MolGNN, a new deep learning model for molecular property prediction. MolGNN applies a graph neural network to computational learning of chemical molecule embedding. Comparing to state-of-the-art approaches heavily relying on labeled experimental data, our method achieves equivalent or superior prediction performance without manual labels in the pretraining stage, and excellent performance on data with only a few labels. Our results indicate that MolGNN is robust to scarce training data, and hence a powerful few-shot learning tool. MolGNN predicted several multi-targeted molecules against both human Janus kinases and the SARS-CoV-2 main protease, which are preferential targets for drugs aiming, respectively, at alleviating cytokine storm COVID-19 symptoms and suppressing viral replication. We also predicted molecules potentially inhibiting cell death induced by SARS-CoV-2. Several of MolGNN top predictions are supported by existing experimental and clinical evidence, demonstrating the potential value of our method.The life-threatening disease COVID-19 has inspired significant efforts to discover novel therapeutic agents through repurposing of existing drugs. Although multi-targeted  therapies are recognized as the most efficient approach to system diseases such as COVID-19, computational multi-targeted compound screening has been limited by the scarcity of high-quality experimental data and difficulties in extracting information from molecules. This study introduces  Although COVID-19 vaccines offer a path to control the spread of coronavirus, there remains the challenge of creating widely available vaccines and rapidly developing updates to match fast-emerging new SARS-CoV-2 strains. Meanwhile, the discovery of novel drugs and therapies against the SARS-CoV-2 infection is critical for tackling the disease. However, discovery and development of effective antiviral therapies can be costly and time-consuming. For this reason, significant efforts have been made toward repurposing drugs for COVID-19 treatment  as a timiscovery .in silico drug design, which provides a powerful tool to speed up chemical compound screening  leverage the power of the graph neural network with a pretraining process to learn molecular embedding, with molecules represented by a heterogeneous graph structure, with atoms as nodes and bonds as edges, and 2) employ a motif self-learning mechanism to encode information extracted from frequent subgraph structures, such as functional groups. In the following, we present evidence that our method represents the molecular structure more efficiently than the traditional sequence model  1/2/3 and the main protease (MPro). JAK is a family of intracellular tyrosine kinases  readouts. Pretrained GNN weights were saved and reused for different downstream classification tasks after fine-tuning with labeled task data.The model was built on a multitask learning framework with three tasks: node- and edge-level embedding learning, self-supervised motif learning, and supervised fine-tuning/graph classification . We follPubChem fingerprint dimension), while the classification MLP had 256 hidden units (same as the dimensions of node embeddings). Our model was built with PyTorch and PyTorch Geometric , average precision (AP), and F1 score as metrics. All metrics were calculated with the  package .MolGNN included a two-stage pretraining method derived from ContextPred . To demonstrate that MolGNN benefited from both pretraining stages, we performed an ablation study  is believed to decrease the rate of SARS-CoV-2 viral infection (MPro binding activity (Among predicted drug candidates, several molecules have already been under study due to their anti–COVID-19 effects, providing a validation to our predictions. For example, we predicted that cyclophosphamide, recently shown to mitigate acute respiratory distress syndrome among COVID-19 patients , could ipatients , may be nfection . Ipidacractivity . Thioctiactivity . Harringactivity . Lopinavactivity .MolGNN to facilitate drug discovery. Compared to state-of-the-art techniques, our implementation showed the following advantages:MolGNN was fully self-supervised. It did not require any extra-labeled data to obtain graph-level embedding as in 1. In the pretraining step, MolGNN not only captured atom- and bond-level information but also substructure information, which was critical for its superior performance in chemical-related tasks.2. Specifically designed for chemicals, MolGNN can successfully handle sparse labeled data. The graph-level label we used in self-supervised pretraining could be much more easily acquired than labels derived from specific experiments, providing our method with a wider range of use.3. We developed a new GNN-based and self-supervised learning method GNN model trained with MolGNN showed robustness, when applied to a small labeled fine-tuning data set, suggesting a potentially powerful few-shot learning method. Even with very little fine-tuning data, pretraining was able to improve final performance by a large margin. This confirms that substructure-based labels can assist neural networks in capturing intrinsic chemical attributes of molecules in their latent space.The in vitro and in vivo. The experimental validation of our testable hypotheses should also be the subject of future collaborative work. Our method may contribute to polypharmacology by predicting candidate molecules for multiple targets, based on various models pretrained and fine-tuned with MolGNN. Finally, we believe that our method may speed up drug development both in the specific case of COVID-19 and of other diseases for which few effective therapies are currently available.Our method provides a powerful tool for new drug development, especially in the case of new and poorly known diseases. Our fine-tuned model successfully identified various molecules exhibiting anti–COVID-19 activity from a large set of chemical compounds. Some of our proposed candidates have already shown potential to contribute to COVID-19 treatment and have been included in the clinical trial. We suggest that such compounds should be tested both


Extensive malignant-appearing calcifications have traditionally been considered a contraindication for breast-conserving surgery. The evaluation of calcifications largely depends on mammography, which is limited by tissue superimposition and is unable to reveal spatial information about extensive calcifications. Three-dimensional imaging modality is needed to reveal the architecture of extensive calcifications. In the present study, a novel cone-beam breast CT-guided surface location technique was investigated to facilitate breast-conserving surgery in breast cancer patients with extensive malignant breast calcifications.Biopsy-proved early breast cancer patients with extensive malignant-appearing breast calcifications were included. A patient will be considered suitable for breast-conserving surgery if the spatial segmental distribution of calcifications is found by 3D images of cone-beam breast CT. Then, the margins of the calcifications were located in contrast-enhanced cone-beam breast CT images. Next, skin markers were located using radiopaque materials, and cone-beam breast CT was reperformed to confirm the accuracy of surface location. During breast-conserving surgery, lumpectomy was performed according to the previous surface location, and an intraoperative specimen x-ray was applied to double-check that the entire lesion was removed. Margin assessment was made for both intraoperative frozen section and postoperative pathology examination.From May 2019 to Jun 2022, 11 eligible breast cancer patients in our institution were included. Breast-conserving surgery was performed successfully in all patients using the surface location approach mentioned before. All patients achieved negative margins and satisfied cosmetic results.This study proved the feasibility of cone-beam breast CT-guided surface location for facilitating breast-conserving surgery in breast cancer patients with extensive malignant breast calcifications. Previous studies have proved that breast-conserving surgery plus radiation is a safe therapeutic option for patients with multifocal DCIS (The indications of breast-conserving surgery (BCS) have expanded over the past two decades . Extensical DCIS . Howevercal DCIS .Mammography (MG) has played a key role in the screening and diagnosis of suspicious calcifications for more than 30 years , 5. UnfoCone-beam breast computed tomography (CBBCT) was approved for diagnostic use in 2015 and had been gaining recognition for providing isotropic three-dimensional (3D) imaging with both high spatial and contrast resolution . MoreoveTo reveal the spatial location and distribution of extensive calcifications, we utilized 3D reconstruction images of CE-CBBCT and developed a novel approach to surface location for breast-conserving surgery in breast cancer patients with extensive malignant calcifications.This exploratory, single-center pilot study was performed on early breast cancer patients from our institution between May 2019 and Jun 2022. Written informed consent was obtained from all patients prior to inclusion. This study was proved by the Ethics Committee of our hospital (registration number: SL-B2022-102-02). The large-scale follow-up study (Registration number: ChiCTR2200060538) is currently recruiting.Included patients were women aged between 18 and 65 years, who had extensive malignant-appearing calcifications on mammography, who had biopsy-proved early breast cancer, and who had a strong desire for breast conservation. Patients who received neoadjuvant chemotherapy were allowed to be included. Patients with evidence of metastatic breast cancer, prior radiation therapy to the breast or chest wall, pregnancy, renal insufficiency, and history of allergic reactions to contrast agents were excluded.All patients received CE-CBBCT  after a biopsy-proved diagnosis was made. Preoperative examinations including routine blood tests, mammography , breast Z axis) was established and then perpendicular axes (X and Y axes) were established from the nipple corresponding to the medial-lateral and inferior-superior dimensions, respectively  patients received neoadjuvant chemotherapy. Most patients  have heterogeneously dense breasts according to the BI-RADS classification. The clinicopathological features of the enrolled patients are listed in CBBCT-guided surface location and oncoplastic breast-conserving surgery were successfully performed for all patients. Negative margins were achieved by postoperative pathology examination. Complete removal of calcifications was confirmed by intraoperative specimen x-ray. Although margin-positivity was found in two (18.2%) patients by intraoperative frozen section, negative margins were achieved after re-excision of the positive margin. The mean sizes of calcifications were 39.1 ± 13.0, 29.4 ± 10.2, and 28.7 ± 9.7 mm upon mammography, CBBCT, and pathology evaluation. Patient-reported cosmetic satisfaction was evaluated 3 months after surgery. All patients were satisfied with the cosmetic outcome. No local recurrence has been observed up to 30 September 2022. Surgical outcomes of CBBCT-guided breast-conserving surgery are presented in in situ is a lobular disease developed within adjacent sick lobes (The breast is an organ with complex 3D architecture and is ideally imaged in its natural anatomy. Unfortunately, 3D information about the breast has been constantly neglected by traditional breast imaging modalities. 3D information including spatial location and distribution of the breast abnormalities, which is crucial in the setting of extensive calcifications, is missing during breast compression and imaging projection in mammography. Previous studies suggested that ductal carcinoma ck lobes , and disck lobes , 11. TheAs a new and promising imaging technique, CBBCT eliminates the superposition of substantial breast tissue and is capable of providing conspicuous 3D images of breast malignancy. CBBCT has shown superior visualization of malignant lesions over digital mammography and its derivative technologies . MoreoveTo the best of our knowledge, our study is the first to use the CE-CBBCT-guided surface location technique for BCS in breast cancer patients with extensive malignant breast calcifications. By using the novel surface location approach, we managed to accurately remove the malignant calcifications, which was confirmed by intraoperative specimen x-ray and pathology examination. All patients achieved negative margins and satisfied cosmetic results. The surface location method in our study accurately mimicked the pathological distribution of extensive DCIS and enabled precise removal of calcifications, making it possible to perform BCS in breast cancer patients with extensive malignant breast calcifications.As a pilot study, the sample size is limited, and a full-scale study needs to be carried out to further optimize the application of this technique.The study suggests that CBBCT can reveal the three-dimensional distribution of extensive malignant calcifications and enable precise removal of microcalcifications through the CE-CBBCT-guided surface location technique, making it possible to perform BCS in breast cancer patients with extensive malignant breast calcifications. Future studies are needed to optimize the application of this technique.


JCI, Linderman and colleagues evaluated neutralizing antibody responses in hospitalized patients with SARS-CoV-2 infections  included unvaccinated individuals (43%). Of the vaccinated patients who required hospitalization, one-third were immunosuppressed. Two-thirds of the immunosuppressed, vaccinated patients had a history of solid organ transplantation or hematologic malignancy with immunosuppression, consistent with a high degree of immunocompromise  or mRNA vaccination (vaccine breakthrough). Reinfections and breakthrough infections in COVID-19 are part of the known challenges of protecting against reinfection from coronaviruses . Vaccina


In recent years, sequence-specific clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems have been widely used in genome editing of various cell types and organisms. The most developed and broadly used CRISPR-Cas system, CRISPR-Cas9, has benefited from the proof-of-principle studies for a better understanding of the function of genes associated with drug absorption and disposition. Genome-scale CRISPR-Cas9 knockout (KO) screen study also facilitates the identification of novel genes in which loss alters drug permeability across biological membranes and thus modulates the efficacy and safety of drugs. Compared with conventional heterogeneous expression models or other genome editing technologies, CRISPR-Cas9 gene manipulation techniques possess significant advantages, including ease of design, cost-effectiveness, greater on-target DNA cleavage activity and multiplexing capabilities, which makes it possible to study the interactions between membrane proteins and drugs more accurately and efficiently. However, many mechanistic questions and challenges regarding CRISPR-Cas9 gene editing are yet to be addressed, ranging from off-target effects to large-scale genetic alterations. In this review, an overview of the mechanisms of CRISPR-Cas9 in mammalian genome editing will be introduced, as well as the application of CRISPR-Cas9 in studying the barriers to drug delivery. Accumulating evidence has suggested membrane transporter proteins play pivotal roles in drug permeability across biological membranes and thus determine the efficacy and safety of drugs ,3,4,5,6.This review elaborates on the principles and application of CRISPR-Cas9 genome editing techniques with a special focus on drug permeability-related membrane proteins.The CRISPR-Cas adaptive immune systems are a natural defence mechanism of bacteria against foreign genetic elements . Some tyStreptococcus pyogenes is one of the best developed and broadly used systems in site-specific genetic engineering of human cells ..Abcb1 (cy cAbcb1  64]. Th. ThAbcb1Mdr1a/b (-/-) rats compared with WT. With the high efficient KI and KO via CRISPR genome editing technologies, more useful in vivo tools  would be generated for studying drug absorption barriers.Drug permeability across the intestine can be easily extrapolated across mammalian species due to the similar composition of the epithelial cell membranes. Laboratory animals are commonly used to predict oral drug absorption in humans by determining absolute bioavailability with the comparison of area under the plasma concentration-time curve (AUC) after intravenous and oral administration. They are also used for the prediction of potential absorption-based drug interactions by comparing oral bioavailability values with and without coadministration of another drug. With the manipulations in embryonic stem cells commonly used to create mouse knockouts, transgenic mice have provided an appropriate model to investigate the roles of specific drug transporter(s) or metabolising enzyme(s). For example, intestinal P-gp limits the bioavailability of a wide range of drugs, including paclitaxel, digoxin, aliskiren, betrixaban, celiprolol, fexofenadine and talinolol, as shown in P-gp knockout mice ,66,67. HMany anticancer drugs are targeting intracellular DNA or proteins and thereby cause DNA damage or inhibition of DNA synthesis and inhibition of cell division. The intracellular concentration of some anticancer drugs is vital, and it has been suggested that the intracellular concentration of a drug is the product of a competition between its passive or active uptake rate and either an active efflux rate or metabolic rate. However, the mechanisms whereby some anticancer drugs enter cancer cells and overcome the biological barriers remain poorly understood. The CRISPR-Cas9 system has provided extra tools to unfold the underlying mechanisms at the genome-scale and simplified the interpretation of results.ABCB1 significantly enhanced the sensitivity to doxorubicin ) in the ADR-resistant ovarian cancer cell line A2780/ADR [ABCB1 in two ABCB1-overexpressing multidrug-resistance (MDR) cell lines, KBV200 and HCT-8/V, remarkedly improved the sensitivity and accumulation of ABCB1 substrate drugs, such as vincristine and doxorubicin. Furthermore, the sensitivity of carfilzomib (CFZ)-resistant myeloma cell line AMO-CFZ [ABCB1 using the CRISPR-Cas9 system.Although the clinical relevance between ABC transporters and multidrug-resistance (MDR) phenotype is controversial and unclear, extensive in vitro studies strongly support their potential roles in the cellular pharmacokinetics of substrate drugs ,69. The 2780/ADR , breast 2780/ADR , osteosa2780/ADR . Similar AMO-CFZ  and acut AMO-CFZ  to CFZ wIn addition, a CRISPR/Cas9 KO of BEN domain-containing protein 3 (BEND3) upregulated efflux transporter breast cancer resistance protein  and reduced the intracellular levels of TAK-243 and induced resistance in acute myeloid leukaemia (AML) cells . SimilarTherefore, CRISPR-Cas9-mediated modulation of ABC transporter genes or their regulator(s) may represent a promising in vitro cancer cell model for substrate identification and molecular pathology research that potentially contributes to uncovering novel therapeutic biomarkers.ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumour cell sensitivity to PCA062, a P-cadherin targeting antibody-drug  conjugate for the treatment of multiple cancer types, including basal-like breast cancer [Historically, genome-wide loss-of-function screening in mammalian cells has employed the RNA interference (RNAi) gene knockdown technology. However, this method is limited by incomplete protein depletion and off-target effects-induced false positives . The appt cancer . ABCC1 ct cancer , and SLCt cancer ,83. SileA genome-wide CRISPR/Cas9 knockout screen identified that SLC1A3 confers L-asparaginase resistance in human prostate cancer PC3 cells . L-asparCTR1 or CTR2 in ovarian carcinoma OVCAR8 cells showed indistinguishable differences in cisplatin sensitivity compared to wild-type cells [BRCA1-mutated ovarian cancer cells in a genome-scale CRISPR-Cas9 knockout screen study [The platinum-containing drugs such as cisplatin, carboplatin and oxaliplatin have been widely integrated into the standard and preferred regimens for various solid cancers. Several SLC transporters, such as CTR1 (SLC31A1) and CTR2 (SLC31A2), have been reported to participate in cisplatin influx in several heterogeneous expression systems  . Howeverpe cells . A studype cells . Loss ofpe cells . To put en study , and a ren study . FurtherEndothelial cells that line the microvasculature of the central nervous system (CNS) constitute the blood-brain barrier (BBB), selectively excluding circulating drugs and toxic agents from entering the majority of the central nervous system but allowing essential nutrients, hormones and certain drugs into the brain. Drug permeability across BBB is mainly determined by the interactions between their physicochemical characteristics and the specialised BBB features, including tight intercellular junctions that markedly limit paracellular permeability, plus a unique expression of ABC and SLC transporters that determine the transcellular permeability. The two most abundant ABC transporters in the human BBB are ABCB1 (MDR1) and ABCG2 (BCRP) , which fSolute carrier family 35, member F2 (SLC35F2), is highly expressed in the BBB and is localised exclusively on the apical membrane of brain microvascular endothelial cells (BMECs), differentiated from human induced pluripotent stem cells (hiPS-BMECs). Silencing SLC35F2, by CRISPR/Cas9 mediated knockout, diminished the apical-to-basolateral transport and intracellular accumulation of its substrate drug YM155 in hiPS-BMECs . By contOatp1a4 promoter [OATP1A2 (SLC21A3) is abundantly expressed in the apical (blood) side of the human BBB ,95,96 anpromoter . Such a The CRISPR/Cas9 system has been applied to edit non-transcribed DNA sequences, including DNA methylation tags. DNA methylation, occurring at the 5-carbon position of cytosine residues located in dinucleotide CpG sites and regions of high CpG density, called CpG islands (CGIs), has been observed in the promoter regions of 60–70% of genes in mammals, which are involved in the epigenetic regulation of genes . ABCC3 iSLC47A1. The CRISPR-Cas9-induced deletions in this CpG area significantly lower SLC47A1/MATE1 mRNA expression in HepG2 cells. This study highlights the importance of epigenetic regulation in pharmacokinetics and the application of CRISPR/Cas9 for editing non-transcribed DNA targets in human cells.Multidrug and toxin extrusion protein 1 (MATE1), which is encoded by solute carrier 47A1 (SLC47A1), functions as the final excretion step of drugs/toxins into bile and urine. Its substrates include vitamin thiamine, cimetidine, metformin, guanidine, procainamide, antiviral agents  and antibiotics  as well as an endogenous substrate creatinine [The breathtaking CRISPR-Cas9-based gene-editing technology is undoubtedly an excellent genetic manipulation method for pharmacology and molecular biology studies. It provides researchers with a convenient tool to identify gene functions with significant advantages, including ease of design, cost-effectiveness, greater on-target DNA cleavage activity, and multiplexing capabilities. The exploitation of CRISPR-Cas9 techniques has expanded the tools to study biological barriers to drug delivery and generated novel insights in the field and may lead to the development of promising strategies to overcome therapeutic failures and minimise drug toxicity. The genome-scale CRISPR-Cas9 KO screen study also facilitates the identification of novel genes in which loss alters drug permeability across the biological membrane and thus, modulates the efficacy and safety of drugs. With the new strategies to enhance KI and KO efficiency via CRISPR genome editing technologies, more interpretable in vitro and in vivo tools are expected for studying barriers to drug delivery.


Insights into the short-term dynamics and micro-longitudinal consequences of subjective age can drive our understanding of its long-term mechanisms across adulthood. Using data from 80 newly retired individuals  collected on 21 days, we made use of a recent methodological advance—multilevel dynamic structural equation modeling. As possible same-day correlates and micro-longitudinal consequences of subjective age, we investigated physical activity, step number, sleep quality, affect, and stress, which were either assessed via wearables (FitBit Charge HR) or daily diaries. Analyses suggest a weak autoregression of subjective age, indicating that how old one feels is determined via daily rather than lasting experiences. Indeed, there were significant same-day relations to all suggested correlates. The one effect lasting across several days was from an older subjective age on subsequent negative affect—a potential short-term mechanism contributing to the detrimental long-term influence of an older subjective age.


Although it is known that anti-PD1/L1 monotherapy does not render significant benefit in patients with EGFR-mutant lung adenocarcinoma, whether the addition of anti-PD1/L1 therapy to chemotherapy can enhance chemotherapy efficacy for TKI-refractory EGFR-mutant lung adenocarcinoma patients is not clear. To address this question, we retrospectively analyzed a cohort of 178 EGFR-mutant lung adenocarcinoma patients who had progressed on EGFR TKIs and received subsequent non-TKI systemic therapy to determine whether the addition of immunotherapy to chemotherapy truly improves clinical outcomes. We found that the addition of anti-PD1 immunotherapy did not add benefit to the platinum-based chemotherapy at the time of TKI progression for EGFR-mutant LUAD. Although underpowered, the anti-VEGF therapy demonstrated a trend towards adding benefit. As ongoing clinical trials with newer agents or combinations demonstrate preliminary efficacy in TKI-resistant EGFR-mutant LUAD patients, the ideal choice for post-TKI treatment is still being evaluated.n = 84), chemotherapy combined with immunotherapy , chemotherapy plus bevacizumab with or without IO , and IO monotherapy . Clinical progression-free survival (PFS) and overall survival (OS) were evaluated. Associations of clinical characteristics with outcomes were assessed using univariable and multi-covariate Cox Proportional Hazards regression models. Results: A total of 178 patients  with a median follow-up time of 42.0 (Interquartile range: 22.9–67.8) months were enrolled. There was no significant difference in PFS between chemoIO vs. chemo groups . Compared to the chemo group, patients who received withBev therapy trended towards better PFS , while patients treated with IO-mono had inferior PFS . Furthermore, PD-L1 level was not associated with PFS benefit in the chemoIO group. Patients with EGFR-mutant LUAD with high PD-L1 (≥50%) had shorter PFS (5.8 months) than non-EGFR/ALK LUAD patients who received chemoIO  as first-line treatment. Chemotherapy-based therapy rendered similar benefit to patients with either EGFR exon19 deletion vs. L858R in the LUAD. Conclusions: This retrospective analysis revealed that immunotherapy provided limited additional benefit to chemotherapy in TKI-refractory EGFR-mutant LUAD. Chemotherapy alone or combined with bevacizumab remain good choices for patients with actionable EGFR mutations.Background: The benefit of chemotherapy combined with immunotherapy in EGFR-mutant lung adenocarcinoma (LUAD) patients whose tumor developed resistance to EGFR tyrosine kinase inhibitors (TKIs) is not thoroughly investigated. The goal of this retrospective cohort study is to assess the clinical efficiency of immunotherapy alone or in combination with chemotherapy in a real-world setting. Methods: This retrospective cohort study enrolled LUAD patients with EGFR sensitive mutations whose tumor had acquired resistance to EGFR TKIs and received systemic treatment with chemotherapy (chemo;  Recent clinical studies have assessed the efficacy of combining immune checkpoint inhibitor therapy with standard first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC). Specifically, chemoimmunotherapy has displayed a marked improvement in clinical outcome in these NSCLC patients. In the Keynote-189 trial, patients without sensitizing EGFR or ALK mutations were treated with pemetrexed and a platinum-based drug in combination with pembrolizumab (anti-PD1) or a placebo. This study found that the addition of anti-PD1 therapy improved both PFS from 4.9 months to 8.8 months and ORR from 18.9% to 47.7% ), osimertinib (30 [16.9%]), afatinib (13 [7.3%]), and gefitinib (12 [6.7%]). No statistical significance of clinical outcome on chemotherapy-based treatment was observed between patients with osimertinib and other generations as first line TKI. Receiving only one line of TKI therapy  indicated a significantly decreased risk of progression on chemotherapy-based treatment vs. more than one prior TKI . For OS among patients with chemotherapy-based treatment, the following factors were significantly associated with an increased all-cause mortality: age > 65 years old , Black racial/ethnic group , and with liver metastasis . No significant deviation of the PH assumption was noted.Last, we performed multicovariate analysis to identify high-risk patient groups. Covariates used for adjustment in the Cox regression model include age, gender, tobacco use, race, if only one type of TKI used, if the first line was osimertinib, the treatment strategy of the non-TKI systemic therapy, with or without liver metastases, with or without brain metastases, with or without bone metastases . On multev group . Among tThis retrospective study evaluated the efficacy of adding anti-PD1/L1 or anti-VEGF therapy to platinum-based chemotherapy in EGFR-TKI-resistant EGFR-mutant adenocarcinoma patients in a single center and real-world setting. We found that immunotherapy adds very limited benefit in terms of PFS and OS to platinum-based doublet. When stratified by PD-L1 levels, even patients with high PD-L1 expression (TPS 50–100%) did not derive as much benefit from chemoIO therapy in EGFR-mutant lung cancers, which is especially disappointing when compared to high PDL1 EGFR-wildtype tumors. This supports the possibility that EGFR mutation itself is a strong biomarker for not responding to chemoIO therapy.p = 0.3). A similar observation was also reported by White et al. in a retrospective analysis cohort [The addition of anti-VEGF therapy showed a trend toward potential benefit compared with chemotherapy only .The intriguing benefit of adding both anti-VEGF and anti-PD1/L1 to chemotherapy warrants further investigation. Anti-VEGF therapy can modulate the tumor immune microenvironment, as VEGF is an immune suppressive cytokine pathway. The combination of anti-VEGF and anti-PD1 therapy has shown promising efficacy both in the preclinical models and clinical trials . When raIn our cohort, liver metastasis is an independent risk factor for both PFS and OS with stronger HR than patients with CNS metastasis. Preclinical models have showed that the efficacy of immunotherapy was restrained in patients with liver metastasis by eliminating the amount of macrophage-mediated T cells . Interesn = 11) is an independent risk factor of overall survival in the current cohort, although no significant difference was observed for PFS.The mortality in patients with Black ethnicity is the highest among all racial/ethnic groups for most cancers . No signOur study was limited by the nature of a single-center retrospective cohort analysis; for example, PD-L1 results were only available in a limited number of patients. A larger multi-institutional collaboration is required to better understand the practice pattern and expand the sample size to further elucidate key questions raised here, including the benefit of anti-VEGF therapy, the optimal choice of therapy for patients with liver metastasis, and the mortality disparity in certain patient ethnic groups. Furthermore, although a Cox regression model was used to adjust for available characteristics, all possible confounding factors could not be controlled, such as comorbidities and changes in the standard care over years, which may impact outcomes. Ongoing trials are investigating immunotherapy with pemetrexed/platinum +/- bevacizumab on NSCLC patients with EGFR mutations after progression on EGFR-TKIs . These perspective studies will provide more data to make this point clearer.We found that the addition of anti-PD1 immunotherapy did not add benefit to platinum-based chemotherapy at the time of TKI progression for EGFR-mutant LUAD. Although underpowered, the anti-VEGF therapy demonstrated a trend towards adding benefit. As clinical trials with newer agents or combinations demonstrate preliminary efficacy in TKI-resistant EGFR-mutant LUAD patients are ongoing, the ideal choice for post-TKI treatment is still being evaluated.


A 37-year-old woman with atypical chest pain was referred to Emergency Department for clinical evaluation. Vital signs at admission were stable, and the physical exam was normal.Panel A). Transthoracic echocardiography showed hypokinesis of the mid-anterior septum, anterior, and inferior walls with preserved ejection fraction (Panel B). Relevant laboratory tests included elevated high sensitivity troponin-I of 3176 ng/L  and B-type natriuretic peptide of 276 pg/mL .ECG showed only biphasic T wave in V3 (Panel C).The patient was diagnosed with non-ST segment elevation myocardial infarction and addressed to invasive coronary angiography, with the evidence of non-obstructive coronary artery disease. Left ventriculography identified hypokinesia of mid-left ventricular segments and hyperkinesia of apical and basal segments (Panel D). At tissue characterization, while traditional T2-weighted (Panels E and F) and late gadolinium enhancement (Panel G) imaging did not show abnormalities, mapping sequences detected a marked increase of native T1 , T2 , and extracellular volume (ECV)  values at all mid-ventricular segments, particularly at anterior, septal, and inferior walls. All these findings were related to the presence of oedema, then supporting the diagnosis of mid-ventricular Takotsubo cardiomyopathy (TC).Cardiac magnetic resonance (CMR) confirmed hypokinesia of all mid-ventricular segments and preserved contraction of apical and basal segments (The apical ballooning pattern in TC is seen in the majority of patients. A very small group of TC presents with atypical features such as mid-ventricular patterns. In this case, CMR was crucial to identify not only peculiar wall motion abnormalities but also to detect, thanks to mapping sequences, the underlying tissue damage, thus leading to the correct diagnosis.


Epigenetic therapies have been difficult to translate to solid tumors, in part because a lack of a full mechanistic understanding has not allowed the selection of tumors most likely to benefit from the therapies. Here we identified a RAS-phenotype that can be targeted by the histone deacetylase inhibitor (HDACi) romidepsin. We showed that the hyperacetylation induced by romidepsin depletes acetyl-CoA, the cell donor substrate for acetylation, and it leads to metabolic stress and death in KRAS-activated cells. Transcriptomic analysis confirmed that perturbation of two acetyl-CoA generation pathways (fatty acid metabolism and branched-chain amino acid (BCAA) metabolism) were correlated with HDACi sensitivity in a 608-cell line panel and in patients treated with belinostat. Our analysis highlights the potential utility of an acetyl-CoA phenotype to sharpen treatment choices for RAS-activated tumors, and it suggests that acetyl-CoA depletion could be a key effect underlying the myriad cellular responses that follow HDAC inhibition. Background: The mechanism of action of romidepsin and other histone deacetylase inhibitors is still not fully explained. Our goal was to gain a mechanistic understanding of the RAS-linked phenotype associated with romidepsin sensitivity. Methods: The NCI60 dataset was screened for molecular clues to romidepsin sensitivity. Histone acetylation, DNA damage, ROS production, metabolic state , and gene expression alterations (transcriptomics) were determined in KRAS-WT versus KRAS-mutant cell groups. The search for biomarkers in response to HDACi was implemented by supervised machine learning analysis on a 608-cell transcriptomic dataset and validated in a clinical dataset. Results: Romidepsin treatment induced depletion in acetyl-CoA in all tested cell lines, which led to oxidative stress, metabolic stress, and increased death—particularly in KRAS-mutant cell lines. Romidepsin-induced stresses and death were rescued by acetyl-CoA replenishment. Two acetyl-CoA gene expression signatures associated with HDACi sensitivity were derived from machine learning analysis in the CCLE (Cancer Cell Line Encyclopedia) cell panel. Signatures were then validated in the training cohort for seven HDACi, and in an independent 13-patient cohort treated with belinostat. Conclusions: Our study reveals the importance of acetyl-CoA metabolism in HDAC sensitivity, and it highlights acetyl-CoA generation pathways as potential targets to combine with HDACi. RAS-mutated tumors are the most common and the deadliest of all human cancers, and they remain among the most refractory to available treatments ,2. In thInterestingly, our group and others ,4,5 haveApart from new G12C genotype targeted therapies, strategies to kill RAS-mutant cells by directly targeting RAS or its downstream effectors have been mostly unsuccessful, mainly due to pathway redundancy and heterogeneities in RAS-induced phenotypes. One therapeutic strategy that has emerged involves the exploitation of tumor metabolic vulnerabilities induced by RAS activation . In factIn this study, we show that romidepsin-induced hyperacetylation caused depletion of stores of acetyl-CoA, the cell donor substrate for acetylation, and leads to lethal metabolic stress in KRAS-activated cells but not in KRAS-WT. We also confirmed that two acetyl-CoA metabolism pathways are hallmarks of sensitivity in a cell panel and in patients treated with an HDACi, validating not only these drugs as metabolism-targeting drugs, but also these pathways as a potential tool to identify a RAS-linked metabolic phenotype in a personalized medicine strategy.The complete NCI-60 panel was obtained from National Cancer Institute (NCI) Anticancer Drug Screen . Pancreatic cell lines were obtained from ATCC . All cells were cultivated in RPMI including 2 mM glutamine and 11.11 mM glucose supplemented by 10% FBS and penicillin/streptomycin .Romidepsin  was provided by the Cancer Therapy Evaluation Program, NCI. Tariquidar was provided by the NCI Anticancer Drug Screen. PD-0325901 (MEK inhibitor), MK-2206 (AKT inhibitor) were purchased from ChemieTek . Glutaminase inhibitor 968 compound was obtained from Millipore ; oligomycinA, NAC, glutathione, dimethyl-α-ketoglutarate, dimethyl-glutamate, sodium citrate, and sodium acetate were obtained from Sigma-Aldrich ; DPI, ML171, and etomoxir was obtained from Selleckchem . MitoQ was provided by Dan L. Sackett . t-tests were performed to evaluate significance of results, and asterisks were added to graphs when the p-value was lower than 0.05. Unless otherwise specified, ® Human Genome U133 Plus 2.0 Array. Microarray data have been deposited in the NCBI Gene Expression Omnibus (accession number GSE133120). Detailed methods are provided in related figure legends.Gene expression basal levels for the NCI-60 and CCLE (Cancer Cell Line Encyclopedia) panels were downloaded via the cBio Cancer Genomics Portal . Gene ext-test t < 0) subtracted from the expression sum of up-regulated genes (t-test t > 0) and divided by the total number of up- and down- regulated genes).Gene expression signature scores were calculated using weighted average expression  were purchased from the Jackson Laboratory . Tumors were generated by injecting 10 million cells subcutaneously into the left flank. Treatment started once the tumor size reached an average of 100 mmCell death was measured by flow cytometry after annexin V-FITC/ propidium iodide staining, according to manufacturer’s instructions . Cell lines with a high expression of the multidrug efflux transporter P-glycoprotein (P-gp) according to the cellMiner database  were co-A western blot was performed as previously described . HistoneOxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using the XF96 or the XFp Extracellular Flux analyzer . The protein amount per well was quantified using the BCA assay.t-test.Metabolic profiling of A549 and ACHN cell lines was performed by Metabolon Inc. . After treatment, cells were pelleted, frozen, and sent for analysis (quadruplicates). All samples were randomly distributed across a single day platform run on GC/MS and LC/MS/MS instrumentation, and they were analyzed using Welch’s two-sample Cells were harvested and sent frozen to the Metabolomics Core at the Translational Research Institute for Metabolism and Diabetes at Florida Hospital for estimation of acetyl-CoA and malonyl-CoA by LC-MS/MS.p-value > 0.0001) between romidepsin alone and romidepsin in combination with the MEK inhibitor , and a 0.92 correlation  for the same analysis with the AKT inhibitor . All but two of the 65 lines were insensitive to AKTi or MEKi alone. These results indicate that RAS downstream inhibitors did not trigger cell death, but amplified romidepsin-induced cell death. We queried mutations in well-known key cancer genes carried by these cell lines from the Cosmic Cancer Database. Eleven relevant genes were grouped in four categories with notable mutation: tyrosine kinase receptor (TKR)/RAS/BRAF pathway , PI3K pathway , CDKN2A, and p53 . Cell doubling time, mutations in the PI3K pathway, CDKN2A, or p53 did not correlate with treatment efficacy  in the two romidepsin-sensitive cell clusters , we screened the NCI-60 panel (58 human cell lines from seven cancer tumor types), adding seven pancreatic cancer cell lines because they have a high prevalence of RAS mutation (97%) . For thi and p53 a. Noticeefficacy . Basal eclusters c. Gene eclusters d. Again,To assess the efficacy of the combination drug therapy in a preclinical model, we injected A549 cells  subcutaneously in athymic mice and treated them with romidepsin and/or MEKi . From 15To understand why RTK/RAS/BRAF mutated cells were sensitive to romidepsin, we focused on the KRAS-mutant cells as a model. Progression in signaling events during romidepsin treatment using clinical dosing was evaluated in at least three KRAS-mutant and three KRAS-WT cell lines. Histone acetylation, the main target of romidepsin, and DNA damage (using the commonly accepted marker γH2AX) were measured by western blot after 6 h combination treatment in six cell lines a. No difNext, total and mitochondrial reactive oxygen species (ROS) production were measured over time in the A549 cell line treated with romidepsin by flow cytometry. Total ROS increased approximately 10–12 h, whereas no increase was detected in mitochondrial ROS over 18 h b. Total N-acetyl-cysteine (NAC) significantly rescued all KRAS-mutant cells from death, whereas neither mitochondrial ROS scavenger (mitoQ) nor NADPH oxidase (DPI) inhibitor did  or itor did . We alsoitor did . These sTaken together, these results indicated that romidepsin induces early cytoplasmic ROS production in KRAS-mutant cells. Mitochondrial ROS production and mitochondrial membrane depolarization appeared late, suggesting that they were apoptotic by-products rather than drivers of apoptosis. Having eliminated the main known causes of ROS production (NADPH oxidase and mitochondria), we therefore explored other possible sources of cytoplasmic ROS imbalance after romidepsin treatment. Four enzymes in metabolic pathways produce cytoplasmic NADPH, an intrinsic ROS scavenger that maintains a cytoplasmic redox balance a. We hypTo assay whether glutamine was a dominant OXPHOS fuel source rather than glucose, the same experiment was performed in 1 h-glucose/glutamine-deprived cells, and glutamine was subsequently added e. In allTogether, these results indicated that cancer cell lines used β-oxidation and glutamine rather than glucose as the main source of fuel for the TCA cycle, independently of KRAS status. Romidepsin impaired OXPHOS via inhibition of β-oxidation in almost all tested cell lines, but impaired glutamine use for OXPHOS only in cells bearing KRAS mutations.To understand the disturbances in β-oxidation and glutamine metabolism induced by romidepsin, a metabolomic analysis was performed in A549 (KRAS-mutant) and ACHN (WT) cell lines. Cells were treated 12 h or 18 h with romidepsin using clinical dosing, and 629 metabolites were quantified. Romidepsin-altered pathways were determined by Metabolync pathway enrichment analysis. Fatty acid metabolism and TCA cycle pathways were significantly modified in both cell lines at 12 h and/or 18 h , as wellMetabolites in the TCA cycle and in its two fueling pathways (glutaminolysis and β-oxidation) are reported in Notably, among all the quantified metabolites in the TCA fueling pathway, only acetyl-CoA and its two direct precursors downstream of β-oxidation (C2- and C4-acylcarnitine) were depleted, whereas almost all other TCA-fueling metabolites were accumulated b. To valTo explore this hypothesis, we returned to the Seahorse experiments. Cells were placed for 1 h in glucose/glutamine-deprived medium, then glutamine, DM-glutamate (cell permeable glutamate), DM-glutarate (cell permeable αKG) or pyruvate were added while OCR was measured d. After When looking at the cytoplasmic NADPH-producing pathways downstream of TCA cycle and/or glutaminolysis , because acetyl-CoA itself cannot cross the cytoplasmic membrane. Various precursor concentrations were tested on three KRAS-mutant cell lines and we selected the optimal conditions for cell death rescue . AdditioWe then investigated the coupling between acetyl-CoA depletion and cell death. In the presence of acetyl-CoA precursors, romidepsin did not induce a decrease in basal OCR levels in the KRAS-mutant cells, contrary to what was observed in the absence of citrate/acetate c. We alsThese results showed that the decrease in acetyl-CoA levels was a leading cause of romidepsin-induced cell death in KRAS-mutant cells. Romidepsin-induced acetyl-CoA decrease caused a reduction in TCA cycle flux and an increase in cytoplasmic ROS production that triggered cell death in KRAS-mutant cells only. Notably, romidepsin-induced damage was reversed by just replenishing acetyl-CoA stocks, and it was not caused by romidepsin-induced inhibition of acetyl-CoA generation pathways, suggesting that it was hyperacetylation-induced acetyl-CoA overconsumption that led to the decrease in acetyl-CoA.Because romidepsin-induced acetyl-CoA decrease was observed in all tested cell lines, whereas only KRAS-mutant cell lines showed a decrease in acetyl-CoA-linked cell death after HDAC inhibition, we hypothesized that KRAS-mutant cells were more acetyl-CoA-dependent than KRAS-WT cell lines. p-values of less significance. To determine whether HDACi-sensitive cells had an acetyl-CoA altered metabolism at baseline, we derived an acetyl-CoA-related signature from transcriptomic datasets. We used the CCLE panel (608 cell lines) with a known response to eight HDACis to gather enough samples to gain a sufficient statistical power . A machip-value = 0.0045 and 0.0004, respectively, while the FA signature alone could not differentiate sensitive and resistant (p = 0.07). These results confirmed that samples with an altered expression profile for acetyl-CoA metabolism at baseline have the strongest sensitivity to HDACi.To validate the importance of acetyl-CoA for HDACi sensitivity in clinical samples, we tested the performance of the three GES in samples obtained from 13 acute myeloid leukemia (AML) patients for whom microarray data prior to treatment and clinical response to belinostat are publicly available d. The coTogether, these results confirmed that alteration in two acetyl-CoA metabolism pathways (FA metabolism and BCAA metabolism) were a hallmark of sensitivity to HDACi in patients. It also validated the two acetyl-CoA derived GES as possible tools to further select patients for whom HDACi could be beneficial.In this study, we discovered that the class I HDAC inhibitor romidepsin caused a depletion in acetyl-CoA, most probably through an overconsumption of acetyl-CoA itself triggered by induced hyperacetylation of histones. We also uncovered a strategy for treating KRAS pathway modified tumors. Indeed, in RAS-activated cells, the depletion in acetyl-CoA triggered a decrease in TCA cycle flux combined with an increase in cytoplasmic ROS that led to cell death. Computational analysis of transcriptomic profiles in a 608-cell panel highlighted inherent alterations in acetyl-CoA generation pathways that could cause a higher dependency on acetyl-CoA depleting drugs, and we confirmed our results in samples from patients treated with belinostat. Together, these findings provide a promising strategy for improving treatments for the RAS pathway-modified tumors, where acetyl-CoA metabolism signatures could be exploited to select patients who are most likely to benefit from HDACi treatment.The tight link between histone acetylation and acetyl-CoA levels is well known but has been described in only one direction: acetyl-CoA levels determine histone acetylation levels. In fact, the abundance of the nucleo-cytosolic pool has a direct impact on the enzymatic activity of HATs . Here, wThis is consistent with the fact that acetyl-CoA is the unique donor of acetyl groups for acetylation, and that even after 6 h treatment followed by 12 h washout, the acetylated histone signal did not fade , implyinIn normal physiology, fluctuations in acetyl-CoA concentration reflect the metabolic state of the cell. Due to this key role, its regulation is tightly controlled by three different mechanisms: the expression of its metabolic enzymes or their regulators, its cellular and subcellular compartmentalization, and the post-translational regulation of the enzymes involved in its production (by acetylation) ,18,20. SAnother point to discuss is the link between TCA cycle and cytoplasmic ROS production. Gansemer et al.  found thIn sum, our results suggest that a dominant effect of HDAC inhibition is the depletion of acetyl-CoA. While the canonical mechanism of opening chromatin to facilitate gene transcription is undoubtedly important in some settings, the rapid cell death in T-cell lymphoma cells is more consistent with a metabolic impact. One future direction will be to determine what role acetyl-CoA could play in the myriad other effects observed following HDAC inhibition, including R-loop persistence, kinetochore assembly and DNA damage.Another direction will be to determine whether this metabolic vulnerability could be exploited in the clinic. Acetyl-CoA metabolism signatures were performant in a small validation cohort of patients, and will have to be tested in a prognostic study in order to confirm their strength for future clinical use as biomarkers to help stratifying patients that should receive HDACi. Our results also open doors to new combinations to treat patients with solid cancer using HDACi with FA or BCCA metabolism inhibitors.


In response to the treatment gap for anxiety and depressive disorders, psychological treatments with innovative modalities and high implementation potential are essential. Internet CBT (iCBT) is a cost/effective approach that could improve access to a low-intensity evidence-based CBT intervention.To assess the feasibility and acceptability of the French adaptation of the physician-prescribed six-lesson This Way Up transdiagnostic iCBT program for mixed anxiety and depressive disorders developed in Australia.Feasibility study with pre- post-intervention evaluations, including an embedded qualitative study in Family Medicine Groups . Inclusion criteria comprise a family physician diagnosis of Major Depression, Panic Disorder, Agoraphobia, Social Anxiety Disorder or Generalized Anxiety Disorder. Primary self-reported outcomes: PHQ-9 (depression) and GAD-7 (anxiety); secondary measures include diagnostic-specific scales and health service utilisation.Family physicians (N=21) from five Family Medicine Groups prescribed iCBT to 45 patients , 31 initiated the program. To date, 20 patients completed 5 or 6 lessons, nine completed between 2 and 4. Intervention and post-treatment assessments are ongoing, results forthcoming. Results of semi-structured interviews with patients (N=15) and family physicians (ongoing) on iCBT acceptability indicate it is beneficial, practical and easy to use. Program adherence requires patient readiness and determination and could be fostered by motivational support from clinicians.Results support this French iCBT program’s scaling-up potential to contribute to reducing the gap in evidence-based treatments for common mental disorders. Its implementation in primary care could improve the effectiveness, efficiency and equity to a rapidly accessible treatment.


The deformation andfracture mechanism of two-dimensional (2D)materials are still unclear and not thoroughly investigated. Giventhis, mechanical properties and mechanisms are explored on exampleof gallium telluride (GaTe), a promising 2D semiconductor with anultrahigh photoresponsivity and a high flexibility. Hereby, the mechanicalproperties of both substrate-supported and suspended GaTe multilayerswere investigated through Berkovich-tip nanoindentation instead ofthe commonly used AFM-based nanoindentation method. An unusual concurrenceof multiple pop-in and load-drop events in loading curve was observed.Theoretical calculations unveiled this concurrence originating fromthe interlayer-sliding mediated layers-by-layers fracture mechanismin GaTe multilayers. The van der Waals force dominated interlayerinteractions between GaTe and substrates was revealed much strongerthan that between GaTe interlayers, resulting in the easy slidingand fracture of multilayers within GaTe. This work introduces newinsights into the deformation and fracture of GaTe and other 2D materialsin flexible electronics applications. Extremely high intrinsic in-plane Young’smodulus (∼1TPa) and strength (∼130 GPa) were revealedin graphene using atomic force microscopy (AFM)-based nanoindentation.18 This AFM-based nanoindentation method combinedwith molecular dynamics (MD) and other density functional theory (DFT)calculations has been extended to measure the nanomechanical propertiesof other 2D materials, such as MoS2 and hexagonal boronnitride (hBN).21 Specifically, detailed knowledge about deformation,fracture, generation of defects and potential phase-transition ofthese 2D materials at nanoscale can be quite different and is essentiallyrequired when their devices are under large strain/stress or undergonumerous loading cycles.22 For example, the strengthof graphene can be significantly influenced by out-of-plane deformationunder external shear loading23 or intrinsictopological defects,24 leading to a failurestrength of >50% reduction.Two-dimensional (2D) materialshave attracted tremendous interest attributed to their extraordinaryelectronic, optical and mechanical properties compared to their bulkcounterparts. Recently, the preparations of 2D materials through mechanicalexfoliation or chemical synthesis have achieved great advances, enablingrenewed investigation into 2D materials beyond graphene.29 However, inaccuracies are noted in this method, mainly due to thefact that the locally concentrated stress near the sharp AFM-tip aftercrack initiation cannot provide sufficient driving force for furthercrack propagation.30 Recently, full-scalenanoindentation techniques with a Berkovich-indenter have been usedto measure the mechanical properties and nanometer-scale structuralchanges in small-volumes of materials and ultrathin-films.32 In the load–displacement curves produced by this Berkovich-tipnanoindentation method, step-like pop-in (PI) is often observed, whichcan be ascribed to dislocation-nucleation, slippage, phase-transition,crack-formation, and so on.37 Discontinuous-like push-out (PO) events in the unloading curve areusually reported as indications of phase-transition.39 Load-drop (LD) events (sudden load decrease at a certain displacement)can also be seen, which have been primarily correlated to the formationof interfacial cracks.36To characterize the local nanomechanicalproperties of 2D materials,the aforementioned AFM-based nanoindentation is commonly used.26 whereas the strength of hBN is insensitive tolayer numbers due to its stronger interlayer vdW-interactions thanthat of graphene.26 Interlayer interactionsare also found can lead to recoverable sliding within graphene multilayersduring the nanoindentation loading.28 However,the critical mechanical properties and effects of interlayer interactionsstill have not been investigated in many other 2D materials includingthe aforementioned GaTe. Specifically, as an exciting emerging material,GaTe multilayers have demonstrated huge advantages with an ultrahighphotoresponsivity for high-performance photodetectors,17 as well as giant potential for desirable optoelectronics, electronics,and nanoelectromechanical system devices with a reported highest anisotropicresistance and a tremendous current on/off ratio within the 2D materialsfamily;43 these highlighted characteristicsmake GaTe a great candidate for future nano- and flexible- optoelectronics,especially when its ambient degradation now can be effectively suppressed.44 GaTe multilayers are also distinguished by anunusually high failure-strain .29 Nevertheless, knowledge about the detailed deformationand fracture behaviors in GaTe multilayers which is crucial for manypractical device applications is still lacking.When comparingthe mechanical properties of reported 2D materials,interlayer interactions are noted playing an important role due totheir different strength of van der Waals (vdW) forces existed betweenindividual layers. For example, a 30% decrease in strength is observedin graphene when the number of layers increases from 1- to 8-layers;In this work,the nanomechanical properties of both substrate-supportedand suspended GaTe multilayers are systematically characterized byfull-scale Berkovich-tip nanoindentation, micro-Raman spectroscopy,AFM, scanning electron microscopy (SEM) and MD simulations. An unusualconcurrence of multiple PIs accompanied by LDs events in loading curvesis first observed in 2D materials family, and the mechanisms of interlayer-slidingand layers-by-layers fracture are unveiled and investigated in detail.P–h) curves obtained from different indentationloadings performed on SiO2/Si-substrate-supported samplescan be classified into three types; a typical curve from each typeis shown in 39The GaTe multilayers samples tested are listed in Table 1 includingthe sample thickness, number of layers and the maximum indentationdepth. It was found that the load–displacement . This implies a sample thicknessindependent P–h curves types and a commonmaterial property driven mechanical mechanism exists in GaTe multilayers.Comparing Type III curves ,the regularly occurring large LDs indicate multiple fracturing orcracking events in the materials during loading which suggests a transitionto irreversible inelastic deformation. Large LDs at lower load, forexample, ∼500 μN in sample 5, resulted in a much lowergradient of the loading curve which is a strong implication of modifiedcompliance of the material. We also note that the observed large LDsare spaced at regular loading steps of ∼400–700 μN,and the accompanied large PI-stage lengths are spaced by ∼10–30nm, respectively . Thisfact implies multiple step-by-step fracture and sliding behaviorsare likely triggered within the GaTe multilayers, which are associatedwith material properties and require further analysis.Theabove indents were compared across different indent-depths;further indents were performed on supported samples at a fixed displacementdepth of 200 nm in random locations and are detailed in P–h curves. As seen from 45).For the indent produced by an indentation depth of 300 nm (45 (45). These preferred crack propagation orientations indicate the anisotropicnature of the in-plane mechanical properties of GaTe multilayers.The morphologyand microstructure of the indents was investigatedby AFM and SEM for further understanding the different types of 0 nm , the following correlation canbe summarized: (i) Pyramidal indent-imprints without macro-cracks,fracture, or pile-ups around the indent result in the Type I P–h curve. As there is no fractureor cracks in the residual-imprints  Pyramidal indent-imprints withfracture, asymmetric pile-ups of the surrounding materials and crackpropagations can be correlated to the Type III P–h curve. In this case, the events of larger PIs and significantLDs can be attributed to the formation of obvious fracture in theindent-center. It is worth noting that, in the residual-indents, areaswith different contrast , similar averagestress of ∼0.2 GPa and largest tension of ∼0.7 GPa appearin both Type II and Type III indents. To conclude, the average residual-stressaround the residual-imprints increased with the indentation depth.In areas away from the indents and cracks, a small tensile-stressof ∼0.1 GPa exists for all samples. Larger tension is normallyfound in the pile-up region and the corners of cracks. The indentationprocess has introduced deformation to the material and as such largelocalized tensile-stress was formed. This has subsequently resultedin the formation of cracks along the weaker directions. Continuedindentation to a higher depth acted as a crack driving force to promotemore crack growth, and this is evidenced by the higher tensile-stressat the crack tips  appeared while some originally existing peaks becameless apparent  or even disappeared in the imprint region. Similarly, for the 80 (sample 1), 200 (samples4 and 5), and 250 nm (sample 2) indent-imprints, new peaks around90 and 99 cm–1 are also apparent in the near-indent-centerregion ; but no significantpeak broadening as that of point B was observed.Micro-Raman spectra along one axis of triangularimprint from 300nm-indent  comprise features of both original phase and transformedphase, further indicating these regions are composed of mixed-phasesrather than single-phase only.Raman spectra with sharp peaks obtained at theindent-center (pointA) after 300 nm-depth-indent (sample 3) indicates the amorphizationor disorder transition is only localized within the fractured pile-uparea; combined with the appearance of multiple LDs, this further impliesthat the GaTe layers are likely partially fractured layer-by-layerand slide away within the interlayers. We do not exclude the possibilityof the formation of new phases during the nanoindentation processinduced by the large local stress. Indeed, the areas with darker contrastin the SEM micrographs , correspondingto the relative larger PI-stages in the experiments. Moreover, thesimulations also unveil that the vdW-interaction-force between GaTemultilayers and the substrates (1.83 eV/nm2) is much strongerthan that between GaTe interlayers (0.56 eV/nm2), thusresulting easier fracture of GaTe layers and interlayer-sliding insteadof slippage on the substrate.Furthermore, it was found that compared with the 10-layer-without-defectssample, the introduction of inevitable void-clusters defects reducedthe loading slope after ∼5 nm-depth and 20 nN-load 4b. This 2/Si-substrates. Indentationwas used to deform the 2D GaTe sample in bending up to 250 nm . This indicates that the multiple small PIs and LDs are inner featuresgenerated within the GaTe multilayers, instead of surface fractures.As the sample was suspended, the maximum load (180 μN) was muchlower than that of supported samples (1800 μN) under the same250 nm displacement. This is plausible as the suspended GaTe filmsare subjected to mainly bending while the supported films were inmore compression as they are supported by a substrate. The absenceof large LDs in suspended GaTe films suggests again that those LDsobserved in supported films are correlated to the macro-cracks andfractures on the surface.However, it is worth noting that no detectablefracture/cracksor a permanent-set indent-imprint could be observed (either by AFMor SEM) even after nanoindentation to 250 nm , indicating a less-degraded crystalquality on the sample surface after the nanoindentation than thatfor the fully supported materials. The Raman spectra evolution alongboth vertical and horizontal directions of the indent-center showsno other significant difference. This result not only validates thegood quality of the crystals in the suspended GaTe after nanoindentation,but also indicates that those PIs and LDs are not attributed to phase-transitionor crystal structure changes. As revealed by MD simulations, concurrenceof multiple small PIs and LDs in suspended GaTe multilayers are alsoascribed to the layer-by-layer fracture, and step-to-step interlayer-slidingbehaviors generated within the GaTe multilayers driven by the lateralforce from the indenter. Some PIs and LDs are also noted to be a bitlarger which can be attributed to defects-mediation.Performing micro-Raman spectra mapping,similar to 2/Si-substrate-supportedand suspended GaTe multilayers wereinvestigated through full-scale Berkovich-tip nanoindentation, micro-Ramanspectroscopy, AFM, SEM, and MD simulations. An unusual concurrenceof multiple PIs and LDs events is observed in the loading curves ofsupported GaTe multilayers, accompanied by fracture and cracks. Thisphenomenon also appears in suspended GaTe multilayers, but withoutany observable fracture or cracks on the surface. Qualitative MD simulationsreveal that such concurrence of multiple PIs and LDs in the loadingcurve originates from layers-by-layers cofracture and step-to-stepinterlayer-sliding within GaTe multilayers during nanoindentation.The vdW-interaction-force between GaTe multilayers and the substratesis also revealed much stronger than that between GaTe interlayers,thus resulting in the easier fracture and sliding of materials withinthe GaTe multilayers instead of slippage on the substrate. This workunveils a new deformation and fracture mechanism within multilayered2D materials and will underpin device designs, especially for nanoflexibledevices based on similar 2D layered materials.In summary,the deformation and fracture behaviors of both SiO


Survival rates of patients with metastatic colorectal cancer (mCRC) have improved in recent years due to an increase in possible treatment options . While cLiquid biopsies have emerged as a highly sensitive method to non-invasively monitor tumor burden and assess response based on tumor-specific genomic information . In multWe assessed ctDNA levels as a prognostic marker for PFS in the Sequencing Triplet With Avastin and Maintenance  Trial, which was a randomized, phase II trial investigating efficacy of bevacizumab (BEV) with FOLFOX and 5-fluorouracil/leucovorin/irinotecan (FOLFIRI), administered concurrently or sequentially, versus FOLFOX-BEV in first-line mCRC. There was a 4-month induction phase with the chemotherapy regimen administered in 2-week cycles, and optional extension of induction up to an additional 2 months at the investigator’s discretion. Induction treatment was followed by maintenance with 5-fluorouracil, leucovorin, and bevacizumab every 2 weeks or capecitabine and bevacizumab every 3 weeks.P = 0.0005). Furthermore, a 10-fold or 100-fold reduction in ctDNA levels between pre and post-induction plasma was associated with better PFS . These results demonstrate that ctDNA quantification in post-induction plasma may serve as a prognostic biomarker for mCRC post-treatment outcomes.As part of the exploratory biomarker analyses in STEAM, retrospective sequencing was performed on tumor tissue and plasma with the AVENIO Expanded Kits , which utilizes a hybrid-capture panel targeting 77 genes with known or emerging value as therapeutic biomarkers. Among the 280 patients enrolled in STEAM, 183 had tumor tissue, 118 had matched pre-induction plasma, and 54 had matched post-induction plasma with evaluable sequencing data. Post-induction plasma had to be collected within 60 days of last drug induction. ctDNA levels in plasma were calculated based on somatic single-nucleotide variants pre-defined by the matched tissue sample. While pre-induction levels of ctDNA did not appear to be associated with PFS following induction therapy, we found that lower post-induction ctDNA levels were associated with better PFS . While more studies are required to define a specific time point for routine ctDNA assessment, one can imagine a possible use of ctDNA readout to personalize duration of induction therapy in this setting.Our results support further investigation of using ctDNA for disease monitoring in mCRC. Taken together with other studies, ctDNA quantification is a robust marker in mCRC that can enable non-invasive assessment of therapy response weeks after initiation of chemotherapy or after completion of induction therapy. ctDNA may also enable disease monitoring in mCRC treated with immunotherapy and multikinase inhibitors , 12. Fur


Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to COVID-19 (COrona VIrus Disease-2019). SARS-CoV-2 acute infection may be associated with an increased incidence of neurological manifestations such as encephalopathy and encephalomyelitis, ischemic stroke and intracerebral hemorrhage, anosmia and neuromuscular diseases. Neurological manifestations are commonly reported during the post-acute phase and are also present in Long-COVID (LCS) and post-COVID-19 syndrome (PCS). In October 2020, the German Society of Neurology  published the first guideline on the neurological manifestations of COVID-19. In December 2021 this S1 guideline was revised and guidance for the care of patients with post-COVID-19 syndrome regarding neurological manifestations was added. This is an abbreviated version of the post-COVID-19 syndrome chapter of the guideline issued by the German Neurological society and published in the Guideline repository of the AWMF . In Germany 23 million patients are officially reported as affected by COVID-19. The actual case positive rate is estimated to be much higher with various models predicting the actual number to be much higher than the number of confirmed cases [The global confirmed case count of coronavirus disease 2019 (COVID-19) reached 500 million as of April 2022 . 5.0% of the patients reported having at least 1 of 12 defined symptoms 12–16 weeks after the SARS-CoV-2 infection, compared to 3.4% in the control group. In children up to the age of 11, the symptoms occurred even more frequently in the non-infected control group (4.1% versus 3.2%). When patients requiring hospitalization because of SARS-CoV-2 infection are compared after 6 months with matched control patients hospitalized for non-COVID-19 illness neuropsychiatric outcomes were not different with the exception of anosmia, which was 4 times more frequent after COVID-19 [The frequently missing control group in the data published to date carries the risk of overestimating the incidence of LCS. A French cross-sectional study showed that people who have not been infected with SARS-CoV-2 report residual symptoms with equal frequencies compared to those with LCS after confirmed infection . The disDiagnostic work up and treatment of affected patients should be performed in an interdisciplinary setting consisting of cardiologists, pneumologists, psychiatrists/psychosomatic doctors and neurologists.https://www.nice.org.uk/guidance/ng188, https://www.who.int/publications/i/item/WHO-2019-nCoV-Post_COVID19_condition-Clinical_case_definition). PCS is present when clinical symptoms occur during or after an illness compatible with COVID-19, persisting for at least 2 months, fluctuating in occurrence and cannot be explained by any other diagnosis. The acute infection dates back at least 12 weeks. Affected are patients who have gone through COVID-19 with a mild to moderate course and remained in quarantine at home, as well as patients who had to be admitted to hospital or were treated on intensive care unit (ICU) [Clinical symptoms occurring during or residually after COVID-19 are defined in relation to the time interval following the acute infection. The post-acute phase includes the period up to 4 weeks after the acute infection. LCS includes clinical symptoms more than 4 weeks after COVID-19 (it (ICU) .The most common neurological complaints are fatigue, concentration and memory disorders, headache, vertigo, myalgia and neuropathy, as well as persistent smell and taste disturbances , 17, 35.Additionally, neurological diseases are reported as sequelae after COVID-19. The risk of (cerebro-)vascular disease, including stroke and transient ischemia, is reported to be approximately 50% higher within the first year after the acute infection with SARS-CoV-2 . EpileptCognitive deficits found both in the subacute stage and in the further course after COVID-19 comprise executive functioning, processing speed, category fluency, memory encoding and recall. This applies to patients with both initially mild and severe COVID-19 courses , 27. CogIf self-reported cognitive deficits occur, a cognitive screening assessment e.g. the Montreal Cognitive Assessment (MoCA) should be performed. If pathological results are detected and the symptoms are persistent for several months with restrictions regarding the activities of daily life (ADLs), further examination is indicated. Diagnostic includes the examination of serum and cerebrospinal fluid (CSF) including CNS autoantibodies against intracellular and surface antigens and neurodegenerative markers, cerebral imaging, and detailed neuropsychological assessment. A significant association between neurocognitive symptoms and anti-nuclear antibodies (ANA) may indicate autoimmunity as an etiological cofactor in post-COVID-19 syndrome . AntineuSelf-report questionnaires such as the Fatigue Scale (FS), the Fatigue Severity Scale (FSS) or the Fatigue Assessment Scale (FAS) should be used to assess the symptoms and severity of fatigue.To date there are no established and effective medical treatment options for post-viral fatigue and cognitive impairment, as well as related conditions such as myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). If there are indications of autoimmunity, therapeutic approaches including corticosteroids, intravenous immunoglobulins or therapeutic apheresis can be administered depending on risk and expected benefit.A causal therapy for fatigue is unknown. Non-drug therapeutic approaches such as relaxation techniques, moderate physical activity, acquisition of adequate coping behavior strategies in addition to psychotherapeutic or psychopharmacological treatment are recommended.A meta-analysis of cohort studies indicates that headache persists in 44% after COVID-19 illness. If headache is already reported during the acute infection, there is an increased prevalence of persistent headache in the context of post-COVID-19 .Self-report instruments (e.g. Brief Pain Inventory) should be used to assess symptoms including the severity of chronic pain. Depending on the clinical presentation and examination, an extended laboratory examination can be carried out to rule out other  causes. MR imaging should be performed in individual cases to exclude structural causes.https://dgn.org/leitlinien/ll-56-ll-therapie-des-episodischen-und-chronischen-kopfschmerzes-vom-spannungstyp/).To date headache related to COVID-19 is treated in analogy to chronic tension headache. Prophylactic treatment options including amitriptyline are recommended. Therapeutic approaches follow the existing guidelines of the German Society of Neurology (DGN) .Subsequent to case history and neurological examination, a laboratory investigation of blood including sedimentation rate, myoglobin, creatine kinase and myositis antibodies if clinically suspected, as well as examination of CSF are recommended. An electrophysiological examination is indicated. These recommendations are in line with existing guidelines regarding muscular and neuromuscular disorder (https://dgn.org/leitlinien/ll-69-ll-myositissyndrome). If laboratory results obtain unremarkable, symptomatic treatment can be tried, e.g. administering gabapentine or pregabaline. Physiotherapy and moderate exercise should be implemented.Therapeutic procedures depend on diagnostic findings and are applied according to existing guidelines  post-infectious inflammation and (virus-triggered) immune-mediated mechanisms including humoral and cell-mediated autoimmunity . In mostLongitudinal MRI studies have demonstrated changes in the structure of the prefrontal cortex and parahippocampal regions .Already the SARS-1 pandemic has proven that individual patients may retain long-lasting clinical complaints, especially pain, fatigue, depression, and sleep disorders. The lack of disease-specific biomarkers impedes etiological assignment as well as the exclusion of other (premorbid) diseases.Neurological manifestations are common in patients with LCS and PCS; commonly reported are cognitive deficits, fatigue, headache, myalgia and neuropathy. A comprehensive—ideally interdisciplinary—diagnostic assessment should be initiated in patients who complain about residual or new symptoms more than 3 months after the acute infection. If immune involvement is detected, immunomodulatory therapy may be considered as an individual treatment attempt. A causal therapy does not yet exist; the symptomatic therapy is carried out according to the guidelines of the DGN. Structured neurorehabilitation programs are urgently needed. In parallel, patients should be offered concomitant psychosomatic co-treatment. Avoiding SARS-CoV-2 infection is the key most important preventive factor, and therefore vaccination against SARS-CoV-2 with one of the available vaccines is highly recommended. An interdisciplinary treatment including internal, psychosomatic, and psychiatric expertise is crucial in diagnostic and treatment of PCS patients.


The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a major health crisis worldwide with its continuously emerging new strains, resulting in new viral variants that drive “waves” of infection. PCR or antigen detection assays have been routinely used to detect clinical infections; however, the emergence of these newer strains has presented challenges in detection. One of the alternatives has been to detect and characterize variant-specific peptide sequences from viral proteins using mass spectrometry (MS)-based methods. MS methods can potentially help in both diagnostics and vaccine development by understanding the dynamic changes in the viral proteome associated with specific strains and infection waves. In this study, we developed an accessible, flexible, and shareable bioinformatics workflow that was implemented in the Galaxy Platform to detect variant-specific peptide sequences from MS data derived from the clinical samples. We demonstrated the utility of the workflow by characterizing published clinical data from across the world during various pandemic waves. Our analysis identified six SARS-CoV-2 variant-specific peptides suitable for confident detection by MS in commonly collected clinical samples. It has been more than two years since the Coronavirus disease 2019 (COVID-19) outbreak, which has since spread worldwide, resulting in almost 6.3 M deaths ; b) deteMost commonly, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is detected using the RT-PCR of patient-derived swab samples, or at-home kits recognizing viral antigens . DespiteIn this study, our MS proteomics-based Galaxy workflows identified six SARS-CoV-2 variant-specific peptides from published clinical samples. This was achieved by extending our previously published workflows by analyzing 12 previously generated and published MS-based proteomics datasets from a variety of geographical areas and infection wave timelines. These datasets cover a timeline from March 2020 to January 2022 and cover seven countries and three continents . We leveTwelve clinical MS datasets  availablhttps://www.gisaid.org/, last accessed on 3 February 2022). As the datasets were from clinical samples, we added human proteins and common contaminants to the COVID-19 protein sequence database.Our previous study  used twohttps://usegalaxy.eu/u/galaxyp/w/coviddiscovery-workflow; last accessed 21 September 2022).The sequence database search parameters used for digestion, modifications, tolerance, and FDR estimation were consistent with the parameters mentioned in the published papers for each of these datasets . Confidep-value ≤ 0.05 assigned by PepQuery were then subjected to spectral visualization and manual inspection using the Multiomics Visualization Platform (MVP) tool [We detected 203 SARS-CoV-2 datasets . peptideVP) tool  within thttps://www.gisaid.org/; last accessed on 3 February 2022. The allprot0203.fasta file included 205,705,355 sequences for a size of 113,874,658 KB. The metadata.tsv file included 7,786,913 accession IDs for a size of 4,586,043 KB. The list of 17 peptide sequences described previously was used to subset the allprot0203.fasta file by keeping only proteins containing at least one of the peptides, with I/L residues undifferentiated. An in-house python script was used on this file for (i) the in-silico digestion of these proteins with two missed cleavages allowed; (ii) mapping of all protein sequences to each peptide from the list of 17 sequences; (iii) retrieval of all matching information (including the Pango Lineage) from the metadata file through the GISAID Accession ID; and (iv) summary of information per peptide using the “groupby” function from the python pandas package. Retained information includes . To further annotate these sequences, a Phylogenetic Assignment of Named Global Outbreak (Pango) lineage analysis; last accessed on 3 February 2022  was perfincludes  the listThe discovery workflow performed sequence database searching using two software platforms (SearchGUI-PeptideShaker and MaxQuant). The sequence database searching of the clinical MS datasets against the database of protein sequences specific to SARS-CoV-2 variants confidently detected 203 unique peptides . These pp-value ≤ 0.05) and the subsequent manual spectral quality inspection using Lorikeet . These We continue to expand our panel to include peptide targets specific to emerging variants and their associated strain lineages. Our current panel contains 906 peptides shared across all strains of SARS-CoV-2, in which six variant-specific peptides useful for monitoring the infection dynamics of clinically distinct forms of the virus were characterized in this work. These peptides align to the nucleocapsid viral particles, which play critical roles in host infection mechanisms, as targets of antigen-based rapid tests, and could also help in therapeutic approaches ,38,39. TIn summary, we demonstrated the use of customized bioinformatic workflows to identify six confident SARS-CoV-2 variant-specific peptides suitable for MS/MS based detection in clinical samples. These peptides should be useful for developing targeted MS-based assays for the rapid and sensitive characterization of variant-specific proteins within clinical samples. Our discovery and verification workflows were developed within Galaxy and are accessible via the public and freely available European Galaxy resource , and as individual tools available in the Galaxy Tool Shed . These w