Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been widely documented as a multi-systemic illness and associated with an increased incidence of thromboses. Likewise, sickle cell disease (SCD) is a hematologic disease responsible for widespread effects on the vasculature and is also associated with elevated thrombotic risk. In this review, we examine the incidence rates of venous thromboembolism (VTE) in SCD and COVID-19 independently and review the mechanisms of coagulopathy associated with both diseases. We describe the possible associations and commonalities between VTE mechanisms, as both diseases cause widespread inflammation that influences each tenet of Virchow’s triad. We also discuss current anticoagulation guideline recommendations for the prevention of VTE events in each of these diseases. We report on current literature to date describing rates of VTE in SCD-COVID-19 patients and outline prospective areas of research to further understand the possible synergistic influence of coagulopathy in these patients. The association between SCD and COVID-19 remains a largely under-researched area of coagulopathy in current hematology and thrombotic literature, and our report lays out potential future prospects in the field. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a multisystemic illness associated with systemic inflammatory response, immune hyperactivity, sepsis, and coagulopathy. Acute respiratory distress syndrome (ARDS) has been well documented as the leading cause of death in patients with COVID-19; however, more recent studies describe the role of venous thromboembolism (VTE) causing pulmonary embolism (PE) having a significant impact on morbidity and mortality . Since tSickle cell disease (SCD) is the most common genetic disorder worldwide, affecting approximately 100,000 people in the United States . It is cInterestingly, both COVID-19 and SCD increase VTE risk and coagulopathy by affecting each of the three parameters of Virchow’s triad. In this study, we review the mechanisms of VTE in COVID-19 and SCD separately and describe the commonalities in coagulopathy risk between the two diseases. We also discuss current literature describing VTE risk in SCD patients affected with COVID-19 and prospects for further studies to continue to investigate this association.Role of COVID-19 in coagulopathySince the onset of the COVID-19 pandemic in 2020, many studies have described the association between elevated VTE risk in COVID-19 patients. It is estimated that hospitalized COVID-19 patients have a 25% higher risk of VTE incidence, while patients with severe disease have an approximately 3.77-fold increased risk of VTE despite the use of prophylactic anticoagulation -8. PulmoCOVID-19 infection mediates coagulopathy by affecting each of the three tenets of Virchow’s triad  are extracellular clusters of decondensed neutrophil DNA complexed with histones and granule proteins, released from dying neutrophils as a mechanism of innate immune defense . NETs alAnnexin A2, a plasminogen receptor with increased expression in inflammatory states, has also been implicated in COVID-19 coagulopathy. Under normal circumstances, it acts to accelerate plasmin generation up to 60-fold. SARS-CoV-2-induced antibodies block Annexin A2 to dampen fibrinolysis and disrupt the balance between thrombosis and clot breakdown .Thromboelastography (TEG) is a viscoelastic test of whole blood that measures clot formation and dissolution in real-time. Whole blood samples from COVID-19 patients have demonstrated increased clot mechanical strength and impaired fibrinolysis, suggesting a hypercoagulable state ,26. The Prophylactic anticoagulation use may be insufficient in VTE prevention in COVID-19 patients. Current data suggests that the overall prevalence of thrombotic complications is 2.6% in acute but non-critical patients and 35.3% in critically ill patients ,27. CurrCoagulopathy in sickle cell diseaseSCD is a chronic inflammatory disease associated with increased lifetime VTE risk compared to the general population. Observational studies indicate a 25% increased VTE likelihood in SCD patients, with the age of incidence similar to high-risk thrombophilia patients (30 vs. 29 years), while the age of incidence of VTE in the general population is much higher (65 years) . BrunsonLike COVID-19, SCD impacts each aspect of Virchow’s triad to trigger coagulopathy , which pooled data from 71 studies, found that SCD patients have a mild-moderate COVID-19 disease course, a two-seven-fold increase in hospitalizations, and a 1.2-fold increase in the risk of death; but when matched with controls with similar comorbidities and end-organ damage, the risks are usually equal . The preGiven the similar mechanisms of coagulopathy and enhanced VTE risk in SCD and COVID-19 patients individually, we hypothesize that SCD patients infected with COVID-19 have higher rates of VTE than the general population affected with COVID-19. To date, there have been few case reports and limited literature addressing rates of VTE in SCD-COVID-19 patients -58.One case series documenting postmortem analysis in three SCD patients in Ghana describes the clinical and pathological findings in COVID-19 patients on autopsy, in which the cause of death was COVID-19 pneumonia. Attoh et al. (2022) found microemboli in the pulmonary vasculature of three SCD-COVID-19 patients despite being managed with prophylactic anticoagulation during their hospitalization . CurrentCurrent literature suggests that all SCD patients with COVID-19 should be given prophylactic dose anticoagulation unless there is another factor that warrants full-dose anticoagulation . The assWe extensively discussed the rates of VTE in SCD and COVID-19 independently and reviewed the mechanisms of coagulopathy associated with both diseases. We described the possible associations and commonalities between VTE mechanisms, as both diseases are causes of widespread inflammation that influence each factor of Virchow’s triad. We also discussed current anticoagulation guideline recommendations for the prevention of VTE events. Moreover, we assessed the current literature to date describing rates of VTE in SCD-COVID-19 patients and outlined prospective areas of research to further understand the possible synergistic influence of coagulopathy in these patients.


Biomechanical forces are of fundamental importance in biology, diseases, and medicine. Mechanobiology is an emerging interdisciplinary field that studies how biological mechanisms are regulated by biomechanical forces and how physical principles can be leveraged to innovate new therapeutic strategies. This article reviews state-of-the-art mechanobiology knowledge about the yes-associated protein (YAP), a key mechanosensitive protein, and its roles in the development of drug resistance in human cancer. Specifically, the article discusses three topics: how YAP is mechanically regulated in living cells; the molecular mechanobiology mechanisms by which YAP, along with other functional pathways, influences drug resistance of cancer cells (particularly lung cancer cells); and finally, how the mechanical regulation of YAP can influence drug resistance and vice versa. By integrating these topics, we present a unified framework that has the potential to bring theoretical insights into the design of novel mechanomedicines and advance next-generation cancer therapies to suppress tumor progression and metastasis. One of the most important lessons from cell biology in the past decades is that biomechanical forces, both endogenous and exogenous, are crucial in regulating the physiology and pathology of the human body. Living cells sense, transduce, and respond to specific biomechanical forces and other physical stimuli in their microenvironments ,3,4,5,6,In this review article, we will present a unified framework  to discuYAP and transcriptional co-activator with PDZ-binding motif (TAZ) are transcriptional co-activators that critically facilitate the signal transduction from biomechanical stimuli into gene expression ,13,14. IIn tumors, YAP plays important roles in regulating neoplasm initiation, growth, metastasis, and drug resistance. Importantly, overexpression and nuclear translocation of YAP occur in multiple tumor types  Figure A. For exSeveral pharmacological inhibitions of YAP or YAP-TEAD interaction show repression effects on tumor progression, although all current inhibitors are not clinically viable ,42,43,44In summary, YAP activity is critical in the drug resistance of cancer cells. Mechanical stimulus shows great potential in regulating YAP in cancer cells and reducing drug resistance . In the In living cells, YAP is sensitive to diverse types of mechanical signals, reflected by its translocation between nucleus and cytoplasm . Cells c2) substrate show nuclear YAP translocation, while the rest 20% of the cells show homogenous intracellular YAP distribution. In contrast, the cells of the same type cultured on soft hydrogel (0.7 kPa) substrate do not show nuclear YAP translocation [In general, ECM stiffness (Young’s modulus) is sensed by transmembrane integrins on the cell surface, which sequentially induce activation of Src, FAK at focal adhesion sites and intracellular Rho-ROCK machinery to increase cytoskeleton contractility, which is mechanical tension in cells generated by the sliding of actin and myosin filaments along each other ,59,60,61location . In adiplocation . Cytoskelocation ,57.Current understanding is that nucleus receives mechanical force from cytoskeletal contractility and induces conformational change in nuclear pores. Such conformational change regulates the active transport rate (both import and export) of YAP through nuclear pores to tune the overall dynamics of YAP nuclear/cytoplasmic translocation . If cytoViscoelasticity of ECM enhances nuclear translocation of YAP. For example, polysaccharide alginate gels with higher viscoelasticity (relaxation time = 30 s) induce nuclear YAP translocation in 10–50% of the cells, while gels with lower viscoelasticity (relaxation time = 350 s) induce nuclear YAP translocation in 0% of the cells. Recent evidence suggests that viscoelasticity regulates YAP through a FAK-Arp2/3-complex-dependent mechanism .Cell spreading area (in 2D culture), i.e., area covered by a cell as it adheres on a 2D substrate, and cell volume (in 3D culture) positively regulate nuclear YAP translocation ,67,68,69Cell contractility, cell spreading area and substrate stiffness often functionally intertwine with each other to influence dynamics of YAP and other cell signaling effectors ,71. posiCells constantly receive mechanical tension and compression forces from the surrounding tissues and cells, especially in the lung and heart tissues. Both static and cyclic stretching of the cells induce nuclear YAP translocation ,64,72,73In a high percentage of solid tumors, YAP/TAZ activities of tumor cells are deregulated, while the conventional Hippo pathway is not ,19,20. BSeveral research groups show that mechanical force regulates YAP nuclear translocation through (1) inducing unique phosphorylation of YAP, and (2) altering conformation of nuclear pores, competing with conventional Hippo-pathway-induced phosphorylation. For example, osmotic pressure increases pYAP-S127 indicating activation of Hippo pathway, and simultaneously induces Hippo-independent pYAP-S128 to disrupt the binding between pYAP-S127 and 14-3-3, eventually resulting in increased nuclear translocation of YAP . Recent Besides phosphorylation of YAP, mechanical stimulus regulates YAP nuclear translocation through regulating nuclear pore conformation. Because YAP lacks nuclear localization sequence, it needs to bind with importin to enter the cell nucleus by active transport through the nuclear pore. Compressing of nucleus changes the conformation of nuclear pore and increases the importing rate of YAP into the nucleus ,77. HoweIn summary, YAP can be regulated by mechanical signals through nuclear mechano-sensing, either endogenous-contractility-mediated or exogenous-force-regulated. Therefore, we propose that possible mechanical strategies that aim to inhibit YAP activity may target (1) changing extracellular mechanical signals; (2) changing force transmission in cytoskeleton; and (3) changing nuclear mechano-sensing efficiency.Drug resistance is one of the most prominent obstacles encountered during cancer treatment ,79,80,81Recent studies indicate that YAP proteins tend to be more activated  in drug-resistant cancer cells than in non-drug-resistant ones. The increased YAP nuclear translocation can induce anti-apoptotic and pro-proliferative phenotypes which can contribute to drug resistance and tumor relapse B,C. BaseIn one group, drug-resistant cancer cells, either immortalized cell lines or primary tumor cells, are found to be intrinsically YAP-rich intrinsically  compared to that of non-drug-resistant cells. One hypothesis is that the intrinsic high YAP expression in these cancer cells results in their drug resistance capability. Another hypothesis is that drug treatment triggers YAP nuclear translocation in these cells thus increases the drug resistance. For instance, Lee TF et al. find that upregulated YAP expression in EGFR-mutant lung cancer cells led to the development of resistance against Epithelial Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib, afatinib and osimertinib . McGowanIn the other group, drug treatment directly increases YAP nuclear translocation in a subpopulation of treated cancer cells and thus actively induce drug resistance. It is worth noting that this second group may overlap with the first one to a certain level. For instance, Kurppa KJ et al. find that YAP nuclear localization increased significantly in EGFR-mutant NSCLC after 10-day combined treatment with EGFR-TKIs osimertinib and trametinib . SimilarIn both groups, the studies propose various direct mechanisms by which YAP nuclear activation increases after drug treatment. For example, by monitoring LATS1 kinase, Kurppa KJ et al. find decreased LATS1 phosphorylation in response to drug treatment, suggesting YAP activation can be drug-treatment-induced by influencing Hippo pathway . In explTo explore a stronger causal link between YAP expression and drug resistance, several studies examine the impact of artificially overexpressing and inhibiting YAP on drug resistance in cancer cells. In general, these studies have found that increased YAP activation induces drug resistance in cancer cells. For instance, Kurppa KJ et al. find that re-expression of wild-type YAP1 in YAP1 KO EGFR-mutated NSCLC re-induces resistance to EGFR-TKIs osimertinib and trametinib . Lee TF In a similar fashion, various studies find that decreased YAP activation causes drug-resistant cancer to become sensitive to drug treatment. For instance, Cheng H. et al. finds that knockdown of YAP by either shRNA or siRNA improves sensitivity to cisplatin treatment in PC9 lung cancer cells; to radiation treatment in PC9, HCC827 and H157 lung cancer cell lines; and to EGFR tyrosine kinase inhibitor (TKI) erlotinib in EGFR-mutated NSCLC PC9 and HCC827 cell lines . They alTogether, the collective evidence strongly supports a causative relationship between YAP activation and drug resistance in cancer cells B,C. MoreNext, we review and discuss the various proposed mechanisms of YAP-mediated drug resistance.In the upstream of YAP activation, drug treatments potentially influence the positive/negative upstream regulators of YAP, such as NF2, Src, and FAK. For example, NF2 inhibits YAP activation through Hippo pathway ,14. NF2 In the downstream of YAP activation, nuclear translocation of YAP potentially contributes to drug resistance, through (1) transcriptional regulation of anti-apoptosis genes; and (2) crosstalk with MAPK pathway and PI3K-AKT pathway. First, in multiple types of human normal and cancer cells, YAP binds to transcriptional factor and upregulates the expression of Connective tissue growth factor (CTGF) and Cyr61 to increase drug resistance . MechaniIn parallel with these upstream and downstream events, the activated YAP coordinates with multiple intracellular cytoplasmic and nuclear molecules to collectively regulate drug resistance. For example, Kurppa KJ et al. assert that YAP mediates the evasion of apoptosis by repressing the induction of the pro-apoptotic protein BMF (Bcl2 Modifying Factor) via engagement of Epithelial-Mesenchymal-Transition (EMT) transcription factor SLUG . Lin L eTogether, these diverse studies highlight new potential pathways, proteins, and mechanisms outside of the known causal effects of YAP which may play a role in YAP-mediated drug resistance in lung cancer. However, prior to effectively targeting YAP to reduce drug resistance, we need to address two questions that remain poorly understood to date: (1) whether mechanically regulated and biochemically regulated YAP contribute to drug resistance in the same mechanism? The phosphorylation sites of YAP due to mechanical signals are different from YAP-S127 in Hippo pathway but its exact sites are unknown (only pYAP-S128 is identified in the osmotic-pressure-induced YAP nuclear tranlocation). Such structural and chemical differences may contribute to distinct binding of YAP with downstream transcription factors and differentially regulated cellular functions. An exact identification of these sites will facilitate the innovation of precise medicine; (2) after inhibition of YAP, how the activation of compensating or redundant pathways  reduces the drug resistance? A deeper understanding of their functional dynamics will promote the design of combinatorial strategies that may be necessary to increase the efficacy of YAP inhibition.Emerging studies have shown that YAP (yes-associated protein) likely has a significant role in regulating the cellular response to immunotherapy in non-small cell lung cancer (NSCLC). Specifically, YAP has been found to regulate the expression of Programmed Death-Ligand 1 (PD-L1), which serves as a predictive biomarker for anti-PD-1/PD-L1 immunotherapy.The binding of PD-L1 with its receptor, PD-1, promotes T-cell tolerance and enables tumor cells to evade immune surveillance. To counteract this undesired behavior, one effective therapeutic method developed has been the anti-PD-1/PD-L1 immunotherapy. The level of PD-L1 expression in a tumor can provide information about its likelihood to respond to anti-PD-1/PD-L1 immunotherapy treatment: Typically, tumors with high PD-L1 expression are more effectively treated with anti-PD-1/PD-L1 immunotherapy, whereas tumors with low or no PD-L1 expression have a lower effectiveness of response to these treatments.However, despite the success of anti-PD-1/PD-L1 immunotherapy in clinical, a considerable number of tumors do not respond to PD-1/PD-L1 inhibitors even though they possess high PD-L1 expression. This clinical challenge highlights the urgent need to understand the underlying mechanisms that contribute to this resistance. Several studies have shed light on the involvement of YAP in resistance to immunotherapy and its relation to PD-L1 expression. Hsu et al. find that, in H2052 and 211H human malignant pleural mesothelioma (MPM) cells, YAP binds to PD-L1 enhancer to promote expression of PD-L1. YAP expression positively regulates PD-L1 expression, examined by both overexpression and verteporfin-inhibition of YAP . HoweverTogether, these findings suggest that YAP nuclear localization and YAP-induced increase in PD-L1 expression contribute to immune evasion, immunotherapy resistance, and drug resistance in lung adenocarcinomas.Besides providing mechanical supports for tissue cells to reside in, extracellular matrix (ECM) functionally regulates physical-chemical interactions between cells and their surrounding microenvironments by influencing various mechanical stimulations, including substrate stiffness (both elastic and viscous properties), patterns of fluid shear stress, or active pressure ,104,105.Increasing studies have demonstrated that YAP has mechanotransducive property which can be regulated by ECM mechanics . MeanwhiA biphasic relationship between drug resistance and their ECM with various stiffness in breast cancer cells. Within 10 kPa (soft), intermediate stiff (38 kPa), and stiff (57 kPa) substrate, intermediate substrate rigidity (38 kPa) induces the highest level of integrin-linked kinase (ILK), nuclear YAP translocation and drug resistance .Negative correlation between ECM stiffness and drug resistance is found in ovarian cancer cells (OCC). YAP has the highest nucleus/cytoplasm ratio (N/C ratio) at stiff substrate (25 kPa). However, OCC on soft substrate stiffness (0.5 kPa) shows higher resistance to cisplatin and paclitaxel, which are drugs widely used in chemotherapies of epithelial ovarian cancer. Whether the drug resistance is regulated by YAP remains to be investigated .Positive correlation between ECM stiffness and drug resistance is found in two groups. First, Gao et al. found that hepatocellular carcinomas cells, which are sensitive to sorafenib (Sora-S cells), gained drug resistance when cultured on stiff substrate (4 kPa). Inversely, cells that have resistance to sorafenib (Sora-R cells) regain the sensitivity of sorafenib after adapting to the soft substrate (0.7 kPa). More importantly, the stiffness-induced YAP activation is confirmed to regulate cells’ mechano-adaption of chemotherapy. In experiments, by quantifying YAP in the nucleus and cytoplasm, researchers find that, first, YAP has higher nuclear localization in Sora-R cells than Sora-S cells; second, in Sora-R cells, the YAP nuclear localization on 4-kPa substrate is higher than that on the 0.7-kPa substrate . After sOverall, no consistent relationship is found between (1) ECM stiffness and YAP; and (2) YAP and drug resistance. When targeting the drug resistance through mechanically regulated YAP, the strategies will be cancer-type- and drug-dependent. In addition to the observations that the changes in ECM’s properties can affect cancer cells’ chemotherapies, recent studies suggest that chemotherapies can change the ECM mechanics and contribute to drug resistance reciprocally. In BRAF-mutant melanoma, BRAF inhibitors (PLX4032) remarkably can remodel surrounding ECM by activating melanoma-associated fibroblasts (MAFs) through phosphorylation of MLC2/MYL9, which is the key regulator of actomyosin contractility. Activated MAFs are sufficient to produce denser collagen fibrils in the ECM and drive drug resistance to PLX4032 in BRAF-mutant melanoma cells. Remodeled ECM drives ERK activation and cancer cells’ resistance through integrin β1 and FAK signaling pathways . AlthougAs discussed in For example, the stiffness of ECM influences cells’ sensitivity and reduces the efficiency of radiotherapies and chemotherapies by impairing DNA double-strand breaks (DSBs)’ repair . This prAs a mechanosensitive transcriptional coactivator , YAP canMore studies have been conducted on integrin and the following signaling pathway. As Wang et al. discuss, the integrin β1/FAK/ERK1/2/NF-κB signaling pathway is activated in liver cancer cells under high-rigidity substrate conditions (16 kPa) . Importa2 ascitic shear stress, tumors could become more resistant to chemotherapies. Hence, these results indicate potential therapeutic targets in ovarian cancers.Besides the Hippo signaling pathway and YAP, the c-Met/PI3K/Akt signaling axis and a mechanosensitive microRNA, miR-199a-3p, were also proven to have critical roles in the mediation of chemoresistance in ovarian cancer cells . ResearcMore effectors that conduct the YAP-independent mechanotransduction need to be investigated and discussed . ResearcYAP promotes drug resistance in cancer cells and can be regulated mechanically . TherefoFirst, besides the Hippo pathway, YAP activation can be regulated mechanically. The mechanical stimuli include: (1) extracellular mechanical signals such as ECM viscoelastic properties, solid stress generated in tissue/tumor microenvironments, and compressive/tensile force applied by body motions; and (2) intracellular mechanical signals such as endogenous cellular contractility and nuclear mechanical properties. These mechanical stimuli regulate the activation of YAP , accompanied by mechanobiological characterizations using molecular tension biosensors and live-cell traction force microscopy, will be instrumental.Based on this notion, we propose three types of methods for the mechanical inhibition of YAP and drug resistance in cancer cells: (1) pharmacological modulation of abnormal ECM mechanical properties in the tumors to suppress the mechano-induced YAP activation; (2) actively applying mechanical signals (e.g., ultrasound ,119 and We envision that the present unified framework  will hel


The isolated rapid-eye-movement sleep behavior disorder (iRBD) is a prodromal condition of Lewy body disease including Parkinson's disease and dementia with Lewy bodies (DLB). We aim to investigate the longitudinal evolution of DLB-related cortical thickness signature in a prospective iRBD cohort and evaluate the possible predictive value of the cortical signature index in predicting dementia-first phenoconversion in individuals with iRBD.We enrolled 22 DLB patients, 44 healthy controls, and 50 video polysomnography-proven iRBD patients. Participants underwent 3-T magnetic resonance imaging (MRI) and clinical/neuropsychological evaluations. We characterized DLB-related whole-brain cortical thickness spatial covariance pattern (DLB-pattern) using scaled subprofile model of principal components analysis that best differentiated DLB patients from age-matched controls. We analyzed clinical and neuropsychological correlates of the DLB-pattern expression scores and the mean values of the whole-brain cortical thickness in DLB and iRBD patients. With repeated MRI data during the follow-up in our prospective iRBD cohort, we investigated the longitudinal evolution of the cortical thickness signature toward Lewy body dementia. Finally, we analyzed the potential predictive value of cortical thickness signature as a biomarker of phenoconversion in iRBD cohort.R = − 0.55, P = 0.024 and R = − 0.56, P = 0.036, respectively) as well as visuospatial impairment . The longitudinal trajectory of DLB-pattern revealed an increasing pattern above the cut-off in the dementia-first phenoconverters  but no significant change in parkinsonism-first phenoconverters . The mean value of the whole-brain cortical thickness predicted phenoconversion in iRBD patients with hazard ratio of 9.33 [1.16–74.12]. The increase in DLB-pattern expression score discriminated dementia-first from parkinsonism-first phenoconversions with 88.2% accuracy.The DLB-pattern was characterized by thinning of the temporal, orbitofrontal, and insular cortices and relative preservation of the precentral and inferior parietal cortices. The DLB-pattern expression scores correlated with attentional and frontal executive dysfunction (Trail Making Test-A and B: Cortical thickness signature can effectively reflect the longitudinal evolution of Lewy body dementia in the iRBD population. Replication studies would further validate the utility of this imaging marker in iRBD.The online version contains supplementary material available at 10.1186/s40035-023-00356-y. Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is associated with synucleinopathy and in most cases, evolves into Lewy body disease (LBD) , 2. DemeDLB patients have progressive cognitive decline with characteristic cognitive fluctuations, vivid hallucination, parkinsonism and RBD as core clinical features . SignifiTo develop an imaging biomarker predicting Lewy body dementia in an iRBD population, we set out to characterize magnetic resonance imaging (MRI)-driven whole-brain cortical signature from DLB and apply it in our prospective iRBD cohort. With longitudinal data, we analyzed the evolution of the cortical signature and the relationship between the imaging marker change and clinical phenoconversion in individuals with iRBD.18F-florbetaben PET . We. We37]. Consistent with a previous longitudinal study in a large cohort , the meaIn this study, the low value of mean cortical thickness at baseline was found to significantly predict overall phenoconversion. This result is in agreement with a previous study by Pereira et al., showing that the mean cortical thickness in ROIs that differed between converters and non-converters, significantly predicted overall phenoconversion in iRBD . The basIn an effort to stratify LBD subtypes in iRBD patients, Rahayel et al. derived a clinical-imaging signature from iRBD patients that could predict dementia-first phenoconversion . The cliIn our study, none of the clinical biomarkers were able to discriminate dementia-first from parkinsonism-first conversion Table . These fThe current study has some limitations. First, the MRI-driven DLB-related cortical thickness pattern needs to be reproduced in large multicenter cohorts. Second, large-sample studies with longer follow-ups would be valuable to evaluate the relationship between the cortical signature and various prodromal markers comprehensively. Nevertheless, the sample size in this study was acceptable for analyzing the predictive performance of our imaging markers because of the relatively high HRs of these markers . Third, The MRI-driven DLB-pattern reflects prodromal clinical features and longitudinal evolution of Lewy body dementia in individuals with iRBD. The MRI-based index consisting of the mean cortical thickness values and the DLB-pattern expression score would help predict and monitor DLB-type phenoconversion in individual patients with iRBD.Additional file 1: Fig. S1. Stable regions of interestin DLB-related cortical thickness pattern. Fig. S2. Direct comparison of the cortical thickness for iRBD versus DLB. Fig. S3. Disease-free survival in iRBD patients with Kaplan-Meier analysis. Fig. S4. Correlation between the DLB-pattern and the AD-pattern. Fig. S5. Longitudinal change of DLB-pattern in the non-converter iRBD group. Table S1. List of ROIs for cortical thickness analysis.


A previously healthy 45-year-old woman presented with seizures. Computed tomography and subsequent magnetic resonance imaging (MRI) revealed calcified brain lesions .MRI revealed multiple cerebral ring-enhancing lesions with cystic components, a scolex, and vasogenic edema . ,Fast imaging, employing a steady-state acquisition MRI sequence also revealed lesions in the fourth ventricle, indicating the racemose form of extra-parenchymal neurocysticercosis (NCC) . The racRice-like ovoid calcifications resembling a starry sky were identified in the muscles , suggestIf rice- or starry sky-like muscular calcifications are detected, cysticercosis must be suspectedThis case highlights the association of NCC with the muscular form of cysticercosis and its peculiar imaging findings in a patient with epilepsy beginning in adult life.


After nearly one century of development, ADCs have emerged as a rather promising approach in the treatment of cancer, especially breast cancer, which is the most common malignant tumor in women2.The concept of antibody–drug conjugations (ADCs) can be tracked back to the early 20HER2), also known as Erb-B2 receptor tyrosine kinase 2 (ERBB2), is a crucial molecular classifier for breast cancer. The HER2 status is determined using immunohistochemistry (IHC) and in situ hybridization (ISH) in pathology laboratories. Approximately 15% of breast cancer patients exhibit HER2 overexpression and are thus defined as HER2-positive breast cancers. HER2 was a pivotal driver and therapeutic target in these patients3. HER2 is not considered to be a driving factor and is conventionally not targetable for breast cancers that display low-to-moderate HER2 expression 4.Human epidermal growth factor receptor 2 ], has revolutionized the precise classification and treatment of HER2-expressing breast cancer, including both HER2-overexpressing HER2-positive breast cancers and HER2-moderately expressed HER2-low breast cancers. By summarizing the unique mechanism of next-generation ADCs and their application in HER2-expressing breast cancers, we hope to examine the great impact of next-generation ADCs on clinicians and researchers, and to stimulate more emphasis on adapting to the era of next-generation ADCs.5. Chemotherapy often lacks tumor specificity, exhibits dose-dependent toxicity, and has a narrow therapeutic window. Conventional targeted therapies necessitate the presence of specific molecular targets , which restricts applicability to a relatively limited population. In contrast, ADCs circumvent these constraints by providing targeted delivery of cytotoxic agents guided by specific antibodies, which reduces damage to healthy cells and enhances tumor cell destruction5.ADCs are comprised of a monoclonal antibody, a linker, and a cytotoxic payload. This unique design enables ADCs to achieve “targeted chemotherapy” and provide advantages over conventional chemotherapy and targeted therapiesrd generation ADCs) have made substantial progress in antibody optimization, linker innovation, and payload selection . Humanized and engineered monoclonal antibodies increase tumor specificity, cleavable linkers enhance selective payload release, and membrane-permeable potent cytotoxic drugs contribute to bystander effects and enhanced efficacy. These advances have expanded the scope of ADC application, improved therapeutic efficacy, and reduced the incidence of adverse events6. Currently, there are many next-generation ADCs on the market or in development for breast cancer, such as T-DXd, SHR-A1811, and RC-48. Comprehensive summaries of these next-generation ADCs have been provided by previous studies8.ADCs have undergone remarkable advancements in recent years. Next-generation ADCs 3. Therefore, we have entered a new era of next-generation ADCs.The prognosis of advanced HER2-positive breast cancer patients has been improved significantly in the past two decades10. This significant positive result has made T-DXd the only priority for second-line treatment of advanced HER2-positive breast cancer, substituting for the previous standard treatment (T-DM1) in the 2023 NCCN guidelines11.In the DESTINY-Breast03 trial, advanced HER2-positive breast cancer patients treated with the next-generation ADC, T-DXd, achieved a > 4-fold increase in the median progression-free survival (PFS) compared to T-DM1, reaching a median PFS of 28.8 months8. Indeed, significant growth and competition for next-generation ADCs may be observed in the foreseeable future.Currently, more than 10 ADCs, most of which are next-generation ADCs, are undergoing clinical trials with the aim to become one of the choices for second-line treatment for advanced HER2-positive breast cancer9. The development of next-generation ADCs provides opportunities for a subset of HER2-negative breast cancers, which are now commonly designated HER2-low breast cancers, that have low-to-moderate HER2 expression.Compared to the significant improvement in survival reported in HER2-positive breast cancer, the survival benefits for HER2-negative breast cancer patients have been relatively limited in the past 20 years12. The phase Ib DS8201-A-J101 trial13 and phase II DAISY trial14 confirmed the efficacy and safety of the next-generation ADC, T-DXd, in advanced HER2-low breast cancers. Then, the randomized control phase III DESTINY-Breast04 trial15 further confirmed the impressive survival benefit of T-DXd treatment for advanced HER2-low breast cancer over chemotherapy, which made T-DXd the preferred treatment for advanced HER2-low breast cancer in the second-line setting for HR-positive and -negative diseases11.HER2-low status is defined as HER2 IHC 1+ or 2+ with ISH-. Based on this definition, HER2-low breast cancer is a prevalent subtype, accounting for a significant proportion (∼50%) of cases12. Notably, for advanced HER2-low breast cancers, the Trop2-targeted next-generation ADC, sacituzumab govitecan (SG), has also been verified to be the preferred treatment in the second-line setting11.The success of T-DXd brought a new therapeutic choice for these HER2-negative breast cancers, for which moderately expressed HER2 was previously not targetable. There are some potential competitors for T-DXd that are undergoing phase I and II clinical trials in advanced HER2-low breast cancersThe application of T-DXd in advanced HER2-positive and -low breast cancers showed the strong potential of next-generation ADCs and raises crucial problems for future clinical trials and translational studies.16. The TALENT study investigated the efficacy of combining T-DXd with endocrine therapy in neoadjuvant treatment for HR+/HER2-low early-stage breast cancer17. The DESTINY-Breast05 study included high-risk HER2+ breast cancer patients who did not achieve pCR after neoadjuvant treatment. The patients were randomized to receive T-DXd or T-DM1 adjuvant therapy to determine the efficacy of the two ADCs18. Next-generation ADCs are being used in an attempt to expand and influence the landscape of early-stage breast cancer treatment, holding tremendous potential for offering patients more treatment choices and hope. Efforts are also made to extend the lower limit of the HER2 level for next-generation ADC use. The results of the DAISY trial suggested the existence of T-DXd responders in HER2-0 tumors14, which exhibited even lower HER2 expression than HER2-low tumors. Thus, defining an accurate lower limit for the HER2 level in the application of T-DXd is urgently needed.The scope of next-generation ADCs represented by T-DXd is continuously broadening. The applicable population of ADCs has extended from patients who had a treatment failure in multiple lines of therapy in the second- or first-line setting and holds potential for early-stage breast cancer. This extension relies on the progression of high-quality randomized controlled trials to provide robust evidence, which are now underway. The DESTINY-Breast11 study explored the effectiveness of T-DXd as neoadjuvant treatment for HER2-positive breast cancer19; however, additional supporting data are needed. Another vital issue is how to treat patients who develop resistance to next-generation ADCs. The mechanisms underlying ADC resistance primarily involve three factors: antibody binding; payload; and internalizing and trafficking processes20. There are currently two main strategies to address ADC resistance: the development of new drugs; and the use of combination therapies21. Dual-site targeting with HER2 antibodies and coupling with novel toxins may offer promising prospects for overcoming resistance in the course of novel drug development22. Furthermore, some studies have explored the combination of ADCs with other therapies to combat resistance, such as ADCs in combination with pertuzumab, TKIs, or immunotherapy24. Resistance is indeed inevitable, but the unique structure and mechanism underlying ADC action provide avenues for addressing resistance, thus prospective clinical trials are needed.Another aspect to consider is how next-generation ADCs can be strategically positioned alongside existing therapies. Breast cancer treatment benefits from a wide array of available therapeutic agents. It is essential to integrate ADCs into the existing treatment regimens, especially for hormone receptor-positive breast cancers, for which there is a rich selection of mature therapies and a relatively good prognosis. Clinical trials have actively explored the combination of T-DXd with existing therapiesJAMA Oncology25 reported that a panel of pathologists had a concordance rate of only 26% in their interpretations of HER2 IHC 0 and IHC 1+. Recent years have witnessed the potential of combining digital pathology and artificial intelligence (AI) models. The development of a more accurate method to accurately diagnose HER2-low tumors might also benefit from this trend, which allows for minimization of interobserver discrepancies26. Other studies are also trying to establish diagnostic pipelines beyond the current system based on IHC and ISH to determine HER2-low status more quantitively and reproducibly27.Accurate pathologic diagnosis of HER2-low is crucial. Several studies have shown that the current diagnostic methods for HER2-low based on IHC and ISH are not entirely robust. Specifically, a multicenter study published in 14. Based on these potential biomarkers, feasible and reliable diagnostic tools can be developed and further validated in prospective clinical trials.Researchers are utilizing various cohorts, especially cohorts from clinical trials, to identify biomarkers for accurate efficacy prediction. The exploration of predictive markers for T-DXd efficacy extended beyond traditional clinical and pathologic indicators in the DAISY trial. By examining digital images of hematoxylin-eosin staining slides with the AI model, researchers found that a baseline spatial feature may predict a worse response, which is characterized by the presence of large areas of HER2 IHC 0 cells distinct from HER2 IHC 3+ tumor cells28. These features allow for high-throughput screening of potential strategies to reverse drug resistance and help accelerate the clinical application of translational research.Investigating the mechanism of primary and secondary drug resistance and how to overcome resistance is a high priority with the increased use of ADCs. By comparing molecular features between responder and non-responder clinical trial cohorts and paired samples before and after treatment, it is possible to effectively identify the underlying mechanisms and overcoming strategies. Notably, leveraging preclinical models, such as patient-derived organoids (PDOs), can recapitulate the molecular characteristics and treatment sensitivity of original patient tumors15. Additional study is warranted to clarify the role of ethnicity in HER2-low breast cancer because these factors may ultimately impact the efficacy of therapeutic interventions.There may be differences in drug efficacy among different ethnicities. In the subgroup analysis of the DESTINY-Breast04 trial, Asian patients with HER2-low breast cancer may be more likely to benefit from T-DXd than patients of other ethnicities in the HR-negative subgroupThe DESTINY-Breast trials are among the best-known clinical trials in the study of next-generation ADCs. The DESTINY-Breast trials proved the impressive effectiveness of T-DXd in breast cancer and provided a new therapeutic choice for patients. The DESTINY-Breast trials, however, have also challenged conventional concepts and persuading clinicians and researchers to establish new knowledge frameworks.18. The clinical status of T-DM1 might be repositioned with the process of DESTINY-Breast series trials. ADCs have opened up an entirely new chapter for the treatment of patients with HER2-low breast cancers, which account for approximately 50% of all metastatic breast cancer cases and were not previously intended for HER2-targeting treatments.The emerging ADCs are gradually changing the treatment landscape of advanced breast cancer in the second-line settings and beyond for HER2-positive breast cancers, and are poised to be adopted as front-line treatments. The status of the previous generation ADC, T-DM1, is being challenged. Although T-DM1 is still the standard treatment for adjuvant therapy of high-risk non-pCR patients after neoadjuvant therapy, especially for early-stage breast cancers, the ongoing DESTINY-Breast05 trial aims to rewrite the guidelinesThe 2-tier HER2-positive/-negative classification has guided the treatment of breast cancer for two decades, with HER2-positive tumors allotted to receive HER2-targeting therapies and HER2-negative tumors to receive other medications. The boundary between HER2-positive and -negative cells in clinical practice seems to be blurred, however, by the successful application of HER2-targeting next-generation ADCs in HER2-low breast cancers. Although it has been commonly acknowledged that HER2-low breast cancer is not a distinct molecular entity, the current 2-tier classification is not sufficient to guide the application of next-generation ADCs in breast cancer. Given the broader application of next-generation ADCs, a modified classification of HER2 status beyond the 2-tier classification might need to be established by introducing new quantitative methods or spatial features.29. This trend suggests that integrating precise classification and treatment outcomes might contribute to more accurate and feasible methods to guide bedside practice. Second, transformation might also occur in the development of novel drugs. In the past, researchers and drug developers have placed more emphasis on searching for new targets, elucidating the underlying mechanisms, and ultimately finding corresponding targeting compounds. With the maturation of ADC technology and the success of T-DXd, drug enterprises may turn to utilizing existing classic targets, such as HER2, HER3, and EGFR, to develop more efficient ADCs6.First, the classic data-driven analysis method might turn to clinical outcome-driven analyses to study the heterogeneity of breast cancers. In data-driven studies, researchers usually employ unsupervised clustering methods regardless of clinical outcomes. Classifications have recently emerged, including clinical outcomes, especially for those outcomes based on cohorts derived from clinical trials, such as ISPY-2Both challenges and opportunities exist in the era of next-generation ADCs. For drug enterprisers, the success of next-generation ADCs have challenged the status of former standard treatments, but also strongly proved the feasibility of ADC technology and opened a new track of drug development. For clinicians, next-generation ADCs added to the toolbox of treatment and provided new opportunities for patients, especially patients who were heavily pretreated. Next-generation ADCs also put advanced vital issues, such as toxicity management. Next-generation ADCs incentivized fundamental researchers to adapt to reshaped research approaches and also offered new topics, such as studying how tumors develop resistance to ADCs and how to treat tumors after ADC resistance. In summary, the emergence of next-generation ADCs has significantly changed the landscape of breast cancer treatment and research. Ongoing research, coupled with regulatory approvals, underscores the promising future of ADCs in combating breast cancer. With continued advancements, ADCs hold the potential to provide more precise and efficacious treatment options, ultimately improving patient survival and quality of life.


Despite intensive multi-agent chemotherapy and hematopoietic stem cell transplantation, cure rates for patients with acute myeloid leukemia (AML) remain poor . One of miRNAs can both promote and inhibit leukemia progression and maintenance by interacting with proto-oncogenes and key signaling pathways . During +CD38− or CD34+CD38+ hematopoietic stem and progenitor cells. In line with our previous findings [To develop a clinically relevant approach for restoring miR-193b function, we encapsulated miR-193b mimics into Dlin-MC3-DMA nanoparticles (LNP/miR-193b) using a semi-automated platform that assures GMP-grade LNPs production, making it suitable for upscaling to clinical applications . The loa+CD11b+ myeloid-derived suppressor cells, B220+ B-cells, or CD3e+ T-cells  cells and their progenies revealed that transient restoration of miR-193b function also spared the hematopoietic stem and progenitor populations  cell population presented with slightly lower expression of c-Kit in the LNP/miR-193b arm than in the control arm, but the difference was not statistically significant  or lineage-primed multipotent progenitors  reduction in hCD45+ cells. LNPs treatment was well tolerated confirming the safety of restoring miR-193b function in immunocompromised hosts . However, spleen size was significantly reduced in mice that received LNP/miR-193b treatment compared to that in control animals (Supplementary Fig. Taken together, by leveraging PDXs, this study overcomes the limitations of cell lines and provides a physiological model of clonal diversity in AML to test the functionality of miR-193b-based therapeutics. Our work supports not only the robust tumor-suppressive functionality of miR-193b but also the utility of miR-mimics for cancer treatment.Interfering with miRNA expression using synthetic oligonucleotides has emerged as a promising treatment approach for leukemia and other malignancies . While tTreatment with LNP/miR-193b was well tolerated and did not cause detectable changes in the HSCs. Mild differences observed in the hematopoietic progenitors and in differentiation of lymphoid and myeloid cells were expected given that miR-193b exhibits divergent cellular functions in different cell contexts. Multiple miR-193b targets repressed in HSCs are activated upon hematopoietic differentiation, specifically in the MAPK/ERK cascade , 7. The In conclusion, our data confirmed that the transient restoration of miR-193b has strong anti-leukemic effects that can be leveraged to treat leukemia in a clinically relevant system based on PDXs. Considering that aberrantly activated MAPK signaling is involved in a plethora of hematological diseases, and that mutations in several miR-193b targets are associated with poor clinical outcomes, our findings suggest that the clinical use of LNP/miR-193b may improve the efficacy and/or tolerability of current chemotherapeutic approaches.SUPPLEMENTAL MATERIAL


We aimed to investigate the relationship between SARS-CoV-2 and other circulating respiratory pathogens. A secondary objective was to assess the correlation between respiratory pathogen detection and antibiotic use at a single medical center during the COVID-19 pandemic.Significant unadjusted correlations from bivariate cross-correlation analysis applying Spearman’s rank method to monthly viral infection prevalence are shown in gray, with negative and positive correlations indicated by − and +, respectively, and noncorrelated virus pairs in white.For viral-viral interaction analysis, data were collected from infection control surveillance reports on all positive respiratory viral pathogen tests  conducted between October 2018 and February 2022. Real-time PCR respiratory panel (RT-PRP) results (negative or positive) were extracted from the VA Corporate Data Warehouse for inpatients and outpatients. A random subset of patients (15-20%) was selected for chart review. Baseline characteristics, signs and symptoms, laboratory results, imaging interpretation, and admission and discharge diagnoses were collected. Spearman's rank correlation was employed to identify viral-viral interactions. Chart review data were analyzed using Wilcoxon-rank sum tests and Fisher's exact test.A total of 883 positive surveillance tests were analyzed for viral-viral interactions. A significant negative relationship was identified between SARS-CoV-2 and the prevalence of 43% of other respiratory pathogen types. Among 1,002 RT-PRP results, 168 patients with an RT-PRP result were chart reviewed. Although numerically a smaller proportion of patients with positive RT-PRP – compared to negative RT-PRP – received antibiotics this difference did not reach statistical significance.This project highlights the diverse influence of SARS-CoV-2 on the prevalence of other respiratory pathogens. No significant impact was detected on antibiotic use of a positive RT-PRP result. A positive RT-PRP result represents only one factor leading to the utilization of antibiotics, with other factors including clinician experience, symptom duration and severity, immunocompetency, and imaging. Based on these findings, we aim to enhance the integration of viral pathogen identification into antibiotic stewardship efforts.All Authors: No reported disclosures


Around 10% of new HIV infections in the US are attributed to injection drug use or male-to-male contact and injected drug use; syringe sharing is the second riskiest behavior for contracting HIV. For many, substance use disorder (SUD) treatment is the primary point of access for receiving HIV-related services. A 2021 survey showed gaps in HIV prevention knowledge and practice among SUD center staff but also interest in education . The BRIDGE PrEP program is a grassroots initiative designed to educate and motivate SUD center staff to increase their clients’ awareness of and access to HIV preexposure prophylaxis (PrEP) services.Ten live continuing medical education (CME) meetings supported by an educational grant from Gilead Sciences, Inc were conducted at SUD centers between August 2022 and May 2023. The 1-hour meetings provided didactic information, real-world insights, faculty-learner interactions, local networking, and take-away print and online staff/patient-education resources . Program goals include:1.Increase HIV testing of people who inject drugs 2. Educate SUD center staff regarding PrEP harm-reduction practices and 3.Build linkages between SUD staff, PrEP providers, and HIV-care networks within local/regional systems. Moore’s Level 1-5 (self-reported) outcomes  were collected via pre-, post-, and 6-week follow-up surveys evaluating knowledge and self-reported frequencies of recommended HIV prevention practice behaviors.To date, there have been 154 participants representing a diverse multidisciplinary learner group extending beyond the clinician-only audience typical of many CME programs . Increases in knowledge were demonstrated across topic areas , and participants reported intent to improve HIV prevention-related practices following the activity . To date, follow-up surveys report specific actions taken on individual and system levels to improve HIV screening and prevention .Positive outcomes related to HIV prevention knowledge and practice among SUD center staff have the potential to impact many at-risk individuals. Additionally, follow-up surveys suggest that improved awareness is prompting collaboration and system-level changes driven by participants .All Authors: No reported disclosures


Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals. Monitoring the transmission of pathogens in the population is crucial for the prevention and control of infectious disease epidemics. Clinical surveillance of pathogens in individuals is vulnerable to sampling bias, favouring those who pursue healthcare intervention and overlooking asymptomatic infections, as well as being resource intensive and reliant on advanced public healthcare services . WastewaThe SARS-CoV-2 pandemic, originating in Wuhan, China in 2019 , has beeAfter the identification of SARS-CoV-2 RNA in patient stool samples , 19, detMonitoring of SARS-CoV-2 in wastewater routinely involves an enrichment step to concentrate viral RNA from large volumes of wastewater, for which a range of ultrafiltration and precipitation-based methods have been trialled , 30. OncHere, we describe a protocol for SARS-CoV-2 quantification and sequencing in wastewater, from processing of raw wastewater samples to library preparation and quality control of the sequencing data . The libWastewater RNA samples used to generate sequencing data presented in this study were selected from those collected as part of the EMHP wastewater programme in England . WastewaSynthetic SARS-CoV-2 RNA was obtained from Twist Biosciences  and used to prepare variant mixtures and serial dilutions, including Control 15 Alpha (EPI_ISL_601443), Control 16 Beta , Control 23 Delta  and Control 29 Delta . Details of concentrations of each variant are provided in the table in Details of the protocol for processing of wastewater samples, including sample clarification, viral enrichment and RNA extraction, and RT-qPCR SARS-CoV-2 quantification can be found in Walker et al. . In brieIn brief, wastewater nucleic acid samples are purified by 1.8x magnetic bead clean-up using Mag-Bind® TotalPure NGS beads (Omega Bio-tek) before cDNA is synthesised using the LunaScript® RT SuperMix Kit . This protocol then utilises the EasySeq™ RC-PCR SARS CoV-2 (novel coronavirus) Whole Genome Sequencing Kit  for library preparation, which generates SARS-CoV-2 amplicons with sequencing adapters and indexes in one step. Wastewater samples for which sequencing data is presented here were processed using the v4.01 probe set from NimaGen. Finally, libraries are purified by two consecutive 0.6x magnetic bead clean-ups with Mag-Bind® beads, to remove primers and PCR artifacts, before being sequenced. Sequencing of samples included in this article was carried out on a NovaSeq 6000 2 × 150 bp SP flowcell .The protocol described in this peer-reviewed article for SARS-CoV-2 amplicon library preparation and sequencing in wastewater RNA samples is published on protocols.io (dx.doi.org/10.17504/protocols.io.81wgb7bx3vpk/v3) and is included for printing as https://github.com/connor-lab/ncov2019-artic-nf), and a BED file supplied by Nimagen containing the primer v4.01 locations. Briefly, reads were trimmed using Trim Galore! V0.6.5  and subsequently mapped to the SARS-CoV-2 reference genome [Raw Illumina sequences were analysed using the Illumina workflow of the ncov2019-artic-nf pipeline , percentage of the genome covered, and mean coverage depth were calculated. Results obtained with the different post-PCR clean-up methods , and standard versus additional RNA clean-up steps, were compared and visualised as scatterplots using custom scripts in R .Medium coverage depths for each of the 154 amplicons were determined for each sample using mosdepth v0.2.6  and heatmaps for the synthetic SARS-CoV-2 lineage mixtures and wastewater samples were generated in R using custom scripts .https://github.com/andersen-lab/Freyja; curated lineage file and UShER global phylogenetic tree downloaded on 31st Dec 2021). Expected and measured abundances as well as percentage genome coverage were plotted using custom scripts in R  magnetic beads. This step was found to significantly improve the quality of sequencing libraries during optimisation of the protocol. To demonstrate this, a set of 74 wastewater samples, collected in June 2021 from sewage network sites across England, were sequenced in parallel through the protocol with and without the RNA clean-up step. The number of raw reads obtained for each sample was not skewed towards either of the two parallel libraries, demonstrating effective normalisation of these libraries during sequencing . When saQuality control (QC) of amplicon libraries generated with this protocol is crucial prior to sequencing. The main method of routine library QC is fragment size analysis using the Agilent D1000 ScreenTape assay on the 4200 TapeStation system . Similar fragment size analysis could be carried out using an Bioanalyzer system , or gel electrophoresis if the former technologies are unavailable. This QC assay captured the success of the RC-PCR reaction in amplifying the desired SARS-CoV-2 amplicon library of around 435 bp, as well as the efficacy of the size selection in purifying these amplicons. A typical size profile of the libraries following post-PCR purification is provided in TM beads supplied, and comparison of these with Mag-Bind® beads has shown equivalent performance for wastewater SARS-CoV-2 library preparation /μl using the variants Alpha and Delta (B.1.617.2), as well as an equal parts Alpha/Delta mixture . The numSARS-CoV-2 lineage calling using Freyja  was ableTo further assess the efficacy of the library preparation protocol and QC pipeline, including differentiating between SARS-CoV-2 variants using Freyja, a range of proportions of the variants Alpha, Beta, Delta and Delta AY.2 were used to prepare 46 samples (“covmix1-46”) at a concentration of 200 gc /μl . Genome Overall, these results demonstrate the efficacy of this protocol for sequencing of SARS-CoV-2, including mixtures of variants expected to be present in wastewater samples. We recommend similar performance evaluation, using synthetic SARS-CoV-2 RNA or clinical samples as positive controls, in any laboratory implementing this protocol.th January 2022 from sewer network sites across England. The resulting libraries were sequenced on a NovaSeq SP flowcell, with an average of 1.5 million 2x150 bp paired-end sequencing reads generated per sample. As expected with equal pooling of PCR products from all samples, regardless of estimated SARS-CoV-2 concentration, samples with lower Ct values generated more sequencing reads and had higher mean coverage depth , meaning the high genome coverage observed is essential for detecting the presence of mutation profiles to support the assignment of variant(s) to samples. Furthermore, with a raw sequencing read depth of >150,000, a mean coverage depth of >200x was obtained for most of the samples  wastewater monitoring programme . To demo samples , providiAlthough some SARS-CoV-2 wastewater monitoring studies have utilised primary settled sewage samples , influenThe NimaGen EasySeq™ RC-PCR SARS CoV-2 (novel coronavirus) Whole Genome Sequencing Kit used in this protocol utilises RC-PCR to integrate PCR amplification and multiplexed library preparation for Illumina sequencing . The firOverall, one-step PCR and adapter incorporation significantly reduces hands-on time over other amplicon library preparation methods. Indeed, RC-PCR preparation takes less than 30 min for one 96 well plate, and hands-on time for the protocol presented here is less than 4 hours, which is significantly less than other protocols such as ARTIC and Midnight . The RC-The primer scheme NimaGen EasySeq™ generates amplicons with a mean insert size of 226 bp, which are shorter than other popular SARS-CoV-2 amplicon schemes such as those in the ARTIC (~400bp)  and the Due to the high-throughput nature of regular wastewater sequencing using this protocol, and the low SARS-CoV-2 concentration commonly associated with wastewater RNA samples, pooling of amplicon libraries was carried out routinely without normalisation. However, when positive control material from clinical samples or synthetic SARS-CoV-2 RNA, which typically contain high SARS-CoV-2 concentrations, is used alongside wastewater samples, post-PCR dilution and/or adjustment of pooling volumes is recommended to prevent controls dominating the obtained sequencing reads. For smaller numbers of samples, or when samples of atypically high SARS-CoV-2 concentration are processed (i.e. <Ct 28), normalisation during pooling of PCR products based on Ct values from SARS-CoV-2 RT-qPCR would improve balancing of read depth across samples.The inclusion of negative controls is important to ensure no contamination has been introduced into the PCR master mix from previous SARS-CoV-2 PCR runs. To avoid introduction of amplicon contamination, PCR master mix should be prepared in a PCR-cabinet, and post-PCR purification and library QC should be performed in a separate area, or preferably room, within the lab and with different equipment . Sequencing reads from negative controls are routinely found to align to the SARS-CoV-2 genome due to the presence of PCR artifacts, the vast majority of which should be filtered out after primer-trimming. However, low levels (<0.1%) of reads from negative controls are routinely found to align to the SARS-CoV-2 genome after primer-trimming, likely due to contamination from previous amplicons with the same barcodes being introduced during post-PCR purification. The use of too many negative controls in pooled libraries may introduce higher levels of PCR artifacts into the final pool, due to high concentrations of these in PCR reactions containing no template, which may reduce the depth of sequencing for wastewater samples in these pools. We therefore recommend that a maximum of two negative controls are included in each 96-well plate.The presence of PCR inhibitors in wastewater is widely recognised as one of the challenges of wastewater-based epidemiology , 44, as We demonstrate that SARS-CoV-2 can be regularly detected through whole-genome sequencing in RT-qPCR-negative wastewater samples, often yielding sufficient genome coverage breadth and depth for variant assignment. This highlights the enhanced sensitivity of targeting amplicons across the genome compared to the short regions targeted by RT-qPCR, which is particularly effective for wastewater monitoring in which samples are expected to contain more degraded viral RNA than clinical samples obtained from nasopharyngeal swabs. Indeed, whole-genome sequencing has been proposed as a highly sensitive alternative diagnostic tool for SARS-CoV-2 detection in clinical samples , 46, altThe protocol described here provides an optimised and efficient method for sequencing of viruses from influent wastewater, utilising reverse complement PCR for amplicon generation and library preparation in one step. This increases efficiency and reduces hands-on time compared to other protocols. We demonstrated the sensitivity of this protocol to provide broad coverage of the SARS-CoV-2 genome from samples containing low template concentrations, and its reliability for high-throughput sequencing. This protocol could be easily applied for the monitoring of other viruses from wastewater, providing cost-effective and population-wide genomic surveillance during disease outbreaks.S1 File(PDF)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(TXT)Click here for additional data file.S4 File(CSV)Click here for additional data file.


Gene editing stands for the methods to precisely make changes to a specific nucleic acid sequence. With the recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, gene editing has become efficient, convenient and programmable, leading to promising translational studies and clinical trials for both genetic and non-genetic diseases. A major concern in the applications of the CRISPR/Cas9 system is about its off-target effects, namely the deposition of unexpected, unwanted, or even adverse alterations to the genome. To date, many methods have been developed to nominate or detect the off-target sites of CRISPR/Cas9, which laid the basis for the successful upgrades of CRISPR/Cas9 derivatives with enhanced precision. In this review, we summarize these technological advancements and discuss about the current challenges in the management of off-target effects for future gene therapy. Genome editing tools hold great promise in treating genetic and non-genetic diseases. Early studies utilize zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for genome editing . ZFNs anThe Cas9/sgRNA complex produces site-specific DNA double-strand breaks (DSBs), stimulating homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways to achieve genome editing. HDR is an accurate but inefficient mechanism, which utilizes a homologous donor template to repair DNA cleavages . By contin silico tools are useful to search for potential off-target sites in the whole genome and calculate the likelihood of an off-target editing , CROP-IT (CRISPR/Cas9 Off-target Prediction and I dentification Tool), CFD (Cutting frequency determination), DeepCRISPR and the Elevation software packages. The algorithm of MIT weights the position effect of the mismatches in the sgRNA to generate a score to evaluate off-target effects (The second group of  effects . CCTop a effects . The CFD effects . DeepCRI effects . Similar effects . The dis effects .in vitro reporting of cleavage effects by sequencing) (Cell-free methods reconstitute nuclease reaction on DNA or chromatin that are extracted from the cells to directly identify genomic cleavages in the test tubes. Representative cell-independent methods are Digenome-seq (digested genome sequencing), DIG-seq (Digenome-seq using cell-free chromatin DNA), Extru-seq, SITE-seq (selective enrichment and identification of tagged genomic DNA ends by sequencing) and CIRCLE-seq (circularization for uencing) .Digenome-seq is a highly sensitive method that can identify indels with 0.1% frequency or lower . In thisA major caveat of Digenome-seq is that the chromatin states are omitted in the assessment of the off-target effects. To solve this problem, an updated version of Digenome-seq called DIG-seq  was deveTo further retain the genome near its intracellular state for better off-target detection, Jeonghun et al. recently reported Extru-seq . In thisAll above methods involve the expensive WGS step. To reduce such cost, scientists developed SITE-seq, a method that adds a selective biotinylation reaction on the cleaved genomic sites and leverages streptavidin pulldown to enrich these sites before sequencing . SITE-seCIRCLE-seq is another method that can detect genome-wide off-target sites without performing WGS. In this method, genomic DNA is first sheared and circularized by intramolecular ligation. With the presence of Cas9/sgRNA complexes, the circular DNA fragments were selectively linearized upon Cas9 nuclease cleavage before they become available for library construction and high-throughput sequencing . In thisin situ and sequencing) have been developed to achieve this goal , IDLVs , GUIDE–seq , LAM-HTGTS (linear amplification-mediated high-throughput genome-wide sequencing), BLESS , and BLISS , which can bind to genome DNA without introducing DSBs and detaching from the edited sites . One stuIntracellular labeling of CRISPR/Cas9-edited loci for the enrichment of these DNA fragments is necessary for more precise determination of the off-target effects in cells. One such labeling tool is called the integrase-defective lentiviral vectors (IDLV), which displays the propensity to integrate into the vicinity of DSBs . IDLV waAnother more popular method to detect off-target sites in cells is called GUIDE-seq . This teBoth IDLV and GUIDE-seq rely on the DNA insertion activity during NHEJ. However, DSBs can also lead to chromosome translocation and rearrangement. To better detect such DSBs, LAM–HTGTS was developed . In thisin situ breaks labeling, enrichment on streptavidin and next-generation sequencing), which captures DSBs in situ via the ligation of biotinylated linkers to cleavage sites in fixed cells  technology was developed  and GUIDE-tag  functions have provided critical insights about the structural basis of off-target effects  at 23/24, 18/24, and 19/24 tested sites, respectively. In addition, GUIDE-seq results demonstrated dramatically decreased number of off-target sites. For example, among the 134 off-target sites of one sgRNA targeting VEGFA with SpCas9, only 19, 24, and 18 of the off-target sites are also detectable with eSpCas9, SpCas9-HF1 and hypaCas9, respectively .SpCas9 variants with enhanced specificity can also be developed by an unbiased high-throughput screen. A good example is a yeast-based screen for SpCas9 mutants with random mutations in the REC3 (REC stands for recognition) domain, the critical Cas9 domain that is responsible for the pairing between the genomic DNA and the Cas9/sgRNA complex. This screen identified four key beneficial point mutations in SpCas9, giving rise to evoCas9, a variant that exhibits fidelity exceeding SpCas9-HF1 and eSpCas9 . AntonioIn addition to protein engineering, the specificity of CRISPR/Cas9 editing can also be enhanced with paired SpCas9 nickases . Cas9 niStaphylococcus aureus requires a more complicated PAM sequence of 5’-NGGRRT-3’  or locked nucleic acids (LNA) into the sgRNAs also reduces the kinetics of Cas9 nuclease reactivity, thereby improving the specificity of genome editing  into the ribose-phosphate backbone of sgRNA and found that MP modifications at certain positions can enhance on-target specificity while dramatically reducing off-target activity . Incorpo editing . The rep editing . The maj editing .Cas9-mediated DSB generation is the main source of CRISPR/Cas9 off-target effect. Therefore the new versions of gene editors that do not create DSBs usually exhibit greater specificity of genome editing. Base editors are one such tool, which couple Cas9 nickases with nucleotide deaminases to achieve single nucleotide conversion without introducing DSBs. The most popular base editors are the adenine base editor (ABE) and the cytosine base editor (CBE). ABE catalyzes the editing from adenine to guanine and CBE converts cytosine to thymine .Although CBE and ABE greatly reduce the classic off-target effects of CRISPR/Cas9 systems, they create new formats of off-target effects such as RNA editing and sgRNA-independent DNA editing . These eThe recent development of EndoV-seq and Detect-seq have greatly facilitated the detection of off-target effects of ABE and CBE on genomic DNA. EndoV-seq leverages the endonuclease V (EndoV) to specifically cut inosine in DNA . Since iIn addition to base editors, CRISPR/Cas9-mediated off-target effects can also be reduced by epigenetic editors . These tvia plasmid transfection, ribonucleoprotein  electroporation, or viral transduction. RNP electroporation exhibits higher on-target editing efficiency and lower off-target mutations than the other delivery methods  and lipid nanoparticles (LNPs) are currently the primary vectors for in vivo gene therapy. AAV-delivered gene expression is known to last for years in terminally differentiated cell types (in vivo, thus LNP is currently the most popular vector for in vivo gene editing (The expression duration of genome editors also influences the choice of vectors for ll types . While tll types . By cont editing , which h editing  (Table 2in vivo and even in patients is particularly challenging. The development of solutions for these problems would give rise to next-generation genome editing tools that accelerate the arrival of the gene therapy era.Efficiency and specificity are the two critical parameters that determine the success of CRISPR/Cas9-based editing of the genome. The tremendous potential of genome editing for gene therapy urges scientists to fully address its safety concerns, particularly the off-target effects. Methods to assess off-target effects of CRISPR/Cas9 have quickly evolved in the last decade. However, limitations still remain in balancing the accuracy versus sensitivity of these new techniques. Direct assessment of off-target effects


Nature article, Lee et al. investigated distinct metabolic dependencies of pancreatic ductal adenocarcinoma (PDA) cells compared to normal tissue, and discovered that PDA cells exhibited a unique reliance on de novo ornithine synthesis from glutamine, rather than arginine. This aberrant polyamine synthesis offers a promising therapeutic target for pancreatic cancer.1In a recently published KRAS mutations, G12D (~50%) and G12V (~30%), respectively. In fact, KRAS mutations are present in almost all PDA, considering this neoplasia as the most RAS-addicted among all solid cancers.PDA, often referred to as the “silent killer,” is a deadly disease that often shows no symptoms until it has already spread to other organs. Current diagnostic tools and (chemo)therapies have not been effective in improving patient outcomes with advanced/metastatic PDA, a genetic disease commonly caused by oncogenic KRAS is mutated, it is “locked” in its active (GTP-bound) state and triggers downstream signaling such as RAF/MEK/ERK and PI3K/AKT/mTOR pathways. This allows oncogenic KRAS to unleash mitogenic processes and develop chemoresistance. There is also significant experimental evidence in PDA supporting the role of oncogenic KRAS signaling in metabolic reprogramming of bioenergetic pathways, specifically involving glutamine and glucose. This metabolic reprogramming is a reversible transcriptional-process influenced by nutrient availability in the tumor microenvironment (TME).2 The TME of PDA is characterized by a highly desmoplastic stroma and abnormal vasculature, which restricts nutrient access. Thus, metabolic adaptation and survival in this hostile environment are critical for PDA aggressiveness.Once 15N(amine)-glutamine or 15N4-arginine, Lee and colleagues demonstrated that glutamine was the main source of ornithine and its polyamine derivatives in PDA cells. In contrast, arginine was the preferred source for polyamine synthesis in non-PDA cancers . Polyamines are essential, low-mass, highly charged molecules that play critical roles in cellular processes related to growth, survival, chromatin structure stabilization, protein and nucleic acid synthesis, and apoptosis in both prokaryotes and eukaryotes. Depleting polyamines leads to cytostasis, emphasizing their importance for normal cell function and growth in mammals.3 In an inducible KRAS(G12D) mouse model of PDA, the researchers confirmed the preference for glutamine metabolism for in-vivo polyamine synthesis, compared to non-tumor-bearing KRAS wildtype control mice. The authors proposed that the unique metabolic rewiring in PDA cells toward glutamine was a consequence of the low-arginine present in TME, which resulted from arginine breakdown by ARG1-expressing myeloid cells. However, it remains unclear how PDA cells maintain this preferential rewiring for glutamine despite the availability of abundant arginine in the culture media in-vitro.Using metabolic tracing with OAT, ARG2, and GATM (glycine amidinotransferase), as well as the rate-limiting enzyme for polyamine synthesis, ornithine decarboxylase 1 (ODC1).4 Interestingly, silencing OAT, but not ARG2 or GATM, significantly reduced glutamine-derived ornithine synthesis and putrescine production. Loss of OAT mimicked the effect of ODC1 knockdown in suppressing AsPC-1 cell-proliferation, which could be rescued by adding putrescine. These results were validated in KRAS(G12D) mouse cells and human orthotopic xenografts, where OAT loss led to a significant reduction in tumor size. Moreover, since polyamines in the TME were primarily derived from PDA cells rather than non-cancer cells, compensatory uptake following decreased intracellular polyamine levels seemed unlikely.To understand potential compensatory mechanisms in PDA, the authors conducted gene knockdown studies in AsPC-1 cells targeting the three enzymes involved in ornithine synthesis: KLF6 decreased expression of polyamine synthesis genes, highlighting the importance of this pathway in PDA.The study also investigated the role of KRAS in polyamine synthesis and identified KRAS-MEK-KLF6 axis that regulates the transcriptional upregulation of polyamine synthesis genes. Inhibition of KRAS or MEK reduced ornithine and putrescine levels, while silencing Comparing the effects of inhibiting OAT and ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, the authors found that OAT inhibition using 5-fluoromethylornithine (5-FMO) suppressed glutamine-derived ornithine and putrescine synthesis specifically in PDA cells, leading to reduced tumor-growth. In contrast, ODC1 inhibition using difluoromethylornithine (DFMO) primarily affected putrescine synthesis, but had off-target effects on both cancer and non-cancer cells. Combining 5-FMO with a polyamine transport inhibitor showed an additive growth-suppressive effect in PDA cells. In an in-vivo setting, OAT inhibition with 5-FMO effectively suppressed PDA growth without observable toxicity-related changes.The study also examined the impact of altered polyamine levels on TME and immune cell populations. Decreased polyamine levels resulted in smaller tumors and reduced immunosuppressive granulocytic myeloid-derived suppressor cells. However, polyamine reduction did not significantly affect immune cell populations in tumors or tumor-draining lymph nodes.OAT or ODC1 knockdown revealed significant changes in gene expression and chromatin accessibility associated with suppressed tumor-growth. These findings highlight the critical role of OAT-mediated polyamine synthesis and suggest it as a potential target for therapeutic intervention in PDA.Further analysis of the transcriptome and chromatin landscape of PDA cells following 5 Silencing OAT or ODC1 resulted in more differentially expressed genes, compared to ARG2 knockdown and unsupervised clustering showed that silencing OAT had greater transcriptional similarity to ODC1 knockdown. Silencing OAT and ODC1 also led to changes in chromatin accessibility, indicating the involvement of OAT in polyamine-induced alterations. Gene-set enrichment analysis revealed pathways associated with suppressed tumor-growth.Finally, the authors explored the impact of polyamines on the transcriptome and chromatin landscape of PDA using RNA- and ATAC-sequencing.Altogether, these findings support the importance of OAT-mediated polyamine synthesis in PDA Fig.  and open