| 1. |
- Akaberi, Dario, 1989-
(författare)
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Identification of protease inhibitors against Flaviviruses and Coronaviruses
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Vector-borne flaviviruses and coronaviruses of zoonotic origins are important human pathogens and represent a serious threat to public health worldwide. Flaviviruses can be found on all continents, apart from Antarctica, where they are transmitted by arthropod vectors causing millions of infections every year. While most of the infections are mild or asymptomatic, flaviviruses like dengue and yellow fever viruses can cause potentially lethal hemorrhagic fever and shock syndrome. Neurotropic flaviviruses like West Nile, Japanese encephalitis, and Tick-borne encephalitis (TBEV) can cause meningoencephalitis with long-term symptoms. Coronaviruses, and in particular betacoronaviruses of zoonotic origin like SARS (2003) and MERS (2012), have been periodically emerging since the early 2000s causing outbreaks of severe respiratory syndrome. The latest example is SARS-CoV-2 that after causing a cluster of infection in the Chinese city of Wuhan, spread all over the world causing at present over 6.9 million deaths. Although vaccines are essential in preventing infections or severe disease and hospitalization in the case of SARS-CoV-2, antivirals represent an extremely valuable tool for treatment and prevention of current and future flavivirus and coronavirus infections. In the work presented in this thesis we have used a combination of in silico and in vitro techniques to identify and test the activity of potential inhibitors of viral proteases. In our first study (paper 1) we unexpectedly identified an HIV protease inhibitor with in vitro activity against ZIKV NS2B-NS3 protease. The inhibitor was identified by virtual screening of a library of known protease inhibitors, evaluated by molecular dynamics simulation and finally tested against recombinant ZIKV protease using a FRET-based enzymatic assay. The same combination of molecular docking and molecular dynamics simulations were also used to correctly predict the activity of a known pan-Flavivirus protease inhibitor against TBEV protease (paper 2). As a result, we were the first to report peptide-based compounds with in vitro activity against TBEV. After the outbreak of the COVID-19 we switched our attention to SARS-CoV-2. We first tested the inhibitory effect of the broad-spectrum antiviral nitric oxide (NO) and found that the NO-releasing compound SNAP had a dose dependent inhibitory effect on SARS-CoV-2 replication in cell-based assays (paper 3). We speculated that SNAP could inhibit SARS-COV-2 protease by trans-nitration of the catalytic Cys145 of SARS-CoV-2 main protease and found that SNAP had a dose dependent inhibitory effect on recombinant SARS-CoV-2 Mpro protease activity in an in vitro enzymatic assay. In our last study (paper 4) we identified a new class of potent SARS-CoV-2 protease inhibitors through the affinity screening of DNA-encoded-chemical libraries containing 4.2 billion compounds. The identified compounds inhibited recombinant SARS-CoV-2 protease with IC50 as low as 25 nM and had a dose dependent antiviral effect in the low micromolar range in infected Calu-3 and Caco-2 cell lines.
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| 2. |
- Ambikan, Anoop T., et al.
(författare)
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Genome-scale metabolic models for natural and long-term drug-induced viral control in HIV infection
- 2022
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:9
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Tidskriftsartikel (refereegranskat)abstract
- Genome-scale metabolic models (GSMMs) can provide novel insights into metabolic reprogramming during disease progression and therapeutic interventions. We developed a context-specific system-level GSMM of people living with HIV (PLWH) using global RNA sequencing data from PBMCs with suppressive viremia either by natural (elite controllers, PLWHEC) or drug-induced (PLWHART) control. This GSMM was compared with HIV-negative controls (HC) to provide a comprehensive systems-level metabo-transcriptomic characterization. Transcriptomic analysis identified up-regulation of oxidative phosphorylation as a characteristic of PLWHART, differentiating them from PLWHEC with dysregulated complexes I, III, and IV. The flux balance analysis identified altered flux in several intermediates of glycolysis including pyruvate, a-ketoglutarate, and glutamate, among others, in PLWHART. The in vitro pharmacological inhibition of OXPHOS complexes in a latent lymphocytic cell model (J-Lat 10.6) suggested a role for complex IV in latency reversal and immunosenescence. Furthermore, inhibition of complexes I/III/IV induced apoptosis, collectively indicating their contribution to reservoir dynamics.
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| 3. |
- Ambikan, Anoop T., et al.
(författare)
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Multi-omics personalized network analyses highlight progressive disruption of central metabolism associated with COVID-19 severity
- 2022
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Ingår i: Cell systems. - : Elsevier BV. - 2405-4712 .- 2405-4720. ; 13:8, s. 665-681
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Tidskriftsartikel (refereegranskat)abstract
- The clinical outcome and disease severity in coronavirus disease 2019 (COVID-19) are heterogeneous, and the progression or fatality of the disease cannot be explained by a single factor like age or comorbidities. In this study, we used system-wide network-based system biology analysis using whole blood RNA sequencing, immunophenotyping by flow cytometry, plasma metabolomics, and single-cell-type metabolo-mics of monocytes to identify the potential determinants of COVID-19 severity at personalized and group levels. Digital cell quantification and immunophenotyping of the mononuclear phagocytes indicated a sub-stantial role in coordinating the immune cells that mediate COVID-19 severity. Stratum-specific and person-alized genome-scale metabolic modeling indicated monocarboxylate transporter family genes (e.g., SLC16A6), nucleoside transporter genes (e.g., SLC29A1), and metabolites such as a-ketoglutarate, succi-nate, malate, and butyrate could play a crucial role in COVID-19 severity. Metabolic perturbations targeting the central metabolic pathway (TCA cycle) can be an alternate treatment strategy in severe COVID-19.
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| 4. |
- Ambikan, Anoop T., et al.
(författare)
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Systems-level temporal immune-metabolic profile in Crimean-Congo hemorrhagic fever virus infection
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 120:37
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever (CCHF) caused by CCHF virus (CCHFV) is one of the epidemic-prone diseases prioritized by the World Health Organisation as public health emergency with an urgent need for accelerated research. The trajectory of host response against CCHFV is multifarious and remains unknown. Here, we reported the temporal spectrum of pathogenesis following the CCHFV infection using genome-wide blood transcriptomics analysis followed by advanced systems biology analysis, temporal immune-pathogenic alterations, and context-specific progressive and postinfection genome-scale metabolic models (GSMM) on samples collected during the acute (T0), early convalescent (T1), and convalescent-phase (T2). The interplay between the retinoic acid-inducible gene-I-like/nucleotide-binding oligomerization domain-like receptor and tumor necrosis factor signaling governed the trajectory of antiviral immune responses. The rearrangement of intracellular metabolic fluxes toward the amino acid metabolism and metabolic shift toward oxidative phosphorylation and fatty acid oxidation during acute CCHFV infection determine the pathogenicity. The upregulation of the tricarboxylic acid cycle during CCHFV infection, compared to the noninfected healthy control and between the severity groups, indicated an increased energy demand and cellular stress. The upregulation of glycolysis and pyruvate metabolism potentiated energy generation through alternative pathways associated with the severity of the infection. The downregulation of metabolic processes at the convalescent phase identified by blood cell transcriptomics and single-cell type proteomics of five immune cells (CD4+ and CD8+ T cells, CD14+ monocytes, B cells, and NK cells) potentially leads to metabolic rewiring through the recovery due to hyperactivity during the acute phase leading to post-viral fatigue syndrome.
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| 5. |
- Appelberg, Sofia, et al.
(författare)
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Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells
- 2020
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Ingår i: Emerging Microbes & Infections. - : Informa UK Limited. - 2222-1751. ; 9:1, s. 1748-1760
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Tidskriftsartikel (refereegranskat)abstract
- How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.
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| 6. |
- Ashokkumar, M., et al.
(författare)
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Unique phenotypic characteristics of recently transmitted HIV-1 subtype C envelope glycoprotein gp120 : Use of CXCR6 coreceptor by transmitted founder viruses
- 2018
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 92:9
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Tidskriftsartikel (refereegranskat)abstract
- Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7- Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.
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| 7. |
- Bai, Xiangning, et al.
(författare)
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Whole-Genome Metagenomic Analysis of the Gut Microbiome in HIV-1-Infected Individuals on Antiretroviral Therapy
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Gut microbiome plays a significant role in HIV-1 immunopathogenesis and HIV-1-associated complications. Previous studies have mostly been based on 16S rRNA gene sequencing, which is limited in taxonomic resolution at the genus level and inferred functionality. Herein, we performed a deep shotgun metagenomics study with the aim to obtain a more precise landscape of gut microbiome dysbiosis in HIV-1 infection. A reduced tendency of alpha diversity and significantly higher beta diversity were found in HIV-1-infected individuals on antiretroviral therapy (ART) compared to HIV-1-negative controls. Several species, such as Streptococcus anginosus, Actinomyces odontolyticus, and Rothia mucilaginosa, were significantly enriched in the HIV-1-ART group. Correlations were observed between the degree of immunodeficiency and gut microbiome in terms of microbiota composition and metabolic pathways. Furthermore, microbial shift in HIV-1-infected individuals was found to be associated with changes in microbial virulome and resistome. From the perspective of methodological evaluations, our study showed that different DNA extraction protocols significantly affect the genomic DNA quantity and quality. Moreover, whole metagenome sequencing depth affects critically the recovery of microbial genes, including virulome and resistome, while less than 5 million reads per sample is sufficient for taxonomy profiling in human fecal metagenomic samples. These findings advance our understanding of human gut microbiome and their potential associations with HIV-1 infection. The methodological assessment assists in future study design to accurately assess human gut microbiome.
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| 8. |
- Banerjee, Indradumna, et al.
(författare)
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MicroCAP: Microfluidic Centrifuge Assisted Precipitation for DNA Quantification on Lab-on-DVD
- 2018
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Konferensbidrag (refereegranskat)abstract
- We report for the first time the MicroCAP technique, for rapid DNA detection and quantification, that does not require any purification or fluorescent labelling of DNA. The invention is based on DNA interacting with a detection dye (Gelred) to form a complex, that forms a visible precipitate within seconds of centrifugation. MicroCAP can be used for DNA quantification, when combined with the Lab-on-DVD with inbuilt centrifugation and sub- micron imaging resolution. We quantify PCR and LAMP assay products using MicroCAP on the integrated Lab-on- DVD platform, and demonstrate a detection limit of 10 ng/!".KEYWORDS: MicroCAP, DNA detection, Centrifuge,Precipitate, LAMP, PCR.INTRODUCTIONDetection of amplified DNA is often based on measurement of turbidity, fluorescence (after staining with a detec- tion dye) or absorbance. Commercially available instruments for DNA quantitation can be broadly divided into two categories: UV instruments based on absorbance (such as spectrophotometers, e.g. Nanodrop or Nanophotometer) and instruments based on measurement of a fluorescent dye (such as plate readers). One bottleneck in quantifying amplified DNA in a nucleic acid amplification test (NAAT) reaction, based on absorbance measurement technique, is the bias introduced due to the presence of the isothermal amplification buffer, dNTPs and other reagents. Each reagent or buffer may have an absorbance density at around 260 nm, elevating the apparent concentration measured by the device compared to the actual value. Hence, for most quantitation based NAATs, it is important to include an extra DNA purification step, which may result in non-negligible loss of the amplified product and increases the cost of the purification kit. Measurements based on fluorescence mostly use fluorescent dyes that are potentially hazardous for handling. In addition, fluorescence based quantitation methods require time consuming labelling and washing steps.In this report, we describe a new method, termed microfluidic centrifugation assisted precipitation (microCAP), involving quantification and detection of DNA based on precipitation of nucleic acids. The basis of the method is formation of a visible precipitate when GelRed, a nucleic acid intercalacting dye commonly used in gel electropho- resis, is mixed with DNA and centrifuged. A visible precipitate is formed after just a few seconds of centrifugation and enables rapid detection of the presence of DNA in a sample. To the best of our knowledge, the visible precipitate formed as a product of centrifuging GelRed mixed with DNA has not been reported before. We showed that the DNA GelRed complex is dense enough compared to water to precipitate upon centrifugation. Further, we extended the μCAP method to the Lab-on-DVD platform1 to quantify the DNA concentration from images generated using the optical DVD reader instrument. The modified DVD player was able to image the precipitate formed up to a detection limit of 10 ng/μl of DNA. For calibration of the images, known quantities of a purified PCR product were used to identify the relationship between the amounts of DNA and precipitate formed. We applied the method to quantify an unknown quantity of LAMP amplicons from a LAMP assay for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. A sensitivity limit of 10 ng/μl of DNA was achieved, comparable with that of a Nanophotometer.18 The results demonstrated that the method is able to quantitatively detect the presence of DNA in a sample in a few seconds without any purification step.EXPERIMENTALThe Lab-on-DVD system was employed for spinning and imaging the precipitate product using a modified DVD drive, as mentioned in our previous report.1 We began by dispensing the sample in the design chamber, adding GelRed dye (at a concentration of 4000X in water) and centrifuging the mixture at 1200 rpm. Figure 1a and 1bshow schematics of the DNA sample precipitation process conducted in test tubes and the DVD platform, respec- tively. We used known amounts of a PCR product to calibrate the quantity of precipitate to the DNA concentration. We used a HIV genome amplified from 50 ng of plasmid pNL4.3 using the primers 0776F and 6231R.2 To evaluate the sensitivity of DNA detection of our system, we used the amplified products from a LAMP assay. The sensitivity of LAMP primers was tested on DNA from pNL4.3 (a HIV-1B genome containing plasmid). A 25X LAMP primer mix was prepared according to Curtis et al.,3 using the same template DNA sequence, set of primers and DNA polymerase. Eight concentrations (each being 5 μl volume) of the HIV-1B genome containing plasmid (pNL4.3) were tested, starting from 1 ng/!" serially diluted to 1 fg/!". Two negative controls were also prepared, one without DNA and primers and one without primers. The total reaction volume was increased to 30 μl (instead of 25 μl used in Curtis et al.3) by multiplying every component volume in the reaction by a factor of 1.2. Fabrication of the multi- layer microfluidic Disc followed the same procedure as described in our previous report.1 The Lab-on-DVD system was used to generate images of the precipitation zone. To quantify the amount of precipitate, an image processing script was written in MATLAB software (Mathworks, USA).RESULTS AND DISCUSSIONMicroCAP was found to be suitable for determining the presence of DNA in a sample, We carried out the LAMP assay in Eppendorf tubes in an oven set at 65°C. After 45 minutes, 3 μl of 10,000X GelRed in water was added to two tubes of 30 μl volume each, one having an unknown concentration of LAMP amplified DNA and the other one with no DNA template as a control. After centrifugation for approximately 5 seconds, a visible precipitate was formed in the tube containing amplified DNA, whereas no precipitate was formed in the control tube (Fig. 2a). 10 μl volume of DNA was inserted into a U shaped channel of the DVD alongwith 1 μl of 10,000X GelRed in water, which was the same ratio of DNA sample to Gelred as used in the test tube. An imageable precipitate was observed in the Lab on DVD custom imaging software (fig.2b).A Matlab script was used for image analysis in which an original image(fig.3a) was transformed into a binary image (fig.3b) by defining a threshold pixel value, exploiting the difference in intensity of the precipitate from its background. The entire area to the left of the threshold line in the histogram (Fig. 3c), i.e. from value 0 to the threshold value (normally 90), was summed to estimate the total area of the precipitate.For DNA quantification, known concentrations of a PCR product was used for calibration. The initial concentration of purified PCR product was 129 ng/μl, measured with a Nanophotometer (in triplicates) after purification with a GeneJet PCR purification kit. The purified PCR product was subsequently diluted serially several times and each diluted concentration was measured again with the Nanophotometer (in triplicate). The measurements were then repeated with the Lab-on-DVD method. Fig. 4a shows four images recorded at four known concentrations together with their binary threshold images. Fig. 4b shows the precipitation area calculated from the images plotted against the known DNA concentrations, showing a linear relationship. 10 ng/μl was the lowest concentration detectable in the DVD images.For quantification of unknown quantities of nucleic acids, we carried out the LAMP assay on HIV-1B genome containing plasmid DNA using serial dilutions (10-fold dilutions from 1 ng/μl to 0.1 fg/μl) to evaluate the limit of detection (Fig.5). Two negative controls were also prepared, one comprising primers and no DNA template and second, no DNA template and no primers.Fig. 6 shows the precipitation area plotted against the starting concentration of DNA template. It shows that the amplification in the LAMP assay is not linear for all the starting concentrations of DNA template. The error bars in the figure show the standard deviation for a particular concentration. For a LAMP assay, which fluctuates somewhat in its yield of amplified prod- ucts, we believe that this error range is acceptable.The precipitation area was converted to an actual yield of DNA products for each of the concentrations. This conversion was based on the linear empirical equation generated from the calibration curve presented earlier in Fig. 4b, given by:y= 9.61x – 4.05 (1) Here, y denotes the precipitation area in arbitrary units while x denotes the DNA concentration.CONCLUSIONWe demonstrated an extremely fast visual DNA quantification method (μCAP) that can be made quantifiable on a Lab-on-DVD platform. The approach was based on DNA forming a precipitate upon centrifugation when in contact with the GelRed dye. Results using HIV-1B genome containing plasmid DNA revealed a detection limit of 0.01 pg/μl or total amount of 0.1 pg of starting DNA template, which is an acceptable standard for resource limited settings. The limit of detection of DNA with the Lab-on-DVD platform was found to be 10 ng/μl, which is almost comparable to the detection limits reported by commercially available instruments, such as the Nanophotometer. However, the μCAP method offers a distinct advantage over other state-of-the-art techniques as it does not require additional purification of the DNA. We believe the μCAP technique combined with the Lab-on-DVD platform provides a simple and low cost technology that can fulfil the need for a point-of-care device for DNA quantification.REFERENCES[1] H. Ramachandraiah, M. Amasia, J. Cole, P. Sheard, S. Pickhaver, C. Walker, V. Wirta, P. Lexow, R. Lione and A. Russom, "Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics."Lab. Chip, 2013, 13, 1578–1585.[2] S. Grossmann, P. Nowak, and U. Neogi, “ Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epide
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| 9. |
- Banerjee, Indradumna, 1986-, et al.
(författare)
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Microfluidic Centrifugation Assisted Precipitation based DNA Quantification
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often require time consuming purification, washing or labeling step. Here, we report a novel microfluidic centrifugation assisted precipitation (uCAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, that can be quantified, when an intercalating dye (GelRed) is added to DNA sample and centrifuged for few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng/ul. Furthermore, using an integrated Lab-on-DVD platform we recently developed, the detection limit was lowered to 10 ng/ul, which is comparable to current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. The simple DNA quantification system could facilitate advanced molecular diagnosis at point of care.
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| 10. |
- Banerjee, Indradumna, et al.
(författare)
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Microfluidic centrifugation assisted precipitation based DNA quantification
- 2019
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 19:9, s. 1657-1664
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Tidskriftsartikel (refereegranskat)abstract
- Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.
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