used for this study. The sternal sensor was positioned on the flat central part of the sternum, the scapular sensor halfway between the scapular trigonum and the acromial angle, in alignment with the upper edge of the scapular spine , and theAfter the calibration, a motion analysis protocol was performed which included the movement tasks which are typically described as difficult to perform by AC patients. The motion analysis protocol included one analytical GH external rotation task, two functional external rotation tasks (grasping the seatbelt and combing hair), and one functional forward flexion task (placing a cup on an overhead shelf) . DetailsEach participant performed three measurement sessions and in each of these sessions the participant performed the motion analysis protocol. One measurement session was performed on the first test occasion and two sessions were performed on the second test occasion. Both test occasions were 2 to 5 days apart, and participants were instructed not to have any medical treatment or perform any physiotherapy/upper extremity exercises between both test occasions. On the first test occasion, the measurement session was accompanied by operator A. On the second test occasion, one measurement session was accompanied by operator A, and the other one by operator B . At least 30 min of rest were foreseen between both sessions on the second test occasion. The specific role of the operators per measurement session was (1) the application of the inertial sensors to the upper extremity, (2) the performance of the static calibration trial, and (3) explanation of the movement tasks to the participant.MVN Studio software, the software included in MVN-Awinda® motion capture system  was used for motion data analysis. For each anatomical model, and generally based on International Society of Biomechanics recommendations , segmentThe first of the five recorded repetitions per session was not selected for data analysis as it could be corrupted by initiation strategies. Movement cycles were defined from start to point of task achievement and checked for erroneous data due to technical errors. Joint angles from all ST, GH, and elbow movements were calculated when the upper extremity was in the start position and at the point of task achievement. Range of motion (ROM), defined as the range of motion between movement start and point of task achievement, was calculated for each ST, GH, and elbow motion for combing hair, grasping a seatbelt, and placing a cup on an overhead shelf. For the analytical GH external rotation task, only ST pro-retraction ROM and GH in-external rotation ROM were calculated.Statistical analysis was done using SPSS version 22 . Kolmogorov–Smirnov tests confirmed data normality. Means and standard deviations were calculated for each ROM.Reliability of ROM was calculated using the intraclass correlation coefficient (ICC) plus a 95% confidence interval (95% CI). Agreement was assessed using the standard error of measurement (SEM) and the minimal detectable change (MDC) between two measurement occasions. Furthermore, a proportional SEM (%SEM) and proportional MDC (%MDC) were calculated for proper data interpretation, by expressing SEM and MDC relative to the mean. The following calculations were used: SEM was based on the square root of the mean square error term from the two-way ANOVA , MDC wasn = 4)) from the first session was used to calculate within-session reliability/agreement ; within-session standard error of the measurement (SEMw)). Averaged data of the four repetitions per session completed by the same operator on different test occasions was used for intra-operator reliability/agreement assessment ; SEMintra-operator; MDCintra-operator). From each of the two sessions on the second test occasion (each accompanied by a different operator), averaged data of the four repetitions per task was used for inter-operator reliability/agreement assessment ; SEMinter-operator; MDCinter-operator) [Single data  ,16,17. Iperator) .For intra-operator and inter-operator agreement assessment, Bland–Altman plots were constructed to graphically display the data of an individual person’s differences between test sessions relative to the respective individual mean, and to examine the data distribution around the zero line. The 95% limits of agreement (LOAs) were calculated (mean difference ± 1.96* SDmean difference) to identify systematic variance  or outliers.Ten participants with AC (mean age (SD) = 54 (±6), 7 females) were included. Mean time since diagnosis (SD) was 12 (± 5) weeks.Means (SD), SEMs, and %SEMs for within-session agreement assessment are reported in Within-session, intra-operator, and inter-operator reliability results are reported for each task and assessed ROM in SEMsw/%SEMsw for ST pro-retraction and GH in-external rotation were 0.4°/17.8% and 2.3°/15.5%, respectively. Intra-operator and inter-operator SEM/%SEM were higher (range 1°–4.6°/range 11.7%–49.2%), with SEMsinter-operator being higher than SEMsintra-operator.ICCsw and ICCsintra-operator for ST pro-retraction and GH in-external rotation, and ICCinter-operator for ST pro-retraction showed substantial reliability, while ICCinter-operator for GH in-external rotation showed slight reliability.Lowest SEMsw were found for all ST, GH, and elbow ROMs, with %SEMsw of ST ROMs being slightly higher than %SEMsw of GH and elbow ROMs. For (%)SEMintra-operator and (%)SEMinter-operator, the highest values were generally found for ST pro-retraction and GH in-external rotation (%SEM range 30.1%–68.1%). (%)SEMintra-operator and (%)SEMinter-operator for the other ST and GH ROMs and for elbow flexion-extension ROM were lower (%SEM range 4.9%–30.4%).Highest (%)SEMsw were found for ST pro-retraction and GH flexion-extension, with reported %SEMsw of 43.7% and 23.3%, respectively. For (%)SEMintra-operator and (%)SEMinter-operator, ST pro-retraction and GH flexion-extension showed the highest values . Lowest (%)SEMsw, (%)SEMintra-operator, and (%)SEMinter-operator were described for GH ab-adduction, GH in-external rotation, and elbow flexion-extension (%SEMs range 5.5% and 22.4%).All ICCsw, ICCsintra-operator, and ICCsinter-operator showed substantial reliability, with the exception of the ICCsinter-operator for ST medial-lateral rotation (0.24), GH ab-adduction (0.74), GH flexion-extension (0.74), and the ICCintra-operator of GH ab-adduction (0.73).Highest (%)SEMsw were found for ST pro-retraction and elbow flexion-extension, with reported %SEMsw of 21.7% and 30.6%, respectively. With regard to (%)SEMintra-operator and (%)SEMinter-operator, the highest values were reported for elbow flexion-extension . For the ST and GH ROMs, %SEMsintra-operator and %SEMsinter-operator were between 9.5% and 31.2%.All ICCsw, ICCsintra-operator, and ICCsinter-operator showed substantial reliability, with the exception of the ICCsinter-operator for ST tilting (0.78), and the ICCintra-operator of GH flexion-extension (0.68) and elbow flexion-extension (0.40).For all tasks, ICCs lower than 0.80  had moreover systematically large confidence intervals. In addition, large confidence intervals were observed for ST pro-retraction during the grasping the seatbelt task and the combing hair task.Nowadays, commercially available inertial measurement systems for human movement analysis are emerging. This study assesses the reliability and agreement of ST, GH, and elbow joint ROM assessment in AC patients using a commercially available inertial measurement system. Such a system is of great value to evaluate treatment effects in persons with AC in daily practice. However, before such a system can be clinically implemented, the establishment of its reliability and knowledge of its measurement errors in the assessment of ST, GH, and elbow joint ROM is essential.The reliability and agreement results, reported in this study, were not sufficient overall, indicating that that the clinical implementation of inertial sensor technology in the assessment of shoulder movement in persons with AC is not straightforward at this moment. The values of the SEMs/MDCs and of the ICCs  were dependent on the assessed joint rotation and assessed task. Furthermore, task-specific differences in within-session, intra- and inter-operator reliability, and agreement were found.However, results were comparable to reported reliability and agreement results of other studies assessing the reliability and agreement of ST or humerothoracic range of motion measurements by means of inertial sensors in healthy persons. Schiefer et al.  reportedIn order to fully investigate the reliability and agreement of the inertial sensor system in the assessment of joint ROM in AC patients, within-session reliability and agreement was assessed together with intra- and inter-operator reliability and agreement. While within-session data provides information on reliability and measurement error caused by intra-subject movement variability , intra-operator (between-session) and inter-operator reliability and agreement provide information on additional sources of error . Specifically regarding the applied inertial sensor technology used in this study, following sources of errors must be considered: calibration inaccuracies when positioning the participant in the neutral calibration position; palpation inaccuracies when placing the sensors on the segments in alignment with the segment orientation; measurement errors when determining anthropometric dimensions which serve as necessary input for the upper body configuration/model; and inaccuracies in task-explanation. The fact that both intra- and inter-operator reliability and agreement are assessed, additionally provides information about the source of error which is seen between sessions: if the error is operator-dependent , better intra-operator than inter-operator reliability and agreement are expected. When intra-operator and inter-operator reliability/agreement are equal but worse than within-session data, natural variability in task-execution by testing on different occasions might be assumed.Furthermore, it is known that the magnitude of the ICC is dependent on between-subject variability  . ConversWhen interpreting the reported results based on the aforementioned information, recommendations for task selection can be made, in order to make specific guidelines on the appropriate use of inertial sensors systems for range of motion measurements in clinical practice. For the analytical GH external rotation, within-session and intra-operator agreement were similar for GH in-external rotation. In contrast, inter-operator agreement was remarkably lower for GH in-external rotation. In addition, for ST pro-retraction, inter-operator agreement was lower than intra-operator agreement. As this difference between intra- and inter-operator agreement is only notable during the analytical GH external rotation, differences in sensor placement between the operators are not expected. The agreement differences might rather be explained by the fact that the analytical external rotation task was the only task without a clear endpoint, making task standardization by thorough task explanation and clear task instructions crucial. As such, the lower inter-operator agreement results are expected to relate to inconsistent task explanation/instructions.For the functional external rotation tasks , different joint ROMs show different reliability and agreement results. For combing hair, better agreement results are reported for ST med-lateral rotation, GH flexion-extension, and elbow flexion-extension, indicating low intra-individual natural movement variability for these motions while combing hair. In contrast, given the lower agreement results for transversal plane motion during combing hair, this task is not recommended to assess ST pro-retraction and/or GH internal-external rotation. It seems a better choice to assess GH in-external rotation during the grasping the seatbelt task. For this task, best agreement results are reported for GH in-external rotation, GH ab-adduction, and elbow flexion-extension. On the other hand, grasping the seatbelt is not the optimal option when assessment of ST motion is of primarily interest, given the worse agreement results for ST medial-lateral rotation, ST tilting, and ST pro-retraction. Placing a cup on an overhead shelf is the task with the best agreement results across all ST and GH joint motions. Only for elbow flexion-extension, worse agreement results are reported. Probably the fact that this task is executed in one movement plane , in contrast to combing hair and grasping the seatbelt which are tasks consisting of movements in all movement planes, adds to the good agreement results for all ST and GH ROMs during placing a cup. Recommendations for parameter selection are summarized in This protocol, consisting of four tasks during which ST, GH, and elbow kinematics are measured by means of a commercially available inertial sensor system, is only reliable and shows agreement within and between operators when specific recommendations for parameters selection  are applied. Given that one measurement session only lasts for 15 minutes, this protocol seems usable in daily physiotherapy or orthopedic practice, when relying on these recommendations. However, when inspecting the magnitude of ST ROMs, one might question their validity. When comparing to literature, reported ST ROM seems underestimated for lateral rotation and overestimated for posterior tilting ,24,25,26There are several limitations of this study. Given that the reported movement protocol was specifically designed for AC patients , one could argue that the reported SEM and MDC values are only applicable to persons with these shoulder movement restrictions, which decreases the generalizability of the results into different patient populations. However, the fact that arm abduction and external rotation movement restrictions often occur in other shoulder joint problems than AC, increases the relevance of the developed protocol for persons with other than AC shoulder joint conditions.Only 10 persons with AC were included in this study which might be a limitation. For between-session reliability assessment, it is essential that all conditions are as similar as possible between the two assessment sessions, to exclude as much ‘noise’ as possible. An example of ‘noise’ is potential improvement in mobility between the two test sessions due to, for example, treatment or the performance of exercises. Given that mobility exercises and physiotherapy are the main treatment in persons with AC, participation in this study interfered with their normal rehabilitation. Therefore, only 10 participants were included.This study indicates that commercially available inertial sensor systems have appropriate reliability and agreement for use in daily practice of persons with AC, when recommendations for parameter selection are followed.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan, China, in late December 2019 and has since spread rapidly around the world. Severe coronavirus disease 2019 (COVID-19) pneumonia patients have abnormal blood coagulation function, but their thromboembolism prevalence is still unknown. We reported a case of a 49-year-old man infected with COVID-19, presenting with fever, chest pain, limb weakness, myalgia, and dyspnea. The patient was diagnosed with severe COVID-19 pneumonia, pulmonary thromboembolism (PTE), deep vein thrombosis (DVT), and cerebral infarction. He received supportive and empirical treatment including anticoagulant treatment, anti-inflammatory treatment, oxygen supply, and inhalation therapy. The patient's symptoms, CT images, and laboratory results improved after treatment, and a throat swab was reported to be negative for SARS-CoV-2 virus by polymerase chain reaction (PCR) test. However, on day 51 of illness onset, CT reexamination demonstrated hemorrhagic infarction. Anticoagulant therapy was discontinued temporarily. After the patient tested negative for SARS-CoV-2 virus by PCR test six more times, he was discharged and remained in home quarantine. This case highlights the importance of clinician attentiveness to the appearance of multiple thromboembolism, especially in patients with severe pulmonary damage. It also emphasizes the diagnostic value of early CT imaging and the need for effective treatment once thrombotic events occur. The coronavirus disease 2019 (COVID-19) pneumonia is a neo-type respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 . SARS-CoV-2 broke out in Wuhan, Hubei, China, in late December 2019 and was subsequently identified as a public health emergency of international concern by the World Health Organization . In thisA 49-year-old man in Wuhan sought care for a half-month history of fever up to 38°C, cough, myalgia, and dyspnea, without chills, diarrhea, nausea, vomiting, or hemoptysis. Results of a pharyngeal swab specimen analysis by the SARS-CoV-2 real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR) test confirmed the patient to be positive for COVID-19 .Lymphocyte count and percentage dramatically decreased.Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were noticeably elevated.Prothrombin time prolongated, and levels of D-dimer and fibrinogen were in the normal range.Myocardial enzyme was in the normal range .The chest computed tomography (CT) scan showed bilateral peripheral ground glass opacification (GGO) .On day 15 of illness onset, he was admitted to the general isolation ward (GIW) in Leishenshan Hospital and was diagnosed with severe COVID-19. Physical examination revealed a body temperature of 38.3°C, a blood pressure of 121/85 mmHg, a pulse of 102 beats per minute, and a respiratory rate of 23 breaths per minute. Laboratory results were summarized as follows:The patient was supplied immediately with persistent low flow oxygen therapy, abidol hydrochloride , and Chinese medicine as antiviral therapy, and moxifloxacin  as antibacterial therapy and symptomatic and supportive treatment. However, the patient's state became progressively aggravated despite active treatments. Five days after admission, he suddenly developed dyspnea with a lower temperature of 36.0°C, as well as a tachycardia and a decreased oxygen saturation value of 52%. He immediately received non-invasive mechanical ventilation (NIMV) because of respiratory failure. A low dose of methylprednisolone and low molecular weight heparin (LWMH) were given concurrently for prophylactic anticoagulation and anti-inflammatory based on the clinical experience. On day 7 of hospitalization, the patient's clinical condition improved and he received high-flow mask oxygen inhalation therapy .On day 8 of hospitalization, the patient experienced weakness in his left upper limb and speech dysfunction. Physical examination indicated Grade 0 muscle strength in the left upper limb while other limbs were normal, the mouth was deviated, and pathological reflection of Babinski's sign was not induced; bilateral pupils were equally in diameter and sensitive to direct or indirect light reflex. Laboratory examination indicated that sensitive troponin I was 0.323 ng/ml (reference 0–0.04 ng/ml). The patient's coagulation function, CK, and CK-MB were normal. These results indicated onset of cerebral infarction. The patient was transferred to the intensive care unit (ICU).On February 24th, the patient subsequently developed severe precordial pain. Urgent electrocardiogram indicated sinus tachycardia rather than cardiac ischemia. After treatment with nitroglycerin, atorvastatin, clopidogrel, and aspirin, the patient still had a chest pain. It was speculated that PTE was occurring. Vascular ultrasound of lower limbs showed deep vein thrombosis of the right lower extremity. Computer tomography pulmonary angiography indicated a filling defect in the trunk and branches of the right inferior pulmonary artery. Cerebral CT showed slightly low-density shadows in the right parietal lobe and the left temporal lobe . LaboratAfter treatments, the patient's symptoms continued to alleviate. Laboratory reexamination indicated that the lymphocyte count and percentage increased reposefully and AST; ALT was mildly decreased while coagulation function was still not improved. Levels of D-dimer and fibrinogen remained high. The patient felt severe chest pain again on March 6. Electrocardiogram indicated sinus tachycardia. Results of a bedside chest radiograph displayed progress of bilateral interstitial infiltrating shadows. To relieve pain, the patient was given diclofenac sodium suppositories and low molecular heparin calcium continuously until day 33 of hospitalization. Amounts of methylprednisolone were gradually decreased to 20 mg on day 32 of hospitalization.On March 11, the patient was transferred to the GIW for intensive consolidation therapy. All symptoms had resolved except for intermittent chest pain and disorder of limb activity. He received ongoing treatment in the GIW, including anticoagulant therapy with LMWH, hepatic functional protection therapy, anti-inflammatory treatment, and Chinese medicinal therapy. A CT of the lungs indicated pulmonary cavitation in the right lower lobe. Meropenem and linezolid were empirically administered as anti-infective treatments for multidrug resistance bacterial infection particularly gram-positive coccus infection. On March 17, cerebral CT indicated hemorrhagic infarction and the anticoagulant therapy was discontinued until March 20. The patient reported less pain and fatigue in his chest in the following days. He was treated with rivaroxaban  when low molecular heparin calcium was discontinued. Physical examination revealed that muscle strength in the left upper limb was Grade 4; task-related dystonia was coming to normal, without numbness of the limbs, inarticulate speech, blurred vision, paresthesia, ataxia, and dystonia. Biochemistry and laboratory examination indicators gradually returned to normal levels. When the patient tested negative for SARS-CoV-2 virus by PCR test six more times, he was discharged and remained in home quarantine.Coagulopathy is regarded as a common complication in patients with severe COVID-19, the clinical syndrome caused by SARS-CoV-2. The overlap in symptoms between COVID-19 and thromboembolism present a challenge for clinical diagnosis, especially for those patients without any high-risk factors. The patient we reported on in this case study was categorized as being at low-risk for venous thromboembolism according to the Padua prediction scale.Previous studies have shown that patients with severe acute respiratory distress syndrome (SARS) have slightly decreased platelet counts and prolonged coagulation profiles and are prone to thromboembolic complications –9. SARS-In the present case, the patient was immediately given low-molecular-weight heparin anticoagulant therapy as soon as the embolization event occurred. However, the therapeutic effect of anticoagulation on the patient was not as remarkable as was to be expected during the course of early treatment. We did not detect a decrease in fibrinogen levels and an improvement in coagulation function until heparin treatment on day 7 of hospitalization, which was slightly different from the curative effect of heparin in pulmonary embolism patients without COVID-19. Notably, the patient presented with hemorrhagic infarction during the subsequent therapy so much that it was necessary for anticoagulant therapy to be discontinued. For COVID-19 patients at high risk of bleeding and severe illness, it is therefore important that appropriate anticoagulation measures be taken to ensure effective treatment of thromboembolism. Zhai et al. recommended pharmacological prevention with low-molecular-weight heparin as a first-line treatment in patients at low or moderate risk of bleeding, as well as a curative anticoagulant with LMWH as a first-line treatment in patients suspected for VTE . TrigoniIn summary, this is a first report of a COVID-19 pneumonia patient with PTE, DVT, and cerebral infarction in Wuhan. Following an active treatment regimen consisting of anticoagulant therapy and anti-inflammatory treatment, the patient recovered well. However, due to the limited nature of this case, further research on predisposing factors and protocols for the treatment of COVID-19 pneumonia patients with multiple thromboembolisms is warranted.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The study is approved by the Medical Ethics Committees, Zhongnan Hospital of Wuhan University (No. 2020099K), and the patient provided consent to participate. Written informed consent for publication of the clinical details and/or clinical images was obtained from the patient.HP and TC contributed to the conceptions and design. GZ, HX, XC, and JL collected clinical data. TC wrote the raw draft. HP and EP MEng contributed to the language editing. All authors have read and approved the manuscript.EP was employed by the company Dascena, Inc. The remaining 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.


It has been well elucidated that multiple types of cancers are at high risk of thrombosis. Several studies have indicated the prognostic value of fibrinogen (Fib) and D-dimer (DD) in prostate cancer (PCa). However, it remains unclear regarding the association of the comprehensive coagulation markers with the clinicopathological features of PCa.  A total of 423 pathologically diagnosed patients with PCa were consecutively collected and stratified as low-intermediate-risk or high-risk groups. The association of coagulation parameters including Fib, DD, prothrombin (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and antithrombin III (AT-III) with clinicopathological features was determined by univariate and multivariate logistic regression analyses. p < 0.001, p < 0.001, and p = 0.043, resp.), while APTT, TT, and AT-III were similar between two groups . Univariate logistic regression analysis demonstrated that Fib, DD, and PT were all positively correlated with high-risk PCa . Nonetheless, after adjusting for PSA, grade, and stage, Fib  but not DD or PT was the unique independent factor associated with high-risk PCa in the multivariate regression analysis.  The levels of Fib, DD, and PT were significantly higher in the high-risk group ( Our study firstly revealed that Fib but other coagulation markers was independently associated with the severity of PCa, suggesting Fib might be useful in PCa risk stratification beyond PSA, stage, and grade. The morbidity and mortality of prostate cancer (PCa) are increasing in recent years all over the world. In China, the proportion of patients with advanced and metastatic PCa when primarily diagnosed is higher than that in western countries . TherefoCurrently, the combination of preoperative prostate-specific antigen (PSA) level, stage, and grade is commonly applied to evaluate the severity of PCa in clinical practice . HoweverPlenty of evidence has shown that cancer was closely associated with hemostasis . MalignaHence, the aim of the current study was to identify the association of comprehensive coagulation parameters with the severity of PCa assessed by the risk stratification system in the Chinese patients with PCa.Patients who had undergone transrectal ultrasound-guided prostate biopsy and been pathologically diagnosed as PCa at our hospital were collected between January 2012 and October 2019.The exclusion criteria of the study were the presence of a medical history of other malignancies or coagulation-related diseases, severe hepatic and/or renal insufficiency, collagen disease, significant cardiovascular disease, or receiving any anticoagulant therapy.All clinicopathological data including coagulation parameters were collected from medical records. All prostate biopsy specimens were routinely sent to the pathological department for diagnosis. Cancer grade assessment was performed according to the 2014 ISUP classification system . The cliVenous blood samples were taken at admission after 8-hour fasting and before any treatment including surgery, ADT, or other therapies. The concentrations of Fib, prothrombin (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and antithrombin III (AT-III) were measured using a Stago auto analyzer, and the plasma DD level was determined on a Hitachi 7600D clinical chemistry analyzer according to the manufacturer's instructions.t-tests, one-way ANOVA, Mann-Whitney U tests, or Kruskal-Wallis tests as appropriate. The qualitative variables were expressed as numbers and percentages and were analyzed by the chi-square statistic tests. All PCa subjects were stratified into two groups: low-intermediate-risk and high-risk groups as previously reported [n = 141), the second tertile group ; n = 142), and the third tertile group ; n = 140), and analyzed clinicopathological and coagulation features in different levels of Fib. Besides, DD and PT were divided into three tertiles and analyzed high-risk patient percentage and PSA levels in different levels of DD and PT, respectively. Correlations between coagulation parameters with high-risk PCa were revealed by univariate logistic regression analysis. The independent factors of high-risk PCa were further determined by multivariate regression analysis with adjustment for confounding factors including PSA, grade, and stage. The differences were considered statistically significant if a p value was less than 0.05. Statistical analysis was carried out using SPSS .The quantitative variables were expressed as mean ± standard deviation (SD) except for DD, which was expressed as median with interquartile range, and were analyzed by Student's reported . Fib wasn = 63, 14.89%) and high-risk group . The clinicopathological characteristics and coagulation parameters of the enrolled subjects are shown in A total of 423 eligible PCa patients were recruited in the present study, with a mean age of 70.8 years. Patients were divided into two groups according to risk severity: low-intermediate-risk group . However, there was no significant difference regarding APTT, TT, or AT-III between the two groups (p > 0.05).Fib, DD, and PT levels of the high-risk group were significantly higher than those of the low-intermediate-risk group . Specifically, there is a statistically significant trend that the late-stage (stage T3+T4) and high-grade (4+5) percentage increased with the elevation of the Fib level as mentioned above. Regarding the coagulation parameters, there were significant differences in DD, PT, TT, and AT-III but not APTT among the three Fib groups . However, no significant correlation between other coagulation parameters and high-risk PCa including APTT, TT, and AT-III was observed . Tissue factor (TF) is the initiation factor of the extrinsic coagulation pathway and plays an important role in prolonging PT. TF was expressed in multiple kinds of cancers including PCa and endothelial cells . As PCa The mechanisms of the correlation of high Fib and adverse clinicopathological features of PCa remain uncertain. There are two sources of high Fib in PCa. PCa cells can secrete tumor-associated cytokines, which stimulate the liver to produce more Fib; besides, PCa cells themselves endogenously synthesize Fib . It was Our study demonstrated Fib was the only coagulation factor that was independently and positively correlated with the risk severity of PCa. The present study might have two potential clinical implications. Firstly, the plasma Fib could be integrated into existing PCa risk stratification systems to further improve treatment decision-making, patient consultation, and prognosis estimation. Secondly, as mentioned above, Fib plays crucial roles in cancer progression and invasion; maybe anticoagulant treatment could be applied to PCa patients with high Fib to decrease the likelihood of metastasis. Besides, anticoagulant treatment could prevent the occurrence of thrombosis and stroke in high Fib patients, which needs further study.In conclusion, this study firstly revealed the association between coagulation profiles and clinicopathological features in PCa patients and finally found that high Fib was positively and independently associated with severity of PCa.There were two main limitations in our study. Firstly, the prognostic significance of Fib was not evaluated in the current study. Secondly, the exact mechanism was not explored in the current analysis.In conclusion, this study provided evidence of an association between comprehensive coagulation markers and clinicopathological features of PCa: patients with high Fib, DD, or PT had more adverse clinicopathological features of PCa. However, only Fib but other coagulation markers was independently associated with the severity of PCa after adjustment for other confounding factors, suggesting Fib might be useful in PCa risk stratification beyond PSA, stage, and grade.


The neural mechanisms underlying subjective responses to meal ingestion remain incompletely understood. We previously showed in healthy men an increase in thalamocortical, and a decrease in insular-cortical connectivity in response to a palatable meal. As sex is increasingly recognized as an important biological variable, we aimed to evaluate sex differences and commonalities in the impact of a well-liked meal on thalamic and anterior insular connectivity in healthy individuals. Participants (20 women and 20 age-matched men) underwent resting-state magnetic resonance imaging (rsMRI) before and after ingesting a palatable meal. In general, the insula showed extensive postprandial reductions in connectivity with sensorimotor and prefrontal cortices, while the thalamus showed increases in connectivity with insular, frontal, and occipital cortices, in both women and men. However, reductions in insular connectivity were more prominent in men, and were related to changes in meal-related sensations (satiety and digestive well-being) in men only. In contrast, increases in thalamic connectivity were more prominent in women, and were related to changes in satiety and digestive well-being in women only. These results suggest that brain imaging may provide objective and sex-specific biomarkers of the subjective feelings associated with meal ingestion. Consumption of a meal induces a sequence of transient sensations before and during ingestion  as well as longer-lasting postprandial sensations. The postprandial experience involves homeostatic sensations  and hedonic sensations , which are independently modulated by various conditioning factors ; for insSex-related differences in the biological responses to food ingestion have been reported. Particularly, women experienced stronger postprandial sensations after a palatable meal, and were more susceptible to the influence of conditioning factors, such as the eating schedule ,8,9. SexBased on these data, we hypothesized that the neural mechanisms underlying the postprandial experience exhibit sex differences. Thus, our aim is to examine sex differences and commonalities in the effects of a palatable meal on brain activity, specifically, on thalamic and anterior insular connectivity, and the relations of brain responses with homeostatic sensations (satiety) and hedonic sensations (digestive well-being).n = 20 each) without history of gastrointestinal symptoms participated in the study. The male participants represent a subset of the participants in a previously reported male-specific [Trials.gov as part of the study NCT02592239. All participants gave written informed consent.Two groups of healthy, non-obese women and men  in response to a palatable probe meal (intervention). Studies were conducted in the afternoon after a 5-h fast. Participants were instructed to refrain from strenuous physical activity on the day before the study and to have their usual breakfast after an overnight fast. Participants completed a structural MRI scan and two 20-min resting state MRI (rsMRI) scans (pre-1 and pre-2) immediately before ingestion of a probe meal, and a third 20-min rsMRI scan after ingestion ; the two pre-meal rsMRI scans were acquired to examine the effect of time. Perception of subjective sensations was measured after the second fasting rsMRI scan , and immediately before and after the postprandial scan . The probe meal was ingested while participants sat in an isolated room adjacent to the brain imaging room.To evaluate the brain activity underlying a positive postprandial experience, a probe meal  was specifically designed by a series of previous studies ; the chaTen centimeter scales graded from −5 to +5 were used to measure hunger/satiety (extremely hungry to completely sated), digestive well-being (extremely unpleasant sensation/dissatisfaction to extremely pleasant sensation/satisfaction), and palatability (very bad/disagreeable to very good/delicious). Subjects received standard instructions on how to complete the scales: The hunger/satiety and digestive well-being scales were scored immediately before, at the end and 20 min after meal ingestion (score the intensity of each sensation you feel now); the palatability scale  was scored only once immediately after ingestion. These scales have been shown to detect differences in postprandial sensations under various conditioning factors ,2,9,24.3). Three 20-min rsMRI scans  were completed, in which participants rested with eyes closed .fMRI acquisition. Images were acquired on a 3.0 T whole-body MR scanner with a 12-channel phased-array head coil and a whole-body transmit coil . The imaging protocol parameters were based on the ADNI initiative.The structural scan involved an axial 3D T1-weighted gradient-echo (MPRAGE) sequence . The first two volumes were discarded to allow for stabilization of the magnetic field. Data were slice-time and motion corrected, spatially normalized to the Montreal Neurological Institute standard template using their structural image, and smoothed with an 8-mm Gaussian kernel. In addition, white matter/cerebrospinal fluid signals were regressed out of the time series data, which were then bandpass filtered (0.01–0.10 Hz).t test, paired tests for intragroup comparisons and unpaired tests for intergroup comparisons; otherwise, the Wicoxon signed rank test was used for paired data within groups, and the Mann-Whitney U test for unpaired data between groups. Differences were considered significant at a p value < 0.05.Mean values (±SE) of the parameters measured were calculated in each group of subjects. Normality of data distribution was evaluated by the Kolmogorov-Smirnov test. Comparisons of parametric normally distributed data were made by Student’s Seed-based correlation has been used in the analysis of rsMRI data, as previously described in detail . In briePartial least squares (PLS) analysis was applied to the normalized correlation maps to examine sex-related and meal-ingestion effects on anterior insula and thalamic connectivity, using freely available code . PLS is p < 0.001) and an extent of at least 150 voxels were considered as reliable.Four seed-PLS analyses were conducted, one for each of the four ROIs . In each analysis, sex was a group factor and there were three meal-ingestion conditions ; the normalized correlation maps were provided as input. As a result, each seed-PLS analysis produced a set of spatial maps with associated weightings for each combination of sex and condition . Significance of each latent variable was assessed using permutation testing (500 permutations), and voxel reliability was assessed using bootstrap estimation (500 samples) . ClusterReliable clusters from the seed-PLS analyses were further analyzed in focused behavioral-PLS analyses, which evaluated the relationships between connectivity changes and changes in perceptual ratings within each sex. As the seed-PLS analyses showed no significant differences between the first and second pre-meal ingestion scan, connectivity difference maps were calculated as the post-meal ingestion connectivity map minus the second pre-meal ingestion connectivity map (post minus pre-2). The ingestion-related changes in satiety and digestive well-being ratings (Δ satiety and Δ well-being) were calculated as the post-meal ingestion rating minus the pre-meal ingestion rating. For each sex, four behavioral-PLS analyses were conducted, one for each of the four ROIs; the connectivity difference maps and both Δ satiety and Δ well-being were provided as input. As a result, each behavioral-PLS analysis produced a set of spatial maps with associated weightings for Δ satiety and Δ well-being . Significance of each latent variable and voxel reliability were assessed in the same manner as in the seed-PLS analyses.2) and men (20–29 Kg/m2); one participant in each group was overweight and the rest were within the normal range. Participants were university students or had a university degree (middle-upper class) and all completed the study and were included in the final analysis.Age range was similar in women (20–43 years) and men (20–40 years). Body mass index was also similar in women (range 19–29 Kg/mp = 0.210). Meal ingestion increased satiety and digestive well-being ratings in men and women  and these effects persisted over the 20-min postprandial period  (p = 0.003 for both) (p = 0.048), but ratings immediately after the meal were higher in women than in men (p = 0.006); hence, the increase in well-being (Δ well-being) was larger in women than in men  . In womeor both) A. Sex din in men B. At 20 n in men BNo significant differences between the two pre-meal ingestion scans were detected both in women and men, indicating the lack of time effects before the intervention ; in contrast, marked and consistent changes were detected after meal ingestion.p < 0.001) and for left anterior insula . Before the meal, i.e., at baseline, significant sex differences in connectivity for the right and left anterior insula were observed (Significant connectivity patterns (latent variables) were detected for right anterior insular  and for left thalamus . During baseline, sex differences in connectivity for the right and left thalamus were observed, reaching statistical significance at the pre-2 scan (Significant connectivity patterns (latent variables) were detected for right thalamus  were detected. However, the increase in satiety and well-being exhibited significant inverse correlations with changes in connectivity of the left anterior insula and direct correlations with right anterior insula, ; thus, gOur study demonstrates that ingestion of a palatable meal induces a response in brain activity and subjective meal-related sensations common to women and men, but with specific sex differences.The probe meal used in the present study was specifically designed to induce a consistent and pleasurable postprandial experience  and, as As in previous studies ,8, compaNotwithstanding the greater subjective responses observed in women, anterior insular connectivity changes in response to meal ingestion (decrease) were more prominent in men. Previous studies showed a more prominent anterior insula response to chocolate satiation , and greOur study further shows that sex differences are involved in the way that specific changes in brain connectivity relate to the subjective experience of the meal effects. Hence, the reductions in anterior insular connectivity were related to changes in perception (satiety and digestive well-being) in men only. Indeed, in men, but in not women, greater Δ satiety and Δ well-being were associated with changes in anterior insula connectivity, specifically, with the TPJ and precentral and prefrontal cortices. The anterior insular cortex is a key node in the brain’s salience network, and is consistently activated in response to salient, homeostatically relevant stimuli associated with conscious feelings such as hunger and satiation . The TPJThe thalamus showed a pattern of results opposite to those of the anterior insula; thalamic connectivity increased in response to meal ingestion and the changes were more prominent in women than in men. As with the anterior insula, thalamic connectivity in the postprandial state was very similar in men and women, while sex differences were observed before meal ingestion. Thus, thalamic connectivity may also be differentially sensitive to fasting conditions in women and men, specifically, with TPJ, precentral, and prefrontal cortices. Additionally, in women, but not in men, greater Δ satiety and Δ well-being were associated with increases in thalamic connectivity, specifically, with the mid and posterior insula and temporal cortices. The thalamus conveys sensory information from the body to the insula, with an extensive relationship between the ventroposterior medial nucleus of the thalamus and the anterior insula. The dorsal mid-insular cortex has been implicated as a region of convergence between gustatory and interoceptive information ,37,38,39We wish to acknowledge that the generalizability of our findings is limited by the small sample size and will require repetition in a larger sample. Our observations are limited to the responses of specific brain regions to a pleasant meal. Based on the literature, brain regions other than insula and thalamus are involved in the brain response to a meal, including the basal ganglia and the hypothalamus, which may also contribute to sex-related differences. Furthermore, sex-differences in the brain responses to non-palatable or aversive meals remain to be explored. As described in another article of this special issue , previouThe results of the present study have general implications for food-related neuroimaging research in healthy individuals, as well as in patient populations  . Our fin


N = 1753 cisgender (CG) healthy participants were used to train (N = 1402) and validate (20% holdout N = 351) a support-vector machine classifying the biological sex. As a second validation, we classified N = 1104 patients with depression. A third validation was performed using the matched CG sample of the transgender women (TW) application sample. Subsequently, the classifier was applied to N = 26 TW. Finally, we compared brain volumes of CG-men, women, and TW-pre/post treatment cross-sex hormone treatment (CHT) in a univariate analysis controlling for sexual orientation, age, and total brain volume. The application of our biological sex classifier to the transgender sample resulted in a significantly lower true positive rate . The TPR did not differ between CG-individuals with  and without depression . The univariate analysis of the transgender application-sample revealed that TW-pre/post treatment show brain-structural differences from CG-women and CG-men in the putamen and insula, as well as the whole-brain analysis. Our results support the hypothesis that brain structure in TW differs from brain structure of their biological sex  as well as their perceived gender . This finding substantiates evidence that TIs show specific brain-structural alterations leading to a different pattern of brain structure than CG-individuals.Transgender individuals (TIs) show brain-structural alterations that differ from their biological sex as well as their perceived gender. To substantiate evidence that the brain structure of TIs differs from male and female, we use a combined multivariate and univariate approach. Gray matter segments resulting from voxel-based morphometry preprocessing of  Being transgender describes the stable feeling of belonging to the opposite sex rather than the biological sex assigned at birth, while the term cisgender (CG) describes the feeling of coherence between biological sex and perceived gender.Although there is an ongoing social and political debate regarding the terms and phrases used to describe gender, little is known about how a divergence between biological sex and perceived gender emerges. A popular view is that sexual brain differentiation and body development diverge in transgender individuals (TIs) . EvidencPrevious research provides extensive information on how brain structure differs as a function of biological sex. Briefly, localized sex differences show higher gray matter volume in CG-men, while the volume of limbic structures is particularly increased in CG-women . HoweverFew ROI-based approaches have studied how the brain structure of TIs differs from CG-individuals. Compared with CG-men, transgender women  show structural alterations of areas associated with body perception. Brain structures that repeatedly showed alterations across multiple studies are the putamen  and the Next to univariate analyses, multivariate analyses offer new insights into the similarities and differences between CG and TIs , 22. In Another methodological motivation for choosing multivariate techniques is that samples of TIs are usually small. Using a multivariate approach trained and validated on larges samples of CG-individuals and applied to TIs allows more valid conclusions about brain-structural differences between TIs and CG-individuals.Multivariate analyses have already been used to investigate whether TIs can be separated from CG-individuals by their brain volumetric patterns , 22. BotThus, an extensively greater generalizability is expected and therefore real-life applicability is enhanced.The classifier trained on healthy CG-participants shows significantly worse performance when applied to a sample of TWThe classifier trained on healthy CG-participants performs equally well in a validation-sample of CG-patients suffering from major depressionFollowing our multivariate approach, we investigated local structural brain alterations in the putamen and the insula , 24–26. CG-women show lower volume in comparison to CG-men .TW-pre and post-CHT show increased volume in comparison to CG-womenTW-pre and post-CHT show lower volume in comparison to CG-menSince we expect CHT to be associated with a further feminization of brain structure and hence reduced volume, we hypothesize thatTW-pre-CHT show higher volume in comparison to TW-post-CHT.Our hypotheses for the multivariate analysis are:To obtain and validate a predictor for biological sex based on structural MRI brain scans, we used three different samples, which purposes are briefly described here prior to sample characteristics: a classifier was trained on a large sample of CG-individuals without any psychiatric disorder using a cross-validation procedure. An independent subsample randomly drawn in advance, served as the first validation set, to avoid overfitting , the Bi[N = 434 ), and th[N = 653 , 33). Ex[N = 653 , 34.N = 1404) of patients diagnosed with MDD were used as second validation-sample. Four hundred and fifty MDD patients exhibited psychiatric comorbidities such as anxiety disorders or substance abuse. Diagnoses were again verified with the structural clinical interview according to DSM-IV criteria [N = 285 participants from the MNC, N = 591 from the BD study, and N = 528 from the FOR2107 study ). TW were recruited during their treatment at the outpatient clinic of the Department of Psychiatry at the University of Münster. Before treatment and study inclusion all participants were carefully tested for chromosomal abnormalities such as Klinefelter syndrome, screened for personality disorders and other psychiatric comorbidities using the structural clinical interview I and II according to DSM-IV criteria , ruling out possible confounding of the classifier due to scanner variability. The TW were in different treatment states, with 18 already treated with hormones , which was not used during classifier training and testing. The remaining data set of N = 1402 subjects was balanced for sex with a random undersampling procedure , and used in a tenfold split procedure resulting in balanced training sets of 1096 subjects in each fold. A principal-component analysis was performed next, to further reduce the dimensionality of the data. The maximum number of principal components is limited to 1096, the number of subjects resulting from the tenfold split. We carried out a Bayes-statistic-based hyperparameter optimization for the support vector classifier , which influences penalties for misclassification, and the gamma parameter , influencing the curvature of the decision boundary. In this iterative Bayes approach, a total of 100 parameter combinations were evaluated. Quality and classifier performance are reported by area under the ROC curve (AUC). The classifier resulting from the best combination of hyperparameters was finally determined using our first validation set, the 20% drawn in advance from the original sample. To exclude potential effects of comorbid depression, this step was repeated with the sample of MDD subjects, as a second validation sample , we applied the true positive rate (TPR), since balanced accuracy (BACC) is a measure not applicable to one-group-only scenarios. Fisher’s exact test was used to clarify whether TPR differs statistically between samples. Interpretation of TPR is based on the hypothesis that TW belong to the category of male biological sex.In order to achieve optimal generalization of our classifier, multiple scanners were deliberately incorporated. A specific correction for possible scanner effects was not intended. Instead, the purpose was to establish a classifier based on scanner invariant features given the large amount of training data and expected excellent classification performances. Comparison of the recognition rates between the individual scanners yielded no significant differences. Hence, an influence of the scanner on the classification results could not be detected—supporting our expectation . Our classifier reached a BACC of 92.06%, and a TPR of 86.93% for CG-men in this sample Table . The resThe BACC for the third validation-sample was 94.03% (CG-part of the TW-sample). The TPR for CG-men was 93.3% and for CG-women 94.7% Table ; see visThe region of interest analysis is summarized in Table In contrast, TW-pre-CHT showed larger volume in both ROI analyses compared to CG-women. Interestingly, TW-pre-CHT also showed higher volume in the putamen compared with CG-men.TW-post-CHT showed lower volume of both regions of interest compared to TW-pre-CHT in both regions of interest. CG-men showed larger volume in both regions of interest compared to CG-women.Detailed results of our exploratory whole-brain analysis can be found in the Supplementary Table In the present study, we developed an SVM using hyperparameter optimization resulting in an accurate classification of biological sex based on structural MRI images. The classifier, trained on a large training set of healthy CG-individuals, performed equally well in three independent validation samples of healthy CG-individuals, and CG-participants suffering from MDD. When applying the same classifier to structural MRI data of TW, the SVM shows a much lower TPR, resulting in significantly more misclassifications of the biological sex of TW  in favor of their perceived gender . Moreover, the descriptive statistics of classification probabilities regarding TW Fig.  indicateHence, our results shed light on two important aspects in biological psychiatry of TIs: (1) The impact of hormonal treatment on brain structure, (2) the separation of psychological distress , hormonal treatment, and trait characteristics of being a TI.Our results replicate the finding that biological sex is increasingly misclassified in TIs, as previously described , 22. ThiOur biological sex classifier shows a higher external validity than other biological sex classifiers. First, it has been tested on controls and MDD-patients, with high and very similar accuracy. Second, the SVM has been trained on large samples that have been collected at different sites. Hence, our SVM can be regarded as more generalizable while preserving performance and accuracy, indicating its robustness to noise.In the present work, we focused on the first application of this SVM on TW. We observed that our SVM was increasingly inaccurate in TW, compared with healthy CG-controls. The explorative analysis revealed that this inaccuracy was particularly increased in TW who had hormonal treatment.Although our TW-pre-CHT sample size was low, we aimed to differentiate structural brain alterations between TW-pre and TW-post-CHT as well as in comparison to CG-women and -men. Our results show brain-structural alterations dependent on the treatment state of TW.Volumes of the insula and putamen were larger in TW-pre-CHT than in CG-women, while TW-post-CHT showed lower volumes of the right insula compared with CG-women.In comparison to CG-men, TW-pre-CHT showed larger volumes of the putamen, while TW-post-CHT showed lower volumes of both insula and putamen. Thus, TW independent of treatment state show brain-structural alterations in our regions of interest in comparison to both, CG-men and -women.Detailed analysis of TW-pre compared with -post-CHT revealed a less pronounced pattern of structural brain alterations in TW-post-CHT compared with CG-women. Comparing TW-pre with TW-post-CHT revealed lower volume of TW-post-CHT in both regions of interest, as well as the whole-brain analysis. This implies that CHT induces a further feminization of brain structure in TW. This result fits with previous longitudinal studies that have shown reductions of cortical thickness in TW-pre to post-CHT . StructuBrain structural alterations of the putamen have been associated with TW across multiple studies and independent of treatment state –13. We eOur combined univariate and multivariate approach revealed associations of CHT with lower accuracy in detecting the biological sex of TW. Our results show that the brain structure of TW aligns with neither their biological sex  nor their perceived gender . This implies that there is a biological basis for being transgender and thus, destigmatizes TIs. Further, this evidence can be used in psychoeducation during treatment of gender dysphoria. The diagnosis of gender dysphoria is new to DSM-5 to allow for treatment if TIs suffers from distress due to incoherence between perceived gender and biological sex. Our results could relieve distress in transgender patients in case of the experience of guilt or shame due to the discrepancy between biological sex and perceived gender.N = 8). Therefore, further studies should follow up on this effect, with higher sample sizes of untreated TW to increase power. An extension of the design with a second control group  should be used to clarify whether misclassification is an effect of treatment only, due to the combination of being transgender and CHT.In line with this idea, hormonal processes, brain-structural development, and the development of gender identity are intertwined . Intrauthttps://photon-ai.com/model_repo/bsc_mri) to test for misclassifications in other clinical diagnoses with high gender imbalance in prevalence rates, such as eating disorders, substance use disorders, or anxiety disorders.The present SVC provides a new tool for research in biological psychiatry. Prevalence of many psychiatric disorders is often higher for one biological sex than for the other. For example, prevalence in autism is higher for biological men than for biological women. Hence, it was hypothesized that female patients with autism might be similar in their brain structure to men. A previous study that developed a biological sex classifier using structural MRI scans and applied it to patients with autism  indeed sNext to our training and validation strategy  and the Interdisciplinary Center for Clinical Research (IZKF) of the medical faculty of Münster (grant Dan3/012/17 to UD). The BiDirect Study is supported by a grant of the German Ministry of Research and Education (BMBF) to the University of Muenster (01ER0816 and 01ER1506).Biomedical financial interests or potential competing interests: TK received unrestricted educational grants from Servier, Janssen, Recordati, Aristo, Otsuka, neuraxpharm.The other authors  declare no competing interests.Supplemental MaterialFigure S1Supplementary Figure S2