Steven Padilla - Academia.edu (original) (raw)

Papers by Steven Padilla

Molecular and cellular biology, 2005

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositi... more The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the...

Journal of Cell Science, 2002

A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identif... more A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P). Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II. We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY. Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively. Interestingly, we measured decreased PtdIns3P levels in Δvps30 andΔ vps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants. Vps5p and Vps17p are subunits of the retromer complex that is requ...

Journal of Clinical Medicine

Background: Although the tissues comprising the ocular conventional outflow pathway have shown st... more Background: Although the tissues comprising the ocular conventional outflow pathway have shown strong viscoelastic mechanical response to aqueous humor pressure dynamics, the viscoelastic mechanical properties of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm’s canal (SC) inner wall are largely unknown. Methods: A quadrant of the anterior segment from two human donor eyes at low- and high-flow (LF and HF) outflow regions was pressurized and imaged using optical coherence tomography (OCT). A finite element (FE) model of the TM, the adjacent JCT, and the SC inner wall was constructed and viscoelastic beam elements were distributed in the extracellular matrix (ECM) of the TM and JCT to represent anisotropic collagen. An inverse FE-optimization algorithm was used to calculate the viscoelastic properties of the ECM/beam elements such that the TM/JCT/SC model and OCT imaging data best matched over time. Results: The ECM of the glaucoma tissues showed s...

Investigative Ophthalmology & Visual Science, Jul 22, 2019

Investigative Ophthalmology & Visual Science, 2017

Purpose : Aqueous out ow system con guration (OSC) determines aqueous ow and IOP that becomes abn... more Purpose : Aqueous out ow system con guration (OSC) determines aqueous ow and IOP that becomes abnormal in glaucoma. We describe a pilot study with an ex vivo system using visually guided positioning and real time observation of OSC changes (Δs) in response to transcleral μP laser (Iridex ) pulses. Methods : M. fasc. primates A (PrA) & B (PrB). PrA: microscope, video recordings, calibration micrometer, 1 mm thick radial limbal segments from 4 quadrants (Q). micromanipulator, single pulse of 810 nm μP laser, duty cycle 31.3%. Paired parameters of stepwise power; range: 500-3000 mw, stepwise duration; range: 125-3,000 msec. Energy range: 0.08-2.35 joules. Clinically, ~1.59 J (CEJ) is applied per single location. Video capture during pulse. PrA: Motion quantitated from still frames with ImageJ. PrB: H&E & trichrome in 4 Q after Rx with CEJ. Results : PrA Visible μP e ects were con ned to longitudinal ciliary muscle (CM) near sclera (Fig. 1) Shortening/Contraction (ShCo) & relaxation was...

Investigative Ophthalmology & Visual Science, 2016

Investigative Ophthalmology & Visual Science, 2016

Investigative Ophthalmology & Visual Science, 2015

Investigative Ophthalmology & Visual Science, 2018

Investigative ophthalmology & visual science, Sep 1, 2017

The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to b... more The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements. Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward. The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glau...

Experimental eye research, May 11, 2016

Glaucoma is a leading cause of blindness worldwide and results from damage to the optic nerve. Cu... more Glaucoma is a leading cause of blindness worldwide and results from damage to the optic nerve. Currently, intraocular pressure is the only treatable risk factor. Changes in aqueous outflow regulate pressure; regulation becomes abnormal in glaucoma. From inside the eye aqueous flows out through the trabecular meshwork into a venous sinus called Schlemm's canal, next into collector channels and finally returns to the episcleral vessels of the venous system. The location of aqueous outflow regulation is unknown. Ex vivo and in vivo studies implicate both pressure-dependent trabecular tissue motion and tissues distal to Schlemm's canal in regulation of aqueous outflow. Technologies have not previously been available to study these issues. New ex vivo imaging in human eyes identifies hinged flaps or leaflets at collector channel entrances using a high-resolution spectral domain optical coherence tomography (SD-OCT) platform. The hinged flaps open and close in synchrony with press...

Experimental Hematology, 2014

MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation.... more MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation. However, our knowledge of miRNA function in terminal erythropoiesis remains limited. To address this issue, we generated a novel animal model, where the miRNA-processing enzyme, Dicer, is selectively inactivated in erythropoietin receptor positive erythroid cells beginning with CFU-e/proerythroblast cells. This results in significant depletion of all miRNAs from the proerythroblast stage onwards, with one exception, miR-451, which is processed by Ago2 in a Dicer-independent manner. We observed that mature Dicer-dependent miRNAs, like miR-451, are dispensable under steady-state conditions, but these mutants have an impaired response to stress erythropoiesis, as demonstrated by a delay in recovery from anemia. This defect was specific to later maturing erythroid cells, as progenitor numbers were unaffected. In addition to generating a novel mouse model to study miRNA function in late erythroid cells, we conclude that miRNAs (both Dicer-dependent and independent) act primarily to regulate the optimal response to stress among late erythroid cells.

Journal of biomedical optics, 2014

The aqueous outflow system (AOS) is responsible for maintaining normal intraocular pressure (IOP)... more The aqueous outflow system (AOS) is responsible for maintaining normal intraocular pressure (IOP) in the eye. Structures of the AOS have an active role in regulating IOP in healthy eyes and these structures become abnormal in the eyes with glaucoma. We describe a newly developed system platform to obtain high-resolution images of the AOS structures. By incorporating spectral domain optical coherence tomography (SD-OCT), the platform allows us to systematically control, image, and quantitate the responses of AOS tissue to pressure with a millisecond resolution of pulsed flow. We use SD-OCT to image radial limbal segments from the surface of the trabecular meshwork (TM) with a spatial resolution of ∼5 μm in ex vivo nonhuman primate eyes. We carefully insert a cannula into Schlemm’s canal (SC) to control both pressures and flow rates. The experimental results demonstrate the capability of the platform to visualize the unprecedented details of AOS tissue components comparable to that d...

Haematologica, 2013

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid disse... more Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors.

Experimental Hematology, 2014

Blood, 2010

To delineate the role of specific members of β1 integrins in stress erythropoiesis in the adult, ... more To delineate the role of specific members of β1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of β1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in β1Δ/Δ reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to β1-deficient mice, α4Δ/Δ mice showed only a kinetic delay in recovery and similar to β1Δ/Δ, terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1Δ/Δ mice. Convergence of information from these comparative studies lends new insight to the...

Investigative Ophthalmology & Visual Science, 2015

Molecular and cellular biology, 2005

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositi... more The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the...

Journal of Cell Science, 2002

A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identif... more A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P). Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II. We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY. Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively. Interestingly, we measured decreased PtdIns3P levels in Δvps30 andΔ vps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants. Vps5p and Vps17p are subunits of the retromer complex that is requ...

Journal of Clinical Medicine

Background: Although the tissues comprising the ocular conventional outflow pathway have shown st... more Background: Although the tissues comprising the ocular conventional outflow pathway have shown strong viscoelastic mechanical response to aqueous humor pressure dynamics, the viscoelastic mechanical properties of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm’s canal (SC) inner wall are largely unknown. Methods: A quadrant of the anterior segment from two human donor eyes at low- and high-flow (LF and HF) outflow regions was pressurized and imaged using optical coherence tomography (OCT). A finite element (FE) model of the TM, the adjacent JCT, and the SC inner wall was constructed and viscoelastic beam elements were distributed in the extracellular matrix (ECM) of the TM and JCT to represent anisotropic collagen. An inverse FE-optimization algorithm was used to calculate the viscoelastic properties of the ECM/beam elements such that the TM/JCT/SC model and OCT imaging data best matched over time. Results: The ECM of the glaucoma tissues showed s...

Investigative Ophthalmology & Visual Science, Jul 22, 2019

Investigative Ophthalmology & Visual Science, 2017

Purpose : Aqueous out ow system con guration (OSC) determines aqueous ow and IOP that becomes abn... more Purpose : Aqueous out ow system con guration (OSC) determines aqueous ow and IOP that becomes abnormal in glaucoma. We describe a pilot study with an ex vivo system using visually guided positioning and real time observation of OSC changes (Δs) in response to transcleral μP laser (Iridex ) pulses. Methods : M. fasc. primates A (PrA) & B (PrB). PrA: microscope, video recordings, calibration micrometer, 1 mm thick radial limbal segments from 4 quadrants (Q). micromanipulator, single pulse of 810 nm μP laser, duty cycle 31.3%. Paired parameters of stepwise power; range: 500-3000 mw, stepwise duration; range: 125-3,000 msec. Energy range: 0.08-2.35 joules. Clinically, ~1.59 J (CEJ) is applied per single location. Video capture during pulse. PrA: Motion quantitated from still frames with ImageJ. PrB: H&E & trichrome in 4 Q after Rx with CEJ. Results : PrA Visible μP e ects were con ned to longitudinal ciliary muscle (CM) near sclera (Fig. 1) Shortening/Contraction (ShCo) & relaxation was...

Investigative Ophthalmology & Visual Science, 2016

Investigative Ophthalmology & Visual Science, 2016

Investigative Ophthalmology & Visual Science, 2015

Investigative Ophthalmology & Visual Science, 2018

Investigative ophthalmology & visual science, Sep 1, 2017

The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to b... more The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements. Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward. The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glau...

Experimental eye research, May 11, 2016

Glaucoma is a leading cause of blindness worldwide and results from damage to the optic nerve. Cu... more Glaucoma is a leading cause of blindness worldwide and results from damage to the optic nerve. Currently, intraocular pressure is the only treatable risk factor. Changes in aqueous outflow regulate pressure; regulation becomes abnormal in glaucoma. From inside the eye aqueous flows out through the trabecular meshwork into a venous sinus called Schlemm's canal, next into collector channels and finally returns to the episcleral vessels of the venous system. The location of aqueous outflow regulation is unknown. Ex vivo and in vivo studies implicate both pressure-dependent trabecular tissue motion and tissues distal to Schlemm's canal in regulation of aqueous outflow. Technologies have not previously been available to study these issues. New ex vivo imaging in human eyes identifies hinged flaps or leaflets at collector channel entrances using a high-resolution spectral domain optical coherence tomography (SD-OCT) platform. The hinged flaps open and close in synchrony with press...

Experimental Hematology, 2014

MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation.... more MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation. However, our knowledge of miRNA function in terminal erythropoiesis remains limited. To address this issue, we generated a novel animal model, where the miRNA-processing enzyme, Dicer, is selectively inactivated in erythropoietin receptor positive erythroid cells beginning with CFU-e/proerythroblast cells. This results in significant depletion of all miRNAs from the proerythroblast stage onwards, with one exception, miR-451, which is processed by Ago2 in a Dicer-independent manner. We observed that mature Dicer-dependent miRNAs, like miR-451, are dispensable under steady-state conditions, but these mutants have an impaired response to stress erythropoiesis, as demonstrated by a delay in recovery from anemia. This defect was specific to later maturing erythroid cells, as progenitor numbers were unaffected. In addition to generating a novel mouse model to study miRNA function in late erythroid cells, we conclude that miRNAs (both Dicer-dependent and independent) act primarily to regulate the optimal response to stress among late erythroid cells.

Journal of biomedical optics, 2014

The aqueous outflow system (AOS) is responsible for maintaining normal intraocular pressure (IOP)... more The aqueous outflow system (AOS) is responsible for maintaining normal intraocular pressure (IOP) in the eye. Structures of the AOS have an active role in regulating IOP in healthy eyes and these structures become abnormal in the eyes with glaucoma. We describe a newly developed system platform to obtain high-resolution images of the AOS structures. By incorporating spectral domain optical coherence tomography (SD-OCT), the platform allows us to systematically control, image, and quantitate the responses of AOS tissue to pressure with a millisecond resolution of pulsed flow. We use SD-OCT to image radial limbal segments from the surface of the trabecular meshwork (TM) with a spatial resolution of ∼5 μm in ex vivo nonhuman primate eyes. We carefully insert a cannula into Schlemm’s canal (SC) to control both pressures and flow rates. The experimental results demonstrate the capability of the platform to visualize the unprecedented details of AOS tissue components comparable to that d...

Haematologica, 2013

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid disse... more Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors.

Experimental Hematology, 2014

Blood, 2010

To delineate the role of specific members of β1 integrins in stress erythropoiesis in the adult, ... more To delineate the role of specific members of β1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of β1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in β1Δ/Δ reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to β1-deficient mice, α4Δ/Δ mice showed only a kinetic delay in recovery and similar to β1Δ/Δ, terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1Δ/Δ mice. Convergence of information from these comparative studies lends new insight to the...

Investigative Ophthalmology & Visual Science, 2015