The virulent and unpredictable nature of COVID-19 combined with a change in reimbursement mechanisms both forced and enabled the rapid adoption of telemedicine around the world. Thus, it is important to now assess the effects of this rapid adoption and to determine whether the barriers to such adoption are the same today as they were under prepandemic conditions.The objective of this systematic literature review was to examine the research literature published during the COVID-19 pandemic to identify facilitators, barriers, and associated medical outcomes as a result of adopting telemedicine, and to determine if changes have occurred in the industry during this time.The systematic review was performed in accordance with the Kruse protocol and the results are reported in accordance with the PRISMA  guidelines. We analyzed 46 research articles from five continents published during the first year of the COVID-19 pandemic that were retrieved from searches in four research databases: PubMed (MEDLINE), CINAHL, Science Direct, and Web of Science.Reviewers identified 25 facilitator themes and observations, 12 barrier themes and observations, and 14 results (compared to a control group) themes and observations. Overall, 22% of the articles analyzed reported strong satisfaction or satisfaction (zero reported a decline in satisfaction), 27% reported an improvement in administrative or efficiency results (as compared with a control group), 14% reported no statistically significant difference from the control group, and 40% and 10% reported an improvement or no statistically significant difference in medical outcomes using the telemedicine modality over the control group, respectively.The pandemic encouraged rapid adoption of telemedicine, which also encouraged practices to adopt the modality regardless of the challenges identified in previous research. Several barriers remain for health policymakers to address; however, health care administrators can feel confident in the modality as the evidence largely shows that it is safe, effective, and widely accepted. The virulent nature of COVID-19 forced social distancing and a decrease of in-person visits to clinics around the world. Telemedicine presented health care providers with solutions that enabled a social-distancing window into the clinical environment and a continuation of the doctor-patient relationship.Telemedicine is defined by the World Health Organization as healing from a distance through information communications technologies by all health care professionals for the “exchange of valid information for diagnosis, treatment and prevention of disease and injuries, research and evaluation” . TelemedA large number of articles were published in the first 12 months of the pandemic (February 2020 to February 2021) on the rapid implementation efforts of telemedicine to enable clinics and hospitals to continue to see patients and care for their needs ,10. HoweAlthough analyses have been published that highlight the advantages to the adoption of telemedicine, with an 8% adoption rate in the United States, the conclusions of these previous studies may not be as robust as possible. The circumstances presented by the pandemic have encouraged wider adoption of this modality of care. Therefore, with proper systematic review techniques, reviewer observations this far into the pandemic will undoubtedly be more robust and widely applicable to medicine.The purpose of this systematic review was to evaluate the facilitators and barriers to the adoption of telemedicine worldwide, including an analysis of health outcomes and patient satisfaction. A brief comparison of the results of this review with those of reviews performed prior to COVID-19 was further performed to identify changes in these factors in light of the pandemic. The Kruse protocol for writing a systematic review was followed, and the findings are reported in accordance with the PRISMA  guidelines ,16. ThisThe search parameters were established to find articles published in 2020 and 2021 concerning telemedicine in all aspects of care and for all ages of patients, published in peer-reviewed journals, using any method of study . Other systematic reviews were excluded because we wanted to compare our results to these previous reviews without confounding the findings. The Johns Hopkins Nursing Evidence-Based Practice Rating Scale (JHNEBP) was used to assess the quality of all articles analyzed . Any stuFour research databases were searched: PubMed (MEDLINE), CINAHL (excluding MEDLINE), Web of Science, and Science Direct. We also performed a journal-specific search of the Journal of Medical Internet Research.Google Scholar was used to determine the general trends of publication on this topic previously and to collect key terms from published articles. These key terms were entered into the US Library of Medicine’s Medical Subject Headings (MeSH) to create an exhaustive search string using Boolean terms. The actual search string used was:  AND (COVID-19 OR coronavirus). The same search string was used in all databases. Similar filters were used in each database .Once the search string was entered into each database, we filtered the results and screened abstracts for applicability. Although filters for the four research databases differ, we generally filtered for the date range (2020-2021), scholarly journals (no theses or opinions), and “full text” to ensure that we would have access to the entire article. Articles were rejected for a variety of reasons: protocol ; opinion (no data); reviews; did not use telemedicine; or did not contribute to our objective statement of identifying facilitators, barriers, or effects on patient satisfaction. The κ statistic was calculated to identify the level of agreement between reviewers .An Excel spreadsheet was used as a data-extraction tool to collect data for reporting and analysis. This spreadsheet was standardized according to the Kruse protocol . We heldIn accordance with the Kruse protocol, PRISMA standard, and JHNEBP, the following fields were collected: database source; date of publication; journal; authors; study title; PICOS ; sample size; bias within study; effect size; country of origin; statistics used; quality metrics from the JHNEBP scale; and reviewer observations as they relate specifically to the objective statement in areas of patient satisfaction, and facilitators and barriers to adoption ,17,20. AThe JHNEBP rating scale was used for assessment of bias within and across studies. Observations of bias and methodological weaknesses were noted . The JHNBecause we included mixed methods and qualitative studies, we were unable to standardize summary measures as would be performed in a meta-analysis.We performed a narrative, or thematic, analysis of the observations to convert them into themes (common threads between articles) . We calcThe database search and study selection process are illustrated in Reviewers collected study characteristics identified by the PRISMA standard such as PICOS see . Of the Many studies used geographically localized samples, which may limit the external validity of the results. Some studies focused only on one gender or race, speaking to the convenience sample or volunteer-basis of their design. Asking for volunteers in a technology-oriented experiment invites bias because the self-selection allows for those who are already technology-oriented or comfortable with technology to participate. This group as the intervention can skew the results because those already comfortable with technology will not experience the frustration experienced by those who are not comfortable with technology. This selection bias also limits the external validity of the results. A comprehensive list of bias, country of origin, sample size, strength, and quality of evidence identified for each study can be found in During the analysis phase of the systematic review process, the reviewers recorded observations to identify instances of patient satisfaction, as well as both facilitators and barriers to the adoption of telemedicine. A thematic analysis was then performed to make sense of the observations . MultiplPatient satisfaction was reported as “strong satisfaction” or “satisfaction” in 9 (20%) and 1 (2%) of the 46 studies, respectively, and 36 studies did not report any measure of patient satisfaction. No studies reported a decline in patient satisfaction as a result of using telemedicine as the intervention.Twenty-five facilitator themes and seven individual observations were identified in the literature by the two reviewers. Only two studies did not identify facilitators. Facilitator themes are listed in The most commonly identified themes were increased self-management, acceptance of the technology from the pandemic, adherence to treatment protocols, access, and social support. For the 46 articles, these themes represent 38% of all 132 occurrences. Other themes included convenience of telemedicine and perceived ease of use, decreased cost, opportunity for education, technical literacy, availability of technology, an increase in patient-provider communication, faster initiation of treatment, increased connectedness, perceived usefulness, and past experience with technology. Health literacy and improved health behaviors were identified less frequently, and increased office efficiencies, medical concerns adequately addressed, enabled social interaction, increased quality of life, improved standard of care, increased flexibility, and increased weight loss were the least frequent themes identified. The following seven individual observations accounted for 5% of the total observations: decreased anxiety, increased technical literacy, televideo enabled reading of body language, fewer miles driven to appointment, long-term use may not be required to develop good habits, decreased emergency room visits, and no significant difference in cost of care.Twelve themes and five individual observations were identified as barriers from the literature by the reviewers; 11 studies did not identify barriers (11%). The most commonly listed barriers were technical literacy, technology needs further development, availability of technology, and patient preference, accounting for 55% of the total 86 occurrences. Cost, connectivity, and confidentiality/security were also identified, as well as health literacy, limits of reimbursement for telemedicine, and lack of personal desire to get better with less frequent occurrences (2 each). The remaining five observations made up a total of 6% of the total occurrences: decrease in quality of life after intervention, discomfort for wearing monitors, workflow issues for providers, lack of data infrastructure, and interoperability.Eighteen of the 46 studies (39%) were performed in North America, 11 (24%) were performed in Europe, 7 (15%) were performed in Asia, 5 (11%) were performed in Australia, 3 (7%) were performed in South America, and 2 (4%) were performed in multiple countries and continents.Eighteen of the studies demonstrated either a clinical or administrative improvement compared to the control group, whereas nine reported no statistically significant results from the control group. Both of these themes demonstrate the efficacy of the telemedicine modality. The remainder of the list in Twelve studies reported 12 statistically significant improvements in clinical outcomes and three reported no statistically significant difference between modalities of care. Both of these themes demonstrated the efficacy of the telemedicine modality. The most commonly observed theme for medical outcomes was decreased body weight, followed by decreased fat mass, improved self-management, increase in controlled asthma, and increased quality of life. The following individual observations contributed to 30% of the total observations: reduction in blood pressure, reduction in mean tacrolimus trough coefficient of variation, improved annual rate of estimated glomerular filtration rate (eGFR) decline, decrease in medication use, decrease incidence of heart failure, decreased mortality, improved mental health inventory, decreased intracranial hemorrhage, and telemedicine identified other areas for intervention.Interventions of mHealth resulted in seven occurrences of a result  and six occurrences of an improvement in at least one clinical outcome. The interventions with telephone or televideo resulted in four instances of improved patient satisfaction and a decrease in eGFR and weight loss. The interventions of eHealth resulted in very few instances of either clinical or administrative outcomes other than improved compliance and health behaviors.Telemedicine is examined in countries worldwide, and it is clear that the COVID-19 pandemic caused a rapid adoption of this modality of medicine to ensure the viability of practices. A key issue for discussion is the differences in findings between this systematic review and another recent similar review . This syThis systematic review exercised a set Boolean search string in four common research databases to analyze 46 articles originating from five continents for themes of facilitators, barriers, and medical outcomes. Nearly 50% of the articles demonstrated the strongest evidence and nearly 60% demonstrated the highest quality of evidence. Various forms of telemedicine were examined: eHealth, mHealth, audio only, telemonitoring, telecoaching, telerehab, robotics or artificial intelligence, and televideo. Twenty-five facilitator themes and individual observations, 12 results themes and observations, and 7 medical outcome themes and observations were recorded and analyzed. Forty-one percent of barrier themes recorded either an improvement or no statistically significant improvement in results compared to the control group. Forty percent of the observations recorded an improvement in at least one medical outcome.Health care administrators can focus on the findings demonstrating that implementation of telemedicine will increase self-management ,49,57,58Health policymakers should focus on several barriers to increase the adoption of telemedicine. Because technical literacy, availability of technology, and connectivity are listed as the most often cited barriers, public programs should be offered to assist those with these difficulties. Technical literacy is often associated along age or socioeconomic lines, and researchers acknowledge the dearth of research in the area of how to overcome this obstacle . HoweverA key similarity between the 2020 systematic review  and thisThis systematic review selected 46 articles for analysis from four commonly available research databases. A larger group for analysis could have yielded richer results. This review also only utilized two researchers to analyze the data; additional researchers could have identified additional themes. Selection bias was controlled through independent analysis of all articles by both reviewers followed by consensus meetings. Publication bias is the largest limitation because we were unable to query and analyze unpublished articles.The COVID-19 pandemic caused huge problems to deliver medicine traditionally. However, these problems created an environment that limited face-to-face medical encounters and fostered legislation to reimburse the telemedicine modality for broad and rapid adoption of telemedicine to expand the access of care beyond the physical walls of the clinic. Physicians should feel confident that the telemedicine modality will be reimbursed and will have very little effect on patient satisfaction. Health care administrators who have not already adopted telemedicine should feel confident in the technology; however, they should ensure that sufficient confidentiality and security measures are in place. Policymakers should enact legislation to remove or mitigate barriers such as availability of technology, technical literacy, and connectivity, as these are commonly referred to in the literature.


Texture analysis (TA) is a newly arisen field that can detect the invisible MRI signal changes among image pixels. Myocardial infarction (MI) is cardiomyocyte necrosis caused by myocardial ischemia and hypoxia, becoming the primary cause of death and disability worldwide. In recent years, various TA studies have been performed in patients with MI and show a good clinical application prospect. This review briefly presents the main pathogenesis and pathophysiology of MI, introduces the overview and workflow of TA, and summarizes multiple magnetic resonance TA (MRTA) clinical applications in MI. We also discuss the facing challenges currently for clinical utilization and propose the prospect. Myocardial infarction (MI) is myocardial necrosis caused by ischemia and hypoxia of cardiomyocytes, an imbalance between oxygen offering and myocardial requirement. It belongs to a part of the clinical manifestation of the acute coronary syndrome (ACS) . MI is aThe European Society of Cardiology (ESC) has conducted the fourth universal definition of MI from cardiac troponin values (cTn) and clinical myocardial ischemia evidence that is ranging from symptoms of myocardial ischemia, ECG abnormalities, and new imaging evidence . HoweverAs the imaging techniques evolve, it plays a more and more important role in MI. Cardiac magnetic resonance (CMR) has advantages, such as non-Radiative, multiparameters and sequences, multiplanar reconstruction capabilities, and high tissue resolution, which composes the “gold standard” tool for evaluating the cardiac structure and function non-invasively and becomes the best available imaging technique for detection of MI , 8. MeanThis article concentrates on the role of MRTA in MI. In this study, we review the major pathogenesis and pathophysiology of MI, the basic concepts and types of MRTA, the clinical applications of MRTA in MI, current challenges, and potential prospects.Myocardial infarction is most commonly owing to coronary thrombosis from the rupture of an atherosclerotic plaque . When thIn MI, once the onset of myocardial ischemia, there is a subsequent decrease in myocardial perfusion, bringing about the reduction of tissue oxygenation, which transforms the hypoxia cardiomyocytes from aerobic to anaerobic metabolism, bringing about edema of cardiomyocyte and ultimately tissue necrosis .The myocardial necrosis process resembles a “wavefront” phenomenon that is ranging from the endocardium to the epicardium  Figure . Within Texture analysis (TA) is a part of radiomics. Through image post-Processing technology, the distribution and relationship of pixel or voxel gray are analyzed to extract many quantitative texture features that are not visible by the naked eyes in medical images . The imaWith high gray and spatial resolution, MRI images possess extensive and similar image information. Therefore, the statistical method becomes the most commonly used method in MRTA. For statistical-based TA, various properties control the distribution and relations of gray-scale values in images to represent texture . First-oStructural-based TA catches intensity changes between the central and adjacent voxels. Local binary patterns (LBP) are a non-Parametric algorithm and also the most frequently used method that depicts the local features of gray-scale connection between image pixels and neighboring pixels .Model-based TA explicates texture in an image with sophisticated mathematical models, for example, stochastic or fractal models. The model parameters are estimated and applied to image analysis. Due to a lack of direction selectivity, this method is unsuited for depicting local image structures .Transform-based TA enables the spatial information of images to convert into spatial frequencies. It contains Gabor, Fourier, and Wavelet transform. As the most widespread method, Wavelet transform can analyze the frequency content of images in different spatial frequency resolutions .Artificial intelligence (AI) is rapidly gaining importance in the medical imaging field and is likely to gradually turn into clinical practice in the next few years , 27. AccMagnetic resonance texture analysis clinical applications in MI emerge as a promising research domain, and many findings have presented encouraging results. The series of studies can be divided into three broad categories: establishing the diagnosis, determining the age of infarct, stratifying risk .Despite the relative importance of various MRI technologies vary, MRI plays a significant role in establishing a diagnosis for both AMI and CMI . The rapApart from being a useful diagnostic tool, MRI can also be applied for differentiation between acute and chronic infarction, which is especially helpful when the patient has multi-infarct in different vascular areas or when an infarct occurs with no clinical symptoms. Distinguishing AMI from CMI has vital clinical significance for treatment and follow-up, especially in patients with pre-Existing CMI, and the probability of locating acute lesions by ECG or coronary angiography is limited. The management of two types of infarcts differs. It is crucial to determine the infarct age, especially when both infarction entities coexist, complicating that will complicate the therapeutic plan and follow-up after treatment. Several imaging features involving the identification of AMI and CMI in previous studies, such as wall thickening and thinning , microvaCompared with those current technologies that rely on image visual evaluation, the quantitative character of TA is a unique advantage. Edema of AMI and fibrosis of CMI hold the most essential characteristics of cardiac pathological changes correspondingly and affect the internal structure of tissues, which indicates there may be some inherent texture discrepancy in the images of tissues influenced by AMI and CMI. Larroza et al.  performeIn recent years, MRTA plays a new role in identifying multiple prognostic indicators that guide risk stratification and prognosis prediction in patients with MI. The applications mainly involve five sub-aspects: (1) differentiation of reversible from irreversible myocardial injury; (2) evaluation of transmurality; (3) detection of MVO; (4) assessment of scar size combined with segmentation; and (5) identification of scar heterogeneity.Differentiating reversible from irreversible myocardial damage is valuable for prognosis prediction. The salvaged myocardium is an independent predictor of prognosis and is associated with mortality, representing the risk area but can be rescued or rescued through revascularization . The myoOne promising result about the differentiation of reversible from irreversible myocardial injury based on MRTA was reported by Bing-Hua Chen et al. . They exThe transmurality of infarction possesses an independent prognostic value in measuring the recovery of contractile function after treatment, and a greater transmural extent is related to poorer recovery , 51.Magnetic resonance texture analysis shows several promising results in the evaluation of transmurality. Ma et al.  combinedMicrovascular obstruction appears in severe microcirculation damage caused by myocyte death, overflow of intracellular substances, severe stasis, and occlusion of end arteries and capillaries . MoreoveMa et al.  applied The extent of infarct can predict left ventricular adverse remodeling (LVAR) and correlates negatively with prognosis –55. BesiMagnetic resonance texture analysis-combined segmentation can better provide the signal intensity features of the scar, then to help better segment the scar and assess scar size. Kotu et al.  segmenteAfter MI, the necrotic tissue is gradually replaced by granulation and fibrous tissue and finally, develops into scar tissue. The myocardium presents heterogeneous nature owing to scarring, and the scar tissue is complex on a histopathological level. In addition, numerous studies have shown that the degree of scar heterogeneity correlates directly with the risk of arrhythmia events, thereby, better guide the implantation of an implantable cardioverter defibrillator (ICD) , 65. In The scar can be described as two areas: (1) the core area, which consists of fibrous tissue and the myocardial fibers which are in a state of complete death, does not respond to the electrical signals transmitted by the myocardium to tell the heart to contract; (2) the border area, also known as peri-infarct area or gray zone area, contains heterogeneous tissue composed of necrotic tissue mix with bundles of viable cardiomyocytes; the electrical signals in these areas will be disturbed, which may lead to reentry and sometimes arrhythmias , 39, 57.Some studies reveal that MRTA helps to identify arrhythmias events for patients with previous MI. Engan et al.  performeDespite these superiorities, extensive clinical application of MRTA in MI remains challenging. First, most of the studies are small sample pilot exploration in a single center, and almost all of them are retrospective studies. The conclusions obtained lack extensive verification support, and the clinical evidence is not sufficient. Therefore, prospective, multicenter, and large sample studies are needed. Second, at present, medical imaging equipment lacks the same image acquisition and imaging algorithm standard, and different acquisition times of the same MR machine and acquisition of different MR machines can affect the stability of radiomics features, and the reproducibility is low. Consequently, strict protocols and standardized methodologies should be followed to maximize the validity of future research. Third, there are many feature parameters in TA, and the prediction accuracy is affected by feature parameters, feature selection methods, and classifiers. In addition, MRTA software is manifold, and there is no evidence to show which software is superior at present. However, 3D TA includes more spatial information and is superior to 2D TA but applies less due to increased complexity. Thus, more accurate and widely applicable feature selection and pattern recognition methods will become the development direction of MRTA.In the era of big data, TA has become a research hotspot in the field of precision medicine and AI . CompareAlthough multiple MRTA clinical applications in MI have shown encouraging results, some aspects are not or rarely referred to and may become future developments.In traditional imaging, the following factors are also related to risk stratification and prognosis in patients with MI that may be helpful for MRTA clinical application, for instance, (1) hemorrhage in the core of infarct has been demonstrated as an adverse prognostic indicator that is relevant to LVAR, large infarct size, and increased LV end-systolic volume ; (2) iscIn addition to the above future developments that MRTA has potential in risk stratification and prognosis prediction in MI, the combined application with MRTA also has a certain prospect. First, except for the cine, mapping, and LGE, applying MRTA to other CMR quantitative techniques, such as strain, diffusion tensor imaging (DTI), perfusion-weighted imaging (PWI), and intravoxel incoherent motion (IVIM), can enrich the clinical research methods of MI. Second, MRTA combined with other imaging techniques, such as ECG, echocardiography, and myocardial radionuclide tomographic imaging may improve early MI diagnosis and prognosis. Third, cTn is the most diagnostic biochemical marker for MI, the combination of MRTA and cTn will increase the understanding of the connection between texture change and progression of MI. Fourth, the combination of radiomics characteristics and genomic data is described as radiogenomics . Gene reAt present, the inferential diagnosis of MI is based on the combination of biochemical markers, ischemic symptoms, or ECG changes. We are entering an era of combined imaging and clinical assessment in disease detection and diagnosis. MRTA can detect the invisible signal changes, thus, provides an additional non-Invasive method to establish the diagnosis, determine the age of infarct, and stratify risk and predict prognosis in MI. Although its current application for MI imaging faces some challenges, MRTA shows good clinical application prospects in MI.FP conducted the reference analysis and wrote the manuscript. LGG and HZ contributed to the topic conception, manuscript revision, and decision to submit for publication and are the co-corresponding authors. The remaining authors contributed to the reference collection and helped in the revision of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (Grand No. 81860316).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As the largest rotator cuff muscle, the subscapularis plays a major role in stabilizing the glenohumeral joint, in conjunction with surrounding rotator cuff structures. Injury to the subscapularis tendon can be isolated, but more commonly is seen in conjunction with supraspinatus tendon pathology. Injury can be associated with biceps pulley instability, superior labral anterior-posterior (SLAP) tears, humeral head subluxation, and anterosuperior and coracoid impingements. The involvement of the rotator interval can lead to what is called “the hidden lesion,” due to its difficulty to diagnose during arthroscopy. Understanding the anatomical relations of the subscapularis tendon with the rest of the rotator cuff and rotator interval, as well as common patterns of injury that involve the subscapularis tendon, can aid in proper diagnosis of these injuries leading to prompt surgical repair. This review describes the anatomy of the subscapularis muscle and tendon, and the magnetic resonance imaging (MRI) patterns of subscapularis tendon injury. The subscapularis muscle is the largest muscle of the rotator cuff. The rotator cuff, consisting of the supraspinatus, infraspinatus, teres minor, and subscapularis muscles, works in concert to facilitate movement and provide stability of the glenohumeral joint . It is tIn this review, we will discuss subscapularis muscle and tendon anatomy, the magnetic resonance imaging (MRI) patterns of subscapularis tendon injury, and associated pathology, and argue that proper diagnosis may influence prompt surgical repair.The subscapularis muscle , trianguThe muscle itself can be divided into two segments, the upper two-thirds and lower one-third, respectively. The muscle provides both passive stabilization and dynamic stabilization of the glenohumeral joint, buttressing the joint from a posteriorly directed force. The upper segment of the muscle has been shown to be more involved with internal rotation, while the lower segment can help in external rotation while the arm is abducted –9. TheseThe superior aspect of the subscapularis tendon provides the inferior border of the rotator interval, a triangular area in the anterosuperior glenohumeral joint capsule that is reinforced by the superior glenohumeral ligament (SGHL) and the coracohumeral ligament (CHL). The anterior aspect of the supraspinatus tendon provides the superior border of the rotator interval, while the base of the coracoid process provides the lateral border –14. The Rotator cuff tears are relatively common, usually occurring in patients over the age of 40, and are often degenerative in nature . Tears oAs described above, patients may present with anterior instability and dislocation; however, many patients do not present with any clinical symptoms at all , 7, 9. BEven upon arthroscopy, subscapularis tendon tears can be difficult to diagnose, especially if they are partial-thickness tears or demonstrate no retraction. Certain arthroscopies have difficulty visualizing the subscapularis tendon attachment at the lesser tuberosity. While the subscapularis tendon is difficult to access and visualize upon arthroscopy, recent attention and improved arthroscopic techniques have increased the incidence of detection of such lesions –23.No concrete MRI grading scheme has been developed for rotator cuff tears. Different classification systems have been suggested for MRI and arthroscopy. Lafosse suggested a grading scheme using arthroscopy, with five different grades based on the extent of the tear . PfirrmaAs the subscapularis plays an important role in the glenohumeral and bicipital stability, failure to address and repair a torn subscapularis tendon can lead to persistent pain. Mall et al. found that concomitant repair of the subscapularis tendon alongside the supraspinatus tendon provided more pain relief than repairing the supraspinatus tendon alone . Other sFailure to repair a concomitant subscapularis tendon tear has been found to compromise the normal biomechanical function of the tendon as described above and potential compromise of an associated posterosuperior rotator cuff tear repair , 26. ChrHowever, small tears are easily missed; Adams et al. described a sensitivity of 36% and specificity of 100% for diagnosing subscapularis tendon tears on MRI . AssociaAdams et al. provided a systemic approach to diagnose a subscapularis tear on MRI, including axial cuts to evaluate subscapularis tears, axial cuts to evaluate subluxation of the biceps tendon, sagittal cuts to evaluate subscapularis atrophy, and sagittal images to evaluate subscapularis tears at the tendinous insertion onto the lesser tuberosity . AccuratMRI and MR arthrography (MRA) of the shoulder joint usually include three planes including the axial, oblique coronal, and oblique sagittal planes. The subscapularis is best evaluated on axial and oblique sagittal planes . Axial iThe pattern of subscapularis tendon tears has been described by Pfirrman et al. to include three types: isolated, involving the supraspinatus tendon, or involving the rotator interval .Isolated tears of the subscapularis muscle have been described in the setting of anterior dislocation of the humeral head and recurrent anterior instability . The mosOsseous changes, however, can unveil a subscapularis injury; lesser tuberosity cysts have been shown to be specific , 3 for tMore commonly, subscapularis tendon tears are associated with supraspinatus tendon tears , at the Injury to the rotator interval, of which the subscapularis tendon forms the inferior border, can produce what has been described as “hidden lesions” , due to The difficulty in diagnosis of rotator interval lesions lies in the variability of rotator interval appearance on MRI . FailureSubscapularis tendon tears can also lead to biceps tendon instability and subsequent subluxation Figures , and 9. MRI-proven abnormalities of the superior aspect of the subscapularis tendon have shown a high sensitivity, specificity, positive predictive value, and negative predictive value with surgically proven biceps pulley lesion, even if the lesions of the biceps pulley were not evident by imaging . In factSubscapularis tendon tears can also lead to anterosuperior impingement, a painful process from impingement of the deep tendinous surface of the subscapularis and the biceps pulley along the anterosuperior glenoid rim. This is elicited by horizontal adduction and internal rotation of the arm , 32. TheOn the other hand, subcoracoid impingement occurs when a part of the rotator cuff finds itself between the coracoid process and the head of the humerus, with a significant decrease in the average coracohumeral distance and the distance between the lesser tuberosity of the humerus and the coracoid process , 38 Fig. A normaSuperior labral anterior-posterior (SLAP) tears may be seen in conjunction with subscapularis and proximal biceps tendon injuries Figures , and 9. As stated above, anterior dislocation may be associated with subscapularis tendon tears, and such injuries coincide with humeral avulsion of the glenohumeral ligament (HAGL), specifically the inferior glenohumeral ligament . Other fSubscapularis tendon and neighboring anatomy pathology can be difficult to diagnose on physical examination and imaging. Failure to address tears to the subscapularis tendon can lead to delayed surgical repair, ongoing pain, and limited function and stability of the glenohumeral joint. Attention to the subscapularis tendon is rising, with a greater understanding aiding a systematic approach for better diagnosis. Increased perception for the subscapularis tendon, the biceps pulley, and rotator interval not only helps patients improve their quality of life but bring this pathology out of hiding.


Hence, biofilm-mediated chronic infections have created a tremendous burden upon healthcare systems worldwide. The development of biofilms upon the surface of medical implants has resulted in the failure of various implant-based surgeries and therapies. Although different conventional chemical and physical agents are used as antimicrobials, they fail to kill the sessile forms of bacterial pathogens due to the resistance exerted by the exopolysaccharide (EPS) matrices of the biofilm. One of the major techniques used in addressing such a problem is to directly check the biofilm formation by the use of novel antibiofilm materials, local drug delivery, and device-associated surface modifications, but the success of these techniques is still limited. The immense expansion in the field of nanoscience and nanotechnology has resulted in the development of novel nanomaterials as biocidal agents that can be either easily integrated within biomaterials to prevent the colonization of microbial cells or directly approach the pathogen overcoming the biofilm matrix. The antibiofilm efficacies of these nanomaterials are accomplished by the generation of oxidative stresses and through alterations of the genetic expressions. Microorganism-assisted synthesis of nanomaterials paved the path to success in such therapeutic approaches and is found to be more acceptable for its “greener” approach. Metallic nanoparticles functionalized with microbial enzymes, silver–platinum nanohybrids (AgPtNHs), bacterial nanowires, superparamagnetic iron oxide (Fe Global mortality and morbidity is maximally associated with infectious diseases and is one of the profound causes for the development of antibiotic resistance. It has been observed that after 1980, pharmaceutical companies stopped manufacturing novel antibiotics due to the lack of returns with respect to the investment, high cost associated with the development of drugs, and prolonged time requirement and for the rapid development of resistances . The devThe field of nanotechnology involves scientific and engineering technologies that aim to synthesize various materials of nano-dimensions that have wide applications in the fields of bioprospecting, pharmaceuticals, human activities, and biomedical applications. The development of nanomaterials is a new and promising strategy for acting as therapeutic agents against various types of biofilm-associated pathogenic infections that are associated with implants and medical devices . VariousAlthough a number of reports are available on the antibiofilm activities of nanomaterials, like carbon nanotubes , oxygen-Nanomaterials, with dimensions lower than 100 nm, have attracted the interest of scientists due to their quantum size effect with the variation of electronic properties. Nanomaterials can have one, two, or three dimensions in the nanoscale, as exemplified by nanotubes, nanorods, nanoflowers, nanowires, nanofibers, fullerenes, dendrimers, and quantum dots . The micFor the last few decades, bacterial cells have been used for the purpose of synthesizing various types of inorganic nanomaterials that include gold, silver, selenium, and silver NPs possessing diverse useful properties . It is tBacillus licheniformis, which was found to be effective in the management of biofilms. The synthesis of AuNPs occurs via the ligands that are produced by the microbial species to prevent the formation of complexes (Rhodococcus sp. were used for the development of monodispersed AuNPs (Deinococcus radiodurans was able to synthesize AuNPs in the presence of high radiation that resulted in the change of Au (III) to Au (I) and finally to Au (0) comprising various types of capping groups which help in stabilizing the AuNPs , which comprises exopolysaccharides, proteins, lipids, nucleic acid, and various other types of biomolecules . EPS helNow, most biofilm eradication approaches involve the development of antibiofilm agents, aimed at preventing the early stages of biofilm construction or acting as biofilm dispersal agents, intended to cause disruption of the mature biofilm. They may follow any of the potential ways like checking of quorum sensing, destruction of eDNA, and affecting swarming and twitching  or can dNanomaterials formed of microbiogenic nanoparticles like nanosilver or nanogold particles efficiently block the active sites and thus hinder the mechanism of quorum sensing . They caThe interaction between NPs and biofilm can be accomplished through the allocation of nanoparticles of the nanomaterials at the vicinity of the biofilm matrix, followed by their attachment. NPs, due to electrostatic interaction, can now bring about mechanical damage to the bacterial cell or can develop oxidative stress followed by the production of reactive oxygen species (ROS) and, as a result of metallic cation release, can interrupt the normal structure and functions of proteins .Alteration in the signaling cascade involves the prevention of the production of EPS that provides an alternate mechanism for preventing the adhesion of bacteria, thus hindering the formation of biofilms. Thus, it forms a very important target for the next-generation therapeutics . In receExtracellular DNA (eDNA) forms an important structural component in stabilizing the biofilm architecture and develops resistance to drugs. It has been observed that substances like amphiphilic cargo and enzymes bring about the cleavage of eDNA, thereby bringing about effective degradation of biofilm . eDNA isThe extracellular matrix provides strength to the indwelling sessile microbial species within the biofilm by virtue of various biomacromolecules known as EPSs, thereby contributing toward shortened antimicrobial susceptibility. So far, EPS targeting for biofilm control has remained underexploited due to lower penetration capabilities of various antibiofilm agents such as antibiotics, biofilm degrading enzymes, and bioactive compounds. Nanoparticles (NPs) have emerged either as EPS matrix disruptors or as carriers of EPS matrix disruptors, and several approaches have recently been proposed . NPs havEPSs  or bacterial cells are, in general, negatively charged, hence providing an efficient way of interaction with positively charged NPs . It has P. aeruginosa and Candida spp. The Stenotrophomonas maltophilia [Sm-SeNPs(-)] and Bacillus mycoides [Bm-SeNPs(+)] had stronger antimicrobial effects than synthetic selenium nanoparticles (Ch-SeNPs) (Desertifilum sp. (D-SNPs) were able to inhibit biofilms of MRSA, resulting in imbalance in CAT, GSH, GPx, and ATPase levels and subsequently forming apoptotic bodies and causing cell wall damage in addition to denaturation of MRSA cellular proteins and genotoxicity (In a study conducted by h-SeNPs) . Thus, btoxicity .P. aeruginosa were found to increase the velocity of wound healing  within the biofilm matrix has been reported to be responsible for the formation of a biomolecular corona-like coating on the surface of NPs due to the phenomenon of adsorption on the NP surface . The phy healing , whereasB.cereus . NanomatB.cereus .The biofilm matrix, also sometimes known as “the dark matter,” is a complex material which creates a barrier shielding the indwelling cells from antimicrobial therapy, immune responses, and environmental challenges and hence prevents eradication strategies. Due to the outstanding challenges presented by the biofilm matrix, a multidisciplinary approach is needed to tackle this problem. Nanotechnology is a plausible solution for antimicrobial and delivery system methodologies for enhanced penetration and targeted delivery of antimicrobials within the biofilm matrix. EPS-targeting strategies involve matrix disruption and enhancing the susceptibility of the biofilm toward antimicrobial therapy.in vivo, and release of NPs into the environment still remain to be answered. Future research work should highlight the complete biofilm eradication by focusing on both the EPS matrix and the microbial cells, minimizing toxicity and resistance development while enhancing the therapeutic effect with the help of nanostructures formed from microbial sources.One of the ways for the synthesis of biogenic NPs involves microbial cells as a reducing, stabilizing, and capping agent in an eco-friendly, sustainable, nontoxic, and inexpensive way. Many researchers have studied the role of bacteria (both Gram-positive and Gram-negative), fungi, or algae in the production of NPs. These methods have resulted in the replacement of various toxic physicochemical methods. However, a few of the questions such as alterations in EPS composition during different environmental/growth conditions, non-commercialization of NP-based antibiofilm technologies, ultimate fate of antibiofilm NPs


This study presents the novelty artificial intelligence in geospatial analysis for flood vulnerability assessment in Dire Dawa, Ethiopia. Flood-causing factors such as rainfall, slope, LULC, elevation NDVI, TWI, SAVI, K-factor, R-factor, river distance, geomorphology, road distance, SPI, and population density were used to train the ANN model. The weights were generated in the ANN model and prioritized. Initial values were randomly assigned to the NN and trained with the feedforward processes. Ground-truthing points collected from the historical flood events of 2006 were used as targeting data during the training. A rough flood hazard map generated in feedforward was compared with the actual data, and the errors were propagated back into the NN with the backpropagation technique, and this step was repeated until a good agreement was made between the result of the GIS-ANN and the historical flood events. The results were overlapped with ground-truthing points at 88.46% and 89.15% agreement during training and validation periods. Therefore, the application of the GIS-ANN for the assessment of flood vulnerable zones for this city and its catchment was successful. The result of this study can also be further considered along with the city and its catchment for practical flood management. As witnessed by [Flood is one of the natural hazards that happens when either the capacity of the river bank or the infiltration capacity of soil is less than the intensity of the rainfall , 2. Thisessed by , the citth and 6th August 2006 were known as black days of the city that very heavy rain caused flooding and both human lives and properties were damaged. According to the national flood report, over 3000 people were displaced, while about 200 people lost their lives.The current was conducted in Dire Dawa city, which is one of the cities in Ethiopia. Dire Dawa is situated in eastern-central of Ethiopia, and it is the city which links Ethiopia with Djibouti. The city is geographically located between 9°25′N and 9°45′N latitude and 41°40′E and 42°50′E longitude . The citTo achieve the objective of this study, data under five criteria, namely, hydrologic criteria, morphometric criteria, permeability criteria, surface dynamic change, and anthropogenic influences were confirmed as flood vulnerability assessing principles, and the detail of these data is presented in X is the normalized input parameters entered into the networks, and Xmin and Xmax are the minimum and maximum values of the normalized input parameters. The raw datasets and the derived input parameters were linked into the NN. The input parameters were selected on the basis of hydrologic, morphometric, permeability, and surface dynamic change and census criteria. Thematic maps were prepared for the individual criteria and prioritized based on their importance of flood vulnerability assessment. The key significant factors of flood vulnerability analysis of the existing data regarding rainfall intensity, land use/land cover (LULC), topographic wetness index (TWI), stream power index (SPI), soil classification, rainfall erosivity factor (R-factor), normalized difference vegetation index (NDVI), soil-adjusted vegetation index (SAVI), population density, areal rainfall, slope, and stream distance of the study area are shown in Figures This study employed the novelty of an integrated ANN model and geospatial analysis approach , 22–24 tThe prioritizing of the selected input parameters was done in the ANN as shown in The application of geospatial for flood vulnerability assessment is intensively used worldwide; however, the application of machine-learning (ANN) is still emerging. Up to now, very limited studies are available in which the artificial neural network (ANN) was applied as a flood assessing tool. The hydrologic, morphometric, permeability, surface dynamic change, and the status of the population were used as information to train the ANN model. The random weight values initially assigned to the NN were systematically trained and updated. The information retrieved from the geospatial characteristics of the criteria was reused in the training processes until the derived flood vulnerability map assessed in the GIS-ANN method agreed with the flood events linked into the results based on the ground-truthing points. The general flowchart of fixing the weights for the selected flood vulnerability assessment criteria is shown in To check the performance of the GIS-ANN method in assessing flood vulnerability, the result obtained in the GIS-ANN and the floodplain areas identified for the testing purpose were compared based on the pie chart plots and overlapping the result with ground-truthing points. The pie chart plots are based on plotting the degree of importance of each criterion with the flood levels generated in the GIS-ANN, where the visualization is based on the percentage of overlapping (equation ) points Points were overlapped with the flood vulnerable areas, and the overlapped points were counted. The ratio of the number of the counted points to the total number of the points was calculated by the intersection analyst tool in the GIS environment .(3)OverlIn this study, the novelty of artificial intelligence in geospatial analysis for flood vulnerability assessment in Dire Dawa watershed, Awash basin, Ethiopia, is presented. The current study used flood causing factors such as hydrologic factors , morphometric factors , permeability factors , surface dynamic factors , and census status (population density around the flood-prone areas) for the assessment of flood vulnerable areas in Dire Dawa city and its catchments. The raw datasets were processed in a GIS environment and sent to the ANN model to prioritize (weighting) based on their importance for the assessment of flood vulnerable areas. The weights of the individual criteria selected in the study were fixed based on the feedforward and backpropagation processes. The reclassified maps of flood-causing factors derived from the existing data Figures  and 3 arAs we can see from the reclassified maps, the categories or classifications presented in all maps is based on the studies conducted in different parts of the world and consulted in different pieces of literature , 28, 29.Five qualitative-based flood vulnerable zones were identified in this study, as shown in The areas under floodplain are due to the torrential rainfall from the upstream of the city. As we have already described the topographic characteristics of the current study area, the upper part of the catchment is surrounded by mountainous regions. As a witness to the flood events of 2006 , 30, theIn this study, the novelty of artificial intelligence in geospatial analysis for flood vulnerability assessment in Dire Dawa watershed, Awash basin, Ethiopia, is presented. To achieve the objective of the study, five major criteria such as hydrologic criteria , and river Euclidean distance from settlement), morphometric criteria , soil permeability criteria (soil erodibility factor (K-factor), topographic wetness index (TWI), and rainfall erosivity factor (R-factor)), surface dynamic charge criteria , and soil-adjusted vegetation index (SAVI)), and census status (population density) were confirmed as flood causing factors to train the ANN model. The raw datasets of the selected flood causing factors were preprocessed and resampled in a spatial analyst tool in a GIS environment and distributed over 30 m × 30 m spatial resolution. The significance of the selected flood causing factors was prioritized in the ANN model with feedforward and backpropagation training processes. The neural networks (NNs) were initially assigned by random values of weights and started the feedforward training processes. With the initial values of weights and feedforward training processes, rough flood hazard zones were prepared, and at this stage, the error was very high. The backpropagation process was started targeting the ground-truthing points collected from the historical flood events, and the process was repeated until the error between the GIS-ANN result and the actual data mage an agreement. Accordingly, flood vulnerable zones classified under five flood levels, namely, very low, low, moderate, high, and very high were identified. The performance of GIS-ANN results was further evaluated by overlapping the ground-truthing points, and about 88.46% agreement was made. Among the flood causing criteria selected for the assessment of flood vulnerable zones, LULC, rainfall, elevation, and slope are the most important, and this is due to the topographic condition of the city. Therefore, the application of an integrated artificial intelligence and geospatial analysis for the assessment of flood vulnerable zones was successful.


Objectives: Cross-limb stent grafts for endovascular aneurysm repair (EVAR) are often employed for abdominal aortic aneurysms (AAAs) with significant aortic neck angulation. Neck angulation may be coronal or sagittal; however, previous hemodynamic studies of cross-limb EVAR stent grafts (SGs) primarily utilized simplified planar neck geometries. This study examined the differences in flow patterns and hemodynamic parameters between crossed and non-crossed limb SGs at different spatial neck angulations.Methods: Ideal models consisting of 13 cross and 13 non-cross limbs were established, with coronal and sagittal angles ranging from 0 to 90°. Computational fluid dynamics (CFD) was used to capture the hemodynamic information, and the differences were compared.Results: With regards to the pressure drop index, the maximum difference caused by the configuration and angular direction was 4.6 and 8.0%, respectively, but the difference resulting from the change in aneurysm neck angle can reach 27.1%. With regards to the SAR-TAWSS index, the maximum difference caused by the configuration and angular direction was 7.8 and 9.8%, respectively, but the difference resulting from the change in aneurysm neck angle can reach 26.7%. In addition, when the aneurysm neck angle is lower than 45°, the configuration and angular direction significantly influence the OSI and helical flow intensity index. However, when the aneurysm neck angle is greater than 45°, the hemodynamic differences of each model at the same aneurysm neck angle are reduced.Conclusion: The main factor affecting the hemodynamic index was the angle of the aneurysm neck, while the configuration and angular direction had little effect on the hemodynamics. Furthermore, when the aneurysm neck was greatly angulated, the cross-limb technique did not increase the risk of thrombosis. Endovascular aortic aneurysm repair (EVAR) is the primary alternative for treating an abdominal aortic aneurysm (AAA). When an AAA has unfavorable anatomy, such as a significant aneurysm neck angulation or widely splayed common iliac arteries, conventional EVAR, which employs contralateral (non-cross) iliac limb deployment, is difficult and time-consuming to cannulate, and it is also followed by poor outcomes . FocusinExperimental and clinical evidence indicates the important role of flow dynamics within the stent grafts in the occurrence of post-EVAR complications, including stent migration and thrombosis . TherefoThe present study investigates the influence of the cross-limb technique on both near-wall and intragraft flow dynamics. In particular, the impact of neck angulation, either coronal or sagittal, on local flow features is analyzed by performing CFD simulations on idealized models of the cross and non-cross limb EVAR. Such an idealized model-based approach will enable  the identification of whether and to what extent a cross-limb EVAR results in the hemodynamic alteration from a conventional limb technique. These results may or may not encourage its use to improve the clinical outcome of EVAR.®, Inc., United States). The model is established under the guidance of clinicians, and the model size is selected through the given SG size parameters in device instructions for use (IFU). It is divided into three parts: the main body part of the bifurcated SG, the limb part of the bifurcated SG, and the iliac limb SG. The inlet and outlet diameters of the bifurcated SG are 25 and 12 mm, respectively, and the diameter of iliac limb SG is 16 mm. The limb and the overall length of the bifurcated SG is 45 and 124 mm, respectively. For a fair comparison, the height of the entire stent graft system was maintained at 225 mm  was adopted for the grid generation. The meshes consisted of a mixture of unstructured tetrahedral and structured hexahedral elements. To improve the computational accuracy of the near-wall hemodynamic parameters, viscous boundary layers  were imposed adjacent to the vessel walls of each model. Finally, the meshes for each configuration contained between 1.4 and 2 million nodes. To determine a sufficient (uniform) mesh refinement level, we applied the grid convergence index (GCI) to the steady-state simulation using the contraction peak condition. kg/m3. In addition, Carreau fluid model which relates approximately with the experimental data was adopted for the properties of non-Newtonian fluids  and oscillatory shear index (OSI) play an important role in understanding the changes in the SG hemodynamic environment after EVAR . Additiowhere T is the time of the cardiac cycle; The cross-limb EVAR was introduced for AAA patients with significant aneurysm neck angulation or widely splayed common iliac arteries. It significantly reduced the cannulation time compared with a conventional non-cross EVAR. However, whether or not the cross-limb stent strategy helped to decrease the incidence of post-EVAR complications (in the face of adverse AAA anatomy) is still in doubt. The current study constructed 26 cross and non-cross limb EVAR models with various coronal and sagittal angulated necks and subsequently numerically investigated the flow patterns in these models. A comparative evaluation of the hemodynamic performance was done in terms of the helical flow strength, flow field, WSS, and pressure drop.First, this study revealed that the pressure drop of the stent increased with the increase in the AAA neck angle for both stent configurations. The blood pressure drop is one of the essential factors affecting blood flow status in the stent. If there is a deviation from the physiological state, the elevated pressure drop will result in disordered blood flow , such asSecond, TAWSS is an essential hemodynamic parameter that directly acts on the endothelial layer. Low TAWSS is accompanied by unstable flow conditions such as turbulence, blood recirculation regions, and blood “stagnation” regions, resulting in an increased risk for thrombosis and intimal hyperplasia . The simThird, previous studies have demonstrated the transport effect of eddies on blood flow and the development of swirls in the cardiac cycle which brings platelets to low-speed wall reflux areas, resulting in thrombosis . From ouFinally, the current study observed that the helicity intensity in a crossed limb SG is significantly higher than that of the non-crossed limb SG when the neck is coronal angulated, which may help to inhibit platelet adhesion and hinder the formation of acute thrombosis . HoweverThe current study has presented TAWSS, OSI, and a few other parameters which are suggested to be related to the initiation and development of thrombosis. Among which, TAWSS represents the WSS magnitude, its particular values are highly related to the inlet flow rate. As a result, if the patient-specific inlet velocity is not available it is difficult to decide the threshold values of abnormal TAWSS for a particular patient. In the contrast, OSI, an indicator of the pulsatility of WSS direction, results from the flow unsteadiness over a cardiac cycle, and if the flow remains laminar, the general flow features of the flow field within the SGs are mainly decided by the geometric features of the computational models. Maybe that’s why a good match between abnormal OSI and thrombosis occurrence has been reported even though no patient-specific boundary condition was available . In addiIn vivo limb stents involve many curved parts, but the current models did not consider the impact of folds, which will be the focus of our future research.There are some limitations to this study. First, the aortic model excluded the suprarenal branch arteries, and it has been reported that the inclusion of the aorta upstream of the renal artery can alter the hemodynamics of the inlet boundary fluid state . Second,In this study, we analyzed the hemodynamic changes in SGs under different configurations, angular directions, and angles. It was concluded that the use of the cross-limbed technique does not increase the risk of thrombosis under poor neck anatomy and the main factor affecting the hemodynamic index was the angle of the aneurysm neck, which suggests that the hemodynamic changes caused by the angle of the aneurysm neck cannot be ignored when the effect of the morphology of SG on hemodynamics is investigated.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.MQ: analysis and interpretation, data collection, writing the manuscript, critical revision, approval of the manuscript, agreement to be accountable, and statistical analysis. YQ: data collection, writing the manuscript, approval of the manuscript, and agreement to be accountable. JW: data collection, critical revision, and approval of the manuscript. TZ and DY: conception and design, analysis and interpretation, critical revision, approval of the manuscript, and agreement to be accountable. All 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The COVID-19 crisis profoundly disguised the vulnerability of human societies and healthcare systems in the situation of a pandemic. In many instances, it became evident that the quick and safe reduction of viral load and spread is the foremost principle in the successful management of such a pandemic. However, it became also clear that many of the established routines in healthcare are not always sufficient to cope with the increased demand for decontamination procedures of items, healthcare products, and even infected tissues. For the last 25 years, the use of gas plasma technology has sparked a tremendous amount of literature on its decontaminating properties, especially for heat-labile targets, such as polymers and tissues, where chemical decontamination often is not appropriate. However, while the majority of earlier work focused on bacteria, only relatively few reports are available on the inactivation of viruses. We here aim to provide a perspective for the general audience of the chances and opportunities of gas plasma technology for supporting healthcare during viral pandemics such as the COVID-19 crisis. This includes possible real-world plasma applications, appropriate laboratory viral test systems, and critical points on the technical and safety requirements of gas plasmas for virus inactivation. I.The name of the event with the most dramatic impact on human societies and financial and healthcare systems across the globe in the 21st century so far is “COVID-19 pandemic.” Particularly, frightening about the pandemic is the speed of its worldwide spread due to the nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus being transmitted in aerosol droplets released during the speaking, breathing, and coughing of infected people. The pandemic ruthlessly dismantled some of the shortcomings in hospitals, disease diagnostics, and public life when it comes to dealing with extremely infectious pathogens. At the same time, many governments have also revealed unpreceded flexibility and willingness to act upon the daily changing situations. It can be hoped that the year 2020 will be marked as the year in which the global community learned that a robust protection strategy is needed to combat health crisis like this, as it will be utterly needed in the event of other pandemic outbreaks, whose lethality, based on social and health context and depending on concurrent adverse events, might also exceed what we are currently experiencing with COVID-19, as demonstrated by the Spanish flu in the early 20th century.The main pillar of fighting a pandemic is to break infection chains. This means disallowing infected people to spread the virus not only by containment but also by performing rigorous disinfection measures of contaminated goods and devices, decontamination of indoor air, especially in operation theaters with mixed ventilation as well as antiseptic prevention and treatment. While the mostly alcohol-based disinfection is suitable for the majority of applications, some cases would benefit from optimized decontamination procedures to help to limit virus spread. During the past months, it was natural to discuss with colleagues within and across scientific disciplines as well as societal stakeholders the options of gas plasma in helping to reduce viral loads wherever this technology might be advantageous over existing methods. We here outline some of the perspectives on how plasma might be useful during a pandemic like COVID-19 after giving a brief introduction on the topics related to this. The aim is to provide the information in a way to be comprehensible to scientists from several disciplines, including physicists, biologists, societal stakeholders, medical doctors, and healthcare practitioners. After all, history has shown that bringing together ideas from several fields often is the key to find new solutions, such as the need for alternative or complementary virus inactivation methods to upgrade existing procedures: a lesson tragically learned by recent viral pandemics, such as COVID-19.II.A.The SARS-CoV-2, the etiological agent of the pandemic called Coronavirus disease 2019 (COVID-19), is a virus associated with infections mainly of the respiratory system as well as other organs and systems. In contrast to eukaryotic cells (such as cells of the human body) and prokaryotic cells (bacteria), viruses do not display a self-maintaining metabolism. This essentially means that eukaryotic and prokaryotic cells can live and multiple based on nutrients from the environment, while viruses are not able to replicate on their own. In contrast, they need a host cell to perform virus replication for them. The virus is highjacking the host cell for producing the proteins and nucleic acids necessary to build new virus particles. For some types of viruses, this also extends to lipids. Eventually, the virus-infected cells turn into virus-producing factories. In a chain reaction, the new virus particles can then enter adjacent cells and infect them, or they can be transported outside the body exploiting existing exit pathways .B.From a biological point of view, it is vital to acknowledge that all living organisms on Earth have co-evolved with viruses. Virus infections are known in, e.g., bacteria , fungi, plants, and vertebrates. It is speculated that the DNA of humans and many other organisms contain many regions originally stemming from viral genomes of ancient times, exemplifying the long-standing interaction of living cells with “parasitic” DNA. Another vital aspect of viruses is their capability, in terms of their nucleic acid sequence, of evolving quickly, compared to eukaryotic organisms. During replication in the host cells, a more than a trillion-fold event in a COVID-19 patient C.Viruses can be classified according to several criteria. One is whether they are enveloped or nonenveloped. Another is whether the information necessary for viral replication is stored on DNA or RNA. Similar to influenza viruses, coronaviruses are enveloped RNA viruses. The envelope, essentially a part of the host cell membranes lined with viral proteins, is one factor determining the virucidal efficacy of disinfection III.A.Physical plasma is an excited gas state, sometimes called “the fourth state of matter,” that can be generated by a continuous supply of energy to the atoms or molecules of a neutral gas. Even if the energy required may be provided separately by thermal, chemical, electrical, and radiative resources or by a combination of all, the predominant ionizing mechanism is the collision process that involves an inelastic collision, electron impact, radiative interactions, and charge exchange. The typical life span of excited states is about 10 ns, i.e., if the energy supply is stopped, a depletion process starts, rapidly quenching the plasma. The most robust procedure of generating a plasma for biomedical purposes is electron impact ionization based on the application of a strong electric field. The electron energy is transferred by inelastic and elastic collisions with the atoms or molecules in the gas, resulting in its full or partial ionization. The temperature of partially ionized gas is always substantially lower than the characteristic ionization temperature. Dependent on the design of a plasma source, an ambient temperature of the plasma can be achieved, making it very interesting for the treatment of heat-sensitive surfaces and materials. The complex physicochemical plasma characteristics depend on a multitude of parameters, including the type and composition of the gas or gas mixture used for plasma generation, the applied energy and electrode configuration, the pressure, and the environment. With regard to its application, especially in the medical context, useful classifications are thermal versus nonthermal plasmas and low pressure versus atmospheric pressure plasmas In the last few years of the steadily growing development of plasma medicine as a new field of disciplines, four cold atmospheric plasma sources have undergone medical clinical trials in Germany and were licensed as medical devices. They operate either as direct dielectric barrier discharge (DBD) or indirect surface DBD or as plasma jet B.The SARS-CoV-2 virus is primarily released from the respiratory tract as an aerosol, which can be increasingly released through intubation, bronchoscopy, rhinoscopy, or surgical interventions, for example, The use of gas plasma technology is successful or promising for hygienic indications when antimicrobial, antiviral, and/or antibiofilm activity is required. This is especially relevant for materials that are thermolabile or sensitive against chemical microbicidal active agents and/or when microbicidal chemical agents cannot or only insufficiently reach the site of action. Because of the highly effective antimicrobial components of gas plasma, the spectrum of its activity includes all vegetative bacteria species, including multidrug-resistant organisms as well as a broad spectrum of viruses C.The application of gas plasma to inactivate, kill, or remove pathogens is under research for more than 50 years. Initially, much work has been done on low-pressure plasmas for antimicrobial treatment of materials and devices. The enhanced availability of atmospheric pressure gas plasma devices from the middle of the 1990s led to intensified investigations for their use for antimicrobial treatments. Even if for these efforts in many cases, the term “plasma sterilization” was claimed, a real plasma-based sterilization process or device that meets the requirements of sterility assurance is not available yet Large-scale plasma devices harbor enormous economic and healthcare potential. The COVID-19 outbreak has led to dangerous shortages of sterilization capacity at hospitals. Plasma-supported hydrogen peroxide gas sterilization using gas plasma technology is an effective alternative to conventional and/or traditional sterilization methods and an indispensable innovation for the sterilization of polymeric and heat-sensitive medical devices Another challenging but promising application is antisepsis using gas plasma technology. Gas plasma tolerability has been shown for skin D.et al.et al.et al.in vitro, on human corneal epithelial cells, and ex vivo, on explanted corneas, without identifying pronounced toxicity. At the same time, a marked antiviral activity was observed. The results demonstrated a good chance of translating plasma to the clinic for use against drug-resistant herpes keratitis et al. investigated the effects of treatment with a helium plasma jet on HIV-1 replication in monocyte-derived macrophages (MDMs). The anti-HIV activity of plasma treatment was observed, inhibiting effect on virus–cell fusion, viral reverse transcription and integration, and virus particles produced by gas plasma-treated cells showing reduced infectivity et al. exposed HIV to a plasma of a helium jet. The results showed increased inhibition of HIV with increasing voltage and plasma treatment time that, however, was already too toxic for the HeLa cells used as an infection model, discouraging this approach in the particular model used. All studies mentioned above were done at atmospheric pressure. At nonatmospheric pressure, a virucidal effect of nitrogen gas plasma on the influenza virus and its components was observed In the frame of gas plasma treatment playing a role in the reduction and control of airborne virus transmission through droplets and to reduce the burden of disease and control the nosocomial spread of respiratory viruses in the hospital setting, the work of Terrier et al. investigated new strategies for the containment of airborne (and waterborne) transmission of viral agents. They studied the plasma-assisted inactivation of MS2 bacteriophages using a DBD plasma source set in an exposure chamber and injected with the aerosolized viruses from an MS2 suspension et al. investigated the mechanism of virus inactivation (water containing surrogate virus-bacteriophages) by reactive oxygen and nitrogen species generated by a surface DBD plasma system. The treatment effectively damaged phage protein and RNA. Moreover, treatment with plasma-treated water was similarly effective than direct plasma treatment, suggesting long-lived reactive species—easily to be manufactured chemically—to be the primary agent of mediating inactivation An easy way to test virucidal activity is through the use of bacteriophages. After a challenge, the infectivity of the phages can be assessed by their potency to lyse bacteria. Wu While most of these studies were rather descriptive in terms of mechanisms and redox chemistry that may be optimal or suboptimal for antiviral activity, the group of Bruggeman has published a sophisticated study, shedding more light on these aspects. A remote radio-frequency plasma source, feline caliciviruses, an array of feed gas compositions, and a dozen of different radical scavengers were used in this article IV.A.in vitro systems as immortalized cell lines, that allow reproducing all stages of the infection cycle, are required to evaluate viral infectivity. This need, per se, represents a limitation to testing, as cell culture systems require dedicated laboratory environments and highly trained personnel to be efficiently implemented.The assessment of the efficacy of gas plasma technology for the inactivation of aerosol-transmitted viruses, such as coronavirus (CoV), relies on the use of laboratory assays for the detection of virus infectivity. Infection of the host by viruses represent a complex process, involving several actions , each one requiring functional integrity of specific viral structures and the virus as a whole. In particular, the functionality of attachment proteins, the integrity of viral capsid, and the absence of significant damage to the viral genome are all required to accomplish the infection of the host cell. As all of these viral structures could be singularly or simultaneously affected by gas plasma treatments, complex 1)Availability of Cell Culture Systems: While several viruses of clinical interest can be readily cultivated in cell lines, some highly relevant human viral pathogens, as for example, norovirus and sapovirus, are not yet efficiently culturable.2)Biosafety Levels (BSLs) of the Viruses of Interest: Diagnostic specimens containing highly pathogenic respiratory CoV, such as SARS, MERS, and SARS-CoV-2, can be manipulated in BSL-2 laboratories with additional personnel protective equipment . However, for the propagation of these viruses in cell cultures (Vero E6 cells), BSL-3 facilities and work practices are strictly required —Version 2; and Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19)—Interim guidance as of February 12, 2020).Further to this, several issues should be fully considered in relation to viral testing associated with gas plasma technology.in vitro cultivation systems or the limitations due to biosafety often prompts the use in experimental settings of surrogate viruses, i.e., viruses that are generally expected to mimic the behavior of the viruses they represent, without posing, however, the same technical and analytical problems. As far as CoV is concerned, given the structural and genetic similarity , other human pathogenic CoV, usually responsible for mild to moderate respiratory illnesses , might be used as surrogates for highly pathogenic CoV. These viruses include the alpha-CoV types 229E and NL63 and the beta-CoV types OC43 and HKU1, which are culturable in BLS-2 environments using, for example, MRC-5, LLC-MK2, HCT-8, and HAE cell cultures, respectively The lack of fusion viruses also in other fields requiring the use of surrogates.Considering these restrictions, animal CoV has been often proposed as surrogates for SARS and MERS. The swine and feline alpha-CoV transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV), for example, have been repeatedly used a surrogate to assess the recovery of CoV from water matrices and air conditioning/ventilation systems Although the use of human/animal CoV surrogates ensures high similarity of viral structures and therefore supports the inference of results to the viruses of interest, the concept of representativeness of surrogate viruses has been challenged phi phages (es. phi phages having a segmented double-stranded RNA). Other approaches to the by-pass cell culture of viruses include the use of the “viability PCR,” i.e., molecular assays specifically designated to assess the integrity of viral capsid or genome . These approaches rely on specific analytical strategies as the use of long-template PCR, covering a large fragment or ideally the whole viral genome to assess its integrity, or the use of nucleic acids intercalating agents, as ethidium monoazide or propidium monoazide (EMA or PMA), to block the PCR amplification of virions in which the loss of capsid integrity has led to the exposure of viral RNA. Some of these strategies have also been applied in association with gas plasma treatment In order to prevent these problems and to reduce the technical complexity of viral testing on cell cultures, alternative strategies have often been proposed, mainly through the use of bacterial viruses (phages), whose detection/enumeration applies conventional bacteriological techniques and is less affected by the presence of matrix residues in the tested portion. As such, different phages have been used as a surrogate for CoV or, more in general, for enveloped viruses, with several studies, including either Enterobacteria phage MS2 (Leviviridae family) or Pseudomonas B.et al.et al.In the last few years, including the period of recent emergency from COVID19, some papers dealing with the role of bioaerosol transport for virus respiratory diseases and the controversy between airborne and droplet transmission might be usefully mentioned. In particular, Bourouiba C.2) and oxygen (O2) containing air results in the generation of ozone (O3) and nitrogen oxides (NOx). Both gas species are essential for pathogen inactivation on the one hand but are highly toxic to the lungs on the other hand, if accumulating in higher concentrations. Therefore, it might be a challenge to balance the concentration of these gas species to achieve a sufficient antiviral effectivity while minimizing any side effects. Consequently, it has to be demanded that all approaches aiming at using plasma-treated breathing air for the inactivation of viruses in the mouth and throat region must guarantee and prove that any inhalation of this air is toxicologically acceptable.There is direct evidence that the probability of infection increases with exposure to viruses, and indirect evidence that the initial viral load influences the severity of the infection Another field repeatedly under discussion is plasma application is hand disinfection In general, any evidence of specific plasma effectivity against SARS-CoV-2 in living tissues like the skin or mucosa has not yet been provided.


The revolutionary evolution in science and technology over the last few decades has made it possible to face more adequately three main challenges of modern medicine: changes in old diseases, the appearance of new diseases, and diseases that are unknown (mostly genetic), despite research efforts. In this paper we review the road travelled by pathologists in search of a method based upon the use of routine instruments and techniques which once were available for research only. The application to tissue studies of techniques from immunology, molecular biology, and genetics has allowed dynamic interpretations of biological phenomena with special regard to gene regulation and expression. That implies stepwise investigations, including light microscopy, immunohistochemistry, in situ hybridization, electron microscopy, molecular histopathology, protein crystallography, and gene sequencing, in order to progress from suggestive features detectable in routinely stained preparations to more characteristic, specific, and finally, pathognomonic features. Hematoxylin and Eosin (H&E)-stained preparations and appropriate immunohistochemical stains have enabled the recognition of phenotypic changes which may reflect genotypic alterations. That has been the case with hepatocytic inclusions detected in H&E-stained preparations, which appeared to correspond to secretory proteins that, due to genetic mutations, were retained within the rough endoplasmic reticulum (RER) and were deficient in plasma. The identification of this phenomenon affecting the molecules alpha-1-antitrypsin and fibrinogen has led to the discovery of a new field of cell organelle pathology, endoplasmic reticulum storage disease(s) (ERSD). Over fifty years, pathologists have wandered through a dark forest of complicated molecules with strange conformations, and by detailed observations in simple histopathological sections, accompanied by a growing background of molecular techniques and revelations, have been able to recognize and identify arrays of grotesque polypeptide arrangements. The term endoplasmic reticulum storage disease (ERSD) was introduced into the medical literature after discovering that, similarly to alpha-1-antitrypsin (AAT) . The twoThe uniqueness of these two diseases resides in the fact that both were described by pathologists from the evaluation of H&E-stained slides, yet they were easily overlooked on clinical grounds. Indeed, AATD individuals in the heterozygous state may have normal AAT serum levels , and HHHIn this paper we review the diagnostic methodology; and we provide an explanation as to how and why H&E-stained slides can provide the correct diagnosis, and by applying the ad hoc protocol, have turned out to be equivalent to molecular genetic analysis when the latter is not available ,7,8.In 1665, Robert Hooke, a botanist, wrote: “By examining with my microscope, sections of cork, I have been able to discover rather easily that the cork is composed of a mosaic of ‘empty polygonal spaces,’ whilst I haven’t reached any certainty with animal tissue. However, it is not unlikely that an observer, some day, with the help of a better microscope could discover them in thinner histological sections” . In 1838Nineteen years later, Rudolph Virchow, professor of pathology in Berlin, prophetically baptized the cell as “the elementary patient” endowed with respiratory (mitochondria), digestive (lysosomes), and locomotor (contractile filaments and microtubules) apparatuses, and excretory and sensory (skin and cell membrane) systems. Based upon physiological and pathological histology, Virchow figured out pathology as “physiology with obstacles,” and invented the cellular pathology .At the end of the 19th century, an Italian pathologist, Camillus Golgi, with rudimentary techniques and a self-made stain, discovered within some neurons’ cytoplasm the “internal reticular apparatus,” a crucial component of the labyrinthine network of channels that are the theater of the intracellular protein traffic—the endoplasmic reticulum (ER) . The celThe Golgi apparatus turned out to be for light traffic, for sorting out and addressing proteins, as demonstrated over the 20th century by histo-autoradiography , electroThe pioneering work on the anatomical distribution of the rough-ER (RER) in hepatocytes by Ma and Biempica ; on protGlycosylation of proteins is a stepwise process by which monosaccharides are covalently bound to a polypeptide chain during its elongation, intracellular transport, and excretion, resulting in a product termed glycoprotein . This meThe attachment of the first sugar (“core” glycosylation) takes place in a special way: a soluble lipid, the dolichol phosphate, plays the role of intermediate between the sugar nucleotide and the peptide. That first accepts the sugar, and subsequently, when the side chain is completed, transfers the “core” oligosaccharide en bloc to the protein . GlycosyThe biosynthesis of glycoproteins starts at the level of polyribosomes attached on ER membranes (RER). During the elongation of the peptide chain (translation), while the latter is still in the membrane, the “core” glycosylation takes place . At this early stage, a sorting out mechanism  determines the fate of the nascent glycopeptide: discharge and segregation into the lumen of the RER or insertion into membranes . During N-acetylneuraminic acid . Finally, they are driven, within secretory vesicles with the cooperation of the microtubule system, towards the cell surface where fusion of the vesicle membrane with the cell membrane occurs. At this time, the internalized proteins are discharged into the blood or to the extracellular space, whilst the proteins to be incorporated into membranes remain there and contribute to the reconstitution of the integrity of the plasma membrane [In the Golgi complex, glycoproteins acquire the completion of the carbohydrate chain by addition of the terminal sugar, membrane .The above scheme works for cells specialized in protein production for export, such as hepatocytes. Some variations are known to occur in other cells ,32.Alpha-1-antitrypsin (AAT), the major component of alpha-1-globulins, is the major serum proteinase inhibitor (Pi), especially active against leucocyte elastases. AAT is also an acute phase reactant, capable of raising its serum level up to quadruple the normal level under conditions of clinical stimulation .The discovery of AAT deficiency (AATD) represents one of the most fascinating examples of scientific intuition, logical clinical reasoning, and research methodology. During an influenza epidemic in 1963 in Sweden, a number of hospitalized patients with pneumonia showed a peculiar abnormality in the serum electrophoresis consisting of a flat or deeply depressed alha-1 globulin pick . InitialSix years later, Harvey Sharp, a pediatrician in Minneapolis, did observe the same electrophoretic pattern in children affected by cryptogenic liver cirrhosis and low serum AAT levels . Sharp pIn 1976 it was shown that the Z AAT was carrying a molecular abnormality consisting of a Glu–Lys substitution , and in In 1984 an EM study on a large series of both homozygous (Pi ZZ) and heterozygous (Pi MZ) AATD individuals , could dIn 1975, Jeppsson et al.  were ablThe above methodology has allowed to distinguish the storing from the non-storing deficiency variants of AAT. The latter indeed are not predisposing to the development of liver disease  thus indEven more interesting was the detection of dark basophilic material inside the eosinophilic AAT globules a. On EM,There is an additional variant of AAT that is stored within hepatocytes and that equally predisposes to liver cirrhosis, the Siiyama, whose mutation, Ser53Phe, has been localized at exon III . By usinAs a sort of scientific butterflies seduced by flowers along our way, we have wandered through a dark magic forest of complicated serine molecules and made detailed descriptions of their strange molecular malformations. Studies on the growing background of multiplying molecular techniques and revelations, the observations have allowed to recognize and identify the array of grotesque polypeptide acrobatics in simple histopathological stains. Most of these observations were challenges we met in diagnostic and research work, with the recurring question: “what could be the reasons for this lesion looking like this?” .The following paragraph will be dealing with a newly discovered disease similar to AATD, regarding a protein of a major hematological interest, the fibrinogen, that was so unexpected and apparently contradicting hematological dogmas, that hematologists were hesitating in trusting it. The disease has been called hereditary hypofibrinogenemia with hepatic storage (HHHS).Light microscopy has been crucial in recognizing this disease, thereby providing a further solid bridge between pathology and molecular pathology. HHHS manifests in the liver and causes cirrhosis in early childhood. In histology, in H&E-stained preparation it is characterized by hepatocytic eosinophilic inclusions resembling AAT globules . In contUnder the EM, fibrinogen inclusions appear mostly in the form of curved tubular structures arranged in a fingerprint-like fashion or elongated tubules with a fiber-glass configuration ,50. ThesBased upon the experience with AATD, a biochemical study on fibrinogen brescia was carried out at Max Planck Institute in Munchen in cooperation with Dr. Agnes Henschen. With the available technique, high performance liquid chromatography (HPLC), no amino acid replacement could be found in circulating fibrinogen brescia . The disFollowing the discovery of fibrinogen brescia, and again starting from the observation in H&E-stained slides of eosinophilic PASD negative cytoplasmic inclusions in liver cells, six further mutations have been identified on the γ chain: aguadilla Arg375Trp) , Al du P5Trp , AlLike in AATD, the aggregation of mutant fibrinogens occurs immediately after the discharge of the protein within the RER. However, in contrast with AATD in which the cell is capable of exporting some 15% of the mutant molecule, the eight mutant fibrinogen gamma chains, and the single case of a mutant alpha chain  cannot tThe defective translocation from the RER to the other sections of the secretory pathway is documented by the absence of aggregated mutant proteins in the SER or Golgi apparatus, either in AATD or HHHS ,22,50.However, a small fraction of AAT mutants can escape the storage phenomenon, reach the circulation, and are detected on serum Isoelectric Focusing (IF). The mechanism of the escape is unknown. Starzl observed a reduction of the amounts of AAT storage in Pi Z cirrhotic patients after porto-caval shunt , therebyInterestingly enough, recently, the formation of allopolymers of mutant fibrinogens and Apo-Beta lipoproteins has been described in patients with both hypofibrinogenemia and hypo-APO-beta-liproteinemia .Mutant fibrinogens and Apo-beta-lipoproteins could be demonstrated in the same inclusions in both H&E a and PASThe analysis of the protein structures by 3D modelling in those cases has unraveled the pathomorphogenesis of the unexpected phenomenon. Fibrinogen gamma chain mutations indeed have been shown to provoke conformational changes in the region of the globular domain of the gamma chain , involveThus, the co-localization of two proteins simultaneously retained within the RER does not contradict the absolute requirement for ERSD (exclusive and selective retention of a mutant protein), as APO-beta-lipoprotein retention is not genetically determined but is an acquired phenomenon secondary to a physical-chemical affinity binding.By analogy, the same holds true for AATD in which heteropolymers (M or S and Z) are entrapped within the RER, as recently demonstrated by confocal microscopy .This review has confirmed the validity of the original definition of ERSD as an accumulation of abnormally conformed proteins due to mutations. The storage is exclusive and selective for the mutant protein and occurs at the site of the early synthesis, the RER. The condition is genetically determined, and hence congenital, hereditary, and permanent.The mechanism of intracellular retention of mutant secretory proteins represents a basic phenomenon in biology and pathology and can potentially affect all secretory proteins.Despite the low plasma level, these diseases are often missed in the clinic. The intracellular protein retention results in cytoplasmic inclusions that can be visualized by a light microscope on H&E-stained slides. The characterization of the inclusions requires stepwise investigations according to an ad hoc protocol.The intracellular storage of mutant proteins causes cell constipation/damage/death, inflammation, and cirrhosis. The discovery of ERSD has led to the identification of the cause of a group of cirrhosis otherwise remaining cryptogenic. The unavoidable intracellular storage of mutant proteins represents the elementary lesion in ERSD, and consequently the true phenotype that strongly correlates with the genotype in both AATD and HHHS . This stThe diagnostic importance of H&E stain in general has been widely acknowledged in an Editorial   by the gHans Popper, the father of modern hepatology, often claimed that the best research tool is “H&E-stained slides connected to the brain” .As fellows and great friends of the two above masters, we can testify to the fact that H&E that has fulfilled the requirements of modern medicine: (a) breaking the walls between the four watertight compartments of the traditional medicine ; (b) favoring the transition from the physician to the MD–researcher, and finally to PhD–scientist; (c) approaching difficult, rare, unknown cases with the habits and techniques previously prerogative of research alone, in this way turning a clinical case into a research project .This background has made it possible for H&E stain to realize the miracle of forging the connection between the diagnostic histopathology of the past and the molecular pathology in the breve new world of the future.It is astonishing that, running through various histological steps backwards and forwards from the most powerful techniques, i.e., IHC, in situ hybridization, EM, molecular histopathology, genetics, and 3D modelling, H&E, an old-fashion technique, properly observed with sensitive eyes, has begun again to tell us a story . It has In perspective, the body of cumulated knowledge should hopefully open the road to future strategies aimed to look for novel small molecules  capable