<p>The MATa XXX-GFP::HIS3 library was grown over night on YPD agar plates and then transfer... more <p>The MATa XXX-GFP::HIS3 library was grown over night on YPD agar plates and then transferred into 50 μl SC-His with 2% glucose in 384-well polystyrene flat-bottom plates, as described in Materials and Methods. Following transfer to water, over 5000 GFP-tagged yeast proteins were visualized during survival in water (after 1 day and 31 days). (A) Proteins that became repressed at an early stage and lost their fluorescent signal and proteins whose levels decreased as they were transported from the cell surface to the vacuolar lumen. (B) Proteins that retained their expression in water but moved from the cytosol to the nucleus. (C) Four proteins that were not expressed in cells growing logarithmically in SC but became expressed upon water transfer. For a complete list, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148650#pone.0148650.s002" target="_blank">S2 Table</a></p
Short tandem repeats (STRs), genomic regions each consisting of a sequence of 1-6 base pairs repe... more Short tandem repeats (STRs), genomic regions each consisting of a sequence of 1-6 base pairs repeated in succession, represent one of the largest sources of human genetic variation. However, many STR effects are not captured well by standard genome-wide association studies (GWAS) or downstream analyses that are mostly based on single nucleotide polymorphisms (SNPs). To study the involvement of STRs in complex traits, we imputed genotypes for 445,720 autosomal STRs into genotype array data from 408,153 White British UK Biobank participants and tested for association with 44 blood and serum biomarker phenotypes. We used two fine-mapping methods, SuSiE and FINEMAP, to identify 119 high-confidence STR-trait associations across 93 unique STRs predicted as causal variants under all fine-mapping settings tested. Using these results, we estimate that STRs account for 5.2-7.6% of causal variants identifiable from GWAS signals for these traits. Our high confidence STR-trait associations impli...
<p>Back of the envelope estimate of the number of cells in an adult human body based on a c... more <p>Back of the envelope estimate of the number of cells in an adult human body based on a characteristic volume and mass.</p
<p>(A) W303-1b cells expressing the 25Q were grown in SC-Ura supplemented with 2% fructose ... more <p>(A) W303-1b cells expressing the 25Q were grown in SC-Ura supplemented with 2% fructose (Fru). Galactose (4%) was added either immediately (Gal 0) or on the indicated following days (Gal 1–9). On the indicated days afterwards, cells were collected and lysed, and proteins were dot-blotted onto nitrocellulose. Levels of 25Q and actin were determined by quantitative immunoblotting using rabbit anti-GFP and mouse anti-actin antibodies, respectively, as descried in Materials and Methods. (B-D) W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 18 hours, cells were either kept in SC-Ura with galactose or in SC-Ura with galactose supplemented with CHX (20 μg/ml), or transferred to water or to water supplemented with CHX. On the indicated days, (B) Aggregation Index was calculated, (C) growth curves were plotted (mean ± SE of 2 biological replicates and technical triplicates within each biological replicate), and (D) CFU viability was tested by spotting on YPD plates. (E) W303-1b or BY4741 cells, logarithmically grown (0.5–0.9 A<sub>600</sub>) in SC containing 2% glucose were either maintained in the same medium or transferred to water at logarithmic phase. Upon transfer (log) or on the following day (day 1), CHX (20 μg/ml) was added. Cells collected on the indicated days were tested for CFU viability on YPD plates. Shown is a representative experiment out of similar three.</p
T ype 1 diabetes is a common chronic disease of childhood with substantial morbidity and mortalit... more T ype 1 diabetes is a common chronic disease of childhood with substantial morbidity and mortality. 1 Optimizing glycemic management is key to preventing complications. 2,3 There are known socioeconomic status (SES) disparities in diabetes management and outcomes. 4,5 For Canadians, financial barriers to medications continue to be an important adverse social determinant of health. 6 This is important because insulin is life sustaining for people with type 1 diabetes. Ontario residents (population 14.5 million) have publicly funded coverage for medically necessary services but not for prescription medications. On Jan. 1, 2018, the Ontario government introduced publicly funded pharmacare (Ontario Health Insurance Plan [OHIP]+), the first payer for drugs for Ontarians younger than 25 years. Before the introduction of OHIP+, only people older than 64 years and those eligible for social assistance had access to publicly funded medications; all others paid out of pocket, through private insurance or were supported, in part, by the Trillium Drug Program (a government program that provides drug coverage for out-of-pocket expenses that are more than 4% of household income). The OHIP+ program covered the cost of medications on the provincial formulary, with no deductible or copayment. Fifteen months after its introduction, the Ontario government changed OHIP+ to exclude those patients with private drug coverage. 7 Thus, Ontario provides a unique "natural experiment" to assess the impact of 15 months of publicly funded pharmacare.
<p>W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose in... more <p>W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 17 hours, cells were either maintained in SC-Ura with galactose, transferred to water, or one volume of their SC medium was diluted with the indicated volumes of water, without changing the cell concentration. On the indicated days, (A) Aggregation Index was calculated, (B) growth curves were plotted (mean ± SE of 3 biological replicates and technical triplicates within each biological replicate), and (C) CFU viability was tested by spotting on glucose or galactose plates.</p
Objectives To determine who spends more energy over a lifetime on maintaining their reproductive ... more Objectives To determine who spends more energy over a lifetime on maintaining their reproductive potential: men or women? Design As a model and energetic equivalent, we set the mass of gametes supported over time from birth until exhaustion of fertility. We calculated gender-specific dynamics of gamete pool mass over time. To this purpose we collated data from existing literature, accounting for gamete volume over stages of development, time in each stage, mass density, and count. Our model generates the integral, or area under the curve (AUC) of the gamete pool mass over a lifetime as a proxy to energetic requirements. Main outcome measures The area under gamete mass curve over a lifetime in men and women. Results The number of gametes over a lifetime is 600,000 in women and close to 1 trillion in men. Accounting for mass and time, women invest approximately 100 gram*days in maintaining the female oocyte pool. Women reach 50% of lifetime AUC by age 10, and 90% by age 25. Men invest...
A GLP-2 analogue is used in individuals with intestinal failure at risk for liver disease, yet th... more A GLP-2 analogue is used in individuals with intestinal failure at risk for liver disease, yet the hepatic actions of GLP-2 are not understood. Treatment of high fat diet (HFD)-fed mice with GLP-2 did not modify the development of hepatosteatosis or hepatic inflammation. In contrast, Glp2r-/- mice exhibited increased hepatic lipid accumulation, deterioration in glucose tolerance, and upregulation of biomarkers of hepatic inflammation. Both mouse and human liver expressed the canonical GLP-2R, and hepatic Glp2r expression was upregulated in mice with hepatosteatosis. Cell fractionation localized the Glp2r to hepatic stellate cells (HSC), and markers of HSC activation and fibrosis were increased in livers from Glp2r-/- mice. Moreover, GLP-2 directly modulated gene expression in isolated HSCs ex vivo. Taken together, these findings define an essential role for the GLP-2R in hepatic adaptation to nutrient excess and unveil a gut hormone-HSC axis, linking GLP-2R signaling to control of h...
Pediatric nephrology (Berlin, Germany), Jan 7, 2017
Sarcoidosis is a multisystem granulomatous disease of unknown etiology that rarely presents in ch... more Sarcoidosis is a multisystem granulomatous disease of unknown etiology that rarely presents in childhood. Here, we report a case of pediatric sarcoidosis, presenting with renal failure and hypercalcemia. A previously well 14-year-old Caucasian boy was admitted to the Hospital for Sick Children, Canada, for hypertension and renal failure following work-up by his family physician for initial concerns of growth failure. On admission, his weight was 35 kg (<3rd percentile), his height was 148 cm (<3rd percentile), and his blood pressure was 154/116 mmHg (>99th percentile for height). Laboratory findings showed elevated creatinine (218 umol/L), hypercalcemia (3.21 mmol/L), and normocytic anemia (hemoglobin 105 g/L). His further assessment showed a urinary concentrating defect with hypercalciuria (calcium/creatinine 1.76 mmol/mmol) and nephrocalcinosis on ultrasound. His eye examination showed uveitis with conjunctival biopsy remarkable for granulomas, which led to pursuit of a d...
Objectives Hyperinsulinism is the most common cause of recurrent hypoglycemia in infants, with tr... more Objectives Hyperinsulinism is the most common cause of recurrent hypoglycemia in infants, with transient and permanent forms. Currently, there are no effective tools to predict severity and time to resolution in infants with transient hyperinsulinism (tHI). Therefore, our objective was to assess whether early glucose trends predict disease duration in tHI. Methods A retrospective, pilot cohort of infants admitted with tHI was phenotyped for clinical and laboratory parameters. Blood glucose (BG) values were collected from the first documented hypoglycemia for 120 h (five days). Results In 27 neonates with tHI, the presence of fetal distress (p=0.001) and higher mean daily BG (p=0.035) were associated with shorter time to resolution of hypoglycemia. In a further sensitivity analysis that grouped the cohort by the presence or absence of fetal distress, we found that in neonates without fetal distress, lower mean daily glucose was associated with longer disease duration (R2=0.53, p=0.01...
Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remod... more Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear ER membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that Erv14 (ER-vesicle protein of 14 kD), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, BMV 1a's perinuclear ER localization is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coats (Sec13, Sec24, or Sec31). The requirement of Erv14 for BMV 1a's localization is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by...
It is often presented as common knowledge that, in the human body, bacteria outnumber human cells... more It is often presented as common knowledge that, in the human body, bacteria outnumber human cells by a ratio of at least 10:1. Revisiting the question, we find that the ratio is much closer to 1:1.
Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, ... more Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process.
<p>The MATa XXX-GFP::HIS3 library was grown over night on YPD agar plates and then transfer... more <p>The MATa XXX-GFP::HIS3 library was grown over night on YPD agar plates and then transferred into 50 μl SC-His with 2% glucose in 384-well polystyrene flat-bottom plates, as described in Materials and Methods. Following transfer to water, over 5000 GFP-tagged yeast proteins were visualized during survival in water (after 1 day and 31 days). (A) Proteins that became repressed at an early stage and lost their fluorescent signal and proteins whose levels decreased as they were transported from the cell surface to the vacuolar lumen. (B) Proteins that retained their expression in water but moved from the cytosol to the nucleus. (C) Four proteins that were not expressed in cells growing logarithmically in SC but became expressed upon water transfer. For a complete list, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148650#pone.0148650.s002" target="_blank">S2 Table</a></p
Short tandem repeats (STRs), genomic regions each consisting of a sequence of 1-6 base pairs repe... more Short tandem repeats (STRs), genomic regions each consisting of a sequence of 1-6 base pairs repeated in succession, represent one of the largest sources of human genetic variation. However, many STR effects are not captured well by standard genome-wide association studies (GWAS) or downstream analyses that are mostly based on single nucleotide polymorphisms (SNPs). To study the involvement of STRs in complex traits, we imputed genotypes for 445,720 autosomal STRs into genotype array data from 408,153 White British UK Biobank participants and tested for association with 44 blood and serum biomarker phenotypes. We used two fine-mapping methods, SuSiE and FINEMAP, to identify 119 high-confidence STR-trait associations across 93 unique STRs predicted as causal variants under all fine-mapping settings tested. Using these results, we estimate that STRs account for 5.2-7.6% of causal variants identifiable from GWAS signals for these traits. Our high confidence STR-trait associations impli...
<p>Back of the envelope estimate of the number of cells in an adult human body based on a c... more <p>Back of the envelope estimate of the number of cells in an adult human body based on a characteristic volume and mass.</p
<p>(A) W303-1b cells expressing the 25Q were grown in SC-Ura supplemented with 2% fructose ... more <p>(A) W303-1b cells expressing the 25Q were grown in SC-Ura supplemented with 2% fructose (Fru). Galactose (4%) was added either immediately (Gal 0) or on the indicated following days (Gal 1–9). On the indicated days afterwards, cells were collected and lysed, and proteins were dot-blotted onto nitrocellulose. Levels of 25Q and actin were determined by quantitative immunoblotting using rabbit anti-GFP and mouse anti-actin antibodies, respectively, as descried in Materials and Methods. (B-D) W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 18 hours, cells were either kept in SC-Ura with galactose or in SC-Ura with galactose supplemented with CHX (20 μg/ml), or transferred to water or to water supplemented with CHX. On the indicated days, (B) Aggregation Index was calculated, (C) growth curves were plotted (mean ± SE of 2 biological replicates and technical triplicates within each biological replicate), and (D) CFU viability was tested by spotting on YPD plates. (E) W303-1b or BY4741 cells, logarithmically grown (0.5–0.9 A<sub>600</sub>) in SC containing 2% glucose were either maintained in the same medium or transferred to water at logarithmic phase. Upon transfer (log) or on the following day (day 1), CHX (20 μg/ml) was added. Cells collected on the indicated days were tested for CFU viability on YPD plates. Shown is a representative experiment out of similar three.</p
T ype 1 diabetes is a common chronic disease of childhood with substantial morbidity and mortalit... more T ype 1 diabetes is a common chronic disease of childhood with substantial morbidity and mortality. 1 Optimizing glycemic management is key to preventing complications. 2,3 There are known socioeconomic status (SES) disparities in diabetes management and outcomes. 4,5 For Canadians, financial barriers to medications continue to be an important adverse social determinant of health. 6 This is important because insulin is life sustaining for people with type 1 diabetes. Ontario residents (population 14.5 million) have publicly funded coverage for medically necessary services but not for prescription medications. On Jan. 1, 2018, the Ontario government introduced publicly funded pharmacare (Ontario Health Insurance Plan [OHIP]+), the first payer for drugs for Ontarians younger than 25 years. Before the introduction of OHIP+, only people older than 64 years and those eligible for social assistance had access to publicly funded medications; all others paid out of pocket, through private insurance or were supported, in part, by the Trillium Drug Program (a government program that provides drug coverage for out-of-pocket expenses that are more than 4% of household income). The OHIP+ program covered the cost of medications on the provincial formulary, with no deductible or copayment. Fifteen months after its introduction, the Ontario government changed OHIP+ to exclude those patients with private drug coverage. 7 Thus, Ontario provides a unique "natural experiment" to assess the impact of 15 months of publicly funded pharmacare.
<p>W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose in... more <p>W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 17 hours, cells were either maintained in SC-Ura with galactose, transferred to water, or one volume of their SC medium was diluted with the indicated volumes of water, without changing the cell concentration. On the indicated days, (A) Aggregation Index was calculated, (B) growth curves were plotted (mean ± SE of 3 biological replicates and technical triplicates within each biological replicate), and (C) CFU viability was tested by spotting on glucose or galactose plates.</p
Objectives To determine who spends more energy over a lifetime on maintaining their reproductive ... more Objectives To determine who spends more energy over a lifetime on maintaining their reproductive potential: men or women? Design As a model and energetic equivalent, we set the mass of gametes supported over time from birth until exhaustion of fertility. We calculated gender-specific dynamics of gamete pool mass over time. To this purpose we collated data from existing literature, accounting for gamete volume over stages of development, time in each stage, mass density, and count. Our model generates the integral, or area under the curve (AUC) of the gamete pool mass over a lifetime as a proxy to energetic requirements. Main outcome measures The area under gamete mass curve over a lifetime in men and women. Results The number of gametes over a lifetime is 600,000 in women and close to 1 trillion in men. Accounting for mass and time, women invest approximately 100 gram*days in maintaining the female oocyte pool. Women reach 50% of lifetime AUC by age 10, and 90% by age 25. Men invest...
A GLP-2 analogue is used in individuals with intestinal failure at risk for liver disease, yet th... more A GLP-2 analogue is used in individuals with intestinal failure at risk for liver disease, yet the hepatic actions of GLP-2 are not understood. Treatment of high fat diet (HFD)-fed mice with GLP-2 did not modify the development of hepatosteatosis or hepatic inflammation. In contrast, Glp2r-/- mice exhibited increased hepatic lipid accumulation, deterioration in glucose tolerance, and upregulation of biomarkers of hepatic inflammation. Both mouse and human liver expressed the canonical GLP-2R, and hepatic Glp2r expression was upregulated in mice with hepatosteatosis. Cell fractionation localized the Glp2r to hepatic stellate cells (HSC), and markers of HSC activation and fibrosis were increased in livers from Glp2r-/- mice. Moreover, GLP-2 directly modulated gene expression in isolated HSCs ex vivo. Taken together, these findings define an essential role for the GLP-2R in hepatic adaptation to nutrient excess and unveil a gut hormone-HSC axis, linking GLP-2R signaling to control of h...
Pediatric nephrology (Berlin, Germany), Jan 7, 2017
Sarcoidosis is a multisystem granulomatous disease of unknown etiology that rarely presents in ch... more Sarcoidosis is a multisystem granulomatous disease of unknown etiology that rarely presents in childhood. Here, we report a case of pediatric sarcoidosis, presenting with renal failure and hypercalcemia. A previously well 14-year-old Caucasian boy was admitted to the Hospital for Sick Children, Canada, for hypertension and renal failure following work-up by his family physician for initial concerns of growth failure. On admission, his weight was 35 kg (<3rd percentile), his height was 148 cm (<3rd percentile), and his blood pressure was 154/116 mmHg (>99th percentile for height). Laboratory findings showed elevated creatinine (218 umol/L), hypercalcemia (3.21 mmol/L), and normocytic anemia (hemoglobin 105 g/L). His further assessment showed a urinary concentrating defect with hypercalciuria (calcium/creatinine 1.76 mmol/mmol) and nephrocalcinosis on ultrasound. His eye examination showed uveitis with conjunctival biopsy remarkable for granulomas, which led to pursuit of a d...
Objectives Hyperinsulinism is the most common cause of recurrent hypoglycemia in infants, with tr... more Objectives Hyperinsulinism is the most common cause of recurrent hypoglycemia in infants, with transient and permanent forms. Currently, there are no effective tools to predict severity and time to resolution in infants with transient hyperinsulinism (tHI). Therefore, our objective was to assess whether early glucose trends predict disease duration in tHI. Methods A retrospective, pilot cohort of infants admitted with tHI was phenotyped for clinical and laboratory parameters. Blood glucose (BG) values were collected from the first documented hypoglycemia for 120 h (five days). Results In 27 neonates with tHI, the presence of fetal distress (p=0.001) and higher mean daily BG (p=0.035) were associated with shorter time to resolution of hypoglycemia. In a further sensitivity analysis that grouped the cohort by the presence or absence of fetal distress, we found that in neonates without fetal distress, lower mean daily glucose was associated with longer disease duration (R2=0.53, p=0.01...
Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remod... more Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear ER membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that Erv14 (ER-vesicle protein of 14 kD), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, BMV 1a's perinuclear ER localization is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coats (Sec13, Sec24, or Sec31). The requirement of Erv14 for BMV 1a's localization is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by...
It is often presented as common knowledge that, in the human body, bacteria outnumber human cells... more It is often presented as common knowledge that, in the human body, bacteria outnumber human cells by a ratio of at least 10:1. Revisiting the question, we find that the ratio is much closer to 1:1.
Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, ... more Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process.
Uploads
Papers by Shai Fuchs