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Background Colorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC. Methods Plasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates. Results From 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts. Conclusions This study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

Activated B-cell (ABC) lymphoma, a distinct molecular entity within diffuse large B-cell lymphoma (DLBCL), remains highly incurable, showing a worse response to standard immunochemotherapy. The discouraging results obtained in several clinical trials using proteasome inhibitors, tyrosine kinase inhibitors, or immunomodulators, lead to an intense search for new, potentially druggable biomarkers in DLBCL. In this study, we designed an experimental strategy for DLBCL to discover high- and low-abundance RNA-seq-derived transcripts involved in the oncogenic phenotype in patients diagnosed with ABC-DLBCL. Based on the results of a comparative analysis, 79 DE genes and two enriched gene sets related to metabolism and immunity were selected. Genes related to drug resistance, anti-inflammatory response, and tumor-cell dissemination were found to be up-regulated, while tumor suppressor genes were down-regulated. Then, we searched for the perturbagens most suitable for gene expression profiling (GEP) by iLINCS-CMap. Herein, we present a novel experimental approach that connects the omics signature of DLBCL with potential drugs for more accurate treatments.

Teetat Kongratanapasert,1,* Supasek Kongsomros,2,5,* Nlin Arya,3 Kripitch Sutummaporn,3 Witthawat Wiriyarat,3 Yada Akkhawattanangkul,4 Tussapon Boonyarattanasoonthorn,5 Nithi Asavapanumas,5 Phongthon Kanjanasirirat,6 Ampa Suksatu,2 Khanit Sa-ngiamsuntorn,7 Suparerk Borwornpinyo,6,8 Pornpun Vivithanaporn,5 Somchai Chutipongtanate,5,9 Suradej Hongeng,6,9 Boonsong Ongphiphadhanakul,10 Arunee Thitithanyanont,2 Phisit Khemawoot,5 Piyamitr Sritara10 1Program in Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand; 2Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 3Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhonpathom, 73170, Thailand; 4Department of Clinical Medicine and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhonpathom, 73170, Thailand; 5Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samutprakarn, 10540, Thailand; 6Excellence Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 7Department of Biochemistry, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand; 8Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 9Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand; 10Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand*These authors contributed equally to this workCorrespondence: Phisit Khemawoot, Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, 10540, Thailand, Tel/Fax +66 28395161, Email phisit.khe@mahidol.ac.thBackground: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems.Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo.Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A.Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals.Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development.Keywords: Boesenbergia rotunda, Zingiberaceae, panduratin A, SARS-CoV-2, COVID-19, hamster

Gaucher disease (GD) is a lysosomal storage disorder, characterized by hepatosplenomegaly, pancytopenia, bone diseases, with or without neurological symptoms. Plasma glucosylsphingosine (lyso-Gb1), a highly sensitive and specific biomarker for GD, has been used for diagnosis and monitoring the response to treatment. Enzyme replacement therapy (ERT) is an effective treatment for the non-neurologic symptoms of GD. Neuronopathic GD (type 2 and 3) accounts for 60%-70% of the Asian affected population.We explored combination therapy of ERT followed by hematopoietic stem cell transplantation (HSCT) and its long-term outcomes in patients with GD type 3 (GD3).Four patients with GD3 and one with GD type 1 (GD1) underwent HSCT. The types of donor were one matched-related, one matched-unrelated, and three haploidentical. The age at disease onset was 6-18 months and the age at HSCT was 3.8-15 years in the patients with GD3. The latest age at follow-up was 8-22 years, with a post-HSCT duration of 3-14 years. All patients had successful HSCT. Chronic graft-versus-host disease occurred in one patient. The enzyme activities were normalized at 2 weeks post HSCT. Lyso-Gb1 concentrations became lower than the pathological value. All of the patients are still alive and physically independent. Most of them (4/5) returned to school. None of the patients with GD3 had seizures or additional neurological symptoms after HSCT, but showed varying degrees of cognitive impairment.ERT followed by HSCT could be considered as an alternative treatment for patients with GD3 who have a high risk of fatal neurological progression.

Recent research indicates that a number of children with autism generate folate receptor alpha autoantibodies (FRAA), which block transportation of folate across the blood-brain barrier, resulting in cerebral folate deficiency syndrome. Plasma FRAA detection permits precision diagnosis and potentially beneficial folinic acid treatment in FRAA-positive children with autism.To investigate FRAA prevalence in Thai children with autism and evaluate the associations between FRAA-positive status, clinical symptom severity, and adaptive functioning.FRAA level was determined in serum samples from 89 children with autism between 2 and 15 years (69 males, 20 females, mean age 7.9 years, SD 3.8). The Childhood Autism Rating Scale-Second Edition (CARS-2) and the Vineland Adaptive Behavior Scales (VABS) were used to evaluate clinical symptom severity and adaptive functioning, respectively.Of 89 children, 30 (33.7%) were FRAA-positive. FRAA-positive children with autism had significantly poorer mean VABS Adaptive Behavior Composite scores (The findings demonstrate the presence of FRAA in children with autism and that FRAA status is associated with poorer adaptive functioning.

Breastfeeding reduces the risk of necrotizing enterocolitis (NEC), one of the most common causes of morbidity and mortality in preterm infants. However, the molecular substrates by which human milk (HM) offers protection against NEC are not well known. Using fetal intestinal epithelial cells treated with known NEC aggravators, namely lipopolysaccharide (LPS) and platelet-activating factor (PAF), we mapped the time-course of changes in targeted expression analysis of 35 NEC-associated genes, so-called the NEC signature. We found, first, that HM treatment fully rescued LPS/PAF-induced fetal intestinal cell death at 12 and 24 h (

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 μg/mL for A. paniculata extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state. Mouse models of severe congenital neutropenia using patient-derived mutations in the GFI1 locus are used to determine the mechanisms by which the disease progresses.

Over the past decades, several study programs have conducted genetic testing in cancer patients to identify potential genetic targets for the development of precision therapeutic strategies. These biomarker-driven trials have demonstrated improved clinical outcomes and progression-free survival rates in various types of cancers, especially for adult malignancies. However, similar progress in pediatric cancers has been slow due to their distinguished mutation profiles compared to adults and the low frequency of recurrent genomic alterations. Recently, increased efforts to develop precision medicine for childhood malignancies have led to the identification of genomic alterations and transcriptomic profiles of pediatric patients which presents promising opportunities to study rare and difficult-to-access neoplasms. This review summarizes the current state of known and potential genetic markers for pediatric solid tumors and provides perspectives on precise therapeutic strategies that warrant further investigations.

The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained a great deal of attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+ charges and an alpha helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause the leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effects against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise, which requires further validation in preclinical and clinical studies.

Simple Summary Cancer cells can detach from a primary tumour and present in peripheral blood as circulating tumour cells, or in the widest sense, as circulating atypical cells (CAC). Although CAC are a promising biomarker for non-invasive cancer screening, they occur at very low frequency and their detection and characterization remains challenging. We here validated isolation and concentration of untouched CAC from spiked cancer cell blood samples and combined this with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). This workflow was optimised to detect as little as six cancer cells per 5000 white blood cells. Future development of our workflow may cover a larger range of cancer types and further improvements to enable the use of MALDI-TOF MS as a cancer-screening platform in clinical settings. Abstract Circulating atypical cells (CAC) are released from a primary tumour site into peripheral blood and are indicators of cancer metastasis. CAC occur at very low frequency in circulating blood, and their detection remains challenging. Moreover, white blood cells (WBC) are the major contaminant in enriched CAC samples. Here, we developed matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a novel CAC characterization platform. Main spectra profiles (MSP) of normal and cancer cells were generated by MALDI-TOF MS, and a cell-main spectra database was then compiled and analysed using the MALDI Biotyper software. Logarithmic scores accurately predicted distinct cell types. The feasibility of this workflow was then validated using simulated samples, which were prepared by 5000 WBC of three healthy individuals spiked with varying numbers (3, 6, 12, 25, 50, and 100) of lung, colon, or prostate cancer cells. MALDI-TOF MS was able to detect cancer cells down to six cells over the background noise of 5000 WBC with significantly higher predictive scores as compared to WBC alone. Further development of cell-MSP database to cover all cancer types sourced from cell lines and patient tumours may enable the use of MALDI-TOF MS as a cancer-screening platform in clinical settings in the future.

The adsorption of proteins on membranes has been used for simple, low-cost, and minimal sample handling of large volume, low protein abundance liquid samples. Syringe-push membrane absorption (SPMA) is an innovative way to process bio-fluid samples by combining a medical syringe and protein-absorbable membrane, which makes SPMA a simple, rapid protein and proteomic analysis method. However, the membrane used for SPMA is only limited to commercially available protein-absorbable membrane options. To raise the method’s efficiency, higher protein binding capacity with a lower back pressure membrane is needed. In this research, we fabricated electrospun polybutylene succinate (PBS) membrane and compared it to electrospun polyvinylidene fluoride (PVDF). Rolling electrospinning (RE) and non-rolling electrospinning (NRE) were employed to synthesize polymer fibers, resulting in the different characteristics of mechanical and morphological properties. Adding graphene oxide (GO) composite does not affect their mechanical properties, however, electrospun PBS membrane can be applied as a filter membrane and has a higher pore area than electrospun PVDF membrane. Albumin solution filtration was performed using all the electrospun filter membranes by the SPMA technique to measure the protein capture efficiency and staining of the protein on the membranes, and these membranes were compared to the commercial filter membranes—PVDF, nitrocellulose, and Whatman no. 1. A combination of rolling electrospinning with graphene oxide composite and PBS resulted in two times more captured protein when compared to commercial membrane filtration and more than sixfold protein binding than non-composite polymer. The protein staining results further confirmed the enhancement of the protein binding property, showing more intense stained color in compositing polymer with GO.

Objective Physicochemical approach such as strong ion difference provides a novel concept in understanding and managing acid–base disturbance in patients. However, its application in pediatrics is limited. This study aimed to evaluate a correlation between the physicochemical approach and blood gas pH for acid–base determination in critically ill pediatric patients. Results A total of 130 pediatric patients were included, corresponding to 1338 paired measures for analyses. Of these, the metabolic subgroup (743 paired measures) was defined. Among physicochemical parameters, the effective strong ion difference showed the best correlation with the blood gas pH in the whole cohort (R = 0.398; p

The third-party umbilical cord blood (UCB)-derived regulatory T cells (Treg) are an alternative to donor-derived Treg as cellular therapy of graft-versus-host disease following hematopoietic stem cell transplantation. However, their suppressive characteristics against autologous and allogeneic T effector cells (Teff) have rarely been documented. The exact role of UCB-Treg in hematologic malignancies is also uncertain. Here, we investigated the direct effects of UCB-Treg on the proliferation of autologous Teff, as compared with allogeneic Teff, and also determined cellular fates of lymphoblasts after UCB-Treg co-culture. UCB-Treg were isolated from 8 UCB samples using 2-step immunomagnetic bead sorting. After 10-day ex vivo expansion, up to 60-fold increase in cell number with 76.7%±4.9% of CD4CD25CD127FoxP UCB-Treg was obtained. Further characterization showed that ex vivo-expanded UCB-Treg contained a higher proportion of CD95CD45RACCR4Treg-B subpopulation compared with the CD95CD45RACCR4Treg-A subpopulation (13.0%±4.8% vs. 0.8%±0.7%; P0.05), along with the detecting of substantial amounts of secretory IL-10 (57.7±17.8 pg/mL) and TGF-β1 (196.5±29.7 pg/mL) in culture supernatants. After 4 days co-culture with UCB-Treg (at the ratio of 1:1), the proliferation of autologous and allogeneic Teff was decreased comparably (43.6%±17.5% vs. 37.6±17.7%; P=0.437). Suppression was independent of HLA-A, B, and DRB1 compatibility between UCB-Treg and Teff. UCB-Treg co-culture with various lymphoblasts showed proliferative suppression of Jurkat T lymphoblasts (45.4%±20.5% at the ratio of 1:1), but not Namalwa and Raji B lymphoblasts. All lymphoblasts had no significant cell apoptosis or death after co-culture. In conclusion, the ex vivo-expanded UCB-Treg had no difference in autologous and allogeneic Teff suppression. UCB-Treg therapy in patients with graft-versus-host disease who have a primary disease of T-cell leukemia may have additional benefits in the prevention of relapsed disease.

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistically friendly are critically needed for global equity, especially for middle to low income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported with the use of the receptor binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, so called “HexaPro”, has been shown for its physical property to possess two RBD in the “up” conformation, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminium hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. In addition, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 μg/mL for A. paniculata extract and 13.2–81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained much attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+charges and alpha-helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effect against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise which requires further validation in preclinical and clinical studies.TeaserIn silico screening of naturally occurring human milk peptides discovers a new anticancer peptide that kills leukemic cells in vitro and ex vivo.

The coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem affecting more than fifty million cases with over one million deaths globally. The effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine anti-SARS-CoV–2 activity of Andrographis paniculata extract and its major component andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Post-infection treatment of A. paniculata and andrographolide in SARS-CoV–2 infected Calu-3 cells significantly inhibited the production of infectious virions with the IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3) and brain (SH-SY5Y), showed the CC50 of >100 μg/mL for A. paniculata extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to the selectivity index over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV–2 infection.

The development of targeted treatment options for precision medicine is hampered by a slow and costly process of drug screening. While small molecule docking simulations are often applied in conjunction with cheminformatic methods to reduce the number of candidate molecules to be tested experimentally, the current approaches suffer from high false positive rates and are computationally expensive. Here, we present a novel in silico approach for drug discovery and repurposing, dubbed connectivity enhanced Structure Activity Relationship (ceSAR) that improves on current methods by combining docking and virtual screening approaches with pharmacogenomics and transcriptional signature connectivity analysis. ceSAR builds on the landmark LINCS library of transcriptional signatures of over 20,000 drug-like molecules and ~5,000 gene knock-downs (KDs) to connect small molecules and their potential targets. For a set of candidate molecules and specific target gene, candidate molecules are first ranked by chemical similarity to their ‘concordant’ LINCS analogs that share signature similarity with a knock-down of the target gene. An efficient method for chemical similarity search, optimized for sparse binary fingerprints of chemical moieties, is used to enable fast searches for large libraries of small molecules. A small subset of candidate compounds identified in the first step is then re-scored by combining signature connectivity with docking simulations. On a set of 20 DUD-E benchmark targets with LINCS KDs, the consensus approach reduces significantly false positive rates, improving the median precision 3-fold over docking methods at the extreme library reduction. We conclude that signature connectivity and docking provide complementary signals, offering an avenue to improve the accuracy of virtual screening while reducing run times by multiple orders of magnitude.