Alfredo Franco-Obregon - Academia.edu (original) (raw)

Papers by Alfredo Franco-Obregon

Proceedings of SPIE, Feb 9, 2012

Digital holographic microscopy (DHM) has been gaining interest from cell biology community becaus... more Digital holographic microscopy (DHM) has been gaining interest from cell biology community because of its label free nature and quantitative phase signal output. Besides, fast shutter time, image reconstruction by numerical propagation of the wave fields, and numerical compensation of the aberrations are other intrinsic advantages of this technique that can be explored for harsh imaging conditions. In the frame of this work, a transmission type DHM is developed with a decoupled epifluorescence microscopy mode for ...

Biomedical Optics Express, Feb 1, 2012

We describe an optimized digital holographic microscopy system (DHM) suitable for high-resolution... more We describe an optimized digital holographic microscopy system (DHM) suitable for high-resolution visualization of living cells under conditions of altered macroscopic mechanical forces such as those that arise from changes in gravitational force. Experiments were performed on both a ground-based microgravity simulation platform known as the random positioning machine (RPM) as well as during a parabolic flight campaign (PFC). Under these conditions the DHM system proved to be robust and reliable. In addition, the ...

Journal of biomedical optics, Mar 1, 2010

Abstract. Previous investigations on mammalian cells have shown that microgravity, either that ex... more Abstract. Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. ...

European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society, Jan 2, 2018

Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. Howeve... more Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. However, the underlying molecular mechanisms are not completely understood. Transient receptor potential canonical (TRPC) channels are implicated in mechanosensing of several tissues, but are poorly explored in IVDs. Primary human IVD cells from surgical biopsies composed of both annulus fibrosus and nucleus pulposus (passage 1-2) were exposed to simulated microgravity and to the TRPC channel inhibitor SKF-96365 (SKF) for up to 5 days. Proliferative capacity, cell cycle distribution, senescence and TRPC channel expression were analyzed. Both simulated microgravity and TRPC channel antagonism reduced the proliferative capacity of IVD cells and induced senescence. While significant changes in cell cycle distributions (reduction in G1 and accumulation in G2/M) were observed upon SKF treatment, the effect was small upon 3 days of simulated microgravity. Finally, downregulation of TRPC6 was shown u...

Scientific Reports

Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common... more Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common to chondrogenesis. Here we document the effects of specified PEMF parameters over mesenchymal stem cells (MSC) chondrogenic differentiation. MSCs undergoing chondrogenesis are preferentially responsive to an electromagnetic efficacy window defined by field amplitude, duration and frequency of exposure. Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (2 mT) exposure to 6 ms bursts of magnetic pulses, at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome and could be attributed to calcium entry after the initial induction. Transient receptor potential (TRP) channels appear to mediate these aspects of PEMF stimulation, serving as a conduit for extracellular calcium. Preventing calcium entry during the repeated PEMF exposure with the co-administration of EGTA or TRP channel antagonists precluded the inhibition of differentiation. This study highlights the intricacies of calcium homeostasis during early chondrogenesis and the constraints that are placed on PEMF-based therapeutic strategies aimed at promoting MSC chondrogenesis. The demonstrated efficacy of our optimized PEMF regimens has clear clinical implications for future regenerative strategies for cartilage. Articular cartilage is an avascular tissue with low potential for self-repair. When left untreated, lesions of the articular cartilage can lead to osteoarthritis 1-3. The success of any technology aimed at repairing chondral defects will thus be based on its ability to produce tissues that most closely recapitulate the mechanical and biochemical properties of native cartilage. To this end many technologies have been advanced yet, none are without drawbacks. The 'microfracture' technique is commonly plagued by the formation of fibro-cartilaginous tissue of low dexterity 4. Autologous chondrocytes implantation and osteochondral autograft transplantation are limited by scarce cartilage production, low proliferative capacity of chondrocytes, chondrocyte de-differentiation and complications due to donor site morbidity 5. Stem cell-based approaches are also being actively pursued in hopes of improved outcome. Mesenchymal stem cells (MSCs) support chondrogenic differentiation and are an attractive cell source for cartilage tissue engineering. However, the neocartilage formed by conventional MSC-based repair

Today there is a big interest in muscle related disorders, such as sarcopenia, the age-related de... more Today there is a big interest in muscle related disorders, such as sarcopenia, the age-related decline in muscle mass. As a result, viable methods for combating muscle wasting disorders are urgently being sought. But in order to achieve satisfactory results new therapies and technologies to enhance muscle regeneration have to be developed based on a better understanding of the exact processes occurring at the transition points, cell division (proliferation) and cell fusion (differentiation), of the different stages in muscle development. Activated myoblasts first undergo many rounds of cell division. Depending on the needs of the cells,

The Journal of physiology, 2002

We studied the effects of membrane stretch and voltage on the gating of single mechanosensitive (... more We studied the effects of membrane stretch and voltage on the gating of single mechanosensitive (MS) channels in myotubes from dystrophin-deficient mdx mice. In earlier studies of MS channels in mdx myotubes, we found a novel class of stretch-inactivated channels. In the present experiments, we used a gentle suction protocol to determine whether seal formation damaged the membrane and altered MS channel gating, since dystrophin-deficiency is known to be associated with an increased susceptibility to mechanically induced damage. In some recordings from mdx myotubes, MS channel open probability gradually increased to levels approaching unity following seal formation. In these recordings, channels remained open for the duration of the recording. In other recordings, MS channel open probability remained low after seal formation and applying weak suction evoked conventional stretch-activated gating. Applying strong suction or very positive voltages, however, caused some channels to enter...

Proceedings of the National Academy of Sciences, 1995

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in f... more We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering 02 tension (P02) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca22+ whose frequency depends on extracellular Ca2+ influx. Switching from a P02 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2' oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low P02 selectively inhibits L-type Ca2' channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low P02 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of 02sensitive, voltage-dependent, Ca2' channels in vascular smooth muscle that may critically contribute to the local regulation of circulation. Oxygen tension (P02) has been recognized for decades as an important factor in the local regulation of vascular tone in vivo (1), and it is known that hypoxia causes relaxation of systemic arteries in vitro (2-4). Nevertheless, the mechanisms underly

PLoS ONE, 2014

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in ... more Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T 1), 12 weeks (T 2), 6 months (T 3) and 12 months (T 4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T 1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.

Kidney International, 1997

Journal of Biomedical Optics, 2010

Previous investigations on mammalian cells have shown that microgravity, either that experienced ... more Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy ͑DHM͒. DHM analysis of living mouse myoblasts ͑C2C12͒ is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.

FEBS Letters, 2013

The goal of the current study was to investigate the effect of heat shock on human in vitro diffe... more The goal of the current study was to investigate the effect of heat shock on human in vitro differentiated monocyte-derived dendritic cells (DCs) and to dissect the role of thermosensitive transient receptor potential (TRP) in the process. We have shown that, of the thermosensitive TRP channels, DCs express TRPV1, TRPV2 and TRPV4. We have furthermore presented evidence that a short heat shock challenge (43°C for 1 hr) decreased the endocytotic activity of the DCs and that this effect could be alleviated by the RNAimediated knockdown of TRPV2 but, importantly, not by the pharmacological (antagonists) or molecular (RNAi) suppression of TRPV1 and TRPV4 activities/levels. Likewise, the heat shock-induced robust membrane currents were selectively and markedly inhibited by TRPV2 "silencing" whereas modulation of TRPV1 and TRPV4 activities, again, had no effect. These intriguing data introduce TRPV2-coupled signaling a key player in mediating the cellular actions of heat shock on DCs.

Clinical and Experimental Pharmacology and Physiology, 2006

1. Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of de... more 1. Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of development. In recordings from membrane patches, MS channels are constitutively active at the resting potential. The channels are selective for cations and have a large single-channel conductance (approximately 25 pS in physiological saline) and a high Ca 2+ permeability (relative permeability of Ca 2+ to K + (P Ca /P K) = 7). 2. Mechanosensitive channel activity recorded from the surface of myotubes from dystrophic mdx mice was substantially greater than the activity recorded from wild-type myotubes. Increased channel activity in the mutant results from the induction in a subpopulation of channels of a novel MS gating mode characterized by markedly prolonged channel openings and inactivation in response to membrane stretch. 3. Membrane stretch or a strong depolarization causes an irreversible switch to the stretch-inactivated gating mode in mdx myotubes. A stretch-induced shift in MS channel gating mode may contribute to stretch-induced elevations in [Ca 2+ ] i during the early stages of disease pathogenesis. 4. Abnormalities of MS channel behaviour are also detected in recordings from patches on flexor digitorum brevis fibres acutely isolated from mdx mice. Mechanosensitive channel opening probability is higher in mdx fibres at all developmental stages. In addition, channel numbers are persistently elevated during postnatal development, failing to undergo a normal process of downregulation during the first 3 postnatal weeks. 5. Two distinct mechanisms may contribute to elevations of [Ca 2+ ] i in dystrophin-deficient skeletal muscle: (i) a membrane stress-dependent switch of MS channels into to a prolonged opening mode; and (ii) a loss of developmental downregulation leading to persistent MS channel expression during postnatal muscle development.

Biophysical Journal, 2003

The nuclear pore complex (NPC) represents the only pathway for macromolecular communication betwe... more The nuclear pore complex (NPC) represents the only pathway for macromolecular communication between the nuclear and cytoplasmic compartments of the cell. Nucleocytoplasmic transport requires the interaction of transport receptors with phenylalanine-glycine (FG)-repeats that line the transport pathway through the NPC. Here we examine the effects of transport receptors and amphipathic alcohols on NPC topology using scanning force microscopy. We show that transport receptors that irreversibly bind FG-repeats increase NPC vertical aspect, whereas transport receptors that weakly interact with FG-repeats increase NPC diameter. Interestingly, small polar alcohols likewise increase NPC diameter. These opposing effects agree with the inhibition or enhancement of nuclear transport, respectively, previously ascribed to these agents. Imaging of nuclear envelope samples was performed with a BioScope AFM (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA) using silicon nitride cantilevers (spring constant 0.16 N/m) with oxide-sharpened tips (Olympus Optical Co., Ltd., Japan

Biomaterials, 2011

Surface biofouling poses an increasing problem in industrial and health care applications, drivin... more Surface biofouling poses an increasing problem in industrial and health care applications, driving research for surface coatings to prevent anti-microbial colonization and characterization of the efficacy of the same. The diversity and increasing sophistication of such coatings, which postulate different types of anti-microbial action on planktonic and surface adhering bacteria, challenge the suitability of current approaches to evaluate and compare the different approaches as well as the speed and accuracy at which screening can be made. We describe and provide proof of principle for a method to use microparticles functionalized with molecular coatings through self-assembly together with flow cytometry readout to evaluate Escherichia coli bacteria surface adhesion and killing efficiency. Advantages of the method are the automation of the method that allows recording an immense number of interactions and the possibility to simultaneously record effects on both surface adhering and planktonic bacteria. We demonstrate and discuss design criteria to obtain this information on two coatings, poly(L-lysine)-graft-C 3 H 6 N þ (CH 3) 2 C 12 H 25 (PLL-g-QAC) and poly(L-lysine)-graft-poly(ethylene glycol)-C 3 H 6 N þ (CH 3) 2 C 12 H 25 (PLL-g-PEG-QAC), which exemplify two different approaches to creating anti-microbial interfaces. Despite an apparent higher killing efficiency of the PLL-g-QAC during brief exposures, the rapid fouling of that surface quickly reduces its efficiency, whereas the PLL-g-PEG-QAC coating showed greater promise in reducing the growth and interfacial colonization of bacteria over longer time scales.

European Cells and …, 2007

Effective Tissue engineering ultimately depends on understanding the interplay between the prolif... more Effective Tissue engineering ultimately depends on understanding the interplay between the proliferation and differentiation stages of development. One of the best systems to examine these processes is myogenesis. Activated myoblasts first undergo many rounds of cell division. ...

Biotechnology Letters, 2011

Peptide therapeutics are of increasing interest due to their biological specificity. We used a si... more Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated