Determining crystal structures through crowdsourcing and coursework

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality

Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence

Human stem cell therapy for type 2 diabetes/obesity (T2D/O) complications is performedwith stem cell autografts, exposed to the noxious T2D/O milieu, often with suboptimal results.We showed in the Obese Zucker (OZ) rat model of T2D/O that when their muscle-derived stemcells (MDSC) were from long-term T2D/O male rats, their repair ecacy for erectile dysfunctionwas impaired and were imprinted with abnormal gene- and miR-global transcriptional signatures(GTS). The damage was reproduced in vitro by short-term exposure of normal MDSC to dyslipidemicserum, causing altered miR-GTS, fat infiltration, apoptosis, impaired scratch healing, and myostatinoverexpression. Similar in vitro alterations occurred with their normal counterparts (ZF4-SC) fromthe T2D/O rat model for female stress urinary incontinence, and with ZL4-SC from non-T2D/O leanfemale rats. In the current work we studied the in vitro eects of cholesterol and Na palmitate aslipid factors on ZF4-SC and ZL4-SC. A damage partially resembling the one caused by the femaledyslipidemic serum was found, but diering between both lipid factors, so that each one appears tocontribute specifically to the stem cell damaging eects of dyslipidemic serum in vitro and T2D/Oin vivo, irrespective of gender. These results also confirm the miR-GTS biomarker value forMDSC damage

Sildenafil Attenuates Inflammation and Oxidative Stress in Pelvic Ganglia Neurons after Bilateral Cavernosal Nerve Damage

Erectile dysfunction is a common complication for patients undergoing surgeries for prostate, bladder, and colorectal cancers, due to damage of the nerves associated with the major pelvic ganglia (MPG). Functional re-innervation of target organs depends on the capacity of the neurons to survive and switch towards a regenerative phenotype. PDE5 inhibitors (PDE5i) have been successfully used in promoting the recovery of erectile function after cavernosal nerve damage (BCNR) by up-regulating the expression of neurotrophic factors in MPG. However, little is known about the effects of PDE5i on markers of neuronal damage and oxidative stress after BCNR. This study aimed to investigate the changes in gene and protein expression profiles of inflammatory, anti-inflammatory cytokines and oxidative stress related-pathways in MPG neurons after BCNR and subsequent treatment with sildenafil. Our results showed that BCNR in Fisher-344 rats promoted up-regulation of cytokines (interleukin- 1 (IL-1) β, IL-6, IL-10, transforming growth factor β 1 (TGFβ1), and oxidative stress factors (Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), TNF receptor superfamily member 5 (CD40) that were normalized by sildenafil treatment given in the drinking water. In summary, PDE5i can attenuate the production of damaging factors and can up-regulate the expression of beneficial factors in the MPG that may ameliorate neuropathic pain, promote neuroprotection, and favor nerve regeneration

Myostatin genetic inactivation inhibits myogenesis by muscle derived stem cells in vitro but not when implanted in the mdx mouse muscle

Abstract Introduction Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). Methods To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis. Results Surprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration. Conclusions Although WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs

Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence Are Damaged by In Vitro Exposure to its Dyslipidemic Serum, Predicting Inadequate Repair Capacity In Vivo.

Female stress urinary incontinence (FSUI) is prevalent in women with type 2 diabetes/obesity (T2D/O), and treatment is not optimal. Autograph stem cell therapy surprisingly has poor efficacy. In the male rat model of T2D/O, it was demonstrated that epigenetic changes, triggered by long-term exposure to the dyslipidemic milieu, led to abnormal global transcriptional signatures (GTS) of genes and microRNAs (miR), and impaired the repair capacity of muscle-derived stem cells (MDSC). This was mimicked in vitro by treatment of MDSC with dyslipidemic serum or lipid factors. The current study aimed to predict whether these changes also occur in stem cells from female 12 weeks old T2D/O rats, a model of FSUI. MDSCs from T2D/O (ZF4-SC) and normal female rats (ZL4-SC) were treated in vitro with either dyslipidemic serum (ZFS) from late T2D/O 24 weeks old female Zucker fatty (ZF) rats, or normal serum (ZLS) from 24 weeks old female Zucker lean (ZL) rats, for 4 days and subjected to assays for fat deposition, apoptosis, scratch closing, myostatin, interleukin-6, and miR-GTS. The dyslipidemic ZFS affected both female stem cells more severely than in the male MDSC, with some gender-specific differences in miR-GTS. The changes in miR-GTS and myostatin/interleukin-6 balance may predict in vivo noxious effects of the T2D/O milieu that might impair autograft stem cell (SC) therapy for FSUI, but this requires future studies

Implanted Muscle-Derived Stem Cells Ameliorate Erectile Dysfunction in a Rat Model of Type 2 Diabetes, but Their Repair Capacity Is Impaired by Their Prior Exposure to the Diabetic Milieu

IntroductionMuscle-derived stem cells (MDSCs) and other SCs implanted into the penile corpora cavernosa ameliorate erectile dysfunction in type 1 diabetic rat models by replenishing lost corporal smooth muscle cells (SMCs) and decreasing fibrosis. However, there are no conclusive data from models of type 2 diabetes (T2D) and obesity.AimTo determine whether MDSCs from obese Zucker (OZ) rats with T2D at an early stage of diabetes (early diabetic SCs isolated and cultured in low-glucose medium [ED-SCs]) counteract corporal veno-occlusive dysfunction and corporal SMC loss or lipo-fibrosis when implanted in OZ rats at a late stage of diabetes and whether MDSCs from these OZ rats with late diabetes (late diabetic SCs isolated and cultured in high-glucose medium [LD-SC]) differ from ED-SCs in gene transcriptional phenotype and repair capacity.MethodsED-SCs and LD-SCs were compared by DNA microarray assays, and ED-SCs were incubated in vitro under high-glucose conditions (ED-HG-SC). These three MDSC types were injected into the corpora cavernosa of OZ rats with late diabetes (OZ/ED, OZ/LD, and OZ/ED-HG rats, respectively). Untreated OZ and non-diabetic lean Zucker rats functioned as controls. Two months later, rats were subjected to cavernosometry and the penile shaft and corporal tissues were subjected to histopathology and DNA microarray assays.Main outcome measuresIn vivo erectile dysfunction assessment by Dynamic Infusion Cavernosometry followed by histopathology marker analysis of the penile tissues.ResultsImplanted ED-SCs and ED-HG-SCs improved corporal veno-occlusive dysfunction, counteracted corporal decreases in the ratio of SMCs to collagen and fat infiltration in rats with long-term T2D, and upregulated neuronal and endothelial nitric oxide. LD-SCs acquired an inflammatory, pro-fibrotic, oxidative, and dyslipidemic transcriptional phenotype and failed to repair the corporal tissue.ConclusionMDSCs from pre-diabetic rats injected into the corpora cavernosa of rats with long-term T2D improve corporal veno-occlusive dysfunction and the underlying histopathology. In contrast, MDSCs from rats with long-term uncontrolled T2D are imprinted by the hyperglycemic and dyslipidemic milieu with a noxious phenotype associated with an impaired tissue repair capacity. SCs affected by diabetes could lack tissue repair efficacy as autografts and should be reprogrammed in vitro or substituted by SCs from allogenic non-diabetic sources

Implanted Muscle-Derived Stem Cells Ameliorate Erectile Dysfunction in a Rat Model of Type 2 Diabetes, but Their Repair Capacity Is Impaired by Their Prior Exposure to the Diabetic Milieu

INTRODUCTION: Muscle derived stem cells (MDSC) and other stem cells implanted into the penile corpora cavernosa ameliorate erectile dysfunction in type 1 diabetic (T1D) rat models by replenishing the lost corporal smooth muscle cells (SMC) and reducing fibrosis. However, no conclusive data on this question in T2/D/obesity models is available. Aim. We studied whether: a) MDSC from T2D Obese Zucker (OZ) rats at an early stage of diabetes (ED-SC), counteract corporal veno-occlusive dysfunction (CVOD) and corporal SMC loss/lipofibrosis when implanted in the OZ rats at a late stage of diabetes; b) MDSC from these late diabetes OZ rats (LD-SC) differ from ED-SC in their gene transcriptional phenotype and repair capacity. METHODS AND OUTCOMES: ED-SC and LD-SC were compared by DNA microarray assays, and ED-SC were incubated in vitro under high glucose conditions (ED-HG-SC). These three MDSC types were injected into the corpora cavernosa of late diabetes OZ rats (OZ/ED, OZ/LD, and OZ/ED-HG rats respectively). Untreated OZ (OZ/UT) and non-diabetic Lean Zucker (LZ/UT) rats were controls. Two months later, rats were subjected to cavernosometry and the penile shaft and corporal tissues were subjected to histopathology and DNA microarray assays. RESULTS: Implanted ED-SC and ED-HG-SC, improved CVOD, counteracted corporal SMC/collagen decrease and fat infiltration in long-term T2D rats, and upregulated nNOS and eNOS. LD-SC acquired an inflammatory/profibrotic/oxidative/dyslipidemic transcriptional phenotype, and failed to repair the corporal tissue. CONCLUSIONS: MDSC from pre-diabetic rats injected into the corpora cavernosa of long-term diabetic T2D rats improve CVOD and the underlying histopathology. In contrast, MDSC from long-term uncontrolled diabetic T2D rats, are imprinted by the hyperglycemic/dyslipidemic milieu with a noxious phenotype associated with an impaired tissue repair capacity. Diabetes-impacted stem cells may lack tissue repair efficacy as autografts, and should either be reprogrammed in vitro, or substituted by stem cells from allogenic non-diabetic sources

Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture

BackgroundPrevious work showed that muscle-derived stem cells (MDSCs) exposed long-term to the milieu of uncontrolled type 2 diabetes (UC-T2D) in male obese Zucker (OZ) rats, were unable to correct the associated erectile dysfunction and the underlying histopathology when implanted into the corpora cavernosa, and were also imprinted with a noxious gene global transcriptional signature (gene-GTS), suggesting that this may interfere with their use as autografts in stem cell therapy.AimTo ascertain the respective contributions of dyslipidemia and hyperglycemia to this MDSC damage, clarify its mechanism, and design a bioassay to identify the damaged stem cells.MethodsEarly diabetes MDSCs and late diabetes MDSCs were respectively isolated from nearly normal young OZ rats and moderately hyperglycemic and severely dyslipidemic/obese aged rats with erectile dysfunction. Monolayer cultures of early diabetic MDSCs were incubated 4 days in DMEM/10% fetal calf serum + or - aged OZ or lean Zucker serum from non-diabetic lean Zucker rats (0.5-5%) or with soluble palmitic acid (PA) (0.5-2 mM), cholesterol (CHOL) (50-400 mg/dL), or glucose (10-25 mM).Main outcome measureFat infiltration was estimated by Oil red O, apoptosis by TUNEL, protein expression by Western blots, and gene-GTS and microRNA (miR)-GTS were determined in these stem cells' RNA.ResultsAged OZ serum caused fat infiltration, apoptosis, myostatin overexpression, and impaired differentiation. Some of these changes, and also a proliferation decrease occurred with PA and CHOL. The gene-GTS changes by OZ serum did not resemble the in vivo changes, but some occurred with PA and CHOL. The miR-GTS changes by OZ serum, PA, and CHOL resembled most of the in vivo changes. Hyperglycemia did not replicate most alterations.Clinical implicationsMDSCs may be damaged in long-term UC-T2D/obese patients and be ineffective in autologous human stem cell therapy, which may be prevented by excluding the damaged MDSCs.Strength & limitationsThe in vitro test of MDSCs is innovative and fast to define dyslipidemic factors inducing stem cell damage, its mechanism, prevention, and counteraction. Confirmation is required in other T2D/obesity rat models and stem cells (including human), as well as miR-GTS biomarker validation as a stem cell damage biomarker.ConclusionSerum from long-term UC-T2D/obese rats or dyslipidemic factors induces a noxious phenotype and miR-GTS on normal MDSCs, which may lead in vivo to the repair inefficacy of late diabetic MDSCs. This suggests that autograft therapy with MDSCs in long-term UT-T2D obese patients may be ineffective, albeit this may be predictable by prior stem cell miR-GTS tests. Masouminia M, Gelfand R, Kovanecz I, et al. Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture. J Sex Med 2018;15:1678-1697