Immune-oncologic (IO) therapy has revolutionized the treatment and management of oncologic disease. Immunotherapy functions by enhancing the host immune-systems ability to endogenously clear malignant cells, however, this activation can also lead to immune-mediated damage to healthy native tissues. These side effects are known as immune-related adverse events or irAEs and can even present with phenotypes similar to autoimmune diseases. IrAEs are the major consequence of checkpoint inhibitors and can have a significant impact on a patient’s cancer treatment and long-term quality of life. The management of these irAEs follows a similar approach to autoimmune diseases. More specifically, the management is akin to that of autoimmune disease exacerbations. While there is an array of immune-suppressing agents that can be used, steroids, immunomodulators and IO discontinuation are cornerstones of irAE management. The exact approach and dosing are based on the severity and subtype of irAE presented. Within recent years, there has been a push to better prevent and manage irAEs when they arise. There has been an additional effort to increase the number of steroid-sparing agents available for irAE treatment given the consequences of long-term steroid therapy as well as patient contraindications to steroids. The goals of this review are to summarize irAE management, highlight significant advances made in recent years and emphasize the future directions that will optimize the use of IO therapy in oncology. Immunotherapy is often seen today as a relatively novel agent in the realm of cancer care. However, immune-acting anti-cancer agents have been used since the late 1970s with BCG in treating bladder cancer as a form of local immune-oncologic (IO) therapy  visuallyIrAEs can occur at any organ system with a wide range of presentations . In a meThe percentage of patients who are eligible for ICI therapy has increased dramatically from 1.54% of cancer patients in 2011 to 43.63% in 2018 . Within The management of these irAEs, as stated earlier, follows similar principles to the treatments used for acute autoimmune pathologies. Corticosteroids and IO treatment discontinuation are the most frequently deployed tactics when it comes to managing irAEs . In addimolecular mimicry with the source of specious antigens coming from malignant cells  and tissue-specific testing/biopsies . CliniciWithin IO clinical trials and in clinical practice, the severity of irAEs are graded using the Common Terminology Criteria for Adverse Events (CTCAE) developed by the US National Cancer Institute (NCI). IrAEs are measured from grade 1-5 correlating to mild, moderate, severe, life-threatening, and deadly, respectively . The appIn this next section, we review the most recent treatment algorithms for the different irAEs using expert consensus guidelines on ICI toxicity management and highlight the advances in each. However, prior to examining the details of management, it is important caveats regarding irAE classification and diagnosis. The grading system is far more useful to organ-specific irAEs and less useful for the ambiguous general (fatigue) and subclinical endocrine  irAEs. Additionally, there lacks a consensus for the documentation and clear classification for irAEs, as many studies have found poor agreement between providers on how to best classify irAE timing, type, and severity .Cardiac reactions require immediate cardiology (preferably cardio-oncology) consultation, ECG, telemetry, echocardiograms as well as biomarkers for cardiac damage  and inflammation  , 32. Newpneumococcus and legionella) and possible chest CT imaging with repeat imaging in 3-4 weeks (Pulmonary irAE or pneumonitis management is divided between mild (grade 1-2) and moderate-severe (grades 3-4) pneumonitis. Grade 1 reactions require IO hold, reassessment in 1-2 weeks  and a possible chest CT with follow-up imaging in 4-6 weeks if symptoms do not resolve . Grade 2-4 weeks , 33. If -4 weeks , 32. Pre-4 weeks , 32. If -4 weeks , 32. Gra-4 weeks , 32. Ora-4 weeks , 32. Maj-4 weeks , 32, 33.Milder skin reactions include maculopapular rashes and pruritis, which are relatively common reactions. Their management is dependent upon the severity of the reaction.For maculopapular rashes grades 1-2, ICI treatment can be continued, and patients can be provided topical emollients, antihistamines/lidocaine patches for pruritis and moderate-high potency topical steroids on affected areas , 32. If More worrisome dermatologic reactions include blistering or bullous dermatitis (BD) and Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) .For BD, SJS and TEN an urgent dermatologic consultation is strongly recommended and, if unavailable, a skin biopsy should be obtained , 32. ForGiardia, Cryptosporidium and E. Histolytica; and a C. difficile toxin  irAEs can be divided into two major categories. Ones that involve the intestines (colitis/diarrhea) and those that involve major digestive organs such as the liver and the pancreas. For all grades, a thorough GI infection panel is required, which includes nucleic-acid amplification tests (NAATs), bacterial cultures; Ova/parasites for le toxin , 32. ForDiarrhea is the most common irAE associated with ICI and is even more common with combination IO regimens . For graFor post-toxicity resolution, grade 3 reactions require discontinuation of CTLA-4 agents and consideration of anti-PD-1/PD-L1 classes for future IO treatments , 32. GraLiver damage from irAEs is often associated transaminitis noticed on laboratory screenings with symptomatic presentations being a sign of more severe damage . GradingFor irAEs associated with the pancreas the general management is split between asymptomatic amylase/lipase elevations and symptomatic pancreatitis, which is further sub-divided by grades 2-4 . For asyIrAEs associated with the kidney are graded based on the degree of creatinine (Cr) elevation. For all grades, it is recommended that patients receive a thorough history for previously nephrotoxic agents  and that nephrotoxic medications are discontinued, or dose reduced. A spot urine protein/creatinine ratio (Up/Cr) IS also recommended. Grade 1 reactions have Cr elevations 1.5-2 times above baseline or have a > 0.3 mg/dL increase . For graNeurologic irAEs are rare but can have significant impacts on patient quality of life. The management for specific subtypes mostly include infection rule-out, CNS imaging/PNS stimulation testing and IO temporary hold (mild/moderate) or permanent discontinuation. Certain rare reactions such as Guillain-Barre syndrome (GBS), myasthenia gravis (MG) and transverse myelitis are managed with the current standard of care and neurology consultation as well as inpatient or ICU admission , 32.Meningitis/encephalitis irAEs require MRI brain with/without contrast (pituitary protocol included), lumbar punctures and neurologic consultation , 32. ForNeuropathy is the most common irAE of the neurologic subtype and is also a common adverse effect of many oncologic treatments, which is an important consideration. Grade of neuropathy are based on the severity and degree of peripheral nerve involvement . Grade 1Visual changes associated with ICIs are rare but should be closely monitored given the impact on patient morbidity and quality of life. Visual irAEs are split into two major categories uveitis and episcleritis . PatientHyperglycemia is a unique irAE that could be linked to an iatrogenic type I diabetes mellitus (T1DM), however, many other cancer treatment agents can cause elevated blood sugar as well. When considering an irAE as the cause of hyperglycemia, the degree of blood sugar elevation and the presence of DKA determine the work-up and management. If patients have fasting hyperglycemia < 200 g/dL and/or a history of T2DM, the elevation can be attributed to pre-existing T2DM or medication-induced hyperglycemia (most commonly steroids) . If the Thyroid irAEs are one of the more common endocrine irAEs. This is why the management of endocrine irAEs can allow for IO continuation despite adverse effects due to the availability of hormone replacement therapy . AccordiFor hypothyroid states, thyroid irAEs are split based on the degree of symptoms at presentation. For asymptomatic/subclinical hypothyroid states, thyroid function tests (TFTs) which include TSH and free T4 are monitored every 4-6 weeks. The following management is based on whether TSH and free T4 adjust appropriately , 32. If For symptomatic thyrotoxic states or presenting with laboratory evidence of low TSH and/or high free T4/total T3, IO can be continued with likely endocrine consultation. Patients should be managed with propranolol 10-20 mg q4-6h or atenolol/metoprolol with repeat TFTs in 4-6 weeks . If normHypophysitis is a significant irAE that has a stronger association with CTLA-4 agents over PD-1/PD-L1 ICIs . PatientPAI is an extremely rare irAE, often not associated with the ICI itself. Thus, PAI should have endocrinology consultation for further work-up of an alternative etiology to irAEs. Similar to many of the other endocrinopathies, the symptom presentation can be subtle requiring keen observation from the treating physician and a comprehensive laboratory panel depending upon the CTCAE grading. Acute hypocortisolemia, in particular, can be life threatening if missed and requires thorough rule out even when symptoms appear mild .Fatigue is a non-specific irAE that is commonly associated with numerous cancer treatments and oncologic disease itself. This makes it particularly relevant to rule out other more insidious causes of fatigue before general irAEs can be definitively diagnosed. The work-up includes a full physical, laboratory panel  and medication review. Severity is graded from 1-4, with management for grades 1-2 (mild-moderate) mostly consisting of IO continuation (can consider hold for grade 2), close monitoring and follow-up for new symptoms . Other cThe major advancements in MSK irAEs includes the use of steroid-sparing immunomodulators for steroid-refractory scenarios, stronger recommendations for non-opioid pain regimens, specific autoantibodies to screen for and greater detail for polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) management. Effectively, PMR and GCA activation or re-activation should be screened for with a history and clinically concerning symptoms, which warrant an urgent rheumatologic and ophthalmologic consultation . This alInflammatory arthritis associated irAEs are split into mild, moderate and severe to determine management. Diagnosis can include rheumatologic consultation if significant. Severity is based on the number of joints affected, functional assessment, imaging findings at joints, presence of auto-antibodies  and the levels of inflammatory markers  . Mild reMyalgias and myositis associated with ICIs require a diagnostic work-up similar to inflammatory arthritis and the severity is divided into mild and moderate-severe. Laboratory work-up includes inflammatory markers , muscle cell biomarkers , strength testing, cranial nerve testing and evaluation for other concomitant irAEs  . Mild reWhile clinicians’ expertise of irAEs management has improved with the wide-use of ICI, several challenges remain. There is a need to clarify the complex network of downstream pathways associated with CTLA-4 and PD-1/PD-L1 augmentation. Specifically, this relates to their impact on irAE profiles as well as oncologic treatment outcomes . AdditioIn addition, steroid-sparing based agents can offer alternative therapeutic options for oncologic patients. This can reduce the long-term adverse effects of steroids, address steroid-resistant irAEs and provide options for patients with contraindications to steroid therapy  . In the With the increasing use of combination regimens, the rates and severity of irAEs are expected to rise compared to ICI monotherapy. This is evident in the rates of irAEs by ICI regimen as seen in Clinical trials that lead to ICI therapy approval frequently excluded patients with pre-existing autoimmune diseases. However, there are nine studies that have monitored these patient populations with pre-existing immunologic pathologies. Polymyalgia rheumatica, myasthenia, rheumatoid arthritis and psoriasis/psoriatic arthritis all appeared to display the highest rates (>50% of patients receiving ICIs) of autoimmune reactivation/flares with ICI therapy , 45, 46.Studies examining the potential for pre-ICI treatments, in which steroids were given before ICI initiation, showed little to no efficacy with regards to the rates of irAEs . In sterWhile some irAEs are associated solely with a negative side effect of ICIs, there is research showing positive correlations between certain irAEs and oncologic outcomes. Certain dermatologic irAEs, such as vitiligo, may be positive prognostic indicators for melanoma patients . SimilarOncologic-specific algorithms may also be necessary for certain aggressive tumors that have complex interactions with the endocrine, humoral, and cellular-based immune systems. Studies examining the impact of IO agents on certain high-grade neuroendocrine (HG-NEN) tumors have shown mixed results raising questions related to the predictive/prognostic value of PD-1/PD-L1 expression alone for IO deployment and side-effect management . While sCurrent controversies within the irAE literature exist mostly in how irAEs are reported, documented and, therefore, managed. Outside of endocrine irAEs, which have specific laboratory cutoffs and have limited alternative diagnosis, there is significant inconsistency between providers and institutions for irAE reporting . Thus, tIrAEs challenge clinicians to weigh the costs and benefits of these new IO therapies. IrAEs are complex and display associations and disassociations with oncologic outcomes. Currently, irAE management algorithms, documentation patterns and pre-ICI screenings appear to be at the forefront of irAE research and could revolutionize the way we manage these side effects.As the use of IO therapy continues to expand to combination regimens and to the adjuvant/neoadjuvant treatment spaces, it becomes an ever-pressing issue that our understanding of irAEs improves and that irAE management evolves. Thus far, significant strides have been made to identify high-risk patients, high-risk irAEs and construct a treatment arsenal consisting mostly of steroids and immunomodulators . With time, this field will continue to expand and allow for further optimization of IO therapy for patients. This can lead to reduced costs, improved oncologic outcomes and maximized quality of life for patients dealing with cancer and IO therapy.TO primarily wrote the manuscript and executed suggested revisions provided by the included authors. BN provided supervision and advisory during the writing and submission process. DM also assisted in revisions and manuscript submission. All authors contributed to the article and approved the submitted version.MB has acted as a paid consultant for and/or as a member of the advisory boards of Exelixis, Bayer, BMS, Eisai, Pfizer, AstraZeneca, Janssen, Genomic Health, Nektar, and Sanofi and has received grants to his institution from Xencor, Bayer, Bristol-Myers Squibb, Genentech/Roche, Seattle Genetics, Incyte, Nektar, AstraZeneca, Tricon Pharmaceuticals, Peleton Therapeutics, and Pfizer for work performed as outside of the current study. BN has acted as a paid member of the advisory board of Exelixis.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) are the two main causes of heart failure (HF). Despite similar clinical characteristics and common “HF pathways”, ICM and DCM are expected to have different personalized treatment strategies. The underlying mechanisms of ICM and DCM have yet to be fully elucidated. The present study developed a novel computational method for identifying dysregulated long noncoding RNA (lncRNA)–microRNA (miRNA)–mRNA competing endogenous RNA (ceRNA) triplets. Time-ordered dysregulated ceRNA networks were subsequently constructed to reveal the possible disease progression of ICM and DCM based on the method. Biological functional analysis indicated that ICM and DCM had similar features during myocardial remodeling, whereas their characteristics differed during progression. Specifically, disturbance of myocardial energy metabolism may be the main characteristic during DCM progression, whereas early inflammation and response to oxygen are the characteristics that may be specific to ICM. In addition, several panels of diagnostic biomarkers for differentiating non-heart failure (NF) and ICM (NF-ICM), NF-DCM, and ICM-DCM were identified. Our study reveals biological differences during ICM and DCM progression and provides potential diagnostic biomarkers for ICM and DCM. Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) are the two most commonly occurring etiologies for heart failure (HF). Despite similar clinical characteristics, ICM and DCM respond differently to the same medication and ICM has a worse prognosis . NowadayCurrently, great effort is being made to investigate common HF pathways and disease-specific characteristics in ICM and DCM –11. At tBoth theoretical and experimental studies have demonstrated that different types of RNA transcripts contain numerous miRNA binding sites, and they can communicate with and regulate each other through competing for shared miRNAs, thus acting as competing endogenous RNAs (ceRNAs) , 13. LncIn the present study, dysregulated lncRNA–miRNA–mRNA ceRNA triplets (LMM-CTs) in ICM and DCM were systematically identified, and time-ordered dysregulated ceRNA networks for the two diseases were constructed. The workflow is shown in Fig. Using sample-matched RNA-seq and miRNA-seq data, the method developed in this study was applied to identify dysregulated LMM-CTs in ICM and DCM. As a result, 1271 dysregulated LMM-CTs including 97 lncRNAs, 85 miRNAs, and 675 mRNAs in ICM were obtained, and 1298 dysregulated LMM-CTs consisting of 107 lncRNAs, 96 miRNAs, and 727 mRNAs in DCM were identified (Supplementary Table p < 0.01 and |log2 fold change (FC) | > 1.2. The results showed that the proportions of SDE genes and DisGenes in dysregulated LMM-CTs were both significantly higher than that of NF LMM-CTs .Subsequently, the proportions of SDE genes and the known disease-associated genes (DisGenes) in dysregulated LMM-CTs were respectively investigated. SDE genes were selected using DESeq2 with To construct TO-BCeN in NF, hsa-miR-3615 and hsa-miR-580-3p were chosen as the initial nodes because these two miRNAs were both in the top 10% lowest dysregulated score in ICM and DCM . The TO-BCeN was then obtained using the breadth-first search (BFS) algorithm, and single gene-based networks were then converted into LMM-CT-based networks.The TO-BCeN consisted of 9 time-ordered levels (denoted L1–L9). As shown in Fig. When constructing the TO-BCeN, it was assumed that its initial nodes should be relatively stable, which would otherwise cause the instability of the entire TO-BCeN. Therefore, the distribution of dysregulated scores of LMM-CTs among nine time-ordered levels for ICM and DCM were respectively examined . Taking ICM for example, the rationality of the TO-BCeN was validated from the perspective of its biological functions. For ICM-dysregulated LMM-CTs at each time-ordered level, based upon the mRNAs at those levels, significantly enriched Gene Ontology biological process (GO BP) terms were acquired with false discovery rate < 0.05 using the R package “clusterProfiler” , and the other was for differentiating ICM from DCM patients (denoted as ICM-DCM).For the identification of NF-ICM diagnostic biomarkers, 1271 significantly dysregulated ICM LMM-CTs were investigated, including 675 mRNAs, 85 miRNAs, and 97 lncRNAs. By applying our method, optimal NF-ICM biomarkers were then obtained. The results are shown in Table The identification of ICM-DCM diagnostic biomarkers underwent the same procedure. As a result, three panels of ICM-DCM biomarkers defined by two mRNAs , one miRNA (hsa-miR-545-5p), and two lncRNAs (LINC01089 and SDCBP2-AS1) were obtained . In the same way, one LMM-CT including TNRC6A, hsa-miR-3065-3p, and OTUD6B-AS1 was identified, showing both accuracy and an AUC value of 0.938 in the training set using LOOCV.To investigate the similarities and differences between ICM and DCM based on ceRNA mechanisms, this study proposed a novel computational approach for identifying dysregulated LMM-CTs in ICM and DCM, which integrated the dysregulation extent of gene expression, gene interactions, and the influence of the dysregulated LMM-CTs. The disease progression of both ICM and DCM was subsequently systematically compared and analyzed by constructing time-ordered dysregulated ceRNA networks. Additionally, multiple panels of diagnostic biomarkers associated with ICM and DCM were identified.Our study has concentrated on the origin and development of the disease. Therefore, ICM and DCM were both compared with NF samples. Time-series gene expression data are capable of providing more valuable information than steady-state, although this type of data for cardiomyopathy was unavailable up to this point. Based on the topological structure of the networks, time-ordered dysregulated ceRNA networks for ICM and DCM were constructed, and the rationality and stability of the networks were verified. This method did not require correction or standardization of expression values among different time points and conditions. Furthermore, the method was able to be combined with time-series sequencing data to reveal even more precise and clear biological processes during disease progression.Biological functions at each time-ordered level for DCM and ICM were analyzed. Based on the results, shared functions among different levels were not observed. However, certain functions did belong to the same functional category, and so some overlap existed among the time-ordered levels. Simultaneously, it was found that inflammation appeared in the early stage of ICM, whereas DCM did not exhibit this phenomenon. This observation, however, did not mean that the early stages of DCM have nothing to do with inflammation since viral myocarditis could lead to the initiation of DCM. One possible reason may be an absence of inflammation-induced DCM in enrolled patients. Additionally, the NF donor heart samples collected for the present study were taken from individuals who had experienced an acute and ultimately fatal event, and they were therefore different from the normal hearts. Due to the limitations of clinical heart biopsies, transcriptome analysis for the study of HF remains a challenge.The present study has also presented a multiple regression model for identifying LMM-CT-based biomarkers to distinguish ICM from DCM. However, owing to a lack of sample-matched mRNA, miRNA, and lncRNA expression data of ICM and DCM, the efficiencies of the identified LMM-CT biomarkers in the test set were not clear, and further studies are required. Our study has provided a strategy for identifying multiple types of combinations of molecules as diagnostic biomarkers, which not only considered the expression variation of the molecules but also reflected potential interactions among them. This idea can be applied to other diseases.In conclusion, we systematically investigated the shared and distinct biological features during ICM and DCM progression by constructing time-ordered dysregulated ceRNA networks and provided a new idea for the identification of multiple types of molecules combined as diagnostic biomarkers. Our results will shed new light on deciphering the underlying pathogenetic mechanisms of ICM and DCM, and provide a basis for developing etiology-specific therapies for HF patients in the future.https://www.ebi.ac.uk/arrayexpress/), which were derived from left ventricular myocardial tissue. The dataset included eight patients with ICM, eight patients with DCM, and eight NF individuals  and DIANA-LncBase 1136]1\docF is the Fisher transformation function, which makes the data approximately follow the normal distribution [rcase and rcontrol are the PCCs of gene expression in case and control samples, respectively.Secondly, the edge score was computed according to Eqs. –6)3\doc3\doc6)3\ribution , and rcaP-value), where the P-value is computed using Fisher’s exact test, which reflects the significance level of the miRNAs directly interacting with the lncRNAs that are enriched in SDE miRNAs (P < 0.01 and |log2FC | >1.2). Similarly, ISmRNAlncRNA|miRNA and ISmiR|mRNA were then computed.Thirdly, the influence score of the dysregulation of an LMM-CT on genes was calculated using Eq. 7\documenS of an LMM-CT was calculated by combining the node score, edge score, and the influence score as followsFinally, the dysregulated score P-value was defined as the proportion of randomly obtained LMM-CT scores larger than the real LMM-CT score, as shown in Eq. (In addition, the statistical significance of each LMM-CT was estimated using a permutation test. RNA labels of samples were randomly shuffled to construct a random LMM-CT, and the LMM-CT score was then recomputed. The above procedure was repeated 10,000 times, and the null distribution for the LMM-CT score was subsequently obtained. For each LMM-CT, an empirical n in Eq. 9\documenP-value < 0.05 were selected as the dysregulated LMM-CTs.In this study, LMM-CTs with a By combining the LMM-CTs in NF identified above, a ceRNA network was obtained. The largest net component was defined as the background ceRNA network in NF, which included 10,835 LMM-CTs consisting of 181 lncRNAs, 143 miRNAs, and 2810 mRNAs. To investigate disease progression, a previous method was employed to construct TO-BCeN in NF . Initiala1 and a2 represent regression coefficients, and c denotes the error. It is worth noting that the effects of interactions among multiple LMM-CTs were not taken into consideration for the sake of maintaining the simplicity of the analysis.In the present study, a method has been presented for identifying disease-associated dysregulated LMM-CTs based on the ceRNA mechanism. To further investigate the classification efficiency of these dysregulated LMM-CTs in distinguishing patients with ICM from those with DCM, a multiple regression model was proposed to yield a dysregulated score for each patient according to the ceRNA hypothesis. For one LMM-CT, the mRNA expression level was presumed to be affected both by the expression level of the miRNA and the interaction between the miRNA and lncRNA; therefore, the multiple regression was modeled according to Eq. 10\documea1, a2, and c under normal conditions using NF samples. For a patient and a given LMM-CT, an estimated mRNA expression value could be calculated by Eq. (As mentioned above, it was assumed that LMM-CTs exist in NF individuals and that a normal biological stable state is maintained. Therefore, it would be possible to obtain d by Eq.  using cod by Eq. 11\documeIn this study, two types of diagnostic biomarkers were identified: one was for distinguishing patients with ICM/DCM from the controls, and the other was for differentiating ICM from DCM patients.A random forest supervised classification model was used to select genes that were strongly associated with the disease diagnosis . At eachRandom forest and SVM algorithms were performed using the R packages “randomForest” and “e1071”, respectively. Classification accuracy and the AUC based on the LOOCV were applied to measure the performance.Supplementary legendsSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6


Laminin, one of the largest glycoproteins of the basement membrane, is an important component of the extracellular matrix. Functions of the basement membrane include regulation of cell signaling behaviors and structural support. Laminin plays a critical role in the regulation of insulin action in muscle, liver, and adipose tissue. The study mainly investigates an association between the change in serum laminin levels and insulin resistance and non-alcoholic hepatosteatosis.This prospective study included a total of 90 participants; 60 patients diagnosed with Grade 2–3 non-alcoholic hepatosteatosis and 30 age- and sex-matched healthy controls between December 2019 and December 2020. Routine laboratory tests including glucose, insulin, homeostatic model of assessment-insulin resistance (HOMA-IR), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and C-reactive protein and laminin levels were measured in the serum of the patient and control groups. Enzyme-linked immunosorbent assay was used for the measurement of laminin levels.The median serum laminin levels were lower in patients with hepatic steatosis, compared to the control group . In the patients with insulin resistance, median laminin levels were lower, regardless of the presence of non-alcoholic hepatosteatosis . There was a weak, negative correlation between the laminin levels and HOMA-IR.Our study results suggest that, although there is no exact link between laminin and non-alcoholic hepatosteatosis, serum laminin levels are lower in patients with insulin resistance by regulating the insulin effect through integrins. The ability of insulin to stimulate glycogen synthesis and suppress gluconeogenesis is diminished in the liver therebyincreasing hepatic glucose production , alcoholic and viral liver diseases), it was shown to be linked to the severity of liver damage and degree of fibrosis  ng/L, respectively; p = 0.003). In addition, the mean serum glucose, insulin, HOMA-IR, ALT, TG, and CRP levels were significantly higher and serum HDL-C levels were significantly lower in patients with non-alcoholic hepatosteatosis (p < 0.001 for both). However, there was no statistically significant difference in the mean AST and LDL-C levels between the groups . However, there was no significant difference in the median serum laminin levels between the patients without non-alcoholic hepatosteatosis and insulin resistance (Group A) and patients with non-alcoholic hepatosteatosis and without insulin resistance (Group B)   and these groups were compared with the control group (Group A). The median serum laminin level was significantly lower in patients with non-alcoholic hepatosteatosis with insulin resistance (Group C) compared to those without insulin resistance (Group B) . However, there was no significant correlation between serum laminin levels and non-alcoholic hepatosteatosis (r = − 0.038 p = 0.321) (Table The multivariate regression analysis revealed an inverse relationship between serum laminin, HOMA-IR, and LDL-C levels (Figure Non-alcoholic fatty liver disease is the leading cause of chronic liver disease in western countries . It may Previous studies demonstrated that laminin is a fibrosis biomarker in alcoholic liver disease, viral hepatitis, and NAFLD. In particular, high laminin levels were shown to be associated with the severity of fibrosis and hepatitis –10. In aIn the present study, we found no significant relationship between the laminin levels and non-alcoholic hepatosteatosis. Therefore, we cannot speculate that laminin is a biomarker fornon-alcoholic hepatosteatosis. This can be explained in two ways: liver parenchyma lacks laminin; however, in advanced stages of liver disease, laminin was shown to be progressively deposited following pathological processes such as capillarization of hepatic sinusoids and fibrosis , 14. TheThe interaction between the ECM and cells is of vital importance for all organs of the human body. ECM remodeling is associated with insulin resistance in several metabolic tissues. However, few studies addressed the link between ECM and glucose metabolism in the literature. Currently, it is still unclear how laminin contributes to alterations in insulin metabolism. One of the most widely adopted theories is that laminin regulates the effect of insulin through structures involved in integrin and integrin signalization . IntegriA review of the literature reveals no head-to-head clinical study showing the exact relationship between laminin and effects of insulin. However, in an experimental study, Hammar et al.  reportedThere are some limitations to this study. The diagnosis of non-alcoholic hepatosteatosis was made based on the ultrasonography, rather than liver biopsy. In addition, the sample size in the group with insulin resistance is limited to draw firm conclusions.The main strength of this study is that it is the first clinical study to investigate the relationship between serum laminin levels and insulin resistance. We believe that these findings will provide additional information forthe limited body of knowledge on this topic in the literature and an insight into the effects of laminin on the mechanism of insulin resistance.In conclusion, our study results suggest that although there is no exact link between laminin and non-alcoholic hepatosteatosis serum laminin levels are lower in patients with insulin resistance possibly by regulating the insulin effect through integrins. Targeting laminin may be beneficial for pathogenesis and associated complications of insulin resistance. However, further large scale, prospective studies are still warranted to draw a firm conclusion.


To compare the early operative outcome of TOF repair with three contemporary repair strategies of RVOTO repair i.e. TAP, Mono-cusp construction (MC) in TAP and pulmonary valve repair.Study is performed at Punjab Institute of Cardiology, Lahore from May 2016 to April 2020. Retrospective analysis of data was performed for patient who underwent TOF repair by three different strategies of RVOT repairs during TOF surgery based on z scoring for pulmonary valve annulus. Group-I underwent trans-annular patch repair, while Group-II and III underwent Mono-cusp repair with autologous pericardium and pulmonary valve repair respectively. Analysis of Variance (ANOVA) and Pearson Chi-Square (PCS) statistics were used to compare the three groups for numeric and categorical variables respectively. Post-hoc t-test and Bonferroni correction were performed for numeric data to compare two groups with each other. Chi-square test was used to perform comparison between groups for categorial variables.ANOVA for aortic cross clamp time, total CPB time, Post-operative mechanical ventilation time, ICU stay and hospital stay showed statistical difference among all three group with p-value less than 0.05 however post hoc T-test showed this variation is limited to post-operative mechanical ventilation only when groups compared with each other. PCS showed there was difference for incidence of difficult weaning from CPB when all three groups compared while there was no difference in operative mortality with p-value of 0.15. However, Group-II comparison with Group-I showed that weaning from CPB was superior in-Group-II with p-value of 0.016. Group-III showed the best statistics for all operative outcome variables among all three groups. Comparison of incidence of post-operative moderate pulmonary regurgitation before discharge between Group-II and Group-III showed significant difference with p-value of 0.0052.PV repair strategy should be employed for RVOT repair of TOF whenever feasible. MC repair showed fewer hours of postoperative mechanical ventilation and higher incidence of easy weaning from CPB when compared to TAP, however its impacts over ICU stay, Hospital stay and operative mortality is not profound in our TOF repair population. Tetralogy of Fallot (TOF) is characterized by ventricular septal defect (VSD), right ventricular outflow tract (RVOT) obstruction, Right ventricle (RV) have diversities hypertrophy and over-riding of aorta.3Severity of TOF is directly related to severity of PV annular hypoplasia. z-scoring is utilized as objective and scientific method to demonstrate the severity of PV annular hypoplasia in surgical practice. Classically, Z score of -2 or less signifies that annular preservation may result in significant residual RVOTO and thus mandate annuloplasty to enlarge RVOT for successful repair. Conversely, Z score of more than -2 favors strategy for PV preservation.7The objective of our study was to compare the early outcome of TOF repair with three contemporary repair strategies of RVOTO repair i.e. TAP, Mono-cusp construction in TAP and pulmonary valve preservation. The purpose is to define the beneficial impact of mono-cusp construction by comparing it to other two repair options.This study was conducted at Punjab Institute of Cardiology, Lahore. Study approval was taken from ethical committee and institute review board . Data was collected retrospectively from May 2016 to April 2020 for patients who underwent surgery for TOF repair. The patient file, operative theater record, intensive care record, preoperative echocardiography and postoperative echocardiography record were utilized to retrieve study related data. During our congenital cardiac surgery practice for above mentioned period of time, every patient of TOF underwent pre-operative echocardiography and cardiac catheterization as pre-operative work-up to clarify the anatomy of TOF and associated cardiac anomalies. We performed the Z score of pulmonary valve annulus for our patient population with online calculator based on Petersen et al study.Age (years), weight (kg), gender , aortic cross clamp time (mins), total cardiopulmonary bypass time , difficulty in weaning from CPB, post-operative ventilation time (hours), ICU stay (hours), hospital stay (Days) were retrieved from hospital record. Difficult weaning from CPB was defined as either need for higher inotropic support  or more than 30 mins of empty heart beating on CPB while initial attempt of liberation from CPB resulted in cardiac dilatation or combination of both. Operative notes were use to get fine operative details including RVOT repair strategy to segregate patient into different categories. Postoperative moderate pulmonary regurgitation was noted for Group-II and Group-III from pre-discharge trans-thoracic echocardiography. Microsoft excel 2016 was utilized to analyze the data. Frequency was measured for categorical data like gender, difficult weaning from CPB, and operative mortality. Mean and standards deviation of mean was measured for numeric data like age, cross clamp time, CPB time, Ventilation time, ICU stay and hospital stay etc. for all three groups. Single factor analysis of variance (ANOVA-test) was performed to find statistical significance  between the groups. P-value of 0.05 or less was specified as significant. Then we performed the post-hoc t-test that compares the statistical difference between two groups . We chose two-sample t-test of equal variance for the purpose. As there were three groups, so Bonferroni correction was used to find corrected post-hoc t-test p-value. For Bonferroni correction, P-value of 0.05 was divided by number of comparison groups that yield p-value of 0.016. So, p-value of 0.016 or less was considered significant for post-hoc t-test between two groups. Pearson chi-square test was utilized to see statistical difference among groups for gender difference, difficulty in weaning from CPB and mortality. Chi-square test was used to perform comparison between groups for categorical variables like weaning difficulty from CPB, mortality and moderate PR between Group-II and III. PCS test was not performed for moderate PR as Group-I has inherent free PR related to operative procedureThese results showed that MC repair do have some beneficial impact in TOF surgery when compared to TAP group with regards to early postoperative outcome i.e. Weaning from CPB, post-operative mechanical ventilation duration, ICU-stay, Hospital stay duration and operative mortality. However, this benefit is limit to ease in weaning from CPB and postoperative mechanical ventilation only when measure statistically by p-value. PV repair showed most impressive impact over early outcome when compared to TAP and MC repair.TAP and PV repair are two extreme strategies of RVOTO repair utilized for TOF surgery. Severe PV annular hypoplasia makes TAP as an inevitable choice particularly when Z score is very low. Higher Z score for PV annulus and good PV leaflets anatomy favors PV sparing for TOF surgery. Some surgical groups even try to preserve PV function even in borderline Z score of PV annulus and accept residual gradient across RVOT (up to 20-30 mmHg).Sasson L et al. had demonstrated that mono-cusp repair showed better clinical outcome in the form of shorter postoperative mechanical ventilation, short ICU & hospital stay in TOF repair when compared to TAP only with no impact over operative mortality.18Literature has shown a clear advantage of PV repair strategy of RVOT repair in TOF with much fewer postoperative hours of mechanical ventilation, shorter ICU & hospital stay and operative mortality.22PV repair strategy should be employed for RVOT repair of TOF whenever feasible. MC repair showed fewer hours of postoperative mechanical ventilation and higher incidence of easy weaning from CPB when compared to TAP, however its impact over ICU stay, Hospital stay and operative mortality is not profound in our TOF repair population.TW: Conceived, designed the research methodology, prepared this manuscript and is accountable for the originality of the research work.MZAA and KK: Did data analysis, helped in writing the manuscript and reviewed the manuscript.


This study aimed to investigate the morphometric alterations in the cortical and subcortical structures in multiple system atrophy (MSA) patients with mild cognitive impairment (MCI), and to explore the association with cognitive deficits.A total of 45 MSA patients  and 29 healthy controls were recruited. FreeSurfer software was used to analyze cortical thickness, and voxel-based morphometry was used to analyze the gray matter volumes. Cortical thickness and gray matter volume changes were correlated with cognitive scores.Compared to healthy controls, both MSA subgroups exhibited widespread morphology alterations of brain structures in the fronto-temporal regions. Direct comparison of MSA-MCI and MSA-only patients showed volume reduction in the left superior and middle temporal gyrus, while cortical thinning was found in the left middle and inferior temporal gyrus in MSA-MCI patients. Cortical thinning in the left middle temporal gyrus correlated with cognitive assessment and disease duration.Structural changes in the brain occur in MSA-MCI patients. The alteration of brain structure in the left temporal regions might be a biomarker of cognitive decline in MSA-MCI patients. Multiple system atrophy (MSA) is a progressive neuro-degenerative disease with a wide range of motor and non-motor symptoms. Current research, assessments, and interventions mainly focus on motor impairment in patients with MSA, such as gait ataxia, limb ataxia, and scanning dysarthria . HoweverStructural magnetic resonance imaging (MRI) is a powerful tool for the study of neuro-degenerative disease. Previous structural MRI studies mainly used voxel-based morphometry (VBM) measures to investigate gray matter (GM) abnormalities. Significant subcortical structural impairment was found in MSA patients with CI . FurtherFreeSurfer is a software suite utilizing surface-based morphological analysis that provides accurate information on cortical thickness and is considered the gold standard for measuring cortical thickness . CollobyThe purpose of our study was to use FreeSurfer analysis combined with VBM to investigate the brain morphological changes that might cause cognitive decline in MSA-MCI patients. The results might help to better understand the relationship between CI and brain structure in MSA patients. We hypothesized that the changes in brain structure, particularly in the fronto-temporal lobules, were associated with the cognitive decline in MSA-MCI patients.Forty-five patients [25 MSA-no cognitive impairment (NCI) and 20 MSA-MCI patients] from the neurology outpatient clinic at the First Affiliated Hospital of China Medical University were enroled. Patients were diagnosed by a movement disorder specialist with “probable MSA” according to the MSA diagnostic criteria  . FurtherCognitive status was assessed using the Montreal Cognitive Assessment (MoCA). We used the Movement Disorders Society Task Force criteria to assess MCI . For the2; flip angle, 12°; dist. Factor, 0.5; matrix size, 256 × 256; slice thickness, 1 mm; voxel size, 1.0 × 1.0 × 1.0 mm; slice number, 176.MRIs were acquired on a 3.0T MRI scanner  equipped with a 32-channel head coil located at the First Affiliated Hospital of China Medical University. High-resolution three-dimensional sagittal T1-weighted images were acquired in a magnetization-prepared rapid acquisition gradient-echo sequence with the following parameters: TR, 5,000 ms; TE, 2,960 ms; FOV, 256 × 256 mm1, was used for cortical reconstruction and thickness analysis on the three-dimensional T1-weighted images data. Details of the methodological have been described previously  registration method  was used to perform demographic, clinical, and cognitive data analysis. The Q-Q plot and the Kolmogorov-Smirnov test were used to check normal distribution assumption of the variables. The chi-square test, the two-sample p < 0.05 and false discovery rate (FDR) method for multiple comparisons was used with age, gender, education, and disease duration as covariates of no interest. If no results were identified through this correction, a less strict threshold of p < 0.01, Monte Carlo corrected, was performed. The whole-brain GM volumes obtained with SPM software were compared among MSA subgroups and the HC group, and post hoc analysis was performed to explore between-group GM volume differences with the total intracranial volume, age, gender, and education added as covariates. Multiple comparisons were performed using the family wise error (FWE) correction, and the cluster-level statistical threshold was set at p < 0.05. Considering that our data do not conform to a normal distribution, Spearman correlation analysis (SPSS 20.0) and the regression models (SPM12) were performed to investigate the correlation of cortical thickness measures and GM volumes with the clinical and cognitive variables. A significance threshold of p < 0.05 was set.Statistical analysis of cortical thickness was performed using FreeSurfer software. A general linear model was used to explore the differences of cortical thickness between the MSA subgroups and the HC group. The cluster-level statistical threshold was set at Forty-five MSA patients  and 29 HCs were enroled. There were no significant differences in age, sex, or level of education between groups. Both patient groups showed significantly lower MoCA scores compared with the HC group, while MSA-MCI patients showed significantly lower MoCA scores and higher UMSARSII scores compared with MSA-NCI patients, as expected. There were no significant differences between MSA-MCI and MSA-NCI patients in terms of UMSARSIII scores and duration of illness .p < 0.05 FDR). Compared to MSA-NCI patients, MSA-MCI patients showed cortical thinning in the left middle and inferior temporal gyrus (p < 0.01 Monte Carlo corrected)  .p < 0.05 FWE)  .In MSA-MCI patients, UMSARSII scores showed significant negative correlation with the extent of cortical thinning in the left precentral gyrus. Significant positive correlations between GM volume and MoCA scores were observed in the right superior and middle frontal gyrus. However, cortical thickness in the left middle temporal gyrus showed significant positive correlation with MoCA scores and negative correlation with duration of disease. No correlations were found with other clinical and cognitive deficits .To explore the contribution of whole-brain morphology alterations on CI in MSA-MCI patients, based on the results of FreeSurfer and VBM, we investigated the relationship between cortical and subcortical structures and clinical measurements. Compared to the HC group, MSA-MCI patients showed widespread structural alterations in the fronto-parieto-temporal lobule and the limbic system. In the direct comparison between MSA-MCI and MSA-NCI, we found that MSA-MCI patients showed extensive GM atrophy in left superior and middle temporal gyrus and cortical thinning in left middle and inferior temporal gyrus. We also observed that cortical thinning of the left middle temporal gyrus was associated with cognitive decline and disease duration in MSA-MCI patients, which was in line with our hypotheses.Several previous studies of a large sample of patients have confirmed the presence of cognitive impairment in MSA patients . Our resThe temporal lobule plays a key role in cognition. Previous functional MRI studies suggested that the left temporal regions were involved in many cognitive processes, including semantic aspects of comprehension  , languagr = 0.41; p = 0.07). Meanwhile no alterations were found in the frontal lobule in the direct comparison between MSA-MCI and MSA-NCI patients. Previous studies (Compared with the HC group, MSA-MCI patients also showed more widespread cortical thinning in the frontal lobule, however correlation results showed that the cortical thinning in the frontal lobule had no significant correlation with MoCA scores ( studies  have sho studies .Of note, the structural alterations in our MSA-MCI patients were mainly in the left rather than right hemisphere, and these results were observed in previous studies . One posThe present study has the following limitations. First, our sample size of patients with this rare condition is relatively small. Therefore, some of the differences we found in the regions between MSA-MCI and MSA-NCI did not exist after multiple comparison correction. Second, our study is a preliminary study on the cognitive impairment and morphological alterations of MSA-MCI patients. We did not examine the different effects of the brain on domain-specific cognitive changes. Future studies should focus on the relationship between various cognitive domains and brain structural alterations. Finally, we noticed that autonomic dysfunction might have an impact on the cognitive assessment in our study, so a rough assessment of autonomic function was achieved using UMSARSIII scales. However, more comprehensive and systematic quantitative tools, such as the Scopa-Aut questionnaire, would be helpful for future research.In conclusion, the present study suggests that cognitive decline due to temporal region alterations might be characteristic of MSA-MCI patients. Since the underlying mechanism and brain alterations of MSA-MCI patients are largely unknown, the results of our study may be helpful to improve the knowledge of health care professionals on the important role of the temporal lobe in the progression of this disease, and this finding may be helpful to the future study of MSA. Future studies need to conduct multiparametric MRI studies on sufficient sample of patients to better understand the brain alterations in MSA patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The First Affiliated Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.CC, QW, and GF contributed to the study conception and design. HYu revised important intellectual content. CC and QW were analyzed and interpreted the data. CC was drafted the manuscript. GF was critically revised the manuscript and involved in study design, data interpretation, manuscript revision, and final approval of the version to be submitted. HYa, MG, YL, and HH performed data acquisition. 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.


Background: Salt substitute, a strategy for salt reduction, has been shown to decrease blood pressure and the incidence of hypertension. However, whether its hypotensive effect will reduce long-term mortality remains unclear. Our study reported an exploratory follow-up of mortality outcomes from previous randomized controlled trial to assess the long-term effect of low-sodium salt on total and cardiovascular disease (CVD) mortality.Methods: Participants who completed a previous 3-year double-blind randomized controlled trial were followed up from 2009 to 2019 to collect mortality data. Multivariable Cox regression models were used to evaluate the association between low-sodium salt intervention and all-cause and CVD mortality.Results: Four hundred and forty participants completed the intervention trial, of which 428 participants had death outcome data recorded after 10 years follow-up: 209 in a salt substitute group and 219 in a normal salt group. Fifty participants died during follow-up, 25 died due to CVD. No significant differences in relative risks were found for all-cause mortality  and CVD mortality  in unadjusted analyses. After adjusted with age and alcohol drinking status, there were significant reductions for stroke mortality among all participants  and for CVD mortality  and stroke mortality  among hypertensive participants.Conclusions: Compared to normal salt, salt substitute might reduce the risk of CVD death, especially stroke among hypertensive patients. Our exploratory follow-up results provide potential evidence that low-sodium salt may be an accessible and effective strategy for prevention of CVD events, but definitive randomized controlled trials are warranted. According to the Global Burden of Disease (GBD) report, cardiovascular disease (CVD) is the leading cause of death globally, accounting for 17.8 million deaths in 2017 , 4. SaltSalt substitutes, as an existing salt reduction strategy available in industrialized China, are formulations where a proportion of the sodium is replaced with potassium or other element. Compared with normal salt with 100% sodium chloride, salt substitutes seek to decrease sodium intake without reducing the perceived total salt consumption, and thus avoid the inherently poor compliance typical of long-term behavioral intervention in salt restriction , 10. In Our previous randomized, double-blind, controlled study provided evidence that an appropriate salt substitute could lower blood pressure during a 3 year intervention , 14. WheOur study was an exploratory follow-up study with participants who completed the previous double-blind, randomized controlled trial. The previous trial explored the hypotensive effect of salt substitute. A detailed description of the previous trial has been reported previously , 14. Bria priori approval by the institution's human research committee.Subsequently, we have undertaken a 10-year observational study among the participants who completed the trial to examine the long-term effects of salt substitute on all-cause and CVD mortality. All surviving participants or relatives who provided information regarding deceased participants gave written informed consent. The institutional review board at the China Medical University approved the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in an During the post-intervention follow-up, village health workers reviewed participants by telephone semi-annually. Multiple sources were analyzed to determine the time and cause of death, including medical records, death certificates and symptoms, as reported by a spouse, sibling, or child, were also cross-checked with the death registration system, where possible. Two physicians blinded to treatment, assessed the underlying cause of death, as obtained from death certificates, and assigned a code according to the International Classification of Disease, Tenth Revision (ICD-10). Causes of death were divided into two broad categories: CVD death  and non-CVD death . The primary study endpoints were all-cause mortality and CVD mortality. The follow-up period started at the end of the intervention  lasting till death, loss to follow-up, or 30 April 2019, whichever occurred first.n) and percentage (%) for categorical variables. T-test and Chi-square test were used to compare differences in baseline characteristics. Cause-specific mortality for all-cause, CVD, and non-CVD deaths were calculated as incidence density and cumulative incidence. The efficacy of salt substitution on mortality was evaluated using absolute risk reduction (ARR), relative risk reduction (RRR), and number-needed-to-treat (NNT). Multivariate Cox proportional hazards models were used to obtain hazard ratios (HRs) and 95% confidence intervals (95% CIs) for mortality, adjusted for baseline age and alcohol consumption. All analyses were undertaken using SPSS statistical software . A 2-sided P < 0.05 was considered statistically significant.Data were presented as mean ± standard deviation (SD) for continuous variables and as number (P = 0.007). At the end of intervention period, differences were shown in medications use (P = 0.001) and SBP level (P = 0.049), and the change in SBP and DBP during the intervention period (2006–2009) differ significantly (P < 0.001) by groups.Of the 462 participants in the 2006 study, 224 were assigned to the salt substitute group and 238 to the normal salt group, with 440 participants completing the 3-year intervention. The principal reasons for participants lost to follow-up were those who moved beyond observation and others reluctant to follow the prescribed schedule. As 6 participants died during intervention and 6 participants could not be traced, valid follow-up information relating to death outcomes was obtained from 428 participants only, 209 in the salt substitute group and 219 in normal salt group . Charact2 = 0.53, P = 0.467, CVD: χ2 = 1.75, P = 0.186). A marginally significant difference in stroke mortality was seen in participants taking salt substitute vs. normal salt . A substantial risk reduction for stroke mortality was observed in the salt substitute group, as estimated by ARR and RRR. NNT for salt substitute intervention was 29. The non-CVD incidence of death per 1,000 person-years was 6.55 in the experimental group and 5.85 in the control group.During the follow-up period, 50 deaths  were recorded, as shown in P = 0.056; P = 0.032; Participants in the salt substitute group had improved survival for all-cause mortality and CVD-related mortality than controls. This protective effect was more obvious in hypertensive patients . In the + and K+ at the end of intervention was measured. Urine data were available for 311 participants , as shown in + excretion and Na+/K+ ratios were significantly different between groups after 3-year intervention, but no difference was identified for urinary excretion of Na+.To evaluate treatment safety, spot urinary samples were collected and the concentration of NaThe results of our previous RCT indicated salt substitute offered significant benefits for lowering SBP and DBP, with potential effects on hypertension , 14. HowSubstantial evidence supports salt restriction as an effective non-pharmacological intervention at the population level for blood pressure management and improved long-term cardiovascular outcomes , 15–17. Despite the long history of salt reduction research, associations between salt reduction and health outcomes remain at issue. In most studies, sodium intake was estimated form urinary sodium excretion. Follow-up of the famous TOHP  report sodium reduction may reduce long-term CVD events (10–15 years after intervention) and all-cause mortality (23–26 years after intervention), suggesting a direct linear relationship between habitual sodium intake and total deaths –22. MetaStroke is strongly associated with blood pressure-lowering intervention , with saThere is also evidence that a high-salt diet might be particularly harmful in hypertensive patients . The DASTo date, there are few reports of long-term trials evaluating the effect of sodium on clinical outcomes, primarily due to logistic and feasibility considerations. The longest intervention period of sodium reduction trials was 36 months , and theThe observed association between sodium reduction and total or CVD mortality can be explained by the “programming” effect. Geleijnse's demonstrated that infants given low sodium formula during their first 6 months had lower SBP than controls after 15 years follow-up, despite no corresponding difference in their urinary sodium excretion . This is+ in our study. Possible explanations include:  may be more motivated to try other health interventions, such as exercise, eating more fruit and vegetables, reducing smoking or alcohol consumption, etc. Unfortunately, such data could not be factored into our analysis. Secondly, because the information was collected by telephone interview, the blood pressure data, which have enormous implications for the progression of cardiovascular disease, were failed to collect in our follow-up study. Third, since over 70% of participants were hypertensive patients and the small number of deaths among those without hypertension, we could not evaluate properly if low-sodium salt reduced total and CVD mortality in the non-hypertensive population. Additionally, considering the low mortality level, we could only adjust baseline age and alcohol drinking status, which may affect the accuracy of our results.Our results provide evidence that replacing normal salt with a low-sodium salt substitute could be a tractable and effective strategy to prevent CVD events. Salt substitute, which typically lowers blood pressure and prevents hypertension, may thus reduce CVD mortality, especially in hypertensive participants. Well-designed multi-center RCTs accessing large, properly-stratified patient populations will be required to elucidate properly and completely the effects of salt substitution on cardiovascular outcomes in the population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the institutional review board at the China Medical University. The patients/participants provided their written informed consent to participate in this study.BZ contributed to the conception and design of the work. HS, BM, and XW contributed to the acquisition, analysis, or interpretation of data for the work. HS drafted the manuscript. HW revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.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.


Imaging is the visual representation of structures and functions of objects, such as biological molecules, biological ultrastructures, tissues, and the spatial organizations of the objects. It is also an indispensable step towards diagnostics and therapeutics in modern medicine. For example, during the current pandemic caused by COVID-19, CT-scans have been used, in addition to nucleic acid detection, as a main criterion for diagnostics. Unlike computers, the human brain has a remarkably strong ability to understand and interpret the information obtained from imaging data, more so than from interpreting numerical or textual data. On the other hand, AI methods may produce more objective and highly reproducible analysis results with increased automation. Therefore, it is beneficial to develop AI methods to complement manual image analysis.Imaging is playing an increasingly significant role in both biological and biomedical sciences. With technologies including optical microscopy, fluorescence microscopy, electron tomography, nuclear magnetic resonance, single particle cryo-EM, and X-ray crystallography, biological imaging has provided rich information about biological systems and molecules at various resolutions, all the way from tissue-level, to cellular-level, to organelle-level, to macromolecular-level, to small-molecular-level, and to atomic-level. Imaging also has many diagnostic and therapeutic applications in medicine with different modalities, such as ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and optical coherence tomography (OCT). Such technologies can provide fast, non-invasive, painless and direct information to clinicians and physicians, which is critical to not only diagnosis, but also prognosis and treatment.With the recent development of AI technologies, especially deep learning, the frontiers on biological and biomedical imaging have been greatly advanced. In this Research Topic, we have collected 16 high quality works on developing or applying state-of-the-art AI techniques for processing, information mining, integrating, diagnosing, comparing, and reviewing biological and biomedical imaging, with their applications in biology, diagnostics and therapeutics.There are in total 16 papers accepted by this Research Topic. Each paper was handled by one guest editor and reviewed by at least two reviewers. We are very grateful to the reviewers in helping us select these high-quality papers for this Research Topic.Seong et al.). Radiology has been a leading technology of digital transformation in healthcare, which is again at the crossroad for the next generation of transformation, possibly evolving as a one-stop integrated diagnostic service. AI promises to offer radiology new powerful new digital tools to facilitate the next transformation. This paper proposes three pathways for AI’s role in radiology: (1) improving the performance of CAD, (2) improving the productivity of radiology service by AI-assisted workflow, and (3) developing radiomics that integrate the data from radiology, pathology, and genomics to facilitate the emergence of a new integrated diagnostic service.The accepted papers focus on developing AI algorithms and systems to process, analyze, interpret, and mine from both biomedical and biological imaging data. We begin by providing a thought-provoking perspective on the future of radiology diagnostic service , which consists of two modules: a hybrid nested U-shaped network (H-part) and a hybrid attention block (A-part). H-part combines a nested multi-depth U-shaped network and a dense network with full resolution to capture more effective features. A-part is used to explore attention information and build correlations between different pixels. With these two modules, Han-Net extracts discriminative features, which effectively segment the boundaries of not only complex and diverse nuclei but also small and dense nuclei. The comparison in a publicly available multi-organ dataset shows that the proposed model achieves the state-of-the-art performance compared to other models.Yang et al. proposed a data-driven diagnostic model for hip dysplasia. Angles including CE, sharp, and Tonnis angle which are commonly measured in clinical diagnosis, are automatically obtained. Samples, each of which consists of these three angle values, are used for clustering according to their densities in a descending order. A three-dimensional normal distribution derived from the cluster is built and regarded as the parametric model for diagnosis of hip dysplasia.We further accepted two papers on X-ray data analysis. Li et al. proposed an interpretable method called Deetal-Perio to predict the severity degree of periodontitis in dental panoramic radiographs. In their method, alveolar bone loss (ABL), the clinical hallmark for periodontitis diagnosis, could be interpreted as the key feature. To calculate ABL, they also proposed a method for teeth numbering and segmentation. First, Deetal-Perio segments and indexes the individual tooth via Mask R-CNN combined with a novel calibration method. Next, Deetal-Perio segments the contour of the alveolar bone and calculates a ratio for individual tooth to represent ABL. Finally, Deetal-Perio predicts the severity degree of periodontitis given the ratios of all the teeth.Strobel et al. developed a semi-automated method that leverages machine learning to identify and quantify vascular metrics in an angiogenesis model imaged with different modalities. Their software, BioSegment, is designed to make high throughput vascular density measurements of fluorescent or phase contrast images.We also accepted one paper on quantifying vascular density in tissue engineered constructs. Dou et al. aimed to test whether chest CT manifestation of 2019 novel coronavirus (COVID-19) can be differentiated by a radiologist or a computer-based CT image analysis system. They conducted a retrospective case-control study that included 52 laboratory-confirmed COVID-19 patients and 80 non-COVID-19 viral pneumonia patients. Their results do not support CT findings replacing microbiological diagnosis as a critical criterion for COVID-19 diagnosis.We then present papers on 3D biomedical imaging data. We start from two papers on CT data analysis. Cai et al. developed and validated a radiomics-based nomogram to predict the prognosis of colorectal cancer (CRC). A total of 381 patients with colorectal cancer were enrolled and radiomic features were extracted from the vein phase of preoperative computed tomography. Their results show that radiomics score derived from the preoperative CT image was an independent prognostic factor and could be a complement to the current staging strategies of colorectal cancer.Fan et al. proposed a framework of a 3D-Mask region-based convolutional neural network (3D-Mask RCNN) computer-aided diagnosis (CAD) system for mass detection and segmentation with a comparative analysis of performance on patient subgroups with different clinicopathological characteristics. The results suggest that the 3D-Mask RCNN CAD framework has advantages over 2D-based mass detection on both the whole data and subgroups with different characteristics.We further accepted four papers on MRI data analysis. Fan et al. predicted responses to NACT in breast cancer by analyzing early changes in tumor heterogeneity modeled by longitudinal dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Their results suggested that changes in DCE-MRI features that reflect a reduction in tumor heterogeneity following NACT could provide early prediction of breast tumor response.Wang et al. proposed a novel model structure to capture 3D MRI images’ essential information and converted them into lower dimensions. The novel CNN model they proposed could automatically differentiate the rare NMOSD from MS, especially, our model showed better performance than traditional 3D CNN models.Gu et al. proposed a multi-head self-attention model (MSAM). By integrating the self-attention mechanism and multilayer perceptron method, the MSAM offers a promising tool to enhance the classification of Temporal Lobe Epilepsy (TLE) subtypes. The robustness of MSAM is extensively assessed with various ablation tests, which demonstrates the effectiveness and generalizability of the proposed approach.Kong et al. proposed a multi-task framework, called the multi-task classification and segmentation network (MTCSN), to achieve joint learning of clearness degree and tissue semantic segmentation. Extensive experiments and ablation studies report the significant performance gains of the MTCSN over state-of-the-art methods.We then included a paper on developing diagnostic algorithm for capsule endoscopy, which is a leading diagnostic tool for small bowel lesions. Huang et al. aimed to provide potential tactile perception methods for MIS by reviewing literatures on tactile sensing in MIS and literatures on industrial robotic tactile perception technologies, especially AI methods on tactile images.We further accepted a paper on reviewing tactile perception technologies on minimally invasive surgery (MIS), which has been the preferred surgery approach owing to its advantages over conventional open surgery. As a major limitation, the lack of tactile perception impairs the ability of surgeons in tissue distinction and maneuvers. Zhou et al. developed a one-shot learning framework, called cryo-ET one-shot network (COS-Net), for simultaneous classification of macromolecular structure and generation of the voxel-level 3D segmentation, using only one training sample per class, from cryo-electron tomography data. Their experimental results demonstrated that COS-Net could efficiently classify macromolecular structures with small amounts of samples and produce accurate 3D segmentation at the same time.We finish our collection of papers by including one paper on biological imaging. To conclude, we thank the authors and the reviewers for their contribution to this Research Topic. We are confident that the collection of articles in this Research Topic will serve as an inspiring compendium for future AI advancement and deployment in biomedical and biological imaging fields.


Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition. Nonself recognition is a prerequisite for the resilient mycelial network that makes up a fungal individual. This network is built by a set of growing hyphal tips that explore their environment in search of resources. Eventually, this growth results in the meeting of two hyphae, whether originating from the same individual or not. If these tips are from the same individual, then fusion will increase the efficiency of the network , while iSystems of nonself recognition are not unique to fungi. Other multicellular organisms such as plants and animals also have such systems, often built on similar molecular pathways . This maIn basidiomycete fungal species, a SI phenotype is consistently observed between heterokaryons . Thus, aTypically, two randomly selected ascomycete individuals from a wild population will show SI following anastomosis. The known molecular determinants of nonself recognition in ascomycete systems are generally based on direct protein interactions . Since aNeurospora species, the mating locus itself acts as a nonself recognition locus, dependent on the TOL protein. During fruiting body initiation, TOL is downregulated in the sexual structures, allowing both mating types to be co-expressed within specific tissues [However, cell fusion between genetically different ascomycete homokaryons should not always result in SI, as fusion is necessary during sexual reproduction. In ascomycete species following fertilization both haploid nuclei briefly co-exist in a heterokaryotic state until meiosis. During this stage, clearly cell death should be prevented despite the presence of two genomes. One possible solution is to reduce the concentration of incompatible proteins by dilution of the cytoplasm of one partner. Mating in ascomycete fungi is typically anisogamous since fertilization often involves male spermatia/conidia donating a nucleus with limited cytoplasm, preventing cytoplasmic mixing and subsequent lethal protein-protein interactions. A second solution is the active toleration of otherwise lethal protein combinations. In several  tissues ,26.Based on the above concepts, a population of an ascomycete species will be polymorphic at a number of loci, either allelic or non-allelic, with each locus being capable of triggering nonself recognition, resulting in SI. The number of polymorphic loci has been experimentally determined for only a few species, but it appears that there can be between 5 and 11 loci responsible . HoweverRhizoctonia solani [Species of basidiomycetes, particularly the mushroom-forming groups, present a challenge for the existing paradigm of fungal nonself recognition. These fungi alternate between a monokaryotic state formed after meiosis and a persistent heterokaryotic state formed from the fusion of two homokaryons, in addition to new outgrowth being heterokaryotic. This outcrossing is facilitated by the extraordinarily high numbers of mating types , ensurina solani ,45.Neurospora sp. to tolerate the brief heterokaryotic phase is through suppressing the action of the tol gene product, which otherwise triggers nonself recognition due to the mating-type alleles [tol expression can be restricted to a specific time and place in the sexual cycle. This reduced tol expression, in theory, permits the spread of parasitic elements, however, the production of the specialized female structures  prior to fertilization limits the potential for the spread of these parasitic elements. It has been shown in Aspergillus nidulans that the fertilizing nucleus can spread to the Hülle cells, accessory multinucleate cells, but only when strains are somatically compatible, indicating that SI limits this spread [tol is not feasible, since prevention of recognition during the heterokaryotic stage is not compatible with the fact that incompatibility occurs between heterokaryons.To achieve near-universal sexual compatibility, any somatic incompatibility between the homokaryons must be overcome . The com alleles . This sus spread ,49. As tIn addition to heterokaryon formation during basidiomycete matings, models of nonself recognition must be compatible with matings between heterokaryons and homokaryons, so-called Buller matings ,51. ThesNeurospora tetrasperma and Podospora anserina have nuclei of both mating types, and in these species, SI occurs between the stable heterokaryons, between homokaryons, as well as between heterokaryons and homokaryons. As long as incompatible protein products are expressed, somatic incompatibility results following hyphal fusion. Due to the pseudohomothallic lifecycle , field isolates of these species are, as a rule, heterokaryotic and highly inbred, with little or no het-gene differences within a heterokaryon, and interactions in nature between homokaryons are likely rare.Somatic incompatibility in ascomycete fungi generally occurs between homokaryons. However, in some pseudohomothallic ascomycete species, incompatibility also functions between heterokaryons. Isolates of N. crassa is a primary ascomycete model for fungal nonself recognition, it seems logical to first examine the closely related N. tetrasperma, which remains heterokaryotic throughout its entire lifecycle. Members of this species produce heterokaryotic conidia, as well as producing four heterokaryotic ascospores per ascus instead of the standard eight homokaryotic ascospores. Thus, the two genomes reside in the same cell continuously, although homokaryotic ascospores are produced at a low rate. It is then interesting to consider how these organisms handle the complications of incompatibility from the co-habitation of two genomes. N. tetrasperma avoids the problem of mating-type associated SI by having inactive tol alleles [N. tetrasperma, SI between homokaryons is not fundamentally different from SI between heterokaryons.As  alleles . Investi alleles . Further alleles ,56. This alleles . Interes alleles . Thus, iP. anserina, may also inform a model for basidiomycete nonself recognition. Several incompatibility systems from this species have been characterized, particularly the prion-forming het-s [het-S which contains a HeLO domain that when exposed causes holes in the plasma membrane, and het-s, which contains a harmless HeLO domain [het-s allele can transition from a harmless non-prion form into the HET-s prion form which can trigger the HET-S protein to cause death [het-s/het-s heterokaryons producing only HET-s or HET-s* are tolerated since they contain a harmless HeLO domain. Additionally, homozygous het-S/het-S heterokaryons are viable since the HET-S is folded to hide the toxic HeLO domain. Heterozygotes het-s/het-S producing both HET-s* and HET-S are tolerated, as the rare transitions of HET-s* to HET-s/HET-S result in local cell death preventing prion propagation while the other cells that have remained HET-s*/HET-S remain viable [het-s/het-s heterokaryons, HET-s* will eventually transform to the prion form and becomes HET-s/HET-s. Such a heterokaryon then triggers cell death if it encounters an individual that produces HET-S, whether that individual is het-S/het-S or het-S/het-s. Thus, such a system can create incompatibility between heterokaryons as well as between homokaryons, without triggering incompatibility within a heterokaryotic mycelium.The other model system for ascomycete SI, ng het-s . This syO domain  Figure 1se death . Homozygn viable . In effen viable ,60. HoweP. anserina may be found in the het-V/het-V1 incompatibility system [het-V ‘allele’ is made of two genes: PaMt1, encoding a methyltransferase (writer), and PaMt2, encoding a protein with 2 domains MLKL  and TUDOR (reader: recognizes methylation status of target) [het-V1 is a null allele, with neither PaMt1 nor PaMt2. If the reader (TUDOR domain of PaMT2) senses a lack of methylation from the writer (methyltransferase of PaMT1) in certain target proteins, it directs cell death using the MLKL domain. Timely co-expression of reader and writer is analogous to restriction-modification in bacteria, with no cell death since all target molecules will receive the protective modification [het-V and het-V1 homokaryons results in an SI reaction, because the target proteins in het-V1 cells that are not methylated, will subsequently be recognized by the reader (TUDOR) leading to cell death [het-V/het-V1 system, co-expression neutralizes the antagonistic alleles. Therefore, a heterokaryotic progeny het-V/het-V1 is stable because the target proteins will be sufficiently methylated, preventing PaMt2 from triggering cell death. This het-V/het-V1 heterokaryon would have a Het-V phenotype and be compatible with het-V/het-V heterokaryons, incompatibility would only result from such individual with Het-V phenotype to a het-V1/het-V1 individual, highlighting the asymmetry in the het-V/het-V1 system [het-V/het-V1 system allows for SI to occur between heterokaryons (as well as between homokaryons), but heterokaryons are tolerated, an important condition of a basidiomycete model. We note that these properties result from the post-translational modification of proteins in the het-V system, and similar modifications to other cytoplasmic molecules would likely have similar results.A second model from y system ,62,63. I target) . The altfication . A confrll death . In thisWe now explore the implications for the different known nonself recognition systems for the typical basidiomycete lifecycle. We consider the lifecycle to include mating between unrelated homokaryons, and reciprocal nuclear migration between homokaryons that are sexually compatible as determined by unlinked mating loci. For the heterokaryotic stage, we assume heterokaryons are incompatible with other heterokaryons, but that nuclear migration from Buller matings is possible. The challenge is how we can explain that SI between two nuclei is avoided in a heterokaryotic cell, which would prevent their stable coexistence during the main stage of the basidiomycete lifecycle.Neurospora tetrasperma [Direct interactions between two protein products from two alleles, whether or not from homologous genes, are not compatible with the heterokaryotic lifecycle. The fusion of two distinct basidiomycete homokaryons would trigger nonself recognition directly inside the heterokaryon. The observation that homokaryons are generally sexually compatible rules out the possibility that mates are chosen based on compatibility at nonself recognition loci. This makes basidiomycete biology fundamentally different from the heterokaryotic rasperma ,55. Whiltol system of N. crassa. This seems like an attractive option since it allows the fusion of unrelated homokaryons, as they would not express the incompatible gene products, or not express part of the downstream pathway. This would allow for reciprocal nuclear migration. However, once nuclear migration is complete, the now dikaryotic cells would express the incompatible gene products, leading to the death of all dikaryotic cells. It could be possible that only one allele was expressed: allele-specific expression similar to genomic imprinting in plants/animals. Such a system would require precise regulation since a single locus that evades this imprinting would trigger cell death. Additionally, such a mechanism is not consistent with observations of nonself recognition in the fusion cells of sexually compatible homokaryons [Another possibility is the presence of an allelic or non-allelic system that is only expressed at the heterokaryon stage, using a mechanism opposite to the okaryons ,44.het-V/het-V1 system of the P. anserina system discussed above in Perhaps a more promising model is found in a post-translational modification system. In such a system, heterokaryons are stable since the writers from the two nuclei modify all the target proteins in the cytoplasm, preventing the corresponding readers from triggering cell death. Such a system would be similar to the The above system could be modified by differential expression of the reader, writer, target, or a mix of these three. Limiting the expression of the target or the reader to the heterokaryon would prevent incompatibilities between homokaryons, but as discussed above, nonself recognition between homokaryons may still be compatible with the basidiomycete lifecycle. Furthermore, the targets of non-self-recognition are often essential genes, with other functional roles . As suchhet-V/het-V1 system, where allelic differences within a heterokaryon are tolerated but incompatibility occurs between heterokaryons based on post-translational modification of cytoplasmic proteins. We consider if such a reader/writer system is consistent with what we know of basidiomycete fungi. We include nuclear migration during mating as a potential explanation for two seemingly conflicting phenomena: Occurrence of cell death, and the formation of a heterokaryon. We first assume a first locus X which encodes the reader and the writer genes, with three alleles; XA, XB, and XC. The genes of XA both modify a set of proteins  and an AC (XAXC) heterokaryon will lead to SI. Inside the AB heterokaryon, for example,  in genetic studies may not be as neutral as assumed. Further experiments are necessary to test if this model is compatible with the genetics and mechanism of SI in basidiomycete fungi.