Background: Multimorbidity, defined as the co-occurrence of ≥2 chronic conditions, is clinically diverse. Such complexity hinders the development of integrated/collaborative care for multimorbid patients. In addition, the universality of multimorbidity patterns is unclear given scarce research comparing multimorbidity profiles across populations. This study aims to derive and compare multimorbidity profiles in Hong Kong  and Zurich .Methods: Stratified by sites, hierarchical agglomerative clustering analysis (dissimilarity measured by Jaccard index) was conducted with the objective of grouping inpatients into clinically meaningful clusters based on age, sex, and 30 chronic conditions among 20,000 randomly selected discharged multimorbid inpatients  aged ≥ 45 years. The elbow point method based on average within-cluster dissimilarity, complemented with a qualitative clinical examination of disease prevalence, was used to determine the number of clusters.Results: Nine clusters were derived for each site. Both similarities and dissimilarities of multimorbidity patterns were observed. There was one stroke-oriented cluster  and one chronic kidney disease-oriented cluster  in each site. Examples of site-specific multimorbidity patterns, on the other hand, included a myocardial infarction-oriented cluster in ZH (2.3%) and several clusters in HK with high prevalence of heart failure (>65%) and chronic pain (>20%).Conclusion: This is the first study using hierarchical agglomerative clustering analysis to profile multimorbid inpatients from two different populations to identify universalities and differences of multimorbidity patterns. Our findings may inform the coordination of integrated/collaborative healthcare services. Multimorbidity is commonly referred to as the co-occurrence of two or more chronic health conditions  and is cMultimorbid patients are clinically diverse  and may Numerous attempts have thus been made to identify common patterns of multimorbidity to simplify the problem , 17. PraThis study aims to describe and compare the patterns of co-occurring chronic conditions among discharged multimorbid inpatients in Hong Kong and Zurich using a hierarchical agglomerative clustering analytic approach, which is typically used for grouping similar individuals based on predetermined ranges of characteristics. Since the healthcare system as well as the cultural and demographic characteristics differ drastically between the two sites, observations of similar multimorbidity patterns may potentially imply universal challenges facing clinicians globally.We conducted a retrospective analysis of clinical records of discharged patients aged ≥ 45 from all public hospitals in Hong Kong  during January 2010-December 2013 and from the University Hospital Zurich  during August 2009-August 2017. The discrepancy of observation period was mainly due to the much smaller amount of data generated from Zurich as it is only one hospital compared with the whole public hospital system in Hong Kong.The data contained information on patients' age, sex, the length of stay, and the first 15 clinical diagnoses made during the hospital stay coded with the International Classification of Diseases, Ninth Revision (ICD-9) for Hong Kong and with the Tenth Revision (ICD-10) for Zurich. Multimorbidity is defined as having two or more chronic conditions using a list of 30 diseases coded either by ICD-9 or ICD-10. The list of chronic conditions is based on validated coding algorithms summarized by Tonelli et al.  and incl30-day readmission and length of stay in hospital were measured to compare health care utilizations between clusters and sites. We considered only the first discharge of each patient during the data collection period to observe their clinical profiles and length of stay, then followed them up 30 days after their baseline discharge to observe any readmission.Stratified by sites (Hong Kong and Zurich), a hierarchical agglomerative clustering analysis implemented with R package “hclust” was conducted to form clusters of patients starting by grouping similar individuals in terms of age groups , sex, and the presence of listed chronic conditions with dissimilarity between patients measured by Jaccard index . Using tWe performed statistical analyses with R and there were no missing data in the anonymized hospital records. An ethics waiver has been granted by Cantonal Ethics Committee of Zurich for the analysis of Zurich inpatient data (Ref: NZ-B-Nr.2017-00882) while the analysis of Hong Kong inpatient data was approved by the Survey and Behavioral Ethics Committee of the Chinese University of Hong Kong (Project Code: Elderly Care – CUHK). As only secondary analysis of anonymized inpatient data was performed, no informed consent was required.Over the corresponding study periods see , there wAccordingly, each site had one stroke-oriented cluster , one among older adults in Hong Kong . Also, the prevalence of peripheral vascular disease is drastically higher in the sample of Zurich than that of Hong Kong. These results may suggest potentially different etiologies of cardiovascular diseases between Hong Kong and Zurich due to different lifestyles, living environments, and economic structures. Nevertheless, such difference may also be attributed to different specialization foci of the hospitals, different referral patterns, and other practices that may differ between sites. More observations are needed to investigate the underlying reasons.In Hong Kong, the clusters featuring high prevalence (>65%) of heart failure also had relatively high prevalence of chronic pain, which was partially the case in Zurich. This may relate to the regular practice of hospital clinicians in the assessment of heart failure which include the report of pain , 28. If In each of the two sites, there existed highly complex clusters (H7 and Z4) in which a wide variety of diseases was featured. However, as these clusters did not constitute a markedly large proportion of multimorbid patients, integrated or collaborative care for the rest of the clusters with obvious characterizing diseases should be the priority for alleviating the healthcare burden of multimorbidity.While the results of this study are context specific, it provides preliminary information on the replicability of multimorbidity profiling between populations. In the literature, there are at least two recent important systematic reviews on the results of multimorbidity profiling from previous research with a huge variety of statistical methods , 30. It Despite this novelty and the methodological strength of the well-validated algorithms to define chronic conditions using ICD-9 and ICD-10, this study has several limitations that require caution in interpreting the results. First, while the Hong Kong data are highly representative of the inpatients of the public sector, we do not have access to patient records in the private sector which may specialize in certain different conditions from those found prevalent in our sample. Also, University Hospital Zurich was our only source of Zurich data; nevertheless, it is a well-established general acute hospital representing diverse patient intake and standard practices. Second, the validity and comprehensiveness of diagnostic codes may differ between sites and may not be sufficiently reflective of the comparison of the true prevalence, especially considering the different healthcare financing mechanisms , infrastructures, medical training, and practice culture, which might have considerable influence on disease coding practices . That miTo conclude, we conducted a hierarchical agglomerative clustering analysis on discharged inpatients from Hong Kong and Zurich based on a list of 30 diseases and provided findings on the universality of multimorbidity patterns across inpatient populations from the two places. Results should be facilitative of the experimentation and development of integrated or collaborative care for multimorbid patients in the healthcare systems of both populations. Future research should adopt more representative samples, longitudinal disease coding, and comprehensive lists of chronic conditions to verify results of this study and make recommendations on the care planning and coordination of services for multimorbid inpatients.hacpaaedr@ha.org.hk.The data analyzed in this study is subject to the following licenses/restrictions: Authorization to access the data may be considered by the Hospital Authority of Hong Kong upon reasonable requests. Requests to access these datasets should be directed to Hospital Authority of Hong Kong, An ethics waiver has been granted by Cantonal Ethics Committee of Zurich for the analysis of Zurich inpatient data (Ref: NZ-B-Nr.2017-00882) while the analysis of Hong Kong inpatient data was approved by the Survey and Behavioral Ethics Committee of the Chinese University of Hong Kong (Project Code: Elderly Care – CUHK). As only secondary analysis of anonymized inpatient data was performed, no informed consent was required.FL and PB conceptualized the study design, conducted the analysis, and drafted the manuscript. EB and SW supervised the analysis and result interpretation and critically commented on the manuscript drafts. All authors contributed to the interpretation of results and helped revise the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Apnea and other breathing-related disorders have been linked to the development of hypertension or impairments of the cardiovascular, cognitive or metabolic systems. The combined assessment of multiple physiological signals acquired during sleep is of fundamental importance for providing additional insights about breathing disorder events and the associated impairments. In this work, we apply information-theoretic measures to describe the joint dynamics of cardiorespiratory physiological processes in a large group of patients reporting repeated episodes of hypopneas, apneas  and respiratory effort related arousals (RERAs). We analyze the heart period as the target process and the airflow amplitude as the driver, computing the predictive information, the information storage, the information transfer, the internal information and the cross information, using a fuzzy kernel entropy estimator. The analyses were performed comparing the information measures among segments during, immediately before and after the respiratory event and with control segments. Results highlight a general tendency to decrease of predictive information and information storage of heart period, as well as of cross information and information transfer from respiration to heart period, during the breathing disordered events. The information-theoretic measures also vary according to the breathing disorder, and significant changes of information transfer can be detected during RERAs, suggesting that the latter could represent a risk factor for developing cardiovascular diseases. These findings reflect the impact of different sleep breathing disorders on respiratory sinus arrhythmia, suggesting overall higher complexity of the cardiac dynamics and weaker cardiorespiratory interactions which may have physiological and clinical relevance. Sleep is a physiological state characterized by noteworthy physiological changes in respiratory and cardiovascular activities, in almost all the stages. As the autonomic nervous system (ANS) activity is strongly related to sleep, several cardiovascular and neurologic disorders may cause sleep disturbances, and conversely disturbances of sleep can produce unbalanced sympathetic or parasympathetic modulation of the cardiovascular functions ,2. SleepThe identification of presence and severity of sleep disorders requires comprehensive sleep evaluation through overnight recordings of multiple physiological signals in a controlled environment. Monitoring and measuring respiration during sleep has undergone many advances in the last decades in terms of quality and validity. Polysomnography allows the synchronous acquisition of multiple physiologic parameters during sleep, e.g., electroencephalogram, electro-oculogram, electromyogram, electrocardiogram (ECG), and pulse oximetry, as well as airflow and respiratory effort, to permit a comprehensive evaluation of the underlying causes of sleep disturbances . DespiteHeart rate variability (HRV)  has beenAlthough the tools of information dynamics are well-defined from a theoretical point of view, their practical implementation remains challenging due to the need of estimating entropy measures in high-dimensional embedding spaces populated by a limited number of process realizations. This issue emerges even more in the analysis of time series acquired during manifestations of sleep disorders, which are typically characterized by short episodes often associated with non-linear dynamics and non-stationary behaviors. To face these issues, the present work introduces an approach to quantify cardiorespiratory interactions during sleep-related breathing disorders which is based on: (i) the design of a processing pipeline which allows extracting observations of the considered dynamic processes  from short stationary time windows centered around single episodes of a given breathing disorder, and then forming an overall observation matrix from several episodes of the same disorder; (ii) the implementation of two strategies for decomposing the predictive information of the target process  into amounts related to the target dynamics  and to the respiratory driver dynamics ; and (iii) the introduction of a new approach for the estimation of entropy measures which improves kernel-based methods weighing probability estimates by means of a fuzzy membership function. Following a preliminary investigation carried out in a conference paper , the anaThe paper is organized as follows. The dataset analyzed in this research is an open-source database provided for the Physionet/CinC Challenge 2018 , consistThe methodology followed in this study for extracting from ECG and AF signals the time series to be analyzed with information theory is described in this subsection. The algorithms were developed using MATLAB R2018a . The analyzed data consist of the signals acquired during nighttime in correspondence with the considered respiratory effort events . The segments for each respiratory event during sleep were identified according to the provided annotations, with an additional restriction to exclude rapid eye movement (REM) sleep periods, as defined by the accompanying sleep stage annotations. Specifically, for any given type of adverse event we considered a time window including the whole duration of the event as well as the epochs of 20 s immediately before (PRE) and after (POST) the event , were monitored and analyzed in the defined periods b. Both sIn order to obtain the RR interval values from the ECG, two R peak detection algorithms ,35 were The described measurement procedure allowed to extract multiple instances of synchronous segments consisting of RR intervals and AF amplitude values. The segments were extracted for each subject and respiratory  event as schematically represented in The RR and RESP time series extracted as described in the previous section were analyzed using measures of cardiorespiratory information dynamics obtained employing the framework designed in . The anarocess X ,23, and The decomposition of the predictive information can be performed in two ways applying the chain rule for mutual information as depicted by the Venn diagrams in The first decomposition a computeP (TX→Y)  as(2)PYown past , and(4)The alternative decomposition b assesseP (SY|X)  as:(5)PYdynamics . On the dynamics .The RR and RESP samples taken from the segments as defined in analysis ,40), i.et period . Using tiability ,43; an enth observation nth and ith observations of The measures of information dynamics were estimated from each observation (sub)matrix using a model-free kernel estimator which extends the kernel approach used to compute the sample entropy ,44 to thfunction c. The corvations :(9)Kx;σ,In our work, the role of the generic variable izations . With thWhen used to determine the conditional entropy, this estimator is usually referred as fuzzy entropy (FuzzyEn)  and reprThe significance level of the kernel-based estimates of the measures of information dynamics was assessed through a surrogate test performed for each pair of RR and RESP time series obtained partitioning the observation matrix in sub-matrices of 300 rows. Specifically, following the procedure described in , surrogap < 0.05.In order to perform a comparative analysis across conditions, the statistical significance of the differences between the distributions of each given information measure computed over the PRE, DURING and POST segments was tested using the nonparametric Kruskal-Wallis test, followed by a post-hoc pairwise nonparametric Wilcoxon signed rank test (two-sided test) with the Bonferroni correction. Moreover, pairwise comparisons between the CONTROL and the time-defined segments were made using the Wilcoxon rank sum test. Statistical significance for all the tests was set at Altogether 735 out of 994 subjects were identified in the selection process exhibiting good representation of some of the observed respiratory events. The number of analyzed subjects per condition that fulfil the minimum length requirement was: 446 patients with RERA , 536 with HA (out of 950), 281 with OSA (out of 861) and 170 with CA (out of 847).The predictive information of the RR dynamics a exhibitThe information storage b displayThe information transfer c decreasThe direction of changes in internal information dynamics d is aligThe changes in cross information e have siThe information storage measure a was sigThe results described in the previous section highlight the usefulness of exploiting information decomposition to investigate the alterations induced in heart rate and cardiorespiratory dynamics by sleep-related breathing disorders. The predictive information a is signDecomposing the predictive information has allowed to highlight that the cSE values d presentX->Y quantifies the part of the information carried by the present of RR that can be predicted exclusively by the past of RESP, and this behavior is also expected given the presumed unidirectional nature of cardiorespiratory interactions [The decomposition of the predictive information evidenced also measures of the information shared between the heart period and the respiratory dynamics, which physiologically reflect cardiorespiratory interactions and the so-called respiratory sinus arrhythmia, RSA ,52). CE ractions ,53. The ractions , and thuIn detail, analyzing the patterns of information transfer c and croX->Y) in any other respiratory sleep disorder. Such results may add clinical relevance to RERA events, which has been debated in the literature [It is physiologically important to evidence that the information transfer and cross information after RERAs are not fully recovered to PRE values, thus documenting the preservation of depressed cardiorespiratory interactions, contrary to what occurs after an OSA or CA event. Just after a RERA event, the information transfer is also statistically different if compared to CONTROL segments, and this behavior is not observed  showed a generally low statistical significance . This beFuture studies may envisage the extension of the proposed analysis taking into account more variables recorded through polysomnography  to account for additional regulatory mechanisms and exploiting in full the network physiology approach ,64. ThisThe aim of this paper was to conduct a cardiorespiratory analysis during breath-related sleep disorders via information dynamics. Our results document that respiration-related breathing disorders during sleep cause noteworthy changes in cardiorespiratory information dynamics and interactions which can be successfully described using information-theoretic measures. Such variations depend on the type of disorder and are particularly evident during the event compared to the baseline condition, but also persist for several seconds after the breathing disorder. These findings can have clinical implications, resulting in perspective useful for the differential classification of respiration-related breathing disorders.Physiologically, the results confirm that sleep disorders are associated with impaired cardiovascular autonomic control, thus provoking an increased probability of developing cardiovascular diseases, also representing a risk factor for the insurgence of other chronic diseases or even more life-threatening events.


Proprioception is a specialized sensory modality encompassing the movement of the joint and its position in space, and it involves the conversion of mechanical deformation of tissues into neural signals. Mechanoreceptors are specialized nerve structures able to transmit mechanical deformation through electrical signals to dorsal root ganglion sensory neurons and are abundant in the muscles, tendons and ligaments of the knee joint. They are believed to play an important role in knee proprioception and dynamic knee stability. Proprioception should always be taken into consideration for successful reconstruction of the cruciate-deficient knee and for pain and function management in the arthritic knee. Advances in histological methods of detection are numerous and continue to highlight the presence and role of mechanoreceptors after ligament reconstruction, depending on choice of graft. In this review, we present the current knowledge of anterior and posterior cruciate ligaments and grafts mechanoreceptors, and their role in proprioception of knee joint, focusing on each type of mechanoreceptors. The knee joint is comprised of a complex configuration of osseous and soft tissue structures that work in conjunction to allow three planes of motion. The static stabilizers of the knee are its ligaments. The main restraints of tibial translation relative to femur are the cruciate ligaments. In particular, the anterior cruciate ligament (ACL) prevents the anterior translation and the posterior cruciate ligament (PCL) prevents the posterior one, acting as a counterpart to the ACL.Both the anterior and posterior cruciate ligaments play an extremely significant role in knee joint stability. Apart from being the main restraint against anterior or posterior translation of the tibia relative to the femur, they also contribute to dynamic stability of the knee joint via proprioception and activation of knee muscles . The preThe aim of this review is to present the current knowledge of anterior and posterior cruciate ligaments mechanoreceptors and their role in the knee.Static awareness of joint position;Awareness of joint movement and acceleration;Reflex responding and regulating muscle activity.Proprioception is a specialized sensory modality encompassing the movement of the joint and its position in space. There are three main functions of proprioception:Moreover, proprioception plays an important role in preventing injuries and maintaining function of the knee joint . The senThere are four types of MRCs classified by Freeman and Wyke :Type I: corpuscles of Ruffini—low-threshold, slowly adapting receptors that respond to mechanical stress. Ruffini endings appear to be stimulated by displacement of the collagen fibers with which they are intertwined. The characteristics of these receptors categorize them as static and dynamic mechanoreceptors, transmitting information about static position, changes in intra-articular pressure, and the direction, amplitude, and velocity of the joint movements.Type II: corpuscles of Vater-Pacini—dynamic, rapidly adapting mechanoreceptors with a low threshold. They are entirely inactive in immobile joints, becoming active only at the onset or cessation of joint movement, moments at which sudden changes of stress occur.Type III: corpuscles of Golgi—high threshold, slowly adapting mechanoreceptors that are completely inactive in immobile joints. They become active only in extreme ranges of movement and when considerable stress is generated in the joint.Type IV: free nerve endings—high-threshold, non-adapting pain receptors.Various histological methods have been used in identifying MRCs mostly using the gold chloride method ,5. RecenThe first histological demonstration of MRCs in human cruciate ligaments was performed by Schultz et al. (using gold-chloride staining techniques), taken at the time of amputation or autopsy . They foUsing an immunocytochemical approach to identify nerve fibers and corpuscular endings, involving a monoclonal antibody directed against the 68-kDa neurofilament protein, Krauspe et al. found two types of corpuscular-like endings; “spiral-like” (type I) and “spray-like” (type II) endings in a child’s ACL mostly near its bony attachments . Adachi In recent studies, the most common performed technique alongside hemotoxin and eosin stains is immunohistological and immunofluorescence using specific antibodies to neurofilament elements, including S100 and neurofilament protein (NFP). Lee et al. harvested 36 tibial remnants during ACL reconstruction and 2 normal ACLs from healthy knees . NineteeDhillon et al. also evaluated the proprioceptive potential in residual ACL remnants using immunohistological methods . A totalIn their study, Sonnery-Cottet et al. investigated the histological features of the remaining fibers of 26 partial ACL tears. Immunohistochemical studies revealed numerous free nerve endings and few Ruffini and Golgi corpuscles . Gao et Sha et al. observed the survival condition and the quantitative variation of MRCs in the tibial remnant of the ruptured ACL in 60 human knees using immunohistochemistry staining with multiple primary antibodies . The patMRCs in PCL are almost equally evaluated in several studies. Katonis et al. were the first to investigate the existence of MRCs in the PCL of the healthy human knee joint . Three tDel Valle et al. obtained samples from 22 PCLs from patients undergoing total knee arthroplasty and 3 PCLs from cadaver specimens. Using immunohistochemical analysis with mouse monodonal antibodies against neurofilament protein (NFP), S-100 protein (S 100P), epithelial membrane antigen (EMA), and vimentin they found all the known MRCs apart from the Golgi-like ones . MartinsCabuk et al. harvested PCLs from 30 patients undergoing total knee arthroplasty and 10 fresh frozen cadavers . Using SThis literature review focuses on histological studies involving both cruciate ligaments. One of the most important findings is that both ligaments are extremely important in terms of proprioception, having a significant number of MRCs that communicate with the central nervous system. Recent immunohistochemical analyses demonstrated that almost all types of MRCs are present in cruciate ligaments. Furthermore, their total number is significantly higher compared to other ligaments and tendons of the knee joint, highlighting their crucial role in knee joint proprioception ,28. The Free nerve endings are found most commonly, followed by Ruffini corpuscles, Pacinian corpuscles and Golgi-like tendon organs. Free nerve endings are mostly involved in the perception of pain and joint inflammation. Ruffini corpuscles are low-threshold, slowly adapting receptors that respond to mechanical stress. Any torque caused by extension, flexion and rotation of the joint can stimulate them to coordinate the overall movement of the knee and to feel position of the still knee joint. Pacinian corpuscles are low-threshold and rapidly adapted pressure receptors, play an important role in joint dynamic movement because they can produce and transmit joint motor sense and are very sensitive to the change of joint position. Golgi–like tendon organs are high-threshold and slow-adapted mechanoreceptors, which play an important role in preventing the joint from extreme flexion, extension and rotation. Of note, most of the MRCs are distributed in the synovium near the attachment points of ligaments with the femur and tibia, especially with the tibia. In a study of knee joint in rabbits, Han et al. found that cruciate ligaments had the higher number of MRCs in comparison with the other knee ligaments. Moreover, they found that gene expression level was positively related to the quantity of MRCs as the levels of NEFM, S-100B and CGRP genes were highest in ACL and PCL, suggesting that these ligaments have the richest sensory nerve endings .Proprioception of the knee should always be taken into consideration for successful reconstruction of cruciate ligaments. Many studies have focused on the presence of MRCs in the torn ACL ,12,13,14Another important issue raised recently is the presence of MRCs in allografts and autografts used for cruciate ligament reconstruction. In 2020, Rebmann et al. studied 26 patients with different types of autografts (semitendinosus and patellar tendon) and three patients with patellar tendon allografts during revision surgery after traumatic rerupture of the ACL graft. Using immunohistochemical analysis, they found Ruffini corpuscles and free nerve endings present in each graft. Comparing the grafts, the highest number of MRCs could be detected in the semitendinosus autograft. With increasing time from implantation, the MRCs, especially in the patellar tendon autografts, showed an increase in their number for both Ruffini and free nerve endings. The authors concluded that the partial increase in the number of receptors over time after ACL reconstruction could indicate a reinnervation of the grafts . AnotherRegarding knee arthrosis, proprioceptive acuity was proposed as a risk factor for the initiation and progression of pain and structural damage in knee . In theiAs the number of total knee replacements (TKR) is expected to increase up to 143% by 2050 in the USA  and up tFurther research is required to fully understand the role of mechanoreceptors. Understanding the role of MRCs in knee kinematics should provide us with more information about the proprioceptive deficiencies associated with ligament ruptures and the pathogeneses of knee arthrosis. Patients at risk for MRCs-deficient knees could be candidates for special rehabilitation protocols, in order to compensate for loss of proprioception and kinesthesia.


Tissue homeostasis is perturbed by stressful events, which can lead to organ dysfunction and failure. This is particularly true for the heart, where injury resulting from myocardial infarction or ischemic heart disease can result in a cascading event ultimately ending with the loss of functional myocardial tissue and heart failure. To help reverse this loss of healthy contractile tissue, researchers have spent decades in the hopes of characterizing a cell source capable of regenerating the injured heart. Unfortunately, these strategies have proven to be ineffective. With the goal of truly understanding cardiac regeneration, researchers have focused on the innate regenerative abilities of zebrafish and neonatal mammals. This has led to the realization that although cells play an important role in the repair of the diseased myocardium, inducing cardiac regeneration may instead lie in the composition of the extra cellular milieu, specifically the extra cellular matrix. In this review we will briefly summarize the current knowledge regarding cell sources used for cardiac regenerative approaches, since these have been extensively reviewed elsewhere. More importantly, by revisiting innate cardiac regeneration observed in zebrafish and neonatal mammals, we will stress the importance the extra cellular matrix has on reactivating this potential in the adult myocardium. Finally, we will address how we can harness the ability of the extra cellular matrix to guide cardiac repair thereby setting the stage of next generation regenerative strategies. Heart failure (HF) is a leading public and clinical health problem of the 21st century with a prevalence of more than 23 million worldwide  when looking at cardiac repair, remodeling, and injury. The presence of aberrant ECM, as is the case in post-infarction HF development/progression, can reduce or even block the effectiveness of cell-based therapies to repair the heart . InteresWith an eye to the future, in this review we will briefly revisit the shortcomings of stem cell-based approaches and stress how understanding the innate ability of the ECM in zebrafish and neonatal mammals for cardiac regeneration may offer alternative approaches to advance curative therapeutic strategies for patients suffering from post-infarction HF.With the goal of being a one-shot solution to cardiac injury, researchers have attempted to identify a cell source capable of inducing cardiac tissue regeneration. This has led to the investigation of several putative stem-cell based therapies to either stimulate the endogenous cells or deliver exogenous stem cells to trigger cardiac regeneration in the preclinical animal setting and in clinical trials; including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), bone marrow-derived stem cells, and most recently cardiac stem cells (CSCs). Despite these immense efforts, only limited and temporary clinical benefits have been observed, and whatever effects have been largely explained by paracrine cues associated with aiding the reduction of the inflammatory response , 7.Of all the stem cell-based approaches, bone marrow-derived stem cells have been the most extensively studied for their cardiac regenerative potential, and over the years a multitude of experimental and clinical studies have been performed. A recent meta-analysis of 43 randomized controlled clinical trials, with 2,635 patients, focused on exploring the results at 6 months, 1 year, 3 years, and 5 years following the exogenously administration of bone marrow stem cells in patients with acute myocardial infarction . The autThe final, and most controversial target for cardiac stem cell-based regeneration are the endogenous CSC. The notion that the adult heart is not just a post-mitotic organ, but has a limited extend for self-renewal, came about from advances in radiocarbon dating . In addiBased on these significant advances in stem cell biology, it has become apparent that although healthy stem cells with cardiomyogenic potential are important, they can by themselves not lead to the long term goal of repairing the injured myocardium. This is in large part due to the toxic effects the pathological micro-environment, specifically the diseased extra-cellular matrix (ECM), has on the regenerative capacity of the myocardium. A recent paradigm shift has suggested that the albeit limit beneficial effect of cell-based approaches may not lie in cell restoration, but may in fact lie in their paracrine actions and more importantly ECM homeostasis . This ECWithin the animal kingdom there are numerous examples of spontaneous organ regeneration following injury. In terms of cardiac regeneration, it is the zebrafish that stands out the most. Zebrafish possess the ability to undergo complete cardiac regeneration without scar formation after the resection of up to 20% of the ventricular apex. Complete re-growth of the amputated region, including the coronary vasculature, myocardium and endothelium is achieved at 60 days post-resection, resulting in a fully functional heart –23. Thiset al induced cardiac damage, by resection of 15% of the ventricular apex, in 1-day-old neonatal mice  model  model . As in tAlthough significant advances have been made in the field of cardiovascular medicine, diseases such as myocardial infarction (MI) and ischemic heart disease, ultimately resulting in heart failure, remain amongst the leading causes of fatalities in the developed world. The mortality of these cardiovascular diseases is in no small part due to the irreversible loss of vessels and cardiomyocytes resulting in a decreased cardiac functionality. Current therapeutic strategies are aimed at alleviating the symptoms rather than repairing the loss of functioning cardiac tissue. Thus, researchers have turned to the field of regenerative medicine to identify novel strategies aimed at regenerating and/or repairing the number of functioning cardiac muscle cells within the failing heart.Based on the current state-of-the-art, it is becoming increasingly evident that the utilization of stem-cell based approaches to achieve regeneration of the adult mammalian myocardium may not be the silver bullet scientists originally hoped for. Over the past decades several breakthroughs have been made in stem-cell biology that have enabled researchers to investigate numerous cell sources for cardiac regenerative purposes, and although some of these studies have demonstrated improvements in cardiac function in the translational setting, actual regeneration of the myocardium has yet to be demonstrated. This suggest that many of the cell sources for cardiac regeneration may hold a paracrine effect on the myocardium post-injury. Therefore, it has become evident that novel approaches have to be explored to attain true regeneration of the damaged myocardium.This is where unraveling the regenerative capacities of the zebrafish and/or neonatal mammalian heart may come in to play. Specifically when looking at the ECM, since several studies have demonstrated that the ECM of zebrafish and neonatal mammalian hearts possess an innate regenerative potential when applied to injured adult hearts. The undisputable potential the ECM has to regenerate the damaged myocardium as also lead to the development of an injectable hydrogel derived from porcine decellularized myocardial ECM (VentriGel), which has recently also been tested in the clinical setting . This stin vitro and in vivo). Finally, and the most crucial step will be the development of these ECM components into a synthetic hydrogel to facilitate the move into clinical application. Advances in the field of synthetic biomaterial have demonstrated the potential for the generation of such synthetic ECM hydrogels (To push the field of cardiac regenerative medicine forward, several hurdles will have to be overcome before we can develop therapeutic strategies to be implemented in a clinical setting. Firstly, we must understand the “why” neonatal mammalian or zebrafish ECM is able to induce adult cardiac regeneration. To this end advances in omics-based approaches, including proteomics and RNA-sequencing, will enable scientist to fully characterize the composition of these early ECM's and identify which components are no longer present in the adult myocardium . Next, wydrogels , 60. Altydrogels . Intramyydrogels . Taken tOne of the greatest obstacles that has to be overcome is our current knowledge gap related to the demonstrated role the ECM, in particular the neonatal ECM, plays in cardiac regeneration , 32–35. AvdP and CB contributed to planning, writing, and critical reading of this manuscript. 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.


For years, fungi have served as repositories of bioactive secondary metabolites that form the backbone of many existing drugs. With the global rise in infections associated with antimicrobial resistance, in addition to the growing burden of non-communicable disease, such as cancer, diabetes and cardiovascular ailments, the demand for new drugs that can provide an improved therapeutic outcome has become the utmost priority. The exploration of microbes from understudied and specialized niches is one of the promising ways of discovering promising lead molecules for drug discovery. In recent years, a special class of plant-associated fungi, namely, fungal endophytes, have emerged as an important source of bioactive compounds with unique chemistry and interesting biological activities. The present review focuses on endophytic fungi and their classification, rationale for selection and prioritization of host plants for fungal isolation and examples of strategies that have been adopted to induce the activation of cryptic biosynthetic gene clusters to enhance the biosynthetic potential of fungal endophytes. Natural products, also known as secondary metabolites (SMs), are a diverse group of mainly low-molecular-weight and structurally diverse chemical entities that are produced by almost all living organisms, but mostly by microbes . The useIn the early days of microbial-based drug discovery, the soil microbiota was the principal source of bioactive compounds ,4. Howevendo = inside; phytón = plant) was coined by Anton de Bary in 1866 to denote fungi that live inside their host plant tissues—as opposed to ‘epiphytes’, a term assigned to fungi that are found on the surface of plants  in addition to four other known compounds that were absent in the two monocultures ) and I (56), that were not produced by the fungus grown in the absence of the two chemical elicitors [Manipulation of the epigenetic environment using chemical elicitors has also been reported to induce the production of toxic SMs such as mycotoxins in fungal endophytes.  culture . These cia toxin . The grolicitors . The strategies for the activation of the silent BGCs described so far are, generally, untargeted in nature and, thus, do not explicitly permit an association between a newly identified secondary metabolite with any particular gene cluster. Instead, these approaches are meant to activate putative biosynthetic pathways whose identity or number may not be known beforehand. Genetic engineering of target strains is a more precise avenue that can be exploited to awaken cryptic BGCs, an approach that relies largely on genome mining. This approach is BGC specific and first involves the ‘mining’ of the whole genome of the target strains for the typical secondary metabolite gene clusters using appropriate bioinformatics tools ,129. BasAspergillus nidulans through the overexpression of transcription factor gene apdR, resulting in the biosynthesis of two novel PKS-NRPS hybrid metabolites designated as aspyridone A and B that had not been previously isolated from this fungus [azaR in A. niger resulted in the production of six new azaphilone compounds designated as azanigerones A-F [xpp1, while resulting in the downregulation of primary metabolism, was found to enhance the upregulation of secondary metabolism, leading to significant increases in the quantity and diversity of SMs in Trichoderma reesei [PfCclA and PfcclA, two epigenetic-related genes in endophytic fungus Pestalotiopsis fici, led to the biosynthesis of fifteen novel polyketides, pestaloficiols T-W, 11 macrodiolide ficiolides A-K and ficipyrone C [Aspergillus terreus BGCs in A. nidulans [McrA) was uncovered as a gene involved in global downregulation of secondary metabolism in A. nidulans [McrA from A. nidulans and its homologs in a number of other fungal strains led to the upregulated production of several secondary metabolites, while its overexpression led to the suppression of SM biosynthesis [LaeA, a positive regulator found in Aspergillus spp., has been shown to enhance SM biosynthesis in members of this genus [Manipulation of pathway-specific regulators is an especially promising approach considering that up to 50% of fungal BCG have been found to harbor cluster-specific transcription factors . The oves fungus . Using tones A-F . Similara reesei . In anotpyrone C . In addipyrone C . This apnidulans . The idenidulans . The delynthesis . The oveis genus . Other mis genus ,138. It is worth mentioning that the choice of any of the approaches discussed herein will depend upon the requirements of individual researchers, the level of expertise and the nature of the fungal strains, among other factors. None of the approaches can be considered as superior, as each has its strengths and limitations.Endophytic fungi have emerged as promising resources with enormous potential in drug discovery. With only a small fraction of the more than 1 million estimated fungal endophytes investigated for their biosynthetic capacity, fungal endophytes remain a largely understudied resource for the discovery novel bioactive molecules. The discovery of endophytic fungal strains capable of producing plant-associated molecules raises the prospects of exploiting such strains as an alternative source of valuable compounds. For example, the discovery of paclitaxel-producing fungal endophytes paved the way for the production of paclitaxel through a semisynthetic process that involves 10-deacetyl-baccatin III as a precursor, plant cell culture and the endophytic fungi. Such an approach for co-cultivation of fungal endophytes with plant cells may offer the possibilities for production of other useful bioactive compounds that are produced in unsustainable quantities in plants.However, to derive the immense benefits and possibilities that fungal endophytes promise, a number of constraints need to be addressed. Among the limitations associated with bioprospecting for bioactives from fungal endophytes is the issue of unculturable fungal strains. While the diversification of isolation media has been shown to enhance the recovery rate of cultivable fungal endophytes, numerous studies have revealed that many endophytic fungal strains are never recovered from plant materials because they are unculturable. Although metagenomics techniques facilitate the detection of such strains, it remains a big challenge to assess such strains for their full SMs biosynthetic competencies using the available technologies. Moreover, even among the culturable strains, loss of viability or capacity for biosynthesis of the compounds of interest either as a result of long-term preservation and repeated sub-culturing is a common challenge. This has often led to the loss of promising fungal strains before they are well studied, and attempts at re-isolation of the same fungal strains from the original habitats are not always met with success. Lastly, the future of fungal endophytes as a source of bioactives is tied to that of their host plants. There is a positive correlation between plant diversity and endophytic fungal diversity. The increase in habitat loss and overexploitation of plants resources in different parts of the world are putting many plant species at a risk of extinction. The loss of such plants means a corresponding loss of an assortment of valuable fungal endophytes, many of which may be uncharacterized and potential producers of interesting chemical molecules.


The Covid 19 pandemia since the first reported case in 2019 had a direct socioeconomic impact related to morbi-mortality and indirect in response to protection and isolation strategies. To our knowledge thrombo-embolic complications can be a mode of revelation complicating the management.We present an exceptional case of a patient with a history of Covid 19,admitted 21 days later for disturbed consciousness, in whom an ischemic occipital stroke ,intestinal and colonic ishemia had been objectified. Our objective through this presentation is to remind the thrombo-embolic particularity of Covid 19, to take the viral attack as a serious antecedent in the periods following theinfection and to put the point on the primordial place of early rehabilitation in patients with stroke.We discuss through this report the recommendations of anticoagulation in Covid 19 patients and the place of early rehabilitation in patients with stroke. We also report a new case among the rare cases described in the literature that associates several thrombo-embolic manifestations secondary to Covid 19, in particular the neurological and digestive association. •Exceptional Clinical Presentation associating an occipital Stroke, a Superior and Inferior Mesenteric Thrombosis following Covid 19 Disease.•We propose take to the viral attack as a serious antecedent.•This manuscript can add new perspectives to the management practice of this very rare presentation. This maWe report according SCARE guidelines  the caseThe particularity of our exceptional case is defined by the rarity of similar cases associating the thromboembolic complication on several organs published in the literature, by the absence of standardization of such a pathology and by and we propose to take into consideration the Covid 19 attack as a serious antecedent in front of the medical affections.2A 28 years old patient native and resident in the Moroccan oriental, having as antecedent an infection Covid 19 during the first wave with light respiratory manifestations; of good evolution under symptomatic treatment. Three weeks later, the patient consulted the emergency room for consciousness disorder, visual and coordination disorders in an apyretic context in whom the clinical examination found an obnubilated patient, hemodynamically and respiratorily stable with a capillary gylcemie level of 1g/l. She was quickly conditioned and a standard infectious biological workup was performed with a hemogram in favor of anemia with Hb at 9g/dl, a lumbar puncture and bacteriological examination of the urine were normal, and a cerebral imaging showed an occipital stroke with free supra-aortic trunks , Fig. 1dtrans-thoracic ultrasound. A new multidisciplinary discussion was held where post Covid 19 thromboembolic complications were evoked as etiology.On the third day of her admission our patient presented an abdominal distension with disturbance of the infectious balance. An abdominal CT scan was performed in favor of entero-mesenteric ischemia and left colonic ishemia motivating the surgical exploration  received no financial support for the research, authorship and/or publication of this article.Siham Elmir: Writing, review and editing of the manuscript.Jabi Rachid, Houda Mirali, Mohammed Noumairi, Mohammed Gartit: Contributed for diagnose and treatment of the patient.Mohammed Bouziane, Brahim Housni and El Oumri Ahmed Amine: Review, Supervision and surgeons of the patient.Registration of research studies: Our paper is a case report; no registration was done for it.Guarantor: Siham Elmir.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.Dr. Siham Elmir.Not commissioned, externally peer reviewed.We don't have any financial sources for our research.All authors disclose any conflicts of interest.


Larix larcina as the highest PIV species in primary assays, Salix planifolia in secondary assays, and Kalmia angustifolia in safety assays, as well as a ranking of other less active species by PIV. Multivariate analysis showed that activity in safety PIV monitored mainly with cytochrome P450 inhibition patterns best reflected patterns of traditional medicine importance in Cree traditional knowledge, whereas potent primary bioactivities were seen in individual plants determined to be most important to the Cree for anti-diabetes purposes. In the secondary anti-diabetes assays, pharmacological variability was better described by plant biology, mostly in terms of the plant part used. Key signal in the metabolomics loadings plots for activity were phenolics especially quercetin derivatives. Traditional Indigenous knowledge in this analysis was shown to be able to guide the identification of plant pharmacological qualities in scientific terms.An ethnopharmacological metanalysis was conducted with a large database available on antidiabetic activities of plant foods and medicines from the northern boreal forest, which are traditionally used by the indigenous Cree of James Bay, Quebec, Canada. The objective was to determine which bioassays are closely associated with the traditional knowledge of the Cree and which pharmacological metrics and phytochemical signals best define these plants and their groups. Data from 17 plant species, ethnobotanically ranked by syndromic importance value for treatment of 15 diabetic symptoms, was used along with 49 bioassay endpoints reported across numerous pharmacological studies and a metabolomics dataset. Standardized activities were separated into primary, secondary and safety categories and summed to produce a Pharmacological Importance Value (PIV) in each of the three categories for each species. To address the question of which pharmacological metrics and phytochemical signals best define the CEI anti-diabetes plants, multivariate analyses were undertaken to determine groupings of plant families and plant parts. The analysis identified  Due to a better understanding of the consequences of loss of indigenous culture through world development, a recent focus of the scientific community is the medicinal value of traditional knowledge . PreviouDeveloping a unique combination of community and laboratory methods in the analysis of traditional knowledge data, the Team in Aboriginal Anti-diabetic Medicine (TAAM) was assembled in 2003 by Dr. Pierre Haddad of the University of Montreal and funded by the Canadian Institutes of Health Research (CIHR) to address the issue of rising rates of Type 2 diabetes (T2D) in Canadian First Nations communities. The work of the TAAM focused mostly on the Cree of Eeyou Istchee (CEI), a group of communities located in the James Bay region of Northern Québec. The team included researchers from three major Canadian research universities and community stakeholders representing both Elders and Public Health professionals. The goal was to answer a call from Cree Elders to their leadership to revitalize traditional medicine as an accessible and culturally relevant method of T2D prevention and management. Through multi-institutional collaborative work, the TAAM investigated the ethnobotany, pharmacology, and phytochemistry of traditional Northern Cree medicinal plants in the context of T2D.After more than 15 years of data collection and analysis focusing on 17 medicinal plant species prioritized based ethnobotanical use for treating symptoms of diabetes , the TAASince 2003, the TAAM has collected data on the use of medicinal plants from the CEI for the prevention and management of T2D. This research began with a targeted ethnopharmacological approach and novel statistical methods to evaluate the CEI ethnobotany with specific focus on traditional medicines used for treating major symptoms of T2D . All repThe 17 medicinal Cree plants were collected under the guidance of Cree elders over the course of more than a decade of collaboration, in the CEI territory of Northern Québec, Canada . BrieflyIn total, 69 bioassay endpoints reported across numerous pharmacological studies were included in this investigation. Of those, 49 contained complete data for all 17 plant species. A full breakdown of the individual endpoints can be found in An effort was also made to estimate a given plant’s overall pharmacological activity in each set of bioassays representing primary antidiabetic potential, secondary antidiabetic potential or herb-drug interaction potential, as described above. For that purpose, the standardized activity described in the previous paragraph was summed over each set of bioassays and then divided by the number of bioassays in this set. In this way, a plant that would be the absolute best in all bioassays of a given set would obtain a value of 1, one that would display half of that maximal activity in the same bioassays would get a value of 0.5 and so on. We termed this value Pharmacological Importance Value (PIV) as a parallel to the SIV obtained from traditional knowledge.In addition to the pharmacological data, phytochemical data collected for m/z) and retention time as well as the intensity (peak area) of that signal for each of the 17 Cree plants.Raw metabolomics data is inherently three dimensional, containing values for signal mass, signal intensity and retention time, and so was condensed here to simplify analysis. Raw data also contains baseline noise signals that are not relevant to analysis and must be filtered out. Further, Waters’ proprietary data format combines normal peak data with lockmass standardization data and signal fragmentation data, which must also be filtered out. To adapt metabolomics data for this study, the fragmentation signal data was removed from the raw data file using ProteoWizard . Data prTo address the question of what biological activities associate with the traditional knowledge of the CEI, the plants were first grouped into quartiles based on their SIV values from the original CEI ethnobotany study of the TAAM . The firPharmacology Dataset above) was generated for each plant to represent its overall level of activity in the bioassays and bioassay subcategories . Groupings by both plant family and plant part used were applied. To investigate key differences in the distributions of metabolic signals between discrete groups, orthogonal partial least square discriminant analysis (OPLS-DA) was used to generate S-plots where the discriminating signals between groups were taken to represent potential biomarkers. Such signals were searched using the Metlin. scripps online database to yield tentative identifications. Search parameters included the signal mass, a mass tolerance of 10 PPM, and adduct possibilities of +H, +Na, and -H2O. Tentative identifications were then based off of the most abundant compound class with lowest mass difference.Upon evaluation of all pharmacological data , PCA anaBased on overlap between the 95% confidence regions of SIV quartiles, it became clear that of the three assay types , the safAssessing potential correlations between the traditional knowledge (SIV) and strength of plant activity in the bioassays (PIV), similar patterns as seen with PCA emerged . Once agActivity distributions among the plants were also be evaluated based on biological relationships, namely plant family and plant part. Within the primary assay data, neither plant family nor plant part relate well with the distribution of plant bioactivities (data not shown). Instead, there is a clear separation in the variable distributions for the two groups of glucose uptake stimulation (muscle and adipose tissue) from the other assays, as well as a loose grouping of the assays involving hepatocytes.R. groenlandicum, R. tomentosum, G. hispidula, K. angustifolia (Ericaceae species), and P. glauca  appear to have a high degree of similarity with respect to their antioxidant and antiglycation activities, both of which relate to radical scavenging capacity. In addition, the two cone extracts, P. banksiana and P. mariana, exhibited similar bioactivity patterns in both the secondary , although this distinction appears to be more driven by the results of the primary bioassays specifically.Considering an active-inactive binary for each plant on each bioassay instead of rank also provided valuable insight. By summing the total number of assays in which statistically significant activity was observed in the previous studies , R. groeHierarchal cluster analysis of the Cree metabolomics data  presenteG. hispidula and V. vitis-idaea plants having more similar growth forms while the P. glauca sample is characterized as a conifer needle.Given the similarity in the phytochemistry of the leaves, discriminant analysis and PCA were used to explore what chemical signals appear to be most representative of this group compared to the rest of the Cree plants. Separate analyses were completed to compare the leaves to their least similar group, the barks , as wellMajor phytochemical signals highly representative of the evaluated groupings in the discriminate analyses separate furthest from the origin, and so are easily determined through visual examination of the plots. These major signals are presented in The objective of this study was to explore the relationship between traditional knowledge within a directed set of uses (treatment of diabetes symptoms) and the patterns of effects in relevant bioassays of such medicinal plants. The strongest detected association was between the importance of CEI medicinal plants (through SIV) and their inhibitory activity on CYP enzymes in the herb-drug interaction assays. This is not surprising given that the role of many CYP enzymes is to respond to chemical challenges coming from the environment . Indeed,In addition, a slightly weaker association was also seen between the most important CEI plants in terms of traditional knowledge and radical scavenging activities displayed in the secondary antidiabetic potential assays. Plants rich in antioxidant polyphenols have received much attention for their positive effects on disease prevention , EvaluatP. glauca was comparable to insulin in its ability to inhibit the activity of G6Pase in hepatocytes while L. laricina was more than twice as effective as insulin at stimulating glycogen synthase activity through GSK-3 phosphorylation , there was almost always at least one plant that either closely matched or exceeded the activity of pharmacologically relevant positive controls. For instance, rylation . Likewislitazone .P. balsamifera. It also was among the lowest five plants for all three PIV categories . In contrast, the majority of secondary antidiabetic bioassays  as well as all herb-drug interaction bioassays used acellular models, the latter based on highly defined recombinant enzymes. Despite the fact that standard operating procedures were generally used across all data sets, the use of cell-based models can provide an explanation for the greater dispersion of the primary antidiabetic potential data set and underlie the difficulty in obtaining stronger associations between such data and Cree traditional knowledge in our multivariate statistical analyses. Conversely, data generated using acellular, most often enzyme-based, bioassays yielded much less variable results; likely enhancing the discrimination of plants along a spectrum of biological activities and enabling stronger statistical association with Cree traditional knowledge. Albeit cell-based models are inherently strong because they integrate several biological events that culminate in the measured outcome.Thirdly, unlike what our team did with the SIV, the PIV used in our analysis considered all bioassays as equivalent in terms of relevant pharmacological activity. Indeed, for the SIV, we had a group of clinical endocrinologists and diabetes researchers provide a rank for the importance or relevance of each symptom in the clinical realm of diabetes; ethnobotanical data being weighed accordingly . A similFinally, our analysis did not take into consideration the period in which plants were collected, nor the geographical ranges of these collections. It must be noted that most of the studies used extracts from uniform collection times and areas. It is well known that rates of phytochemical biosynthesis vary significantly in relation to seasonal and latitudinal variations in light intensity, climate, and soil composition . Our teaOne of the purposes of this investigation was to explore the power and potential for multivariate analysis of multidisciplinary data in the context of ethnopharmacology. A useful application of the work would have been to develop predictive models where the general activities of a given plant could be projected based on its taxonomic family, the part used, or a quick screen of its phytochemical profile. Perhaps unsurprisingly, despite the patterns discussed here, it is apparent that there was still quite a long way to go before a model of such power can be fully realized. These results, however, can inform the design of future studies with fewer limitations and greater statistical power for investigating potential relationships between ethnobotany, phytochemistry, and pharmacology.Overall, this work highlights the depth and insight of traditional knowledge within indigenous communities with regards to plant medicines. Specifically, CYP inhibition patterns as a whole best reflected patterns of traditional medicine importance in the CEI traditional knowledge, whereas potent primary bioactivities were seen in individual plants determined to be most important to the CEI for anti-diabetes purposes. In the secondary anti-diabetes assays, pharmacological variability was better described by plant biology, mostly in terms of the plant part used. Thus, traditional indigenous knowledge should be cherished for its ability to guide the deciphering of plant qualities that may have previously been overlooked by science.


Non-coding RNAs (ncRNAs) comprise a set of abundant and functionally diverse RNA molecules. Since the discovery of the first ncRNA in the 1960s, ncRNAs have been shown to be involved in nearly all steps of the central dogma of molecular biology. In recent years, the pace of discovery of novel ncRNAs and their cellular roles has been greatly accelerated by high-throughput sequencing. Advances in sequencing technology, library preparation protocols as well as computational biology helped to greatly expand our knowledge of which ncRNAs exist throughout the kingdoms of life. Moreover, RNA sequencing revealed crucial roles of many ncRNAs in human health and disease. In this review, we discuss the most recent methodological advancements in the rapidly evolving field of high-throughput sequencing and how it has greatly expanded our understanding of ncRNA biology across a large number of different organisms. Escherichia coli in 1984 [Caenorhabditis elegans [The central dogma of molecular biology that Francis Crick proposed in 1957 described a model for the flow of information between genes and proteins . Additio in 1984 , and Xis in 1984 , and mic elegans , in the In parallel to the discoveries of diverse RNAs and their roles in the field of molecular biology, a novel technology for the determination of the sequences of nucleic acids appeared on the horizon. The first DNA sequencing approach has been developed by using a chain termination approach established by Sanger and colleagues in 1977 . Based oThe latest major advance in sequencing technology is the development of long-read sequencing . Long-read sequencing is largely brought about by two novel technologies: Single-molecule real-time (SMRT) sequencing and nanopore sequencing, which are commercialized by Pacific Biosciences and Oxford Nanopore respectively . SMRT seThe development of different sequencing techniques enables faster and more cost-efficient sequencing and improved quality of sequencing data . AdditioThis review summarizes the recent development in ncRNA sequencing and describes the impact sequencing technology has had on the field of ncRNA biology. We focus on how RNA sequencing has been an important tool to uncover novel ncRNAs  as well While genome sequencing efforts came to fruition upon the turn of the century, one major question which remained open was: How much of the genome is transcribed? In order to address this question, the RIKEN institute decided to start a consortium called FANTOM consortium . The goaIn general, progress in understanding biology through RNA sequencing rests heavily on the shoulders of technological advancements. An enduring goal of the field is to increase the detection sensitivity in order to gain information when only small quantities of RNA or DNA are available. Within the last decades, substantial progress has been made on that front, making it possible to sequence the RNA content even from single cells. The field of single cell transcriptomics has subsequently greatly enhanced our understanding of cellular heterogeneity across many tissues . This leThis started to change with the first demonstration of single-cell sequencing of the small RNAs . With thBeyond simply probing which ncRNA molecules are present in any given sample, sequencing technology can also give insights into the functional role of ncRNAs. On the computational side, tools are being developed to help us to understand what the myriad of ncRNA sequences obtained by sequencing reveals about ncRNA function. Recent computational tools based on deep learning were developed to improve sequence classification into ncRNA classes or infer function of short ncRNAs transcripts based on the sequences alone ,104.One of the most studied roles for ncRNAs is the regulation of the transcriptome. This can take place at the different stages such as the transcriptional, post-transcriptional or chromatin level . Since tncRNAs have the ability to directly interact with the transcriptional machinery and thereby regulate gene expression. One of the most comprehensively studied examples of that is the mechanism of sex-chromosome dosage compensation . The inaIn contrast to transcriptional silencing, sequencing ncRNAs also led to important insights into transcriptional activation. One example for this is the discovery of eRNAs. Enhancers are genomic regions that activate transcription from a distance and are often marked by histone modifications such as H3K27Ac . InteresA major determinant of transcriptional regulation is the chromatin state . Hence, One of the first methods that aimed to map the global RNA-chromatin interactome in an unbiased fashion was named global RNA interactions with DNA by deep sequencing (GRID-seq) . GRID-seAnother set of methods that have been termed “hybridization capture methods” detect interaction sites of ncRNA with chromatin in a genome-wide fashion . MethodsBesides direct interaction with the proteins that bind to DNA, ncRNAs have also been shown to directly interact with DNA and thereby affect chromatin accessibility. One example of these direct interactions are triple helices called RNA–DNA–DNA triplexes . RecentlAnother form of RNA-DNA hybrid, which can involve mRNA as well as ncRNAs, resulting in the displacement of single-stranded DNA are so called R-loops. Mounting evidence accumulated in recent years suggests a number of regulatory functions of R-loops in gene expression . InitialOne of the most extensively studied ways to regulate the transcriptome post transcription is through miRNAs . AccordiBeyond the mere description of which miRNAs are expressed under any given circumstances, sequencing technology also showed how miRNAs regulate the transcriptome on a genome-wide scale. One technique that aims to accomplish this is called crosslinking, ligation, and sequencing of hybrids (CLASH) . CLASH bMore recently, advances in sequencing protocols enabled the investigation of the intracellular miRNA kinetics . The conBesides the above described roles of ncRNAs in the regulation of transcript production and translation, there is also a more indirect way in which ncRNAs can influence the transcriptome. lncRNAs, for example, have been shown to act as molecular scaffolds in cells . The lncAs transcriptional and posttranscriptional regulators of gene expression, ncRNAs are involved in many cellular physiological and pathological processes. Thus, they represent a promising target in medical research. In particular, RNA sequencing has uncovered many ncRNAs that are differentially expressed in the context of various diseases. Accordingly, ncRNA research has the potential to elucidate pathological mechanisms, identify novel prognostic and diagnostic markers or represent therapeutic targets . This poIn order to lay out the ncRNA landscape, sequencing approaches are needed to broadly uncover which ncRNAs are expressed in any given tissue. Thus, RNA sequencing greatly helped to unravel the transcriptomic landscape and thus generate reference atlases for comparison of pathological conditions . As an eIn general, there are large differences in the abundance of ncRNAs within different cell types and tissues. Especially circRNAs and lncRNAs exhibit tissue-specific expression ,138,139.Due to their complex regulatory functions, aberrantly expressed ncRNAs may contribute to the pathogenesis and progression of disease. This has been reported in the context of numerous pathologies in particular cancer  and neurOnce identified, sequencing of circRNAs can also enable insights into their potential function based on their sequence. According to the competing endogenous RNA hypothesis , circRNAInterestingly, in addition to these endogenous interactions between e.g., miRNAs and their mRNA targets, there are also interactions between pathogen-derived RNAs and human host RNAs. In a recent study, Yang et al. report that the genomic RNA of the SARS-CoV2 virus interacts with a number of host RNAs . Using sLiquid biopsy is a promising approach in clinical medicine that involves the collection of body fluids for diagnostic and prognostic analysis and monitoring. The aim is to replace more invasive tissue sampling and examinations as well as to enable earlier diagnosis and continuous monitoring especially for diseases that benefit from early detection. In order to find biomarkers for these purposes, RNA, DNA or proteins can be isolated and analyzed from cells found in e.g., blood, bronchial lavage, urine or cerebrospinal fluid. Especially for the investigation of neurological diseases, liquid biopsies offer a promising opportunity, given that the human brain is one of the most difficult organs to access for tissue biopsy in the human body. These benefits are exemplified in a recent study which analyzed small RNA sequencing data obtained from Parkinson’s disease patients’ whole blood . The autOne advantage of analyzing RNA from whole blood samples is that it is technically rather simple, yet very informative. A recent study has demonstrated that whole blood transcriptome predicts gene expression for other tissues in the body . InteresIn addition to these approaches, analysis of extracellular RNA emerges as a promising field for medical diagnostics. Sequencing cell-free RNA has demonstrated encouraging results for the diagnosis of several diseases ranging from cancer to cardiovascular diseases . In contAlongside advancements in the field of ncRNA research in human health and disease, in recent years there was also a surge in research of non-human ncRNAs and their potential roles. Thanks to new sequencing methods and novel approaches to data analysis, it has become possible to elucidate ncRNAs and their roles in viruses and various organisms ranging from archaea over scorpions to mice.Here, we provide a general overview of some of the recent advancements in research fields beyond human biology with the help of sequencing technology and computational analysis.Viral ncRNA studies are a rapidly evolving research area that was propelled by the rise of sequencing technologies . Since tMore recently, RNA-seq was used to assess the ncRNA repertoire produced by Ebola virus (EBOV) and Marburg virus. It was found that EBOV encodes small ncRNAs that act independently of the host miRNA machinery and do not display the ability to suppress viral replication or silence gene expression, thus leaving the question of their role in the host cell open .Besides human viruses, other viral pathogens have also been investigated. A recent study examined the African Swine Fever virus (ASFV), which lethally infects pigs going along with substantial economic ramifications ,183. SevSequencing has also opened up new horizons in prokaryotic ncRNA research. The development of metatranscriptomics has proven particularly useful, wherein the transcriptome of a complex microbiome is sequenced as a whole. This not only makes it possible to detect even lowly expressed genes, such as some ncRNAs but enables the investigation of non-culturable organisms using high-throughput sequencing ,185. TheAn important class of ncRNAs found in prokaryotes are small RNAs (sRNA) that range in length from 50–500 nt and regulate gene expression through RNA-RNA interactions . They weHalobacteria. Shotgun metagenomic sequencing was employed and resulted in the identification of over 1500 ncRNAs, 54% of which were of archaeal origin. Interestingly, significantly more intergenic sRNAs were found in archaea than in bacteria and the reverse was observed for antisense sRNAs [A recent study looked at the metatranscriptomic landscape of extremophiles inhabiting halite nodules  in the Atacama desert. Most of the extremophiles belonged to the domain of Archaea and specifically the class of se sRNAs .The rise of sequencing technologies has also enabled more specific and focused plant-based studies of ncRNAs. A particular focus has been laid on the role of ncRNAs in plant immunity, development and stress response  ,193. SeqGenerally, studies of plants used as crops have great economic significance and are therefore of particular value to society. Notably, crop species such as Chinese cabbage and rice have come under scrutiny recently. By employing NGS based approaches  the roles of ncRNAs in conferring resistance against infections ,199,200,Arabidopsis thaliana [A good example of a specialized sequencing approach being used in plant ncRNA research is the study by Ariel et al. where by employing CHiRp sequencing the function of lncRNA Auxin-Regulated Promoter Loop (APOLO) was studied in thaliana . The resthaliana .Further advancements in ncRNA research in plants have been made by the use of long-read sequencing technology. One example is the study of lncRNA expression in seed development of wheat and triticale (hybrid of wheat and rye) by nanopore sequencing. The study highlighted the utility of nanopore sequencing for genomes with high complexity and identified a large number of novel lncRNAs . In the Finally, considering the characteristics specific to plant lncRNAs ,205,206 Saccharomyces cerevisiae), one of the most studied fungi species, with many ncRNAs acting as important gene expression regulators [ncRNAs account for up to 25% of the genome of budding yeast  took advantage of readily available sequencing data. By combining datasets with data generated by themselves, the authors were able to identify more than 10,000 novel lncRNAs in A. albopictus including ncRNAs with potentially modulating effects on Dengue and Zika virus replication [Invertebrates comprise ~95% of animal species , and alm elegans . RNA seqfly wing . This ncfly wing . Similarlication . StudiesAndroctonus crassicauda [Another recent study investigated the poorly defined lncRNA landscape of scorpions, in particular, the venom glands of the species ssicauda . In the ssicauda . This stIn comparison to other taxa, the state of ncRNA research is more advanced in vertebrates. Mammalian organisms, in particular rodents and humans, have been studied extensively. But other vertebrates have traditionally not been the focus of vertebrate research. Examples include the study of the repertoire and function of circRNAs in the liver of the Whitespotted Bamboo shark , and theAmongst avians, chicken has been extensively researched. Studies utilizing sequencing technologies have focused on the roles ncRNAs play in embryogenesis , environStudies focused on livestock represent another example of understudied vertebrate research given its large economic impact. An example is the investigation of ncRNAs in cattle considering that cattle and buffalo meat and milk are the sources of 45% of global animal protein supply . ComputaBeyond tackling food supply issues for a growing world population, the generation of transgenic animals for production of biopharmaceuticals presents another incentive for increased research efforts in vertebrates. One of the main methods used to this end is somatic nuclear cell transfer (SCNT) , which sHomo sapiens. By analyzing DNA sequencing data from Homo sapiens, Neanderthals and chimpanzees, the authors found that chimpanzees had no lncRNAs amongst the genes that have an association with modern human personality and a much higher proportion of lncRNAs was detected in Homo sapiens compared to Neanderthals. In total, 94% of those genes found only in Homo sapiens were ncRNAs, most being lncRNAs [Interestingly, the increasingly broad application of sequencing to discover the ncRNA landscape of traditionally understudied species is not limited to species that are alive today. Ancient DNA sequencing offers insights into genomic landscapes of extinct life forms . A recen lncRNAs .Although most of the above described findings were based on short-read sequencing technology, the development of long-read sequencing has also left its mark on mammalian research. For instance by employing nanopore sequencing, human and mouse brain circRNAs were characterized, revealing widespread splicing events in circRNAs, highlighting the importance of developing sequencing technologies . It willThe rise of sequencing technologies has revolutionized the study of the transcriptome, including ncRNAs. Much has been understood about ncRNAs and recent years have seen a rapid increase of published ncRNA papers . Thanks The development of single-cell sequencing has allowed the study of ncRNAs at the single-cell level and with the more specialized and targeted sequencing methods it has become possible to study not only the landscape of ncRNAs but also their functionalities in the cell. The recently developed long-read sequencing technology demonstrated its potential for the field of ncRNA research, for example by elucidating circRNA exon composition . These tAlthough the field of ncRNA sequencing has come a long way since the discovery of the first ncRNAs in the 1950s and 1960s, exciting technological advances will continually advance our understanding of the complexity of the ncRNA transcriptome. For instance, the performance of nanopore sequencing technology has been steadily improving in the last years . The abiin vivo. Detection of ncRNAs still remains challenging. Reasons for this include the low expression levels of many ncRNAs [Nevertheless, challenges remain. With the discovery of novel ncRNA species, increased efforts will be needed to elucidate their functions y ncRNAs , unique y ncRNAs . Due to y ncRNAs , Thomas y ncRNAs  and Warry ncRNAs . Similary ncRNAs ,252. Anny ncRNAs . The laty ncRNAs ,255. Fur


Numerically small breeds have often been upgraded with mainstream breeds. This historic introgression predisposes the breeds for joint genomic evaluations with mainstream breeds. The linkage disequilibrium structure differs between breeds. The marker effects of a haplotype segment may, therefore, depend on the breed from which the haplotype segment originates. An appropriate method for genomic evaluation would account for this dependency. This study proposes a method for the computation of genomic breeding values for small admixed breeds that incorporate phenotypic and genomic information from large introgressed breeds by considering the breed origin of alleles (BOA) in the evaluation. The proposed BOA model classifies haplotype segments according to their origins and assumes different but correlated SNP effects for the different origins. The BOA model was compared in a simulation study to conventional within-breed genomic best linear unbiased prediction (GBLUP) and conventional multi-breed GBLUP models. The BOA model outperformed within-breed GBLUP as well as multi-breed GBLUP in most cases. The efficiency of breeding programs for local breeds is often compromised by the limited number of individuals and has resulted in a decreasing competitiveness with high yielding breeds, especially with the advent of genomic selection (GS). In GS, large reference populations are required to accurately predict breeding values of the individuals  and are Several studies were conducted using different approaches to enable GS in numerically small breeds using the reference population of a second breed (across-breed prediction) or extending the own reference population by adding the reference population of the second breed (multi-breed prediction) as reviewed by In many numerical small dairy cattle breeds sires from a large and high yielding breed were used in order to speed up genetic gain in the small breed. This resulted in some cases in a substantial amount of introgressed genes and in a mosaic-like haplotype pattern with a mix of native and introgressed haplotypes. For example, in the German Angler breed located in the northern part of Germany, admixture plays a substantial role in the population structure and the proportion of migrant alleles from other breeds is remarkable . A very This study proposes a method for the computation of genomic breeding values for small admixed breeds that incorporates phenotypic and genomic information from large introgressed breeds. The start and the end of the introgression events are considered to be in the past, which is applicable to many small local admixed breeds. A multi-breed BOA model is derived for multi-breed genomic selection that is suitable for application when the individuals have fragmented genomes. It classifies haplotype segments according to their origins and assumes different SNP effects for the different origins.For validation, it was compared with models that did not consider the breed-origin of QTL alleles. All models were applied to simulated datasets. In the simulation, the genotypes of the small admixed breed were derived from German Angler cattle, while the genotypes of the introgressed breed were derived from German Holstein cattle. Different scenarios were investigated in which the number of genotyped animals of the target breed, i.e., the numerically small Angler breed, varied, while the number of genotyped animals of the large introgressed breed, i.e., the German Holstein, remained constant.The data basis for the simulation study were 50k SNP-chip  genotypes of Angler (AN)  and HolsSeveral subsets of the total Angler data set were sampled to mimic different population sizes for the small breed. Subset simAN1 consisted of 750 individuals, simAN2 consisted of 1,500 individuals and simAN3 contained all 3,000 simulated Angler individuals. The different simAN populations are referred to as breed size scenarios. The sample sizes represent 12.5, 25 and 50% of the number of simHF individuals. In each of the subsets, all sires had the same number of offspring.From the 23,448 SNPs that segregated in both breeds, 1,000 SNPs were randomly selected as QTLs, while the remaining SNPs were used as markers for genomic prediction.The QTL effects for the simAN datasets and the simHF dataset were correlated. The additive effects Hence, the correlation of QTL effects between the two simulated breeds was 0.95. Dominance was not modelled. The additive effects were scaled to represent a trait with an additive variance of formulas . The pheThe true breeding values (TBV) were calculated as optiSel  was usedThe BOA model was compared with two conventional methods for the prediction of genomic breeding values, which are within-breed prediction with GBLUP for the simulated Angler cattle, and a multi-breed prediction with GBLUP.It is assumed that genotypes and phenotypes from several breeds or crosses are available, which includes the target breed. The number of SNP is denoted as andThe model equation for the phenotypic value of individual Alternative representations of the model and the mixed model equations are given in the The BOA model requires the breed origins  optiSel  was usedThe covariance matrix For within-breed genomic prediction we used the modelThe SNP markers for genomic prediction were chosen as follows. From the 22,448 SNPs that were not chosen as QTLs, all SNPs that segregated with a minor allele frequency (MAF) <0.03 within one of the simulated breeds and SNPs that did not segregate in both breeds were omitted. Across all replicates, on average 21,670 SNPs remained and were used for genomic prediction.The genomic predictions were done separately for each breed-size scenario and each replicate. The accuracies of prediction were assessed by a 5-fold cross validation. The individuals of the respective simAN dataset were assigned to five different classes such that individuals from different classes had no sires in common. Hence, each class included the offspring of 10 sires. In each cross-validation cycle, one class was used as the validation set, and the four remaining classes were used as the reference population.For multi-breed GBLUP and for the BOA model, the respective simAN reference set was joined with the simHF individuals. Consequently, the number of individuals from the simAN population in the reference population varied, while the number of simHF individuals was constant.An overview on the sample sizes is given in The marker effects were estimated with all three models for each cross-validation cycle.The genomic breeding values of the individuals in the validation set were computed for the BOA model asFor each method, the prediction accuracy was calculated as the correlation between the GEBVs and the TBVs of the validation individuals. The accuracies presented in the results are the averages, taken over all cross-validation cycles and replicates.The mean proportion of SNPs with Holstein origin across all replicates was 0.157 ± 0.007. It was shown in this study that a multi-breed genomic prediction with the proposed BOA model can increase the accuracies of the GEBVs for numerically small admixed populations over multi-breed and within-breed genomic predictions with GBLUP under certain conditions. The model considers the breed origin of each haplotype in individuals with admixed genomes consisting of native and introgressed haplotype segments. This makes it especially interesting for numerical small breeds with historic introgression from high yielding breeds, as it was observed e.g., in the German Angler or Vorderwald breed , 2015.The simulated QTL positions were the same in both breeds. Recent mutations that could have created new QTLs were ignored. The QTL effects of both genetic groups were assumed to be highly correlated with a correlation of 0.95. The QTL positions were chosen from the 50k chip, so the simulated QTLs are common variants whereas a large fraction of the additive variance is expected to come from rare QTL variants e.g., . This caThe LD structures in the simulation are expected to be similar to those investigated in real populations  because This paper focused on methods to improve GS in small admixed populations. It compared the prediction accuracies of various methods in a simulated population that had a similar LD structure as the target breeds. A detailed quantification of the impact of influencing factors  on the accuracies was beyond the scope of the study. The study explicitly focused on admixed populations where the events of introgression were in the past and breeding programs aim at reducing migrant contributions from other (high yielding) breeds or at least keep the amount of migrant contributions constant e.g., . TherefoIn this study, the multi-breed BOA model was compared with conventional multi-breed GBLUP and within-breed GBLUP. The multi-breed BOA approach led to an increase in the accuracy of the genomic breeding values when the number of genotyped AN individuals was small and medium, and showed similar results as the within-breed GBLUP method for the large reference sets. The difference between the prediction accuracies of the models, however, tend to decrease with an increasing number of genotyped AN individuals. The multi-breed prediction with GBLUP was not superior to within-breed prediction when the number of genotyped AN individuals was large. The reason is possibly that the multi-breed GBLUP model assumes a perfect correlation between the marker effects of both breeds. This assumption was certainly violated in the simulation. The BOA model, which accounts for the correlation between marker effects, could improve upon single-breed evaluations and outperformed multi-breed GBLUP in all cases.For the prediction of genomic breeding values of Angler in practice, to date, a joint reference population of several Scandinavian red dairy breeds  is used. To increase the accuracies of the GEBVs for Angler, about 170 genotyped and progeny-tested German Angler bulls have been included to this reference set as well (private communication In the past decades, the Angler breed has been upgraded with other breeds, such as Red Holstein and Holstein Friesian to increase its economic value. This has led to relatively high kinships between them . HoweverA multi-breed genomic prediction with the proposed BOA model increased the accuracies of the estimated genomic breeding values for numerically small admixed populations over multi-breed and within-breed genomic predictions with GBLUP. The BOA model assumes that the additive effect of an allele depends on the genetic group from which the respective haplotype segment originates. It is of special interest for multi-breed genomic predictions for numerically small breeds with past introgression from high yielding breeds.


Apis mellifera). Hygienic worker bees are able to detect and destroy diseased brood, thereby reducing the reproduction of economically important pathogens and parasites such as the Varroa mite (Varroa destructor). Video observation of this behaviour on infested combs has many advantages over other methods of measurement, but analysing the recorded material is extremely time-consuming. We approached this problem by combining automatic tracking of bees in the video recordings, extracting relevant features, and training a multi-layer discriminator on positive and negative examples of the behaviour of interest. Including expert knowledge into the design of the features lead to an efficient model for identifying the uninteresting parts of the video which can be safely skipped. This algorithm was then used to semiautomatically identify individual worker bees involved in the behaviour. Application of the machine-learning method allowed to save 70% of the time required for manual analysis, and substantially increased the number of cell openings correctly identified. It thereby turns video-observation of individual cell opening events into an economically competitive method for selecting potentially resistant bees. This method presents an example of how machine learning can be used to boost ethological research, and how it can generate new knowledge by explaining the learned decision rule in form of meaningful parameters.Machine-learning techniques are shifting the boundaries of feasibility in many fields of ethological research. Here, we describe an application of machine learning to the detection/measurement of hygienic behaviour, an important breeding trait in the honey bee ( Social insects are dominant species in many ecosystems. They are thought to represent more than 50% of global insect biomass, although they only account for approximately 2% of species diversity . The keyApis mellifera) workers against the parasitic mite, Varroa destructor. Pollination by honeybees is crucial to many ecosystems as well as the agricultural production of many crops .Conflict of interest statement. P.B., S.T. and A.R. are co-founders of the start-up company Adaptiv Lernende Maschinen GmbH and participated in the present work within the framework of a commercial contract that enabled them to found this company, which exploits the software developed and the know-how gained during the project described here. The other authors declare no conflict of interest.bpac005_Supplementary_DataClick here for additional data file.