The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large ... more The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alaninelysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.
Working with cultured dermal fibroblasts derived from control individuals and patients with syste... more Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-δ (PKC-δ) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-δ, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides-804 to-675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-δ expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-δ participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-δ inhibitors could suppress fibrosis in this disease.
Over the last 10 years there have been only a handful of publications dealing with the oral virom... more Over the last 10 years there have been only a handful of publications dealing with the oral virome, which is in contrast to the oral microbiome, an area that has seen considerable interest. Here, we survey viral infections in general and then focus on those viruses that are found in and/or are transmitted via the oral cavity; norovirus, rabies, human papillomavirus, Epstein-Barr virus, herpes simplex viruses, hepatitis C virus, and HIV. Increasingly, viral infections have been diagnosed using an oral sample (e.g. saliva mucosal transudate or an oral swab) instead of blood or urine. The results of two studies using a rapid and semi-quantitative lateral flow assay format demonstrating the correlation of HIV anti-IgG/sIgA detection with saliva and serum samples are presented. When immediate detection of infection is important, point-of-care devices that obtain a non-invasive sample from the oral cavity can be used to provide a first line diagnosis to assist in determining appropriate c...
During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise... more During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located ف 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.
Limited information is available about the effects of HIV and subsequent antiretroviral treatment... more Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans , and Candida , in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4 + T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification ...
Understanding of the human microbiome continues to grow rapidly; however, reports on changes in t... more Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of "crosstalk" between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against H...
The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and... more The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Large segments of the sequence are conserved but there are also considerable variations which range in extent from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and largescale deletions and insertions. In general, smaller differences are found among mammalian tropoelastins and greater ones between chicken and mammalian tropoelastins. Although only a single elastin gene is found per haploid genome, the primary transcript is subject to considerable alternative splicing, resulting in multiple tropoelastin isoforms. Functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the gene. The introns of the human gene are rich in Alu repetitive sequences, which may be the site of recombinational events, and there are also several dinucleotide repeats, which may exhibit polymorphism and, therefore, be effective genetic markers. The 5' flanking region is G+C rich and contains potential binding sites for numerous modulating factors, but no TATA box or functional CAAT box. The basic promoter is contained within a 136 bp segment and transcription is initiated at multiple sites. These findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.
We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RN... more We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user i...
Systemic sclerosis (SSc) is a fibroproliferative autoimmune disease characterized by the excessiv... more Systemic sclerosis (SSc) is a fibroproliferative autoimmune disease characterized by the excessive production and deposition of collagen in skin and internal organs. Acting through specific transmembrane receptors, transforming growth factor-? (TGF-?) is known to play a crucial role in the development of tissue fibrosis. The authors had previously identified several signaling molecules other than Smad proteins including protein kinase C-d (PKC-d) as candidates downstream of TGF-? receptors. The aim of the present study was to examine the possible role of PKC-d in the upregulation collagen gene expression in SSc dermal fibroblasts. Significant findings Compared to normal fibroblasts, SSc dermal fibroblasts had increased type I and III collagen mRNA and protein as well as upregulation of PKC-d protein. Rottlerin, a specific inhibitor of PKC-d, inhibited collagen gene expression in both normal and SSc fibroblasts. Moreover, this study identified a 129-bp promoter region of the type I collagen gene encompassing nucleotides-804 to-675 which was responsive to the transcriptional inhibition by rottlerin and dominant-negative PKC-d expression. Comments These results indicate that PKC-d is a key molecule in the upregulation of type I collagen expression. However, the number of samples obtained from normal and SSc skins was too small to show a significant difference in the amounts of collagen and PKC-d expressed. In addition, most of the results presented in this paper relied on a single "selective" inhibitor of PKC-d, rottlerin. The following points should be addressed in future studies: 1) identification of transcription factors downstream of PKC-d that interact with a promoter segment encompassing nucleotides-804-675; 2) comparison of PKC-d activity between SSc and normal fibroblasts; 3) possible differences in collagen and PKC-d expression among subgroups of SSc (e.g., diffuse cutaneous SSc and limited cutaneous SSc). Methods ELISA, collagenase digestion assay, northern blotting, in vitro nuclear transcription assay, chloramphenicol acetyl transferase gene assay, high-resolution fluorescence immunomicroscopy, western blotting Additional information
Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae famil... more Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae family. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood greatly simplifying the assay. Loop-mediated Isothermal Amplification (LAMP) is a rapid assay that detects nucleic acids, including ZIKV RNA. Aim: The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. Method: A total of 131 samples (68 saliva and 63 urine samples) from 69 subjects in the acute phase of ZIKV infection, and confirmed positive for ZIKV by blood analysis through real time-PCR, were collected and analyzed by Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP). Results: From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 min, and from the 63 urine samples, 25 (39.7%) were positive with the average Tp of 15.8 min. Saliva detected more samples (p = 0.0042) and had faster Tp (p = 0.0176) as compared with urine. Conclusion: Saliva proved to be a feasible alternative to diagnose ZIKV infection during the acute phase by LAMP.
Journal of visualized experiments : JoVE, Jan 12, 2017
High-density peptide microarrays allow screening of more than six thousand peptides on a single s... more High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.
In recent years, there have been increasing numbers of infectious disease outbreaks that spread r... more In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 s...
The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large ... more The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alaninelysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.
Working with cultured dermal fibroblasts derived from control individuals and patients with syste... more Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-δ (PKC-δ) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-δ, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides-804 to-675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-δ expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-δ participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-δ inhibitors could suppress fibrosis in this disease.
Over the last 10 years there have been only a handful of publications dealing with the oral virom... more Over the last 10 years there have been only a handful of publications dealing with the oral virome, which is in contrast to the oral microbiome, an area that has seen considerable interest. Here, we survey viral infections in general and then focus on those viruses that are found in and/or are transmitted via the oral cavity; norovirus, rabies, human papillomavirus, Epstein-Barr virus, herpes simplex viruses, hepatitis C virus, and HIV. Increasingly, viral infections have been diagnosed using an oral sample (e.g. saliva mucosal transudate or an oral swab) instead of blood or urine. The results of two studies using a rapid and semi-quantitative lateral flow assay format demonstrating the correlation of HIV anti-IgG/sIgA detection with saliva and serum samples are presented. When immediate detection of infection is important, point-of-care devices that obtain a non-invasive sample from the oral cavity can be used to provide a first line diagnosis to assist in determining appropriate c...
During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise... more During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located ف 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.
Limited information is available about the effects of HIV and subsequent antiretroviral treatment... more Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans , and Candida , in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4 + T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification ...
Understanding of the human microbiome continues to grow rapidly; however, reports on changes in t... more Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of "crosstalk" between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against H...
The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and... more The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Large segments of the sequence are conserved but there are also considerable variations which range in extent from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and largescale deletions and insertions. In general, smaller differences are found among mammalian tropoelastins and greater ones between chicken and mammalian tropoelastins. Although only a single elastin gene is found per haploid genome, the primary transcript is subject to considerable alternative splicing, resulting in multiple tropoelastin isoforms. Functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the gene. The introns of the human gene are rich in Alu repetitive sequences, which may be the site of recombinational events, and there are also several dinucleotide repeats, which may exhibit polymorphism and, therefore, be effective genetic markers. The 5' flanking region is G+C rich and contains potential binding sites for numerous modulating factors, but no TATA box or functional CAAT box. The basic promoter is contained within a 136 bp segment and transcription is initiated at multiple sites. These findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.
We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RN... more We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user i...
Systemic sclerosis (SSc) is a fibroproliferative autoimmune disease characterized by the excessiv... more Systemic sclerosis (SSc) is a fibroproliferative autoimmune disease characterized by the excessive production and deposition of collagen in skin and internal organs. Acting through specific transmembrane receptors, transforming growth factor-? (TGF-?) is known to play a crucial role in the development of tissue fibrosis. The authors had previously identified several signaling molecules other than Smad proteins including protein kinase C-d (PKC-d) as candidates downstream of TGF-? receptors. The aim of the present study was to examine the possible role of PKC-d in the upregulation collagen gene expression in SSc dermal fibroblasts. Significant findings Compared to normal fibroblasts, SSc dermal fibroblasts had increased type I and III collagen mRNA and protein as well as upregulation of PKC-d protein. Rottlerin, a specific inhibitor of PKC-d, inhibited collagen gene expression in both normal and SSc fibroblasts. Moreover, this study identified a 129-bp promoter region of the type I collagen gene encompassing nucleotides-804 to-675 which was responsive to the transcriptional inhibition by rottlerin and dominant-negative PKC-d expression. Comments These results indicate that PKC-d is a key molecule in the upregulation of type I collagen expression. However, the number of samples obtained from normal and SSc skins was too small to show a significant difference in the amounts of collagen and PKC-d expressed. In addition, most of the results presented in this paper relied on a single "selective" inhibitor of PKC-d, rottlerin. The following points should be addressed in future studies: 1) identification of transcription factors downstream of PKC-d that interact with a promoter segment encompassing nucleotides-804-675; 2) comparison of PKC-d activity between SSc and normal fibroblasts; 3) possible differences in collagen and PKC-d expression among subgroups of SSc (e.g., diffuse cutaneous SSc and limited cutaneous SSc). Methods ELISA, collagenase digestion assay, northern blotting, in vitro nuclear transcription assay, chloramphenicol acetyl transferase gene assay, high-resolution fluorescence immunomicroscopy, western blotting Additional information
Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae famil... more Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae family. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood greatly simplifying the assay. Loop-mediated Isothermal Amplification (LAMP) is a rapid assay that detects nucleic acids, including ZIKV RNA. Aim: The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. Method: A total of 131 samples (68 saliva and 63 urine samples) from 69 subjects in the acute phase of ZIKV infection, and confirmed positive for ZIKV by blood analysis through real time-PCR, were collected and analyzed by Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP). Results: From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 min, and from the 63 urine samples, 25 (39.7%) were positive with the average Tp of 15.8 min. Saliva detected more samples (p = 0.0042) and had faster Tp (p = 0.0176) as compared with urine. Conclusion: Saliva proved to be a feasible alternative to diagnose ZIKV infection during the acute phase by LAMP.
Journal of visualized experiments : JoVE, Jan 12, 2017
High-density peptide microarrays allow screening of more than six thousand peptides on a single s... more High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.
In recent years, there have been increasing numbers of infectious disease outbreaks that spread r... more In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 s...
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Papers by William Abrams