The World Health Organization recommends providing calcium supplements to pregnant women through ... more The World Health Organization recommends providing calcium supplements to pregnant women through antenatal care (ANC) along with iron-folic acid (IFA) supplements to reduce maternal mortality. Alth...
Alcoholism: Clinical and Experimental Research, 2022
BACKGROUND There is growing evidence that the gut microbiota can be shaped by early-life experien... more BACKGROUND There is growing evidence that the gut microbiota can be shaped by early-life experiences/exposures, with long-term consequences for brain, behavior, and health. Changes in the gut microbiota have also been identified in neurodevelopmental disorders including Autism Spectrum Disorder and schizophrenia. By contrast, no studies to date have investigated whether the gut microbiota is altered in individuals with Fetal Alcohol Spectrum Disorder (FASD), the neurodevelopmental disorder that results from prenatal alcohol exposure (PAE). The current study was designed to begin to address this gap. METHODS To assess the impact of PAE on the fecal microbiota we used a rodent model in which pregnant Sprague-Dawley rats were provided with an ethanol-containing diet or a control diet throughout gestation. Fecal samples were then collected from adult male and female animals and 16s rRNA sequencing performed. RESULTS Overall, PAE rats showed increased richness of bacterial species, with community structure investigations demonstrating distinct clustering by prenatal treatment. In addition, prenatal treatment and sex-specific alterations were observed for many specific microbes. For example, in males, Bacteroides, and Bifidobacterium, and in females, Faecalitalea and Proteus, were differentially abundant between PAE control rats, respectively. CONCLUSIONS Taken together, these results show for the first time that PAE has a long-lasting and sex-specific impact on the fecal microbiota with further research needed such the microbiota could be considered in the development of new interventions in FASD.
The expression analysis of recombinant proteins is a challenging step in any high-throughput prot... more The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.
Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply... more Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.
To rapidly find "best-in-class" antibody therapeutics, it has become essential to devel... more To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, w...
THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for s... more THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB ant...
Transient gene expression in mammalian cells is an efficient process for producing recombinant pr... more Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by ∼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and ...
Transient gene expression in mammalian cells is an efficient process to produce recombinant prote... more Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR-26a-5p and miR-337-5p, that increased human IgG1 (huIgG1) yields by 50% and 25%, respectively. The titer increase was achievable by expressing miR-26a-5p from oligonucleotides or a plasmid. Furthermore, combining miR-26a-5p with valproic acid (VPA) treatment doubled huIgG1 titers. Assessment of miR-26a-5p and VPA treatment across a panel of 32 human and murine antibodies demonstrates that the level of yield enhancement was molecule-dependent, with most exhibiting a range of 50 - 100% titer increase. These findings exemplify that combining genetic and chemical manipulation...
For some antibodies intended for use as human therapeutics, reduced effector function is desired ... more For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vi...
Objectives Mild cognitive impairment (MCI) is the key symptom of early dementia. Except cognitive... more Objectives Mild cognitive impairment (MCI) is the key symptom of early dementia. Except cognitive training, there is early evidence showing that physical exercise could enhance brain function in elderly with MCI. This study evaluated how far an 8-week exercise program, combining work-out and yoga, could effectively improve the brain function of the elderly with mild cognitive impairment. Methods Elderly persons, aged over 60, with mild cognitive impairment and no physical disabilities are assigned to three groups (N = 90, n = 30 in each group): 1) YO Gym work-out Program, 2) YO Gym plus "Smart Eight-Style" and "Brain Yoga", 3) Comparison Group. The two exercise groups participated in the activities for at least 3 times per week, 40 minutes each session. The cognitive and physical outcomes were assessed using the Montreal Cognitive Assessment (MoCA) and the Senior Fitness Test, and blinded assessments were conducted by research staff. Results The exercise groups h...
The World Health Organization recommends providing calcium supplements to pregnant women through ... more The World Health Organization recommends providing calcium supplements to pregnant women through antenatal care (ANC) along with iron-folic acid (IFA) supplements to reduce maternal mortality. Alth...
Alcoholism: Clinical and Experimental Research, 2022
BACKGROUND There is growing evidence that the gut microbiota can be shaped by early-life experien... more BACKGROUND There is growing evidence that the gut microbiota can be shaped by early-life experiences/exposures, with long-term consequences for brain, behavior, and health. Changes in the gut microbiota have also been identified in neurodevelopmental disorders including Autism Spectrum Disorder and schizophrenia. By contrast, no studies to date have investigated whether the gut microbiota is altered in individuals with Fetal Alcohol Spectrum Disorder (FASD), the neurodevelopmental disorder that results from prenatal alcohol exposure (PAE). The current study was designed to begin to address this gap. METHODS To assess the impact of PAE on the fecal microbiota we used a rodent model in which pregnant Sprague-Dawley rats were provided with an ethanol-containing diet or a control diet throughout gestation. Fecal samples were then collected from adult male and female animals and 16s rRNA sequencing performed. RESULTS Overall, PAE rats showed increased richness of bacterial species, with community structure investigations demonstrating distinct clustering by prenatal treatment. In addition, prenatal treatment and sex-specific alterations were observed for many specific microbes. For example, in males, Bacteroides, and Bifidobacterium, and in females, Faecalitalea and Proteus, were differentially abundant between PAE control rats, respectively. CONCLUSIONS Taken together, these results show for the first time that PAE has a long-lasting and sex-specific impact on the fecal microbiota with further research needed such the microbiota could be considered in the development of new interventions in FASD.
The expression analysis of recombinant proteins is a challenging step in any high-throughput prot... more The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.
Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply... more Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.
To rapidly find "best-in-class" antibody therapeutics, it has become essential to devel... more To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, w...
THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for s... more THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB ant...
Transient gene expression in mammalian cells is an efficient process for producing recombinant pr... more Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by ∼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and ...
Transient gene expression in mammalian cells is an efficient process to produce recombinant prote... more Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR-26a-5p and miR-337-5p, that increased human IgG1 (huIgG1) yields by 50% and 25%, respectively. The titer increase was achievable by expressing miR-26a-5p from oligonucleotides or a plasmid. Furthermore, combining miR-26a-5p with valproic acid (VPA) treatment doubled huIgG1 titers. Assessment of miR-26a-5p and VPA treatment across a panel of 32 human and murine antibodies demonstrates that the level of yield enhancement was molecule-dependent, with most exhibiting a range of 50 - 100% titer increase. These findings exemplify that combining genetic and chemical manipulation...
For some antibodies intended for use as human therapeutics, reduced effector function is desired ... more For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vi...
Objectives Mild cognitive impairment (MCI) is the key symptom of early dementia. Except cognitive... more Objectives Mild cognitive impairment (MCI) is the key symptom of early dementia. Except cognitive training, there is early evidence showing that physical exercise could enhance brain function in elderly with MCI. This study evaluated how far an 8-week exercise program, combining work-out and yoga, could effectively improve the brain function of the elderly with mild cognitive impairment. Methods Elderly persons, aged over 60, with mild cognitive impairment and no physical disabilities are assigned to three groups (N = 90, n = 30 in each group): 1) YO Gym work-out Program, 2) YO Gym plus "Smart Eight-Style" and "Brain Yoga", 3) Comparison Group. The two exercise groups participated in the activities for at least 3 times per week, 40 minutes each session. The cognitive and physical outcomes were assessed using the Montreal Cognitive Assessment (MoCA) and the Senior Fitness Test, and blinded assessments were conducted by research staff. Results The exercise groups h...
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