Publications by authors named "Robert Chua"

13 Publications

  • Page 1 of 1

Functional States in Tumor-Initiating Cell Differentiation in Human Colorectal Cancer.

Cancers (Basel) 2021 Mar 4;13(5). Epub 2021 Mar 4.

Translational Functional Cancer Genomics, National Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center (DKFZ) Heidelberg, 69120 Heidelberg, Germany.

Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.
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http://dx.doi.org/10.3390/cancers13051097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961698PMC
March 2021

Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19.

Nat Biotechnol 2020 Dec 24. Epub 2020 Dec 24.

Center for Digital Health, Berlin Institute of Health (BIH) and Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

In coronavirus disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying causes and the effects of the main anti-hypertensive therapies-angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)-remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical COVID-19 progression. ACEI treatment was associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic antiviral responses, whereas ARB treatment related to enhanced epithelial-immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.
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http://dx.doi.org/10.1038/s41587-020-00796-1DOI Listing
December 2020

Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19.

Nat Neurosci 2021 02 30;24(2):168-175. Epub 2020 Nov 30.

Department of Neurology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.

The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata.
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http://dx.doi.org/10.1038/s41593-020-00758-5DOI Listing
February 2021

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis.

Nat Biotechnol 2020 08 26;38(8):970-979. Epub 2020 Jun 26.

Institute of Virology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health (BIH), Berlin, Germany.

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.
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http://dx.doi.org/10.1038/s41587-020-0602-4DOI Listing
August 2020

SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells.

EMBO J 2020 05 14;39(10):e105114. Epub 2020 Apr 14.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.
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http://dx.doi.org/10.15252/embj.20105114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232010PMC
May 2020

The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells.

Clin Epigenetics 2019 07 2;11(1):98. Epub 2019 Jul 2.

Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.

Background: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro.

Methods: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK.

Results: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis.

Conclusions: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.
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http://dx.doi.org/10.1186/s13148-019-0698-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604314PMC
July 2019

Loss of RNF40 Decreases NF-κB Activity in Colorectal Cancer Cells and Reduces Colitis Burden in Mice.

J Crohns Colitis 2019 Mar;13(3):362-373

Department of General, Visceral and Pediatric Surgery, Göttingen Center for Molecular Biosciences, University Medical Center Göttingen, Göttingen, Germany.

Background And Aims: Inflammatory bowel diseases are linked to an increased risk of developing colorectal cancer [CRC]. Previous studies suggested that the H2B ubiquitin ligase RING finger protein-20 [RNF20] inhibited inflammatory signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB]. However, the role of RNF40, the obligate heterodimeric partner of RNF20, in the context of inflammation and CRC has not been addressed. Here, we examined the effect of RNF40 loss on CRC cells in vitro and on inflammation and inflammatory signaling in vitro and in vivo.

Methods: We evaluated H2Bub1 levels in human and murine colorectal tumors by immunohistochemistry. Moreover, we correlated H2Bub1 and RNF40 levels in vivo and assessed the consequences of RNF40 depletion on cellular phenotype and gene expression in CRC cells in vitro. Finally, we examined the effect of a colon-specific loss of Rnf40 in a murine model of colitis, and assessed both local and systemic inflammation-associated consequences.

Results: In vitro studies revealed that the tumorigenic phenotype of CRC cells decreased after RNF40 depletion and displayed gene expression changes related to chromosome segregation and DNA replication, as well as decreased induction of several NF-κB-associated cytokines. This effect was associated with decreased nuclear localization of NF-κB following tumor necrosis factor alpha treatment. Consistently, the colon-specific loss of Rnf40 exerted a protective local, as well as systemic, effect following acute colitis.

Conclusions: Our findings suggest that RNF40 plays a central role in the maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.
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http://dx.doi.org/10.1093/ecco-jcc/jjy165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599279PMC
March 2019

Valine/isoleucine variants drive selective pressure in the VP1 sequence of EV-A71 enteroviruses.

BMC Infect Dis 2017 05 8;17(1):333. Epub 2017 May 8.

Cirad, UMR 17, Intertryp, TA-A17/G, Campus International de Baillarguet, 34398, Montpellier Cedex 5, France.

Background: In 2011-2012, Northern Vietnam experienced its first large scale hand foot and mouth disease (HFMD) epidemic. In 2011, a major HFMD epidemic was also reported in South Vietnam with fatal cases. This 2011-2012 outbreak was the first one to occur in North Vietnam providing grounds to study the etiology, origin and dynamic of the disease. We report here the analysis of the VP1 gene of strains isolated throughout North Vietnam during the 2011-2012 outbreak and before.

Methods: The VP1 gene of 106 EV-A71 isolates from North Vietnam and 2 from Central Vietnam were sequenced. Sequence alignments were analyzed at the nucleic acid and protein level. Gene polymorphism was also analyzed. A Factorial Correspondence Analysis was performed to correlate amino acid mutations with clinical parameters.

Results: The sequences were distributed into four phylogenetic clusters. Three clusters corresponded to the subgenogroup C4 and the last one corresponded to the subgenogroup C5. Each cluster displayed different polymorphism characteristics. Proteins were highly conserved but three sites bearing only Isoleucine (I) or Valine (V) were characterized. The isoleucine/valine variability matched the clusters. Spatiotemporal analysis of the I/V variants showed that all variants which emerged in 2011 and then in 2012 were not the same but were all present in the region prior to the 2011-2012 outbreak. Some correlation was found between certain I/V variants and ethnicity and severity.

Conclusions: The 2011-2012 outbreak was not caused by an exogenous strain coming from South Vietnam or elsewhere but by strains already present and circulating at low level in North Vietnam. However, what triggered the outbreak remains unclear. A selective pressure is applied on I/V variants which matches the genetic clusters. I/V variants were shown on other viruses to correlate with pathogenicity. This should be investigated in EV-A71. I/V variants are an easy and efficient way to survey and identify circulating EV-A71 strains.
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http://dx.doi.org/10.1186/s12879-017-2427-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422960PMC
May 2017

Chikungunya as a cause of acute febrile illness in southern Sri Lanka.

PLoS One 2013 2;8(12):e82259. Epub 2013 Dec 2.

Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America ; Hubert-Yeargan Center for Global Health, Durham, North Carolina, United States of America.

Background: Chikungunya virus (CHIKV) re-emerged in Sri Lanka in late 2006 after a 40-year hiatus. We sought to identify and characterize acute chikungunya infection (CHIK) in patients presenting with acute undifferentiated febrile illness in unstudied rural and semi-urban southern Sri Lanka in 2007.

Methodology/principal Findings: We enrolled febrile patients ≥ 2 years of age, collected uniform epidemiologic and clinical data, and obtained serum samples for serology, virus isolation, and real-time reverse-transcriptase PCR (RT-PCR). Serology on paired acute and convalescent samples identified acute chikungunya infection in 3.5% (28/797) patients without acute dengue virus (DENV) infection, 64.3% (18/28) of which were confirmed by viral isolation and/or real-time RT-PCR. No CHIKV/DENV co-infections were detected among 54 patients with confirmed acute DENV. Sequencing of the E1 coding region of six temporally distinct CHIKV isolates (April through October 2007) showed that all isolates posessed the E1-226A residue and were most closely related to Sri Lankan and Indian isolates from the same time period. Except for more frequent and persistent musculoskeletal symptoms, acute chikungunya infections mimicked DENV and other acute febrile illnesses. Only 12/797 (1.5%) patients had serological evidence of past chikungunya infection.

Conclusions/significance: Our findings suggest CHIKV is a prominent cause of non-specific acute febrile illness in southern Sri Lanka.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082259PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846738PMC
February 2015

Scrub typhus with sepsis and acute respiratory distress syndrome.

J Clin Microbiol 2013 Aug 12;51(8):2787-90. Epub 2013 Jun 12.

Mount Elizabeth Hospital, Singapore.

Scrub typhus is a major infectious threat in the Asia-Pacific region. We report an unusual case of scrub typhus in a patient in Singapore who presented with sepsis and acute respiratory distress syndrome but lacked the pathognomonic eschar. The patient recovered after appropriate diagnosis and doxycycline treatment. Rickettsial diseases should be included in the differential diagnosis of febrile illnesses in regions where the diseases are endemic, and absence of eschar should not be the criterion used to rule out scrub typhus.
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http://dx.doi.org/10.1128/JCM.00463-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719619PMC
August 2013

The early clinical features of dengue in adults: challenges for early clinical diagnosis.

PLoS Negl Trop Dis 2011 31;5(5):e1191. Epub 2011 May 31.

Communicable Diseases Centre, Tan Tock Seng Hospital, Singapore, Singapore.

Background: The emergence of dengue throughout the tropical world is affecting an increasing proportion of adult cases. The clinical features of dengue in different age groups have not been well examined, especially in the context of early clinical diagnosis.

Methodology/principal Findings: We structured a prospective study of adults (≥ 18 years of age) presenting with acute febrile illness within 72 hours from illness onset upon informed consent. Patients were followed up over a 3-4 week period to determine the clinical outcome. A total of 2,129 adults were enrolled in the study, of which 250 (11.7%) had dengue. Differences in the rates of dengue-associated symptoms resulted in high sensitivities when the WHO 1997 or 2009 classification schemes for probable dengue fever were applied to the cohort. However, when the cases were stratified into age groups, fewer older adults reported symptoms such as myalgia, arthralgia, retro-orbital pain and mucosal bleeding, resulting in reduced sensitivity of the WHO classification schemes. On the other hand, the risks of severe dengue and hospitalization were not diminished in older adults, indicating that this group of patients can benefit from early diagnosis, especially when an antiviral drug becomes available. Our data also suggests that older adults who present with fever and leukopenia should be tested for dengue, even in the absence of other symptoms.

Conclusion: Early clinical diagnosis based on previously defined symptoms that are associated with dengue, even when used in the schematics of both the WHO 1997 and 2009 classifications, is difficult in older adults.
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http://dx.doi.org/10.1371/journal.pntd.0001191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104968PMC
September 2011

Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans.

PLoS Pathog 2010 Aug 19;6(8):e1001051. Epub 2010 Aug 19.

Singapore Institute for Clinical Sciences, A*STAR, Singapore.

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2(low)) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-gamma during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.
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http://dx.doi.org/10.1371/journal.ppat.1001051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924358PMC
August 2010

In vitro inhibition of human influenza A virus replication by chloroquine.

Virol J 2006 May 29;3:39. Epub 2006 May 29.

Defence Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, #09-01, 117510, Singapore.

Chloroquine is a 9-aminoquinolone with well-known anti-malarial effects. It has biochemical properties that could be applied to inhibit viral replication. We report here that chloroquine is able to inhibit influenza A virus replication, in vitro, and the IC50s of chloroquine against influenza A viruses H1N1 and H3N2 are lower than the plasma concentrations reached during treatment of acute malaria. The potential of chloroquine to be added to the limited range of anti-influenza drugs should be explored further, particularly since antiviral drugs play a vital role in influenza pandemic preparedness.
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http://dx.doi.org/10.1186/1743-422X-3-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1481635PMC
May 2006