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Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood,but low concentrations at early stages pose challenges. To detect them,next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein,a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand,post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood,surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse,anticipating clinical relapse by 34.61,9.10,and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients. View Publication

Adoptively transferred T cells and agents designed to block the CD47–SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge,we engineered the CD47 variant CD47(Q31P) (47E),which engages SIRPα and provides a ‘don’t eat me’ signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies,and mediate substantial,sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3,the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages,offering enhanced potency against solid tumours. Combination of TCR or CAR T cells expressing the engineered CD47 variant 47E with anti-CD47 antibody therapy results in synergistic antitumour efficacy due to T cell resistance to clearance by macrophages,while maintaining macrophage recruitment into the tumour microenvironment. View Publication

BackgroundMany cardiomyopathy‐associated FLNC pathogenic variants are heterozygous truncations,and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood.Methods and ResultsWe describe an individual with biallelic FLNC pathogenic variants,p.Arg650X and c.970‐4A>G,with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X,Glu2458Serfs71X,Phe106Leu,and c.970‐4A>G with hypertrophic and dilated cardiomyopathy,atrial fibrillation,and ventricular tachycardia. Induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) were generated. The FLNC truncation,Arg650X/c.970‐4A>G,showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C,gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control,we assessed the necessity of filamin C in iPSC‐CMs for response to the proteasome inhibitor bortezomib. After exposure to low‐dose bortezomib,FLNC‐null iPSC‐CMs showed an increase in the chaperone proteins BAG3,HSP70 (heat shock protein 70),and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC‐CMs had prolonged electric field potential,which was further prolonged in the presence of low‐dose bortezomib. FLNC null engineered heart tissues had impaired function after low‐dose bortezomib.Conclusions FLNC pathogenic variants associate with a predisposition to arrhythmias,which can be modeled in iPSC‐CMs. Reduction of filamin C prolonged field potential,a surrogate for action potential,and with bortezomib‐induced proteasome inhibition,reduced filamin C led to greater arrhythmia potential and impaired function. View Publication

Inflammation induced by lung infection is a double-edged sword,moderating both anti-viral and immune pathogenesis effects; the mechanism of the latter is not fully understood. Previous studies suggest the vasculature is involved in tissue injury. Here,we report that expression of Sparcl1,a secreted matricellular protein,is upregulated in pulmonary capillary endothelial cells (EC) during influenza-induced lung injury. Endothelial overexpression of SPARCL1 promotes detrimental lung inflammation,with SPARCL1 inducing ‘M1-like’ macrophages and related pro-inflammatory cytokines,while SPARCL1 deletion alleviates these effects. Mechanistically,SPARCL1 functions through TLR4 on macrophages in vitro,while TLR4 inhibition in vivo ameliorates excessive inflammation caused by endothelial Sparcl1 overexpression. Finally,SPARCL1 expression is increased in lung ECs from COVID-19 patients when compared with healthy donors,while fatal COVID-19 correlates with higher circulating SPARCL1 protein levels in the plasma. Our results thus implicate SPARCL1 as a potential prognosis biomarker for deadly COVID-19 pneumonia and as a therapeutic target for taming hyperinflammation in pneumonia. The molecular basis underlying infection infection-mediated lung pathology is not fully revealed. Here the authors report that SPARCL1 expressed in pulmonary capillary endothelial cells contributes to immune pathology in mouse model via pro-inflammatory macrophage induction,while circulating SPARCL1 levels corelate with COVID-19 lethality. View Publication

The MUC1 gene evolved in mammals for adaptation of barrier tissues in response to infections and damage. Paraspeckles are nuclear bodies formed on the NEAT1 lncRNA in response to loss of homeostasis. There is no known intersection of MUC1 with NEAT1 or paraspeckles. Here,we demonstrate that the MUC1-C subunit plays an essential role in regulating NEAT1 expression. MUC1-C activates the NEAT1 gene with induction of the NEAT1_1 and NEAT1_2 isoforms by NF-κB- and MYC-mediated mechanisms. MUC1-C/MYC signaling also induces expression of the SFPQ,NONO and FUS RNA binding proteins (RBPs) that associate with NEAT1_2 and are necessary for paraspeckle formation. MUC1-C integrates activation of NEAT1 and RBP-encoding genes by recruiting the PBAF chromatin remodeling complex and increasing chromatin accessibility of their respective regulatory regions. We further demonstrate that MUC1-C and NEAT1 form an auto-inductive pathway that drives common sets of genes conferring responses to inflammation and loss of homeostasis. Of functional significance,we find that the MUC1-C/NEAT1 pathway is of importance for the cancer stem cell (CSC) state and anti-cancer drug resistance. These findings identify a previously unrecognized role for MUC1-C in the regulation of NEAT1,RBPs,and paraspeckles that has been co-opted in promoting cancer progression. View Publication

ObjectiveAlthough cancer therapy suppresses recurrence and prolongs life,it may be accompanied by strong side effects; thus,there is a strong demand for the development effective treatments with fewer side effects. Cancer therapy using plant-derived essential oils is attracting attention as one promising method. This study investigated the antitumor effects of essential oil volatiles on breast cancer cells and identifies four essential oils that display antitumor activity.MethodsBreast cancer cells were cultured in a 96-well plate,then one of twenty essential oils was added dropwise to the central well. The plate was incubated at 37 °C for 48 h and the effect of the volatile components of each essential oil on the surrounding breast cancer cell growth ability was examined using an MTT assay. Gas chromatography was used to investigate the concentration of the transpiration components that may affect cancer cells.ResultsOf the 20 essential oils,Lemongrass,Lemon myrtle,Litsea,and Melissa displayed strong anti-tumor effects. These essential oils inhibited the growth of nearby breast cancer cells,even when diluted more than 500-fold. The transpiration component of lemon Myrtle showed the strongest antitumor effect,but was the least cytotoxic to mononuclear cells in normal peripheral blood (PBMC). Each of these essential oils contained a very large amount of citral. The IC50 against breast cancer cells when citral was volatilized from each essential oil was 1.67 µL/mL for geranial and 1.31 µL/mL for neral. Volatilized citral alone showed strong anti-proliferation and infiltration-inhibiting effects.ConclusionThe transpiration components of Lemongrass,Lemon myrtle,Litsea,and Melissa are thought to inhibit breast cancer cell proliferation due to their high levels of citral. View Publication

Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs),including melanoma differentiation-associated gene 5 (MDA5). MDA5 recognizes the genome of dsRNA viruses and replication intermediates of single-stranded RNA viruses. MDA5 also plays an important role in the development of autoimmune diseases,such as Aicardi-Goutieres syndrome and type I diabetes. Patients with dermatomyositis with serum MDA5 autoantibodies (anti-CADM-140) are known to have a high risk of developing rapidly progressive interstitial lung disease and poor prognosis. However,there have been no reports on the soluble form of MDA5 in human serum. In the present study,we generated in-house monoclonal antibodies (mAbs) against human MDA5. We then performed immunohistochemical analysis and sensitive sandwich immunoassays to detect the MDA5 protein using two different mAbs (clones H27 and H46). As per the immunohistochemical analysis,the MDA5 protein was moderately expressed in the alveolar epithelia of normal lungs and was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and acinar cells of the pancreas. Interestingly,soluble MDA5 protein was detectable in the serum,but not in the urine,of healthy donors. Soluble MDA5 protein was also detectable in the serum of patients with dermatomyositis. Immunoblot analysis showed that human cells expressed a 120 kDa MDA5 protein,while the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min after MDA5 agonist/double-strand RNA stimulation. Hydrogen deuterium exchange mass spectrometry revealed that an anti-MDA5 mAb (clone H46) bound to the epitope (415QILENSLLNL424) derived from the helicase domain of MDA5. These results indicate that a soluble MDA5 protein containing the helicase domain of MDA5 could be rapidly released from the cytoplasm of tissues after RNA stimulation. Highlights•In this study,we established five in-house mAbs against human MDA5.•We performed immunohistochemical analysis and developed a sensitive sandwich immunoassay system to detect soluble MDA5 protein in sera.•MDA5 protein was detectable in the sera,but not in the urine of healthy donors. The MDA5 protein was also detected in the sera of patients with dermatomyositis.•In addition,levels of the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min of dsRNA stimulation. View Publication

SummaryStem cell organoids are powerful models for studying organ development,disease modeling,drug screening,and regenerative medicine applications. The convergence of organoid technology,tissue engineering,and artificial intelligence (AI) could potentially enhance our understanding of the design principles for organoid engineering. In this study,we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs. We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We clustered and refined the cardiac organoids based on their functional similarity using unsupervised machine learning approaches,thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium transient rising time in distinguishing organoids based on geometric patterns and clustering results. This integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids,paving the way for more controlled and optimized organoid design. Graphical abstract Highlights•A cardiac organoid library with 7 geometric designs created by micropatterning technique•Single-organoid analytics identifies the geometry-determining physiological properties•Unsupervised organoid refinement reduces the organoid heterogeneity without human bias MotivationWith the significant advancement in organoid technology,there is a critical need for an optimization pipeline that can evaluate the designs of organoid systems under controllable engineering parameters. By integrating micropatterning-based organoid engineering and machine learning techniques,we can quickly analyze the total functional outputs of a single organoid and evaluate the impact of engineering inputs on organoid physiology from a cardiac organoid library with 7 geometric designs. AI-driven analytical approaches were applied to not only understand functional heterogeneity from single cardiac organoids but also provide mechanistic insights into physiological enhancement associated with different geometric designs. By combining a micropatterning technique and machine learning,Kowalczewski et al. develop a single-organoid analytics workflow to analyze a cardiac organoid library with 7 geometric designs. Their approach can be used as a guide for design optimization for engineered cardiac organoids with specifically tailored physiological functions. View Publication

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species−mediated signaling,it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1),a key enzyme in EtOH metabolism,promotes alcohol-induced hepatic steatosis and inflammatory liver disease,at least in part by mediating changes in intestinal permeability. For instance,gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation,the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs,including miR-132,miR-212,miR-378,and miR-552,have been shown to repress the expression of CYP2E1,suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here,we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation,as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs,including CYP2E1-targeting miRNAs,and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species−mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus,our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1,the stabilization of CYP2E1 mRNA in the presence of EtOH,and the relationship of this pathway to subsequent intestinal injury. View Publication

BackgroundStrategies toward HIV-1 cure aim to clear,inactivate,reduce,or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir,robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible HIV-1.MethodsWe developed Specific Quantification of Inducible HIV−1 by RT-LAMP (SQuHIVLa),leveraging the high sensitivity and specificity of RT-LAMP,performed in a single reaction,to detect and quantify cells expressing tat/rev HIV-1 multiply spliced RNA (msRNA) upon activation. The LAMP primer/probe used in SQuHIVLa was designed to exclusively detect HIV-1 tat/rev msRNA and adapted for different HIV-1 subtypes.ResultsUsing SQuHIVLa,we successfully quantify the inducible viral reservoir in CD4+ T cells from people living with HIV-1 subtypes B and C on cART. The assay demonstrates high sensitivity,specificity,and reproducibility.ConclusionsSQuHIVLa offers a high throughput,scalable,and specific HIV-1 reservoir quantification tool that is amenable to resource-limited settings. This assay poses remarkable potential in facilitating the evaluation of potential interventional strategies toward achieving HIV-1 cure. Plain language summaryHIV infection remains challenging because the virus hides in certain cells,making it invisible to the immune system. This hidden virus forms what is called a latent HIV reservoir. If someone with HIV stops their antiviral therapy,the virus quickly re-emerges. Because of this,researchers are exploring various strategies to eliminate this reservoir and cure HIV. To evaluate these strategies,we need a method to measure the reservoir’s size before and after trials. Our study introduces SQuHIVLa,a highly sensitive and specific method for quantifying the latent reservoir. SQuHIVLa could become a vital tool for monitoring HIV patients and assessing treatment effectiveness,bringing us closer to finding a cure. Hossain,Lungu et al. develop SQuHIVLa,a sensitive and specific method to quantify the frequency of cells expressing HIV-1 tat/rev msRNA as a surrogate for the inducible HIV-1 reservoir. SQuHIVLa offers a high throughput,scalable HIV-1 reservoir quantitation tool that is amenable to resource-limited settings. View Publication

SummaryRetinal ganglion cell (RGC) differentiation is tightly controlled by extrinsic and intrinsic factors. Growth and differentiation factor 15 (GDF-15) promotes RGC differentiation,opposite to GDF-11 which inhibits RGC differentiation,both in the mouse retina and in human stem cells. To deepen our understanding of how these two closely related molecules confer opposing effects on retinal development,here we assess the transcriptional profiles of mouse retinal progenitors exposed to exogenous GDF-11 or -15. We find a dichotomous effect of GDF-15 on RGC differentiation,decreasing RGCs expressing residual pro-proliferative genes and increasing RGCs expressing non-proliferative genes,suggestive of greater RGC maturation. Furthermore,GDF-11 promoted the differentiation of photoreceptors and amacrine cells. These data enhance our understanding of the mechanisms underlying the differentiation of RGCs and photoreceptors from retinal progenitors and suggest new approaches to the optimization of protocols for the differentiation of these cell types. Graphical abstract Highlights•GDF receptors are expressed in various cell types in the retina•GDF-11 and GDF-15 play different roles in RGC/AC development•GDF-11 induces photoreceptor and amacrine differentiation•GDF-15 promotes RGC maturation Cell biology; Developmental biology; Transcriptomics View Publication

IntroductionHuman Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I),hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells,resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB,ULBP1,and ULBP3,which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1,but the precise mechanism remains undetermined.MethodsWe set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells,as well as HHV-6B infection,to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches.ResultsWe demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels,either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex. View Publication