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Papers by Brian Grimberg

Additional file 1: Table S1. PvDBP and PvAMA-1 primer sequences.

The line bisecting the graph identifies where points would fall if identical results were observe... more The line bisecting the graph identifies where points would fall if identical results were observed between the two diagnostic methods (> 1).<b>Copyright information:</b>Taken from "Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for transmission"http://www.malariajournal.com/content/7/1/66Malaria Journal 2008;7():66-66.Published online 25 Apr 2008PMCID:PMC2373791.

This article cites 24 articles, 16 of which can be accessed free

IEEE Transactions on Magnetics, 2021

We have designed, developed, and evaluated an innovative portable magneto-optical detector (MOD) ... more We have designed, developed, and evaluated an innovative portable magneto-optical detector (MOD) in which a light beam with variable polarization passes through a fluid sample immersed in a variable magnetic field. The light intensity is measured downstream along the forward scattering direction. The field is turned on and off through the in-and-out motion of nearby permanent magnets. As a result, for sufficiently, magnetically, and optically anisotropic samples, the optical absorption is sensitive to changes in the light polarization. Both detection and characterization applications are, therefore, available. For instance, both the degree of malaria infection and hemozoin crystalline properties can be measured and studied, respectively. We present experimental results for synthetic hemozoin and describe them in terms of the basic physics and chemistry underlying the correlations of the directions of the external magnetic field and the light beam polarization. We connect this work t...

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antib... more Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of ...

Vaccines, Apr 7, 2020

Zika virus (ZIKV) is a significant public health concern due to the pathogen's ability to be tran... more Zika virus (ZIKV) is a significant public health concern due to the pathogen's ability to be transmitted by either mosquito bite or sexual transmission, allowing spread to occur throughout the world. The potential consequences of ZIKV infection to human health, specifically neonates, necessitates the development of a safe and effective Zika virus vaccine. Here, we developed an intranasal Zika vaccine based upon the replication-deficient human adenovirus serotype 5 (hAd5) expressing ZIKV pre-membrane and envelope protein (hAd5-ZKV). The hAd5-ZKV vaccine is able to induce both cell-mediated and humoral immune responses to ZIKV epitopes. Importantly, this vaccine generated CD8 + T cells specific for a dominant ZIKV T cell epitope and is shown to be protective against a ZIKV challenge by using a pre-clinical model of ZIKV disease. We also demonstrate that the vaccine expresses pre-membrane and envelope protein in a confirmation recognized by ZIKV experienced individuals. Our studies demonstrate that this adenovirus-based vaccine expressing ZIKV proteins is immunogenic and protective in mice, and it encodes ZIKV proteins in a conformation recognized by the human antibody repertoire.

Malaria journal, Jan 3, 2018

Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing bec... more Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite's haemozoin to inhibit parasite viability. Paramagnetic haemozoin crystals, a byproduct of the parasite's haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most...

Malaria Journal, 2016

Background: Conventional malaria parasite detection methods, such as rapid diagnostic tests (RDT)... more Background: Conventional malaria parasite detection methods, such as rapid diagnostic tests (RDT) and light microscopy (LM), are not sensitive enough to detect low level parasites and identification of gametocytes in the peripheral blood. A modified and sensitive laboratory prototype, Magnetic Deposition Microscopy (MDM) was developed to increase the detection of sub-microscopic parasitaemia and estimation of gametocytes density in asymptomatic school children. Methods: Blood samples were collected from 303 asymptomatic school children from seven villages in Bagamoyo district in Tanzania. Participants were screened for presence of malaria parasites in the field using RDT and MDM whereas further examination of malaria parasites was done in the laboratory by LM. LM and MDM readings were used to calculate densities and estimate prevalence of asexual and sexual stages of the parasite. Results: Plasmodium falciparum parasites (asexual and sexual stages) were detected in 23 (7.6 %), 52 (17.2 %), and 59 (19.5 %) out of 303 samples by LM, RDT and MDM respectively. Gametocytes were detected in 4 (1.3 %) and 12 (4.0 %) out of the same numbers of samples by LM, and MDM, respectively. Likewise, in vitro results conducted on two laboratory strains of P. falciparum, 3D7 and NF54 to assess MDM sensitivity on gametocytes detection and its application on concentrating gametocytes indicated that gametocytes were enriched by MDM by 10-fold higher than LM. Late stages of the parasite strains, 3D7 and NF54 were enriched by MDM by a factor of 20.5 and 35.6, respectively. MDM was more specific than LM and RDT by 87.5 % (95 %, CI 71.2-89.6 %) and 89.0 % (95 % CI 82.9-91.4) respectively. It was also found that MDM sensitivity was 62.5 % (95 % CI 49.5-71.8) when compared with RDT while with LM was 36.5 % (95 % CI 32.2-60.5). Conclusions: These findings provide strong evidence that MDM enhanced detection of sub-microscopic P. falciparum infections and estimation of gametocyte density compared to current malaria diagnostic tools. In addition, MDM is superior to LM in detecting sub-microscopic gametocytaemia. Therefore, MDM is a potential tool for low-level parasitaemia identification and quantification with possible application in malaria transmission research.

PloS one, 2015

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence v... more Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies ex...

Infectious Diseases in Clinical Practice, 1998

ABSTRACT

Infectious Diseases in Clinical Practice - INFECT DIS CLIN PRAC, 1998

ABSTRACT

Journal of Immunological Methods, 2011

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using d... more Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.

Experimental Parasitology, 2012

h i g h l i g h t s " This is a simple and inexpensive method to increase the reticulocyte count ... more h i g h l i g h t s " This is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. " By exposing cord blood to hypotonic saline for 5 min, normocytes are lysed. " This method increases reticulocytemia by over 3-fold. " The recovered blood has intact hemoglobin and is still able to support malaria parasites growth.

Evolution, 2005

Some hypotheses for the evolution of sex focus on adaptation to changing or heterogeneous environ... more Some hypotheses for the evolution of sex focus on adaptation to changing or heterogeneous environments, but these hypotheses have rarely been tested. We tested for advantages of sex and of increased mutation rates in yeast strains in two contrasting environments: a standard and relatively homogeneous laboratory environment of minimal medium in test tubes, and the variable environment of a mouse brain experienced by pathogenic strains. Evolving populations were founded as equal mixtures of sexual and obligately asexual genotypes. In the sexuals, cycles of sporulation, meiosis, and mating were induced approximately every 50 mitotic generations, with the asexuals undergoing sporulation but not ploidy cycles or recombination. In both environments, replicate negative control populations established with the same pair of strains were propagated with neither mating nor meiosis. In test tubes with no sex induced, sexuals were fixed in all five replicates within 250 mitotic generations, whereas in mice with no sex induced, asexuals were fixed in all four replicates by 170 generations. Inducing sex altered these outcomes in opposite directions in test tubes and mice, decreasing the fixation frequencies of sexuals in test tubes but increasing them in mice. These contrasts with asexual controls suggest an advantage for sex in mice but not in test tubes, although there was no difference between test tubes and mice in the numbers of populations fixed-for sexuals. In analogous experiments testing for an advantage of increased mutation rates, wild-type genotypes became fixed at the expense of mutators in every replicate of both test tube and mouse populations, indicating a disadvantage for mutators in both environments. Increased rates of point mutation do not appear to accelerate adaptation.

Journal of immunological methods, 2011

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using d... more Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.

Antimicrobial Agents and Chemotherapy, 2000

High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene ... more High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the re...

Pharmaceuticals, 2011

The number of available and effective antimalarial drugs is quickly dwindling. This is mainly bec... more The number of available and effective antimalarial drugs is quickly dwindling. This is mainly because a number of drug resistance-associated mutations in malaria parasite genes, such as crt, mdr1, dhfr/dhps, and others, have led to widespread resistance to all known classes of antimalarial compounds. Unfortunately, malaria parasites have started to exhibit some level of resistance in Southeast Asia even to the most recently introduced class of drugs, artemisinins. While there is much need, the antimalarial drug development pipeline remains woefully thin, with little chemical diversity, and there is currently no alternative to the precious artemisinins. It is difficult to predict where the next generation of antimalarial drugs will come from; however, there are six major approaches: (i) re-optimizing the use of existing antimalarials by either replacement/rotation or combination approach; (ii) repurposing drugs that are currently used to treat other infections or diseases; (iii) chemically modifying existing antimalarial compounds; (iv) exploring natural sources; (v) large-scale screening of diverse chemical libraries; and (vi) through parasite genome-based ("targeted") discoveries. When any newly discovered effective antimalarial treatment is used by the populus, we must maintain constant vigilance for both parasite-specific and human-related factors that are likely to hamper its success. This article is neither comprehensive nor conclusive. Our purpose is to provide an overview of antimalarial drug resistance, associated parasite genetic factors (1. Introduction; 2. Emergence of artemisinin resistance in P. falciparum), and the antimalarial drug development pipeline (3. Overview of the global pipeline of antimalarial drugs), and highlight some examples of the aforementioned approaches to future antimalarial treatment. These approaches can be categorized into "short term" (4. Feasible options for now) and "long term" (5. Next generation of antimalarial treatment-Approaches and candidates). However, these two categories are interrelated, and the

Gene, 1999

The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned an... more The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned and sequenced from Candida albicans by homology with the Saccharomyces cerevisiae ERG3. The ERG3 ORF contained 1158 bp and encoded 386 deduced amino acids. The ...

Additional file 1: Table S1. PvDBP and PvAMA-1 primer sequences.

The line bisecting the graph identifies where points would fall if identical results were observe... more The line bisecting the graph identifies where points would fall if identical results were observed between the two diagnostic methods (> 1).<b>Copyright information:</b>Taken from "Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for transmission"http://www.malariajournal.com/content/7/1/66Malaria Journal 2008;7():66-66.Published online 25 Apr 2008PMCID:PMC2373791.

This article cites 24 articles, 16 of which can be accessed free

IEEE Transactions on Magnetics, 2021

We have designed, developed, and evaluated an innovative portable magneto-optical detector (MOD) ... more We have designed, developed, and evaluated an innovative portable magneto-optical detector (MOD) in which a light beam with variable polarization passes through a fluid sample immersed in a variable magnetic field. The light intensity is measured downstream along the forward scattering direction. The field is turned on and off through the in-and-out motion of nearby permanent magnets. As a result, for sufficiently, magnetically, and optically anisotropic samples, the optical absorption is sensitive to changes in the light polarization. Both detection and characterization applications are, therefore, available. For instance, both the degree of malaria infection and hemozoin crystalline properties can be measured and studied, respectively. We present experimental results for synthetic hemozoin and describe them in terms of the basic physics and chemistry underlying the correlations of the directions of the external magnetic field and the light beam polarization. We connect this work t...

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antib... more Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of ...

Vaccines, Apr 7, 2020

Zika virus (ZIKV) is a significant public health concern due to the pathogen's ability to be tran... more Zika virus (ZIKV) is a significant public health concern due to the pathogen's ability to be transmitted by either mosquito bite or sexual transmission, allowing spread to occur throughout the world. The potential consequences of ZIKV infection to human health, specifically neonates, necessitates the development of a safe and effective Zika virus vaccine. Here, we developed an intranasal Zika vaccine based upon the replication-deficient human adenovirus serotype 5 (hAd5) expressing ZIKV pre-membrane and envelope protein (hAd5-ZKV). The hAd5-ZKV vaccine is able to induce both cell-mediated and humoral immune responses to ZIKV epitopes. Importantly, this vaccine generated CD8 + T cells specific for a dominant ZIKV T cell epitope and is shown to be protective against a ZIKV challenge by using a pre-clinical model of ZIKV disease. We also demonstrate that the vaccine expresses pre-membrane and envelope protein in a confirmation recognized by ZIKV experienced individuals. Our studies demonstrate that this adenovirus-based vaccine expressing ZIKV proteins is immunogenic and protective in mice, and it encodes ZIKV proteins in a conformation recognized by the human antibody repertoire.

Malaria journal, Jan 3, 2018

Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing bec... more Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite's haemozoin to inhibit parasite viability. Paramagnetic haemozoin crystals, a byproduct of the parasite's haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most...

Malaria Journal, 2016

Background: Conventional malaria parasite detection methods, such as rapid diagnostic tests (RDT)... more Background: Conventional malaria parasite detection methods, such as rapid diagnostic tests (RDT) and light microscopy (LM), are not sensitive enough to detect low level parasites and identification of gametocytes in the peripheral blood. A modified and sensitive laboratory prototype, Magnetic Deposition Microscopy (MDM) was developed to increase the detection of sub-microscopic parasitaemia and estimation of gametocytes density in asymptomatic school children. Methods: Blood samples were collected from 303 asymptomatic school children from seven villages in Bagamoyo district in Tanzania. Participants were screened for presence of malaria parasites in the field using RDT and MDM whereas further examination of malaria parasites was done in the laboratory by LM. LM and MDM readings were used to calculate densities and estimate prevalence of asexual and sexual stages of the parasite. Results: Plasmodium falciparum parasites (asexual and sexual stages) were detected in 23 (7.6 %), 52 (17.2 %), and 59 (19.5 %) out of 303 samples by LM, RDT and MDM respectively. Gametocytes were detected in 4 (1.3 %) and 12 (4.0 %) out of the same numbers of samples by LM, and MDM, respectively. Likewise, in vitro results conducted on two laboratory strains of P. falciparum, 3D7 and NF54 to assess MDM sensitivity on gametocytes detection and its application on concentrating gametocytes indicated that gametocytes were enriched by MDM by 10-fold higher than LM. Late stages of the parasite strains, 3D7 and NF54 were enriched by MDM by a factor of 20.5 and 35.6, respectively. MDM was more specific than LM and RDT by 87.5 % (95 %, CI 71.2-89.6 %) and 89.0 % (95 % CI 82.9-91.4) respectively. It was also found that MDM sensitivity was 62.5 % (95 % CI 49.5-71.8) when compared with RDT while with LM was 36.5 % (95 % CI 32.2-60.5). Conclusions: These findings provide strong evidence that MDM enhanced detection of sub-microscopic P. falciparum infections and estimation of gametocyte density compared to current malaria diagnostic tools. In addition, MDM is superior to LM in detecting sub-microscopic gametocytaemia. Therefore, MDM is a potential tool for low-level parasitaemia identification and quantification with possible application in malaria transmission research.

PloS one, 2015

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence v... more Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies ex...

Infectious Diseases in Clinical Practice, 1998

ABSTRACT

Infectious Diseases in Clinical Practice - INFECT DIS CLIN PRAC, 1998

ABSTRACT

Journal of Immunological Methods, 2011

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using d... more Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.

Experimental Parasitology, 2012

h i g h l i g h t s " This is a simple and inexpensive method to increase the reticulocyte count ... more h i g h l i g h t s " This is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. " By exposing cord blood to hypotonic saline for 5 min, normocytes are lysed. " This method increases reticulocytemia by over 3-fold. " The recovered blood has intact hemoglobin and is still able to support malaria parasites growth.

Evolution, 2005

Some hypotheses for the evolution of sex focus on adaptation to changing or heterogeneous environ... more Some hypotheses for the evolution of sex focus on adaptation to changing or heterogeneous environments, but these hypotheses have rarely been tested. We tested for advantages of sex and of increased mutation rates in yeast strains in two contrasting environments: a standard and relatively homogeneous laboratory environment of minimal medium in test tubes, and the variable environment of a mouse brain experienced by pathogenic strains. Evolving populations were founded as equal mixtures of sexual and obligately asexual genotypes. In the sexuals, cycles of sporulation, meiosis, and mating were induced approximately every 50 mitotic generations, with the asexuals undergoing sporulation but not ploidy cycles or recombination. In both environments, replicate negative control populations established with the same pair of strains were propagated with neither mating nor meiosis. In test tubes with no sex induced, sexuals were fixed in all five replicates within 250 mitotic generations, whereas in mice with no sex induced, asexuals were fixed in all four replicates by 170 generations. Inducing sex altered these outcomes in opposite directions in test tubes and mice, decreasing the fixation frequencies of sexuals in test tubes but increasing them in mice. These contrasts with asexual controls suggest an advantage for sex in mice but not in test tubes, although there was no difference between test tubes and mice in the numbers of populations fixed-for sexuals. In analogous experiments testing for an advantage of increased mutation rates, wild-type genotypes became fixed at the expense of mutators in every replicate of both test tube and mouse populations, indicating a disadvantage for mutators in both environments. Increased rates of point mutation do not appear to accelerate adaptation.

Journal of immunological methods, 2011

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using d... more Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.

Antimicrobial Agents and Chemotherapy, 2000

High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene ... more High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the re...

Pharmaceuticals, 2011

The number of available and effective antimalarial drugs is quickly dwindling. This is mainly bec... more The number of available and effective antimalarial drugs is quickly dwindling. This is mainly because a number of drug resistance-associated mutations in malaria parasite genes, such as crt, mdr1, dhfr/dhps, and others, have led to widespread resistance to all known classes of antimalarial compounds. Unfortunately, malaria parasites have started to exhibit some level of resistance in Southeast Asia even to the most recently introduced class of drugs, artemisinins. While there is much need, the antimalarial drug development pipeline remains woefully thin, with little chemical diversity, and there is currently no alternative to the precious artemisinins. It is difficult to predict where the next generation of antimalarial drugs will come from; however, there are six major approaches: (i) re-optimizing the use of existing antimalarials by either replacement/rotation or combination approach; (ii) repurposing drugs that are currently used to treat other infections or diseases; (iii) chemically modifying existing antimalarial compounds; (iv) exploring natural sources; (v) large-scale screening of diverse chemical libraries; and (vi) through parasite genome-based ("targeted") discoveries. When any newly discovered effective antimalarial treatment is used by the populus, we must maintain constant vigilance for both parasite-specific and human-related factors that are likely to hamper its success. This article is neither comprehensive nor conclusive. Our purpose is to provide an overview of antimalarial drug resistance, associated parasite genetic factors (1. Introduction; 2. Emergence of artemisinin resistance in P. falciparum), and the antimalarial drug development pipeline (3. Overview of the global pipeline of antimalarial drugs), and highlight some examples of the aforementioned approaches to future antimalarial treatment. These approaches can be categorized into "short term" (4. Feasible options for now) and "long term" (5. Next generation of antimalarial treatment-Approaches and candidates). However, these two categories are interrelated, and the

Gene, 1999

The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned an... more The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned and sequenced from Candida albicans by homology with the Saccharomyces cerevisiae ERG3. The ERG3 ORF contained 1158 bp and encoded 386 deduced amino acids. The ...