Furthermore, soluble micrograft treatment leads to increased expression and posttranslational phosphorylation of Fra-1 and c-Jun, resulting in the upregulation of wound healing-associated genes mainly involved in the regulation of cell migration. Collectively, our work provides insights into the molecular mechanisms behind the cell-free micrograft treatment, which might contribute to future advances in wound repair therapies.Impaired wound healing and tissue regeneration have severe consequences on the patient's quality of life. Micrograft therapies are emerging as promising and affordable alternatives to improve skin regeneration by enhancing the endogenous wound repair processes. However, the molecular mechanisms underpinning the beneficial effects of the micrograft treatments remain largely unknown. In this study, we identified the active protein-1 (AP-1) member Fos-related antigen-1 (Fra-1) to play a central role in the extracellular signal-regulated kinase- (ERK-) mediated enhanced cell migratory capacity of soluble micrograft-treated mouse adult fibroblasts and in the human keratinocyte cell model. Accordingly, we show that increased micrograft-dependent  The skin is the largest organ of the human body providing protection, immune defense, and sensation. Traumatic injuries and impaired wound healing greatly affect the quality of life of patients and eventually contribute to mortality. Therefore, optimal tissue regeneration and proper wound management are required to maintain skin integrity.β signaling and the classical transcription factor NF-κB have been described as regulators which elicit the immune response as the initial step of the wound healing process. This results in the immune cell and platelet-derived production of a plethora of growth factors including transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) at the site of injury . AP. APin vi Figures . Similar Figures .Interestingly, transwell migration assay of both MG-treated fibroblasts and keratinocytes showed significantly reduced cell migration upon AP-1 inhibition  assay in the presence of the AP-1 inhibitor T-5224. Interestingly, no changes in cell cycle phases were observed in MG-treated fibroblasts and the overall proliferation rate remained unchanged . In contin vitro scratch assay to the MG solution treatment with and without AP-1 inhibition. The conditioned medium collected from these conditions was then used to perform a fluorescence-based assay to determine the enzymatic activity of the MMPs. Remarkably, enzymatic activity was greatly hindered upon AP-1 inhibition in fibroblasts and keratinocytes  remodeling represent crucial events for successful wound healing. Therefore, we aimed to assess the impact of AP-1 activity on the enzymatic function of MMPs. Recently, it has been shown that essential wound healing-associated genes, such as cytokines and MMPs, were strongly upregulated upon AMG treatment . Upon tr Figures . These fWound healing processes enhanced by the soluble MG have been shown to be induced in an ERK-dependent manner . To furtVegfa and Flt1) were altered by this inhibition, a multitude of genes involved in cell migration were significantly affected upon treatment with PD0325901, confirming that the soluble MG treatment mainly increased cell migration. Additionally, high levels of Fosl1 and Junb were downregulated after MEK inhibition, possibly linking this reduction to the changes in cell migration.Interestingly, upon ERK inhibition in MG-treated cells, reduced levels of pFra-1/pFRA-1 were observed while pJun/pJUN did not decrease . This coin vitro and in vivo [As skin injuries and impaired tissue regeneration have drastic financial and medical consequences for patients, micrografts (MG) provide an affordable treatment to aid the endogenous wound healing. Although multiple MG systems have been shown to be successful in clinical trials, the molecular mechanisms behind these techniques remain unclear . Previou in vivo . HoweverCAAT enhancer-binding protein gamma (C/EBPγ) silencing [Forkhead box protein C2 (Foxc2) is a known regulator of angiogenesis in both wound healing and oral squamous cell carcinoma [Fosl1.In the present work, we identified AP-1 among other transcription factors responsible for establishing a regenerative transcriptional profile in both fibroblasts  and kerailencing . Aside farcinoma , 31. HowFosl1/FOSL1 transcript levels and phosphorylated Fra-1 and FRA-1 contribute to an elevated AP-1 activity in both fibroblasts and keratinocytes  phosphorylated by ERK enables its binding together with SRE-binding factor (SRF) to serum response elements (SRE) in the promoter region of nd c-Jun –41. In and c-Jun , 43. Stund c-Jun , 44. Simnd c-Jun . In addind c-Jun , 47. In wounding . FinallyFosl1, resulting in a higher amount of the transcription factor Fra-1/AP-1. This in turn becomes posttranscriptionally phosphorylated by the active ERK signaling. Phosphorylated AP-1 subsequently triggers a migratory phenotype in keratinocytes and fibroblasts through the activation of MMP activity and a transcription program favoring chemotaxis and cell motility. Although other signaling pathways and transcription factors are likely to contribute to this phenotype, we show that inhibition of both ERK and AP-1 is sufficient to completely revert in vitro wound closure by the soluble MG treatment. Altogether, our results highlight the crucial role of ERK-dependent transcriptional activity of AP-1 in coordinating the beneficial effects of the MG treatment, opening new opportunities for future wound healing studies.Collectively, we propose a mode of action behind the accelerated wound healing using the cell-free MG treatment . Our fin


Disease-associated mutations in the FUS gene were sufficient to disrupt formation of the FUS-SFPQ complex, resulting in deregulated alternative splicing of MAPT pre-mRNA and a consequent increase in the 4-repeat (4R)-tau/3-repeat (3R)-tau (4R-T/3R-T) ratio, ultimately leading to neurodegeneration that recapitulated key aspects of FTLD , amyotrophic lateral sclerosis (ALS), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) have distinct clinical features from presenting symptoms through to management. However, accumulating evidence suggests some convergence in molecular and genetic pathology, which may argue against their taxonomy as completely distinct entities. Indeed these disorders are often referred to as the FTLD spectrum, for which fused in sarcoma (FUS), transactive response (TAR) DNA-binding protein 43 (TDP-43), and tau are pathological hallmarks  context. Notably, in their previous study, Ishigaki and co-workers demonstrated that both FUS-silenced and SFPQ-silenced mice similarly exhibited increased 4R-T/3R-T ratios along with FTLD-like behaviour, hippocampal neuronal loss and phosphorylated tau accumulation  protein aggregates. However, deregulation of RBPs such as TDP-43, SFPQ and FUS, and other aspects of RNA metabolism, are increasingly recognized as crucial players in these disorders. The mechanisms by which these defects in protein homeostasis and RNA metabolism conspire to cause neuronal degeneration are of key importance to resolve. Indeed, a common site of molecular convergence here is the ribonucleoprotein (RNP) complex, which is composed of both RNA and proteins (RBPs). RBPs implicated in FTLD spectrum disorders commonly contain prion-like low complexity domains (LCDs) and intrinsically disordered regions, which render them aggregation-prone. These RBPs possess the important physiological capacity to undergo liquid-liquid phase separation (LLPS), which refers to the condensation of molecules into liquid-like non-membrane delimited organelles (the aformentioned nuclear paraspeckles are an example of this phenomenon). LLPS likely occurs through tightly regulated prion-like polymerization driven by weak, transient molecular interactions between LCDs and other multivalent protein or RNA interaction domains. LLPS can be regulated by specific protein:protein, protein:RNA, and/or RNA:RNA interactions and is a molecular principle governing transient intracellular compartmentalization. LCD mutations can promote the fibrillization of molecular assemblies from  liquid-like droplets. Such perturbation in the kinetics of LLPS (i.e. hyperassembly and/or defective disassembly) may ultimately lead to some of the most recognized features of FTLD spectrum disorders including insoluble protein aggregates within the cytoplasm. Additionally, deregulated LLPS may lead to the sequestration of RBPs , in turn causing their functional deficiency and the misregulation of key splicing events, such as Specifically, future studies might aim to extend the important findings by Ishigaki and co-workers by addressing the potential relationship between spatial dissociation  of RBPs and deregulated LLPS. Another key consideration is the cell type specificity of such molecular events, particularly with respect to glia given their well recognized roles in neurodegeneration. Furthermore, it would be important to understand whether the molecular phenomena reported in this study relate in some way to the increasingly recognized prion-like spread of pathology between cells in these disorders (


Background: The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. Methods: We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR.Results: In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs.Conclusions: The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.  To identify patients and carriers with SARS-CoV-2 infection, widespread diagnostic testing is of paramount importance. Among other factors, the diagnostic performance of applicable tests depends on the clinical stage of disease and the quality of harvested material. Currently, the most commonly applied method in SARS-CoV-2 testing is using swab material harvested through the nasopharyngeal route or deep pharyngeal brushing. Both procedures must be performed by sufficiently trained healthcare workers (HCWs) and may result in sampling discomfort for the patients and potential exposition of HCW to contagious droplets or even aerosols. The pre-analytical value of different sampling methods and locations have yet to be evaluated. Copan eSwabs and dry swabs  were used to simultaneously brush mucosal cells at different locations of the pharynx Figure 1  in hospiThe pharyngeal lavage was harvested by providing the patient with a receptacle containing 10 mL of 0.9% saline solution. The patients were instructed to gargle intensely with the solution for 10 seconds and then spit the recovered liquid into the same receptacle, immediately followed by closure and transport to the laboratory. Pharyngeal lavage samples were analyzed directly after arrival or stored at 4°C and re-analyzed after 48 h (stability testing).All procedures and material extractions were performed by a single, well-trained, experienced investigator. Within a few minutes, the following procedures were accomplished: nasopharyngeal eSwab left; nasopharyngeal dry swab right; buccal eSwab; eSwab of the palatoglossal arch, eSwab of the left posterior pharyngeal wall ; dry swab of the right posterior pharyngeal wall ; eSwab of the left posterior pharyngeal wall left (brushing); dry swab of the right posterior pharyngeal wall (brushing), Saliva (patient spits into a receptacle); pharyngeal lavage; pharyngeal lavage 5 minutes after 10); pharyngeal lavage after eating and drinking.Additionally, we performed 23 paired-tests of nasopharygeal eSwabs and pharyngeal lavage in known COVID-19 patients with early-stage SARS-CoV-2 infection as defined by a previous RT-PCR with a Ct value <30 within the past 24 hours.Furthermore, eSwab samples from 8 randomly chosen hospitalized COVID-19 patients were resuspended in DMEM medium supplemented with 1% penicillin/streptomycin and immediately subjected to VERO cell transduction assays within 4 hours. To detect SARS-CoV-2 infection, either Copan Liquid Amies eSwabs or conventional dry swabs as well as pharyngeal lavage were subjected to cellular lysis and RNA extraction, followed by RT-PCR. RNA extraction was performed on the CyBio Felix 96-well plate system , and real-time RT-PCR was conducted using LightCycler Multiplex RNA Virus Master Mix on a Lightcycler 480 RT system  or a ViiA7 system .in vitro transcribed E gene RNA), as well as SARS-CoV-1 strain Frankfurt 1 RNA control, and a SARS-CoV-2 strain RNA control (European Virus Archive GLOBAL). EAV extraction control  was used as internal PCR control. For all analyses, a Sarbecovirus-specific LightMix Modular SARS-CoV COVID-19) E gene assay was used   or  or 5] orThe cell culture analysis showed no correlation between CPE and RT-PCR positivity. This indicates that the observed CPEs are not caused by SARS-CoV-2 infection of the Vero cells. The BetaCoV/Germany/BavPat1/2020 transduced positive-control strain showed a strong correlation of 100% with clear cut CPEs and very low Ct-Values of SARS-CoV2-PCR. The positive RT-PCR results with high Ct-Values in 3 of the supernatants may be due to remaining inactive virus from the original patient swab. In agreement with recent reports, the results showed that specimens of patients with low SARS-CoV-2 concentrations (Ct >30) are unable to infect cell cultures [The authors declare that they have no competing interests.We would like to thank the brave nurses of the Clinic for Infectiology in our hospital for their untiring commitment during the difficult pandemic times.The authors did not receive any external fundingRalph Wendt and Olaf Nickel have shared first authorship.


Myocardial hemorrhage is a common complication following reperfusion of ST-segment-elevation acute myocardial infarction (MI). Although its presence is clearly related to infarct size, at present it is unknown whether post-reperfusion hemorrhage affects left ventricular (LV) remodeling. Magnetic resonance imaging (MRI) can be used to identify myocardial infarction, myocardial hemorrhage and microvascular obstruction (MVO), as well as measure LV volumes, function and mass.Ninety-eight patients  with MI reperfused with percutaneous coronary intervention (PCI) were studied in the first week and at 4 months after the event. T2-weighted MRI was used to differentiate between hemorrhagic  and non-hemorrhagic infarcts . MVO and infarct size were determined on contrast-enhanced MRI, while cine MRI was used to quantify LV volumes, mass and function. Twenty-four patients (25%) presented with a hemorrhagic MI. In the acute phase, presence of myocardial hemorrhage was related to larger LV end-diastolic and end-systolic volumes and infarct transmurality, lower LV ejection fraction as well as lower systolic wall thickening in the infarcted myocardium . Infarct size, size of area at risk and size of MVO were significantly larger in patients with hermorrhagic MI. At 4 months, a significant improvement in LV ejection fraction in patients with non-hemorrhagic MI was seen . LV ejection fraction did, however, not improve in patients with hemorrhagic MI . Multivariate analysis showed myocardial hemorrhage to be an independent predictor of adverse LV remodeling at 4 months (defined as an increase in LV end-systolic volume). This pattern was independent of initial infarct size (See Table Myocardial hemorrhage, the presence of which can easily be detected with T2-weighted MRI, is a frequent complication after successful myocardial reperfusion, and an independent predictor of adverse LV remodeling regardless of initial infarct size.


COVID-19 pandemic will continue to pose a major public health threat until vaccination-mediated herd immunity is achieved. Most projections predict vaccines will reach a large subset of the population late in 2021 or early 2022. In the meantime, countries are exploring options to remove strict lockdown measures and allow society and the economy to return to normal function. One possibility is to expand on existing COVID-19 testing strategies by including large-scale rapid point-of-care diagnostic tests (POCTs). Currently, there is significant variability in performance and features of available POCTs, making selection and procurement of an appropriate test for specific use case difficult. In this review, we have used the World Health Organization’s (WHO) recently published target product profiles (TPPs) for specific use cases of COVID-19 diagnostic tests to screen for top-performing POCTs on the market. Several POCTs, based on clinical sensitivity/specificity, the limit of detection, and time to results, which meet WHO TPP criteria for direct detection of SARS-CoV-2 (acute infection) or indirect diagnosis of past infection (host antibodies), are highlighted here. Despite recent successes in vaccine development, the COVID-19 pandemic will continue to pose a major public health threat until a significant number of the global population is vaccinated and herd immunity is achieved. In the meantime, countries are exploring options to balance between preventing the further spread of SARS-CoV-2 and softening the societal lockdown that has caused major political and financial crisis. Most projections predict reaching herd immunity to SARS-CoV-2, primarily by mass vaccination ,4.The current gold standard for the diagnosis of acute SARS-CoV-2 infection is the reverse transcription polymerase chain reaction (RT-PCR) test that can detect small amounts of viral nucleic acid (SARS-CoV-2 RNA) in clinical specimens  with high accuracy ,6. HowevPOCTs or near-patient tests are rapid decentralized  tests that can diagnose acute or prior SARS-CoV-2 infection within minutes of specimen receipt, allowing for rapid decisions concerning patient care and management to prevent further spread see . POCTs cIt is important to highlight that significant variability in the SARS-CoV-2 incubation periods and kinetics, host immune and antibody response to the virus, and COVID-19 clinical phenotypes among individuals can limit the effectiveness of diagnostic tests. In addition, the performance of tests can be influenced by several confounding factors such as disease severity and stage, patient age, sampling site and collection method, virus strain subtype, presence of other respiratory pathogens, and technical errors which can lead to false negative or false positive results . According to the World Health Organization (WHO), a TPP outlines the desired profile or characteristics of a target product that is aimed at a particular disease or diseases . The TPPBox 1Definitions:▪Rapid Test: a qualitative or semi-quantitative in vitro diagnostic medical device, intended to be used singly or in a small series, which involves non-automated procedures and has been designed to give a fast result.▪Point of Care Testing: testing that is performed near or at the site of the patient, outside a general laboratory environment, with the result leading to possible change in the care of the patient.Potential advantages:▪Improved turnaround time▪Improved monitoring during pandemics where frequent testing is desirable▪Smaller sample (may be less invasive) and reagent volumes▪Advantages in remote regions where access to laboratory is limited▪Economic—POCTs may offer wider economic benefit with a reduced number of clinical visit and fewer hospital admissions▪Greater patient involvement in their own care, improved patient experience▪Availability outside core laboratory normal hoursPotential disadvantages:▪Reduced quality of analysis ▪Poor record keeping▪Lack of result interpretation▪Unnecessary duplication of equipment▪Data recording may be complex and less robust▪Incompatibility with laboratory results▪POCT can be expensive in absence of economies of scale that come from centralized laboratory testingPoint-of-care test for suspected COVID-19 cases and their close contacts to diagnose acute SARS-CoV-2 infection;Test for diagnosis or confirmation of acute or subacute SARS-CoV-2 infection, suitable for high-volume needs;Point-of-care test for prior infection with SARS-CoV-2;Test for prior infection with SARS-CoV-2 for high volume needs.In addition to key parameters that measure the analytical and clinical performance of a diagnostic test see , other pBox 2Analytical Sensitivity: or limit of detection (LOD) is frequently defined as the lowest amount of analyte  that can be accurately measured by the assay. The LOD of a COVID-19 POCTs is typically determined as the lowest SARS-CoV-2 analyte concentration (titrated at increasing concentration into a background) that was detected ≥95% of the time (at least 19 out of 20 replicates tested positive). Analytical Specificity: or cross reactivity is the ability to unequivocally detect a specific analyte  and differentiate it from other interfering substances . The cross-reactivity and potential microbial interference of a COVID-19 test is typically evaluated by testing a panel of commensal and respiratory pathogenic microorganisms  in the absence or presence of heat inactivated SARS-CoV-2 virus. Clinical Sensitivity and Specificity: clinical or diagnostic sensitivity is the ability of a test to return a positive result when the patient has the disease. Clinical specificity is the ability of the test to produce a negative result when the patient sample does not have the disease. The clinical performance of a COVID-19 POCT is evaluated by using confirmed positive and negative SARS-CoV02 clinical specimen. Positive SARS-CoV-2 specimen are typically from patients who presented within 7 days of COVID-19 symptom onset.Clinical specimen: for antigen tests, the quality and relevant abundance of SARS-CoV-2 in collected clinical specimens, heavily dependent on the collection site and disease timeline, are critical for the performance evaluation of the assays. For example, the sensitivity of RT-PCR in detection of SARS-CoV-2-infected patients is ~93% in bronchoalveolar lavage fluid, 63% in nasopharyngeal swabs, 72% in sputum, 32% in pharyngeal swabs, and 29% in stool [in stool . FurtherPOCT performance variability: the significant variability observed in performance between COVID-19 tests can be explained by:▪Differences in test population▪Time of testing; proportion of early versus late COVID-19 disease stage▪Specimen type ▪Differences in RT-PCR protocols used as reference assayThe emerging POCTs will not necessarily meet all the criteria outlined in the WHO TPP, but the TPP will provide a framework to assist in the manufacturing of products that will meet a use case. In addition, the TPPs can be utilized to compare key features of COVID-19 POCTs and select products that best respond to the public health needs of each region. In this review, we focus on features of non-PCR COVID-19 POCTs intended for the diagnosis of acute SARS-CoV-2 infection (WHO TPP #1) and detection of prior SARS-CoV-2 infection (WHO TPP #3). 6 and 104 genomic copies/mL, respectively. However, due to a lack of standard LOD unit, we have reported the LOD for antigen-based tests in TCID50/mL  and the LOD for RNA-based tests in genomic copies/mL for   and RNA-based  POCTs have the potential to detect patients with high viral loads, often during the first week of COVID-19 infection, which are most likely to transmit the virus to others . Here, wpies/mL) . The TCIfect see . Finallyfect see .SARS-CoV-2 viral load in airways becomes low or undetectable beyond two weeks post-infection, leading to diminished sensitivity of molecular tests. COVID-19 serological or antibody tests are immune-based assays  that detect immunoglobulins produced by the host in response to SARS-CoV-2 specific antigens. Studies suggest that human immunoglobulins including IgA/IgM/IgG titers remain low in the early stages of COVID-19 disease and typically become detectable (seroconversion) beyond one to two weeks after COVID-19 symptom onset, at which time, most likely, the infectiousness is decreased and some degree of immunity has developed. The IgA, IgM, or IgG antibodies reveal distinct kinetics and seroconversion timeline in response to SARS-CoV-2 that can affect the performance of antibody tests . The information provided by the FDA and FIND databases can be useful starting points for choosing the most appropriate COVID-19 diagnostic test to use when implementing a testing strategy. Relating critical performance characteristics to how well a product meets the relevant TPP will ensure that suboptimal tests are not used. The limitations of the use of these databases must be acknowledged, however, before finally selecting the appropriate test(s) for a specific diagnostic use case. The FDA database contains details of the data submitted by the manufacturer and reviewed for suitability by the FDA. Therefore, it is not independently confirmed data. Although the data presented from the FIND database has been generated independent of the manufacturer, it too has its limitations. There is no consensus on a standard protocol for independent validation of COVID-19 diagnostic tests. However, as noted, this information does provide an excellent starting point for considering what test to choose. Once a selection is made of potential candidates based on performance data, further considerations will be necessary before choosing the best option(s). These may include further verification of critical performance characteristics, pricing, resourcing  of the testing, quality control, as well as connectivity issues, where results can inform further public health measures, to name a few.To date, testing for SARS-CoV-2 infection has mostly relied on RT-PCR assay. However, diagnostic technology for COVID-19 POCTs has improved at a tremendous rate in recent months. As highlighted in this review, there are several POCTs on the market that meet critical performance criteria defined by the WHO TPPs. Top-performing direct (antigen/RNA) and indirect (antibody) POCTs cited here see  can poteBox 3The following COVID-19 POCTs have met the WHO TPP ‘desirable’ criteria for clinical sensitivity/specificity, LOD, and Time to Results :Direct (antigen/RNA) POCTs:▪DetectaChem MobileDetect Bio BCC19 Test (RT-LAMP)▪Mammoth Biosciences SARS-CoV-2 Detectr Test (RT-LAMP/CRISPR)▪Quidel Sofia-2 Flu+SARS Antigen Test▪Seasun Biomaterials AQ-TOP Plus COVID-19 Rapid Test (RT-LAMP)▪Shenzhen Bioeasy Biotechnology Bioeasy Diagnostic Kit COVID-19 Antigen TestIndirect (antibody) POCTs:▪Guangzhou Wondfo Biotech Wondfo SARS-CoV-2 Ab Test ▪Hangzhou Biotest Biotech RightSign COVID-19 IgG/IgM Rapid Test▪Hangzhou Alltest Biotech AllTest COVID-19 IgG/IgM Rapid Test ▪NG Biotech NG IgG/IgM Rapid Test▪Sugentech SGTi-flex COVID-19 IgG▪VivaCheck Biotech COVID-19 IgM/IgG Rapid Test


The rapid development and remarkable success of checkpoint inhibitors have provided significant breakthroughs in cancer treatment, including hepatocellular carcinoma (HCC). However, only 15-20% of HCC patients can benefit from checkpoint inhibitors. Cancer stem cells (CSCs) are responsible for recurrence, metastasis, and local and systemic therapy resistance in HCC. Accumulating evidence has suggested that HCC CSCs can create an immunosuppressive microenvironment through certain intrinsic and extrinsic mechanisms, resulting in immune evasion. Intrinsic evasion mechanisms mainly include activation of immune-related CSC signaling pathways, low-level expression of antigen presenting molecules, and high-level expression of immunosuppressive molecules. External evasion mechanisms are mainly related to HBV/HCV infection, alcoholic/nonalcoholic steatohepatitis, hypoxia stimulation, abnormal angiogenesis, and crosstalk between CSCs and immune cells. A better understanding of the complex mechanisms of CSCs involved in immune evasion will contribute to therapies for HCC. Here we will outline the detailed mechanisms of immune evasion for CSCs, and provide an overview of the current immunotherapies targeting CSCs in HCC. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated deaths worldwide, accounting for approximately 75-85% of primary liver cancers Cancer stem cells (CSCs) have been shown to be responsible for recurrence, metastasis, and local and systemic therapy resistance in HCC Figure 1) and provide an overview of the current immunotherapies targeting CSCs in HCC .In this review, we summarize the role of CSCs in the tumor immunosuppressive environment (+/CD38- myeloid leukemia (AML) cells have the ability to initiate tumors in NOD/SCID mice et al. confirmed that as few as 1000 HCC SP cells have tumorigenic ability in NOD/SCID mice, whereas up to 1×106 non-SP cells were unable to initiate tumors CSCs are a small population of cells that can self-renew and differentiate to initiate and maintain tumor growth Figure 1) The existence of HCC CSCs indicates tumor heterogeneity and hierarchy, which is a hallmark feature of resistance to immunotherapy In HCC CSCs, signaling pathways involved in self-renewal and differentiation characteristics mainly include the Wnt/β-Catenin signaling pathway, Notch signaling pathway, Hedgehog signaling pathway, TGF-β signaling pathway, and AKT signaling pathway et al. found that glioblastoma CSCs were weakly positive and negative for MHC-I and MHC-II, leading to a lack of a T cell-mediated immune response A major mechanism by which CSCs avoid being attacked by the immune system involves minimization of antigenicity by downregulating key components of the cellular antigen processing and presentation machinery, mainly including transporters associated with antigen processing (TAP) and/or major histocompatibility complex (MHC) molecules Additionally, CSCs have been found to express high levels of CD47 , which inhibits macrophage phagocytosis by binding to its cognate ligand, signal-regulatory-protein-α (SIRPα) et al. demonstrated that epithelial-mesenchymal transition (EMT) enriched more PD-L1 in CSCs of breast and colon cancer cells by the EMT/β-catenin/STT3/PD-L1 signaling axis than non-CSCs + cells (CSCs) compared to CD44- cells (non-CSCs), which was found to be dependent on the constitutive phosphorylation of STAT3 in CSCs Moreover, accumulating evidence has indicated that CSCs express high levels of PD-L1, which induce T cell apoptosis by binding to its cognate receptor PD-1. Hsu etc.), contributing to tumor initiation and migration HBV and HCV infections are major risk factors for HCC development and are also associated with the acquisition of a stem-like phenotype in HCC +/CD49f+ tumor stem cells isolated from alcohol-fed HCV Ns5a or core transgenic mice, are tumorigenic based on the roles of TLR4 and Nanog, which is correlated with TGF-β signaling pathway due to Nanog-mediated expression of IGF2BP3 and YAP1 et al. found that alcoholic steatohepatitis accelerates early HCC by increasing the stemness and miR-122-mediated hypoxia-inducible factor 1α (HIF-1α) activation high) CSC-like HCC cells have more unsaturated fatty acids, and lipid desaturation-mediated endoplasmic reticulum (ER) stress signaling regulates the expression of MYCN gene in HCC CSCs et al. showed that saturated fatty acid can induce the properties of CSC in HCC through NF-κB activation + T cells, cytotoxic CD8+ T cells, natural killer (NK) cells and Th17 cells As we known, alcoholic, and nonalcoholic steatohepatitis (NASH) have emerged as an important risk factor in the development of HCC et al. demonstrated that HIF-1a-induced EMT led to the creation of an immunosuppressive TME to promote the metastasis of hepatocellular carcinoma cells. They found that hypoxia-induced EMT of hepatoma cells recruited and educated suppressive indoleamine 2,3-dioxygenase (IDO)-overexpressing TAMs to inhibit T-cell responses and promote immune tolerance in a CCL20-dependent manner Hypoxia is common in HCC, especially in patients with liver cirrhosis + CSCs of HCC can promote tumor angiogenesis through neurotensin/interleukin-8/CXCL1 signaling. Moreover, HCC CSCs preferentially secrete exosomes to promote VEGF secretion from endothelial cells, which in turn promotes tumor angiogenesis et al. found that VEGF increases the proportion of CD133+ CSCs by activating VEGFR2 and enhances their self-renewal capacity by inducing Nanog expression in HCC An overwhelming number of studies have described the close crosstalk between CSCs and angiogenesis in the TME of various tumors, including HCC Intratumoral hypoxia is a key driver of tumor angiogenesis + CSCs via the STAT3 signaling pathway + TAMs to inhibit T-cell responses and promote immune tolerance Over the decades, a large number of studies have been accumulated that extensively describe the interaction of CSCs with the immune system et al. found that drug-resistant HCC cell-derived IL-6 can enhance the expansion and immunosuppressive function of MDSCs MDSCs are another type of suppressive immune cell that seems to enable CSCs to escape antitumor immunity During the decades, several monoclonal antibodies (mAbs) have been successfully used in clinical patients for the treatment of human cancer, such as antagonists of VEGF, bevacizumab for colorectal cancer, ramucirumab for HCC and so on et al. indicated that EpCAM+ circulating tumor cells were associated with poor prognosis in HCC patients after curative resection in vitro and vivo etc.) have shown that BiTE catumaxomab (Anti-EpCAM x Anti-CD3) can effectively improve the quality of life and survival time of malignant ascites (MA) from ovarian and nonovarian  cancer patients + tumor cells in a manner associated with the release of proinflammatory Th1 cytokines EpCAM is a common marker of CSCs in HCC. Sun Given that CD47 acts as a marker for HCC CSCs and is crucial for evading phagocytosis by macrophages; thus, targeting CD47 is a promising approach to affect CSCs + HCC CSCs in vitro and in vivoet al. showed that CD44 antibody-targeted liposomal nanoparticles can target and eliminate HCC CSCs in preclinical models et al. suggested that anti-CD24 antibody conjugating doxorubicin can improve antitumor efficacy and has less systemic toxicity in an HCC preclinical model in vivoBased on other HCC CSC markers, such as CD133, CD44, and CD24, related mAbs have demonstrated their effectiveness in eliminating HCC CSCs in preclinical models. Jianhua Huang and colleagues demonstrated that cytokine-induced killer (CIK) cells bound with anti-CD3/anti-CD133 bispecific antibodies can effectively target and kill CD133The rapid development and remarkable success of checkpoint inhibitors in the activation of CTLs led to cancer immunotherapy being named the “Breakthrough of the Year” by Science in 2013 As one of the most vascular solid tumors, the role of angiogenesis has been extensively studied in HCC, and CSCs play an important role in promoting angiogenesis in HCC. Multiple kinase inhibitors with anti‐angiogenic activity, such as sorafenib and lenvatinib, have been approved by the FDA for the treatment of advanced HCC https://clinicaltrials.gov/), the most registered type of HCC-related CAR-T clinical trial is GPC-3-targeted CAR-T, mainly because GPC-3 is the specific cell surface marker of HCC et al. conducted a phase I clinical study (NCT02541370) using autologous CAR-CD133 T-cells to treat 23 patients with advanced and CD133-positive tumors, including 14 advanced HCC patients. The results showed that CAR-CD133 T-cell therapy was feasible and had controllable toxicities; 3 patients achieved partial remission (including 1 HCC patient), and 14 patients (including 9 HCC patients) acquired stable disease; the 3-month disease control rate was 65.2%, and the median progression-free survival was 5 months The advent of chimeric antigen receptor (CAR) T cell immunotherapy opens a new avenue in adoptive cell therapy, indicating the next breakthrough in immunotherapy Another promising adoptive cell therapy, T-cell receptor (TCR)-engineered T-cell immunotherapy, has attracted widespread attention and been extensively studied. Compared with CAR-T cells, TCR-T cells can recognize intracellular tumor-associated antigens depending on the MHC complex. In HCC, targeting alpha-fetoprotein (AFP) or HBV/HCV-associated antigens with TCR-T therapies has shown powerful antitumor effects in preclinical models As mentioned previously, CSCs of HCC have low expression of MHC molecules, which contribute to immune escape. Interestingly, the inhibitory receptors of NK cells can recognize MHC-I molecules, hence, NK cells do not usually attack normal cells in vitro and in vivoin vivohttps://clinicaltrials.gov/), a series of clinical trials are ongoing based on DC vaccines (loaded with HCC neoantigens or virus-associated antigens) or combined PD-1 monoclonal antibodies. However, DC vaccination using CSC-associated antigens against HCC needs to be further investigated in future preclinical and clinical trials.CSC-directed immunotherapies to promote tumor cell recognition and elimination by the immune system are mainly focused on the use of DC vaccines Figure 1). Currently, immunotherapeutic approaches targeting HCC CSCs mainly include antibody immunotherapy based on CSC markers, immune checkpoint inhibitors, antiangiogenic therapy, CAR-T/TCR-T cell therapy, NK cell-based cancer immunotherapies, and DC vaccines .In summary, accumulating evidence has suggested that CSCs can create an immunosuppressive microenvironment through certain intrinsic and extrinsic mechanisms, resulting in immune evasion in HCC. The intrinsic mechanisms mainly include the following: 1. the activation of immune-related CSC pathways; 2. low-level expression of TAP and/or MHC molecules; and 3. high-level expression of CD47 and PD-L1. The external mechanisms mainly include the following: 1. HBV/HCV infection; 2. alcoholic/nonalcoholic steatohepatitis; 3. hypoxia stimulation; 4. abnormal angiogenesis; and 5. infiltration of suppressive immune cells  the identification of unequivocal CSC-specific molecular markers through multiomics analyses, such as the combination of proteomics and single-cell analysis; b) dissection of the complex mechanisms of the crosstalk between CSCs and immune cells; and c) validation of the effects of combinatorial treatments in future preclinical and clinical trials, such as DC vaccination  in combination with checkpoint inhibitors, CAR-T/TCR-T therapy in combination with antiangiogenic therapy, anti-CD47 antibody combined with CAR-T, CAR-T combined NK cell-based cancer immunotherapies, and so on. In conclusion, based on the heterogeneity, plasticity and scarcity of HCC CSCs, it is suggested that combinatorial treatments will be more efficacious than anti-CSC treatment alone. Overall, future immunotherapy should serve as a model for combined therapy.


Vitellogenesis is pre-requisite to insect egg production and embryonic development after oviposition. During insect vitellogenesis, the yolk protein precursor vitellogenin (Vg) is mainly synthesized in the fat body, transported by the hemolymph through the intercellular spaces (known as patency) in the follicular epithelium to reach the membrane of maturing oocytes, and sequestered into the maturing oocytes via receptor-mediated endocytosis. Insect vitellogenesis is governed by two critical hormones, the sesquiterpenoid juvenile hormone (JH) and the ecdysteriod 20-hydroxyecdysone (20E). JH acts as the principal gonadotropic hormone to stimulate vitellogenesis in basal hemimetabolous and most holometabolous insects. 20E is critical for vitellogenesis in some hymenopterans, lepidopterans and dipterans. Furthermore, microRNA (miRNA) and nutritional (amino acid/Target of Rapamycin and insulin) pathways interplay with JH and 20E signaling cascades to control insect vitellogenesis. Revealing the regulatory mechanisms underlying insect vitellogenesis is critical for understanding insect reproduction and helpful for developing new strategies of insect pest control. Here, we outline the recent research progress in the molecular action of gonadotropic JH and 20E along with the role of miRNA and nutritional sensor in regulating insect vitellogenesis. We highlight the advancements in the regulatory mechanisms of insect vitellogenesis by the coordination of hormone, miRNA and nutritional signaling pathways. Apis mellifera, Vg also protects the queen and long-lived worker bees from oxidative stress and further extends their life span  for lipid binding, a 1943 domain with unknown function (DUF1943), and a von Willebrand factor type D domain (vWFD) in the C-terminus   is mostly synthesized in the fat body and deposited into developing oocytes. In addition to the fat body, Vg synthesis has been reported in follicle cells  and the ecdysteroid 20-hydroxyecdysone . Both JH and 20E can stimulate various aspects of vitellogenesis but vary across insect orders depending on reproductive traits  and insulin  are involved in biosynthesis of JH and 20E and crosstalk with JH and 20E pathways, which in turn regulate various aspects of insect vitellogenesis , a primary JH early-response gene that codes for a zinc finger transcription factor, transduces JH signaling in regulating adult reproduction , a member of basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family. JH induces the heterodimerization of Met with another bHLH-PAS protein, Taiman (Tai) to form an active JH-receptor complex  in the follicular epithelium, and facilitates the uptake of Vg into developing oocytes , cell-division-cycle 6 (Cdc6), cyclin-dependent kinase 6 (Cdk6) and adenovirus E2 factor-1 (E2f1) expressed in response to both JH and Met  and origin-recognition-complex subunit 5 (Orc5). Knockdown of LCMT1, PP2A, FoxO, Cdc2, or Orc5 in vitellogenic female locusts resulted in lower ploidy and significantly reduced Vg expression in the fat body as well as arrested oocyte maturation and suppressed egg development  and Ribosomal protein L32 (RpL32), consequently enhancing ribosomal biogenesis in the fat body essential for massive Vg synthesis , Met and Kr-h1, further stimulating Vg expression and oocyte maturation  or FoxO in fed adult females resulted in markedly reduced Vg expression along with impaired oocyte maturation  are two key enzymes involved in miRNA biogenesis and functioning. Depletion of Ae. aegypti, lineage-specific miR-1890 targets the 3′UTR of JH-regulated Chymotrypsin-like serine protease (JHA15). After a blood meal, 20E suppresses miR-1890 expression to prevent adult females from impaired blood digestion in the midgut and Vg synthesis in the fat body /mitogen-activated protein kinase (MAPK) signaling is activated in the yolk cells  and assembled into the secretory vesicles via membrane fusion with the Golgi apparatus. The 78-kDa Glucose-regulated protein (Grp78), a heat shock protein 70 kDa family member is one of the most abundant chaperones in fat body ER of H. armigera, depletion of Met severely suppressed Vg and VgR transcription, disrupted yolk protein uptake into oocytes, and eventually led to reduced fecundity  gene, coding for a calcium-binding glycoprotein that forms part of the extracellular membranes is critical for follicle cell morphological changes and patency induction that facilitate Vg uptake by developing oocytes   activator in R. prolixus have shown that PKC activation is required for the induction of JH-dependent Na+/K+-ATPase activity  and PKC , C-type muscarinic acetylcholine receptor (mAChR-C) and Cirl-like (CirlL) played key roles in ovarian Vg transportation and uptake  family , which enters the developing oocytes through VgR-mediated endocytosis , 75 (E75), 93F (E93), Broad-Complex (Br-C), and Ftz-f1. The 20E pathway appears to be evolutionarily conserved in vitellogenesis and oogenesis across insect orders  and Ultraspiracle (USP), leading to transcriptional activation of 20E-response genes including ZW, LY, and QH collected the references. ZW, LY and SZ wrote the manuscript. 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.


Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has presently become a rapidly spreading and devastating global pandemic. Veno-venous extracorporeal membrane oxygenation (V-V ECMO) may serve as life-saving rescue therapy for refractory respiratory failure in the setting of acute respiratory compromise such as that induced by SARS-CoV-2. While still little is known on the true efficacy of ECMO in this setting, the natural resemblance of seasonal influenza’s characteristics with respect to acute onset, initial symptoms, and some complications prompt to ECMO implantation in most severe, pulmonary decompensated patients. The present review summarizes the evidence on ECMO management of severe ARDS in light of recent COVID-19 pandemic, at the same time focusing on differences and similarities between SARS-CoV-2 and ECMO in terms of hematological and inflammatory interplay when these two settings merge. COVID-19 is a disease caused by the novel SARS-CoV-2 virus which appeared in December 2019 and is now a worldwide pandemic ) were significantly elevated compared with patients with ARDS who survived; elevated D-dimers were elevated and prognostic of worse outcome in other reports as well. In another study comparing COVID-19 survivors to non-survivors, non-survivors had significantly higher D-dimer and fibrin degradation product (FDP) levels and 71.4% of non-survivors met clinical criteria for disseminated intravascular coagulation (DIC) during the course of their disease [Coagulopathies of diverse etiologies were described in COVID-19 patients; of importance is the augmented risk of venous thromboembolism (VTE). Though there are no published case series thus far, there exist reports of abnormal coagulation parameters in hospitalized patients with severe COVID-19 disease. In a multicenter retrospective cohort study from China, elevated D-dimer levels (> 1 g/L) were strongly associated with in-hospital death, even after multivariable adjustment  . In the = 0.003)  involvin disease .Other laboratory parameters for the routine assessment of blood coagulation appear to be normal in COVID-19 patients regardless of the severity of infection, except for a single case of prolonged prothrombin time (PT)  and reduWhile the optimal anticoagulation regimens to prevent VTE and DIC in the setting of critically ill, immobilized COVID-19 patients remain unknown, V-V ECMO is a double-edged sword; on one side, it promotes thrombosis and hypercoagulable state; on the other, ECMO circuits are eliminating coagulation factors binding them irreversibly to surface coating material. In fact, iv. administered unfractionated heparin (UFH) which is required for the ECMO run  is furthJAMA Viewpoint [Table iewpoint  stating iewpoint .Table 2Finally, ELSO will continue to collect data through member centers through the ELSO Registry and provide recommendations as additional information becomes available from ongoing studies .Because ECMO therapy and COVID-19 itself are associated with certain, often synergistic changes in hematological and inflammatory status of the patients, the efficacy of ECMO is largely dependent on centers’ experience with such therapies.Additional file 1. Supplementary references.


Since March 2020, large-scale efforts to reduce transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), have continued. Mitigation measures to reduce workplace exposures have included work site policies to support flexible work site options, including telework, whereby employees work remotely without commuting to a central place of work.This multistate case-control study assessed possible exposures to COVID-19. Methods have been described elsewhere  were ineligible , and 18 (2%) were excluded because of nonresponse to the telework and work-from-home question. The final analytic sample (314) included 153 (49%) case-patients and 161 (51%) control-participants. An unmatched analysis was performed because of the strict inclusion criteria that resulted in many participants being ineligible for the investigation. This activity was reviewed by CDC and participating sites and conducted consistent with applicable federal law and CDC policy.**Data collected in this investigation included self-reported demographic characteristics, underlying chronic medical conditions,Unconditional logistic regression models, accounting for site-level clustering, were used to estimate odds ratios and 95% CIs for associations between telework status and case-patients and control-participants, adjusting for age, sex, race/ethnicity, and presence of one or more underlying chronic medical conditions , have a college degree or higher (p<0.01), and report at least one underlying chronic medical condition (p = 0.02) . In the A total of 110 (35%) participants reported teleworking or working from home at least part of the time, and 138 (44%) reported going into an office or school regularly 2 weeks before illness onset . ParticiAmong the 248 participants who reported telework status and some form of employment during the 2 weeks before illness onset, case-patients were more likely to have reported exclusively going to an office or school setting  in the 2 weeks before illness onset than were control-participants. The association persisted when the analysis was restricted to the 175 participants who reported working in a profession outside the selected critical infrastructure sectors .This investigation provides evidence of the potential health benefits of teleworking associated with the COVID-19 pandemic. Among participants who reported being employed during the 2 weeks preceding illness onset, the percentage who reported teleworking on a full- or part-time basis was significantly lower among case-patients (35%) than among control-participants (53%). For case-patients and control-participants, the percentage who reported teleworking is higher than national estimates that suggest 26% of U.S. adults were teleworking because of COVID-19 during July 2020 , as well as enhancing policies to protect employees and the community.Since March 2020, large scale measures to reduce workplace transmission of SARS-CoV-2, including workplace closures and providing telework options, have been implemented.Adults who received positive test results for SARS-CoV-2 infection were more likely to report exclusively going to an office or school setting in the 2 weeks before illness onset, compared with those who tested negative, even among those working in a profession outside of the critical infrastructure.Businesses and employers should promote alternative work site options, such as teleworking, where possible, to reduce exposures to SARS-CoV-2. Where telework options are not feasible, worker safety measures should continue to be scaled up to reduce possible worksite exposures.


Trials of intermittent preventive treatment (IPTp) of malaria in pregnant women that compared dihydroartemisinin-piperaquine with the standard of care, sulfadoxine-pyrimethamine, showed dihydroartemisinin-piperaquine was superior at preventing malaria infection, but not at improving birthweight. We aimed to assess whether sulfadoxine-pyrimethamine shows greater non-malarial benefits for birth outcomes than does dihydroartemisinin-piperaquine, and whether dihydroartemisinin-piperaquine shows greater antimalarial benefits for birth outcomes than does sulfadoxine-pyrimethamine.We defined treatment as random assignment to sulfadoxine-pyrimethamine or dihydroartemisinin-piperaquine before pooling individual participant-level data from 1617 HIV-uninfected pregnant women in Kenya  and Uganda . We quantified the relative effect of treatment on birthweight (primary outcome) attributed to preventing placental malaria infection (mediator). We estimated antimalarial (indirect) and non-malarial (direct) effects of IPTp on birth outcomes using causal mediation analyses, accounting for confounders. We used two-stage individual participant data meta-analyses to calculate pooled-effect sizes.Overall, birthweight was higher among neonates of women randomly assigned to sulfadoxine-pyrimethamine compared with women assigned to dihydroartemisinin-piperaquine , despite placental malaria infection being lower in the dihydroartemisinin-piperaquine group . Mediation analyses showed sulfadoxine-pyrimethamine conferred a greater non-malarial effect than did dihydroartemisinin-piperaquine , whereas dihydroartemisinin-piperaquine conferred a slightly larger antimalarial effect than did sulfadoxine-pyrimethamine , although more frequent dosing increased the antimalarial effect .IPTp with sulfadoxine-pyrimethamine appears to have potent non-malarial effects on birthweight. Further research is needed to evaluate monthly dihydroartemisinin-piperaquine with sulfadoxine-pyrimethamine  to achieve greater protection against malarial and non-malarial causes of low birthweight.Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bill & Melinda Gates Foundation, and Worldwide Antimalarial Resistance Network. Plasmodium falciparum sequester in the placenta, causing inflammatory cellular responses that lead to increased risk of preterm delivery  and intrauterine growth restriction, which are both causes of low birthweight (<2500 g).In sub-Saharan Africa, malaria infection during pregnancy is a major cause of low birthweight. For pregnant women, red blood cells infected with hweight <500 g.2, Parasite resistance to sulfadoxine-pyrimethamine in eastern and southern Africa has led researchers to evaluate alternative drug regimens for IPTp. Dihydroartemisinin-piperaquine remains the most promising candidate, given its long-acting prophylactic effect and highly efficacious antimalarial activity. To date, three trials11Evidence before the studyClinicalTrials.gov and PubMed for original articles using the search terms “intermittent preventive treatment” AND “sulfadoxine-pyrimethamine” AND “malaria in pregnancy” OR “non-malarial effect” OR “bacterial vaginosis” OR “reproductive tract infections” OR “sexually transmitted infections”. No language or time restrictions were used in this search. We identified two observational studies, done in malarious regions of Zambia and Burkina Faso, and one editorial that addressed the potential non-malarial effect of sulfadoxine-pyrimethamine. Both observational studies found intermittent preventive treatment with sulfadoxine-pyrimethamine in pregnant women (IPTp) had a dose-dependent effect on birth outcomes, with one study reporting that IPTp with sulfadoxine-pyrimethamine was associated with reduced odds of sexually transmitted and reproductive tract infections, and overall adverse birth outcomes. The editorial, which cited a study done in a low malaria prevalence (<1%) setting of Zambia, reported that IPTp with sulfadoxine-pyrimethamine conferred a protective effect on birth outcomes. However, none of these studies differentiated between the non-malarial and antimalarial effects of IPTp with sulfadoxine-pyrimethamine on adverse birth outcomes.We searched Added value of this studyTo our knowledge, this is the first study to use mediation analysis on individual-participant data from randomised controlled trials of IPTp to deconstruct the non-malarial and antimalarial effects of sulfadoxine-pyrimethamine relative to dihydroartemisinin-piperaquine. Dihydroartemisinin-piperaquine is one of the most promising candidates to replace sulfadoxine-pyrimethamine in IPTp regimens. Our findings show that IPTp with sulfadoxine-pyrimethamine confers a greater non-malarial effect on birthweight than does IPTp with dihydroartemisinin-piperaquine, but IPTp with dihydroartemisinin-piperaquine has moderately greater antimalarial effects on birthweight than does IPTp with sulfadoxine-pyrimethamine if given at monthly intervals.Implications of all the available evidenceIn areas with high parasite resistance to sulfadoxine-pyrimethamine, IPTp with sulfadoxine-pyrimethamine could be more beneficial in improving birthweight through preventing the non-malarial causes of adverse pregnancy outcomes compared with IPTp with dihydroartemisinin-piperaquine. However, IPTp with dihydroartemisinin-piperaquine, when given monthly, has moderately greater antimalarial effects on birthweight than does IPTp with sulfadoxine-pyrimethamine. The combination of dihydroartemisinin-piperaquine plus a partner compound that confers protection against the non-malarial causes of lower birthweight  could increase the public health effect of IPTp more than dihydroartemisinin-piperaquine or sulfadoxine-pyrimethamine alone. However, further IPTp trials are needed to validate whether the combination of dihydroartemisinin-piperaquine and sulfadoxine-pyrimethamine  would be safe and efficacious.To test this alternative hypothesis, we aimed to use data from three randomised trials in Kenya and Uganda to assess whether sulfadoxine-pyrimethamine shows greater non-malarial benefits for birth outcomes than does dihydroartemisinin-piperaquine, and whether dihydroartemisinin-piperaquine shows greater antimalarial benefits for birth outcomes than does sulfadoxine-pyrimethamine. We used mediation analysespfdhfr 51I, 59R, and 108N and pfdhps 437G and 540E) and 5·8% have the sextuple mutation (pfdhps A581G).pfdhf/pfdhps mutations in Busia, although Tororo and Busia are adjacent districts.We collected individual participant-level data from three trials in Siaya County, Kenya (Kenya-STOPMiP),Trial eligibility was restricted to HIV-uninfected pregnant women who were resident in the study region or health facility catchment area with no history of receiving IPTp during their current pregnancy.In the Kenya-STOPMiP trial,In the Ugandan studies,For all studies, each dose of sulfadoxine-pyrimethamine was three tablets of 500 mg sulfadoxine and 25 mg of pyrimethamine given as a single dose. In Kenya-STOPMiP,Our mediation analysis included women who had singleton livebirths, received at least one IPTp dose, and a known status of either past or active placental malaria infection. Placental malarial infection was determined by including women who either had a histopathological assessment of placental malaria using placenta tissue or women who tested positive for placental malaria by either microscopy, loop-mediated isothermal amplification, or PCR methods using placental blood. Women were excluded if they were assigned to the Kenya-STOPMiP ISTp group, assigned to the Uganda-BC1 IPTp with dihydroartemisinin-piperaquine every 4 weeks group, or had an unknown status of either past or active placental malaria .Ethics approvals were granted by the Kenya Medical Research Institute, Makerere University School of Biomedical Sciences, the Uganda National Council for Science and Technology, the US Centers for Disease Control and Prevention, and the University of California, San Francisco.We defined treatment as random assignment to IPTp with either sulfadoxine-pyrimethamine or dihydroartemisinin-piperaquine. The mediator in our analysis was defined as the presence of previous or active placental malaria infection. A woman was determined to have a previous or active placental malaria infection if she had pigment or parasites in her placenta determined by histopathology of the placental tissueConfounding variables were identified a priori based on causal assumptions represented in a directed acyclic graph . BecauseThe primary outcome was a continuous measure of birthweight at delivery measured in g. Secondary outcomes were low birthweight <2500 g) and preterm delivery . Further details on measurement of these outcomes are reported in the trials.00 g and 7We used causal mediation analysisWe estimated crude differences in birth outcomes between IPTp regimens using linear or log-binomial regression models with random assignment as the sole predictor. For mediation analyses, we used the mediation R packageinteraction) of these terms were less than 0·10. We modelled continuous predictors as three-knot restricted cubic splines if the p value of the F test for the joint-effect of the non-linear components was less than 0·05. CIs around mediation effect estimates were generated for each study with a quasi-Bayesian approach using 1000 simulations. Effect modification by gravidity of indirect and direct effect estimates was tested using the test.modmed functiondifference. For the mediator and primary outcome , we report effect estimates separately for each study and by gravidity, regardless of whether there was evidence of a statistical interaction. Analyses of secondary outcomes (low birthweight and preterm delivery) were not reported separately by gravidity as they were relatively uncommon.For all models, treatment–gravidity and treatment–mediator interaction terms were tested wherever possible and incorporated if the p values . In Kenya-STOPMiP,I2=92%; p<0·0001) and effects differed between primigravidae and multigravidae in the Ugandan studies (interaction=0·68). In the Ugandan studies, dihydroartemisinin-piperaquine was associated with a significantly lower risk of placental malaria compared with sulfadoxine-pyrimethamine  and effects were larger in multigravidae compared with primigravidae (interaction=0·0095 for Uganda-BC1 and pinteraction=<0·0001).In the pooled analysis, dihydroartemisinin-piperaquine was associated with a lower risk of placental malaria infection compared with sulfadoxine-pyrimethamine, but this finding did not reach statistical significance . There was substantial heterogeneity between studies . In Kenya-STOPMiP, the mean difference was 87 g (95% CI 24–150) and effects were similar between primigravidae and multigravidae (pinteraction=0·82). In Ugandan studies, sulfadoxine-pyrimethamine was not associated with a significantly higher birthweight compared with dihydroartemisinin-piperaquine and there was insufficient evidence that effects differed by gravidity . However, risks of low birthweight  and preterm delivery did not significantly differ between IPTp regimens, overall or for any of the three individual studies .In mediation analyses, dihydroartemisinin-piperaquine did not show a significantly larger antimalarial effect on birthweight than did sulfadoxine-pyrimethamine in the pooled analysis , although effect estimates varied between studies , although effect estimates were heterogeneous between studies  and we found no evidence that the non-malarial effects differed between primigravidae and multigravidae in the Kenya-STOPMiP and Uganda-BC1 study (pdifference>0·33). In the Uganda-BC3 study, sulfadoxine-pyrimethamine conferred a greater non-malarial effect than did dihydroartemisinin-piperaquine in multigravidae, but not in primigravidae .In the pooled analysis, sulfadoxine-pyrimethamine conferred a greater non-malarial effect on birthweight than did dihydroartemisinin-piperaquine .The non-malarial effect on low birthweight had a similar relationship to the continuous measure of birthweight . In the I2=48%; p=0·15), particularly between Kenyan and Ugandan studies.In the pooled analysis, sulfadoxine-pyrimethamine was associated with a 13% (or 100×[1–1/1·15]) greater non-malarial effect on preterm delivery risk than was dihydroartemisinin-piperaquine , although CIs around all effect estimates included the null . EffectsWe did sensitivity analyses to test the robustness of our effect estimates. First, we restricted the definition of our mediator to active placental infections only, as past infections include those that might have been present before study enrolment. Dihydroartemisinin-piperaquine was associated with a lower risk of active infections compared with sulfadoxine-pyrimethamine , and we By pooling data from three randomised controlled trials, we found evidence that IPTp influences birthweight through both antimalarial and non-malarial mechanisms. Crude analyses of data from these trials showed that, despite the substantially larger protective effect of dihydroartemisinin-piperaquine on placental malaria, birthweight was higher for neonates of women randomly assigned to sulfadoxine-pyrimethamine. We did mediation analyses to investigate this seemingly paradoxical relationship. We found, via mechanisms mediated by malaria prevention, that monthly IPTp with dihydroartemisinin-piperaquine (as observed in the Uganda-BC3 study) was associated with a modest, but significant increase in birthweight (31 g) compared with sulfadoxine-pyrimethamine. By contrast, there was little difference in the antimalarial effect on birthweight between IPTp groups in studies with less frequent IPTp dosing . We found that, via mechanisms not mediated by malaria, sulfadoxine-pyrimethamine was associated with a significant increase in birthweight (87 g) compared with dihydroartemisinin-piperaquine, and this effect was similar across studies. Antimalarial effects on preterm delivery risk did not follow birthweight or low birthweight trends, suggesting the mechanism by which IPTp affects birthweight might be via promotion of intrauterine fetal growth, rather than timing of delivery. We observed some evidence in Uganda that non-malarial effects were greater in multigravidae, which might reflect a greater attributable fraction of non-malarial causes of low birthweight compared with primigravidae, for whom malaria might be a more predominant cause of low birthweight.Trichomonas vaginalis,Gardnerella vaginalis ,Neisseria gonorrhoeae,Chlamydia trachomatis.Although we do not know the exact non-malarial mechanisms by which sulfadoxine-pyrimethamine is improving birthweight, it is likely that the antibiotic properties of sulfadoxine are, at least partly, responsible for these observed effects.In the Uganda-BC3 study, 457 (73%) of 633 women received at least six IPTp doses, whereas in the Kenya-STOPMiP and Uganda-BC1 study, 592 (73%) of 806 and 178 (100%) of 178 women received three or less IPTp doses, respectively. More frequent dosing could explain why the antimalarial effect of dihydroartemisinin-piperaquine was larger in the Uganda-BC3 study than the other studies. Our findings support the results of the original Uganda-BC1 study,This study has limitations. First, mediation effect estimates might have been subject to unmeasured confounding, although our sensitivity analyses suggest non-malarial effects were fairly robust. Second, placental malaria (mediator) could have been measured with error, which would have probably, on average, have biased the antimalarial and non-malarial effect toward the null. However, a sensitivity analysis using a more specific definition of the mediator  showed similar results. Third, our meta-analysis effect estimates were derived from only three studies and should be interpreted with caution. Finally, our study might have had low statistical power to detect true differences between gravidity subgroups and further studies are needed to support our findings.In conclusion, mediation analyses enabled us to quantify the greater benefits of sulfadoxine-pyrimethamine against the non-malarial causes of lower birthweight compared with dihydroartemisinin-piperaquine, and the greater benefits of monthly dihydroartemisinin-piperaquine against placental malaria as a cause of lower birthweight compared with sulfadoxine-pyrimethamine. These findings have two important policy implications. First, this study suggests that IPTp with sulfadoxine-pyrimethamine might be beneficial in areas of low malaria transmission, where IPTp is not currently recommended, as long as the prevalence of these non-malarial causes are high. Second, the study suggests that in areas of high sulfadoxine-pyrimethamine resistance or high malaria burden, rather than replacing sulfadoxine-pyrimethamine with dihydroartemisinin-piperaquine, a combination of these two regimens for monthly IPTp administration might be more efficacious in improving birthweight. Future IPTp trials need to validate the efficacy and safety of this combination. Provided IPTp with sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine is safe and efficacious, this regimen or other combinations that target both malarial and non-malarial causes of lower birthweight (eg, dihydroartemisinin-piperaquine plus azithromycin


Chronic liver diseases, characterized by an excessive accumulation of extracellular matrix (ECM) resulting in scar tissue formation, are a growing health problem causing increasing morbidity and mortality worldwide. Currently, therapeutic options for tissue fibrosis are severely limited, and organ transplantation is the only treatment for the end-stage liver diseases. During liver damage, injured hepatocytes release proinflammatory factors resulting in the recruitment and activation of immune cells that activate quiescent hepatic stellate cells (HSCs). Upon activation, HSCs transdifferentiate into highly proliferative, migratory, contractile and ECM-producing myofibroblasts. The disrupted balance between ECM deposition and degradation leads to the formation of scar tissue referred to as fibrosis. This balance can be restored either by reducing ECM deposition (by inhibition of HSCs activation and proliferation) or enhancing ECM degradation ). MMPs play an important role in ECM remodeling and represent an interesting target for therapeutic drug discovery. In this review, we present the current knowledge about ECM remodeling and role of the different MMPs in liver diseases. MMP expression patterns in different stages of liver diseases have also been reviewed to determine their role as biomarkers. Finally, we highlight MMPs as promising therapeutic targets for the resolution of liver diseases. The liver is the largest organ in the human body and performs a variety of functions, including metabolic functions and detoxification. Liver functions are mainly carried out by the hepatocytes, the parenchymal liver cells that are abundantly present in the liver and are involved in metabolic processing of proteins, carbohydrates and lipids; detoxification of xenobiotic compounds; and secretion of essential molecules . The nonAmong the nonparenchymal cells, HSCs are the key cell type in the liver. Also known as vitamin A-storing cells, they are located in the space of Disse, i.e., the space between parenchymal cells and sinusoidal endothelial cells. HSCs maintain liver homeostasis by preservation of hepatocyte mass, vasoregulation, ECM remodeling and liver regeneration; to a lesser extent they regulate metabolism and detoxification . These cLiver disease affects millions of people, accounting for about 1.2 million deaths per year, and therefore poses a significant health-related burden worldwide . Liver dHepatocellular damage followed by liver inflammation and fibrosis culminates into liver cirrhosis and/or HCC. Chronic exposure to insults  forces the hepatocytes to undergo apoptosis and necrosis, secreting reactive oxygen species (ROS) and proinflammatory growth factors, cytokines and chemokines, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β). Secretion of these proinflammatory factors induces recruitment and activation of resident and infiltrating immune cells, especially macrophages (resident KCs and circulating monocytes), leading to chronic liver inflammation. The immune cells secrete proinflammatory and profibrogenic factors that activate quiescent HSCs, resulting in an excessive accumulation of ECM and loss of liver architecture and function ,14. ActiWhile acute liver injury—in most cases—can be reversed, chronic liver injury often results in irreversible and progressive cirrhosis . Based oMMPs belong to the large metzincin superfamily that includes four subfamilies: matrixins (MMPs); astracins ; bacterial serralysins; and adamalysins , disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) and snake venom proteins, reprolysins) ,27. MMPsBesides ECM components, MMPs can also cleave cell surface molecules and pericellular nonmatrix proteins, thereby regulating cell behavior . MoreoveHuman MMPs can be categorized into six different groups based on their substrate specificity and homology: (i) collagenases ; (ii) stromelysins ; (iii) gelatinases ; (iv) matrilysins ; (v) membrane-type MMPs ; and (vi) others , -27, -28) ..101].MMP-25, also known as MT6-MMP, contains a glycosyl phosphatidyl inositol (GPI) anchor to attach to the plasma membrane  and has MMP-28, also called epilysin, has shown to be upregulated during alcohol-mediated hepatocyte damage . UpregulMMPs play a pivotal role in ECM remodeling in normal physiology; however, dysregulation of MMP expression and/or activity has been found to directly or indirectly contribute to progression of liver diseases. Different MMPs are involved in the different stages, with their expression varying among acute liver injury, liver inflammation, fibrosis/cirrhosis and hepatocellular carcinoma, while some MMPs are also involved in the disease resolution . PreclinSeveral MMPs can degrade fibrillar collagens; among these, the most potent MMPs are collagenases, including MMP-1, MMP-8 and MMP-13. Delivery of MMP-1 for the resolution of liver diseases has been extensively studied and has shown to be a highly promising approach in different experimental animal models. In 2003, Iimuro et al. demonstrated that adenoviral-vector-mediated delivery of human pro-MMP-1 (Ad5MMP-1) attenuated established liver fibrosis in a long-term thioacetamide (TAA)-induced liver fibrosis rat model. In this study, the authors found that intravenous administration of Ad5MMP-1 resulted in intrahepatic pro-MMP1 expression and MMP-1 activity, suggesting that pro-MMP1 was successfully converted into the active form in vivo. Consequently, Ad5MMP-1 ameliorated fibrosis after two weeks and remained attenuated for an additional four weeks. Significantly, ECM degradation via MMP-1 delivery was accompanied by inhibition, disappearance and/or apoptosis of activated HSCs, subsequently resulting in reduced TIMP expression (thereby reducing ECM accumulation) and increased hepatocyte proliferation (favoring liver regeneration) . This st4)-induced rat model of liver fibrosis [4-induced liver fibrosis by inducing production of MMP-1 (and MMP-14 and thioredoxin) with subsequent suppression of HSCs activation [In another study, Du et al. investigated a different MMP-1 delivery approach via transplantation of MMP-1-overexpressing bone-marrow-derived stem cells (BMSCs/MMP-1). In this study, isolated primary rat BMSCs were transfected with a recombinant adenoviral vector containing human MMP-1 gene, and these transfected cells were then transplanted in a carbon tetrachloride (CClfibrosis . BMSCs/Mfibrosis  and lackfibrosis . Recentltivation . In anottivation .4- and bile-duct ligation (BDL)- induced liver cirrhosis rat models. The authors found that in vivo administration of AdMMP-8 resulted in intrahepatic expression of pro-MMP-8 and its functional active form, consequently resulting in the reversal of fibrosis accompanied with an improvement in liver function tests and intrahepatic blood pressure in both animal models. Furthermore, authors found that TGF-β mRNA expression was decreased, while MMP-9 and HGF expression was highly increased. Overall, the results suggested AdMMP-8 as a promising antifibrotic therapy [4-induced liver fibrosis and protected liver function following 70% hepatectomy [Besides MMP-1, MMP-8 delivery has been also explored using adenoviral vector. Garcia-Bañuelos et al. investigated the therapeutic efficacy of adenoviral vector mediated delivery of MMP-8 (AdMMP-8) in CCl therapy . In anotatectomy .Apart from collagenases, gelatinases MMP-2 and -9 are also exploited as potential targets for the treatment of liver fibrosis. As mentioned previously, MMP-2 is involved in fibrosis progression as well as regression. Li et al. investigated HSC-specific delivery of MMP-2 siRNA using vitamin-A-coupled cationic liposomes. HSCs are known to express high levels of vitamin A receptors on their cell surface. Li et al. prepared vitamin-A-coupled liposomes to deliver MMP-2 siRNA (VitA-lip-MMP-2 siRNA) and demonstrated that VitA-lip-MMP-2 siRNA effectively delivered MMP-2 siRNA to the HSCs. VitA-lip-MMP-2 siRNA inhibited HSC activation and proliferation and subsequently decreased the deposition of collagen-I . Althoug4-induced fibrosis, as confirmed by reduced portal and periportal deposition of collagen. MMP-9 mutants inhibited gene expression of fibrosis-related markers and suppressed HSC-to-myofibroblast transdifferentiation in vitro and in vivo. Furthermore, adenoviral-vector-mediated delivery of MMP-9 mutants led to increased HSCs apoptosis, as reported previously by vector-based delivery of MMPs. This study concluded that MMP-9 mutants are a novel therapeutic strategy for liver fibrosis by scavenging TIMPs [In another study, Roderfield et al. examined MMP-9 mutants for TIMP-1 scavenging properties. It is known that the imbalance in MMP/TIMP ratio regulates fibrosis development . Interesng TIMPs .Although MMPs have shown promising results in the preclinical models, none of the MMPs have been used as therapeutics in clinical trials for liver diseases. However, several clinical studies have explored MMPs as biomarkers and/or MMP genotype polymorphism as a risk factor in chronic liver diseases including HCC and colorectal liver metastasis. Before exploring MMPs as therapeutic targets, it is important to understand the role and mechanism of MMPs during the course of the disease. Both MMPs and MMPIs (MMP inhibitors) can be used as potential therapeutics. Although MMPs represent promising and interesting therapeutic targets, the following considerations should be kept in mind before designing MMPI or MMP delivery approaches: (i) MMPs are associated with normal physiologic processes, including ovulation, trophoblast invasion and embryonic development; (ii) ECM fragments resulting from MMP degradation are biologically active and therefore can also mediate secondary effects impacting the physiological and pathological processes; (iii) specificity and selectivity, poor pharmacokinetics, dose-limiting side effects/toxicity, instability and poor bioavailability should be considered with respect to MMPIs ; (iv) MMLiver injury due to excessive alcohol intake, unhealthy lifestyle or other risk factors that progresses to fibrosis and eventually cirrhosis and HCC affects millions of people worldwide and is the major cause of liver-related mortality and morbidity. Surprisingly, no therapeutically effective therapies are available, with liver transplantation representing the only treatment available for the end-stage liver failure. This review shines light on important mediators of liver diseases, the MMPs. MMPs play an important role in not only the initiation and progression, but also in the resolution of different liver diseases. Insight in the specific roles and expression patterns of different MMPs elucidates possibilities for new noninvasive biomarkers and therapeutic targets. Different MMPs and their inhibitors have been explored in preclinical studies for the treatment of liver diseases by degrading the most abundant fibrotic ECM protein, col-I. On the other hand, the serum levels of MMPs have been used as noninvasive biomarkers for staging liver diseases, including BA, ALD-induced fibrosis, HCC and metastasis. These examples demonstrate the importance of gaining knowledge about the possibilities of using MMPs for the diagnosis and treatment of liver diseases.