Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) regulates the concentration of all-trans retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. The mechanisms regulating Cyp26b1 expression in postnatal Sertoli cells, the main components of the stem cell niche, are so far unknown. During gonad development, expression of Cyp26b1 is maintained by Steroidogenic Factor 1 (SF-1) and Sex-Determining Region Y Box-9 (SOX9), which ensure that RA is degraded and germ cell differentiation is blocked. Here, we show that the NOTCH target Hairy/Enhancer-of-Split Related with YRPW Motif 1 (HEY1), a transcriptional repressor, regulates germ cell differentiation via direct binding to the Cyp26b1 promoter and thus inhibits its expression in Sertoli cells. Further, using in vivo germ cell ablation, we demonstrate that undifferentiated type A spermatogonia are the cells that activate NOTCH signaling in Sertoli cells through their expression of the NOTCH ligand JAGGED-1 (JAG1) at stage VIII of the seminiferous epithelium cycle, therefore mediating germ cell differentiation by a ligand concentration-dependent process. These data therefore provide more insights into the mechanisms of germ cell differentiation after birth and potentially explain the spatiotemporal RA pulses driving the transition between undifferentiated to differentiating spermatogonia.-Parekh, P. A., Garcia, T. X., Waheeb, R., Jain, V., Gandhi, P., Meistrich, M. L., Shetty, G., Hofmann, M.-C. Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Regulation of GDNF expression in Sertoli cells.

Sertoli cells regulate male germ cell proliferation and differentiation and are a critical component of the spermatogonial stem cell (SSC) niche, where homeostasis is maintained by the interplay of several signaling pathways and growth factors. These factors are secreted by Sertoli cells located within the seminiferous epithelium, and by interstitial cells residing between the seminiferous tubules. Sertoli cells and peritubular myoid cells produce glial cell line-derived neurotrophic factor (GDNF), which binds to the RET/GFRA1 receptor complex at the surface of undifferentiated spermatogonia. GDNF is known for its ability to drive SSC self-renewal and proliferation of their direct cell progeny. Even though the effects of GDNF are well studied, our understanding of the regulation its expression is still limited. The purpose of this review is to discuss how GDNF expression in Sertoli cells is modulated within the niche, and how these mechanisms impact germ cell homeostasis.

Biomarkers in cancer therapy related cardiac dysfunction (CTRCD).

Biomarkers are at the cornerstone of preventive measures and contribute to the screening process. More recently, biomarkers have been used to gauge the biological response to the employed therapies. Since it is ubiquitously used to detect subclinical disease process, biomarkers also have found its place in cancer therapy related cardiac dysfunction (CTRCD). The aim of this review is to comprehensively present up-to-date knowledge of biomarkers in CTRCD and highlight some of the future biomedical technologies that may strengthen the screening process, and/or provide new insight in pathological mechanisms behind CTRCD.

Advances in nanoprobes for molecular MRI of Alzheimer's disease.

Alzheimer's disease is the most common cause of dementia and a leading cause of mortality in the elderly population. Diagnosis of Alzheimer's disease has traditionally relied on evaluation of clinical symptoms for cognitive impairment with a definitive diagnosis requiring post-mortem demonstration of neuropathology. However, advances in disease pathogenesis have revealed that patients exhibit Alzheimer's disease pathology several decades before the manifestation of clinical symptoms. Magnetic resonance imaging (MRI) plays an important role in the management of patients with Alzheimer's disease. The clinical availability of molecular MRI (mMRI) contrast agents can revolutionize the diagnosis of Alzheimer's disease. In this article, we review advances in nanoparticle contrast agents, also referred to as nanoprobes, for mMRI of Alzheimer's disease. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease.

Biostable ssDNA aptamers specific for Hodgkin lymphoma.

As a "chemical antibody", oligonucleotide aptamers can specifically bind to their target molecules. However, clinical potential of aptamers in disease diagnosis is not yet fully explored. Using a tumor cell-based selection protocol, we developed single-stranded DNA aptamers for Hodgkin lymphoma (HL) tumor cells. The aptamers specifically bound to HL cells with a high affinity, reaching maximal cell binding at 10 nM final concentration. Importantly, the aptamers were able to selectively detect HL cells and did not react to other tumor or blood cells in mixed samples, indicating that the aptamers can be used as a specific probe for in vitro analysis of HL cells. Moreover, due to the inherent properties of DNA, the aptamers were stable in human serum, suggesting potential for in vivo detection of HL tumor cells.

Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate.

Vascular endothelial growth factor A (VEGF-A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemet's membrane. Descemet's membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10-h time course in the absence of SOS. Diffusion studies with VEGF121, a non-heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell-free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin-coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin-rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin-rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemet's membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues.

A surrogate marker for very early-stage tau pathology is detectable by molecular magnetic resonance imaging.

The abnormal phosphorylation of tau is a necessary precursor to the formation of tau fibrils, a marker of Alzheimer's disease. We hypothesize that hyperphosphorylative conditions may result in unique cell surface markers. We identify and demonstrate the utility of such surrogate markers to identify the hyperphosphorylative state. Methods: Cell SELEX was used to identify novel thioaptamers specifically binding hyperphosphorylative cells. Cell surface vimentin was identified as a potential binding target of the aptamer. Novel molecular magnetic resonance imaging (M-MRI) probes using these aptamers and a small molecule ligand to vimentin were used for in vivo detection of this pre-pathological state. Results: In a mouse model of pathological tau, we demonstrated in vivo visualization of the hyperphosphorylative state by M-MRI, enabling the identification at a pre-pathological stage of mice that develop frank tau pathology several months later. In vivo visualization of the hyperphosphorylative state by M-MRI was further validated in a second mouse model (APP/PS1) of Alzheimer's disease again identifying the mutants at a pre-pathological stage. Conclusions: M-MRI of the hyperphosphorylative state identifies future tau pathology and could enable extremely early-stage diagnosis of Alzheimer's disease, at a pre-patholgical stage.

Aptamers recognizing glycosylated hemagglutinin expressed on the surface of vaccinia virus-infected cells.

Traditional methods for detection and identification of pathogenic viruses or bacteria tend to be slow and cumbersome. We have developed aptamer probes with the capacity to rapidly detect the presence of viral infection with specificity and sensitivity. Vaccinia virus (VV) was chosen as the model because it is closely related to variola virus that causes smallpox. A method known as cell-SELEX (systematic evolution of ligands by exponential enrichment) was used to generate very selective and highly specific aptamers designed to recognize proteins expressed on the surface of VV-infected cells. Characterization of the aptamers showed that the virus-encoded hemagglutinin, a protein expressed on the surface of infected cells, is the preferential binding target. These studies show the feasibility of generating aptamers against a given specific infectious agent and will enable further development of aptamers as diagnostic and/or therapeutic tools against a broad range of infectious agents.

Generating aptamers for recognition of virus-infected cells.

BACKGROUND: The development of molecular probes capable of recognizing virus-infected cells is essential to meet the serious clinical, therapeutic, and national-security challenges confronting virology today. We report the development of DNA aptamers as probes for the selective targeting of virus-infected living cells. METHODS: To create aptamer probes capable of recognizing virus-infected cells, we used cell-SELEX (systematic evolution of ligands via exponential enrichment), which uses intact infected live cells as targets for aptamer selection. In this study, vaccinia virus-infected and -uninfected lung cancer A549 cells were chosen to develop our model probes. RESULTS: A panel of aptamers has been evolved by means of the infected cell-SELEX procedure. The results demonstrate that the aptamers bind selectively to vaccinia virus-infected A549 cells with apparent equilibrium dissociation constants in the nanomolar range. In addition, these aptamers can specifically recognize a variety of target infected cell lines. The aptamers' target is most likely a viral protein located on the cell surface. CONCLUSIONS: The success of developing a panel of DNA-aptamer probes capable of recognizing virus-infected cells via a whole living cell-SELEX selection strategy may increase our understanding of the molecular signatures of infected cells. Our findings suggest that aptamers can be developed as molecular probes for use as diagnostic and therapeutic reagents and for facilitating drug delivery against infected cells.

Electron microscopic investigation of the lens capsule and conjunctival tissues in individuals with clinically unilateral pseudoexfoliation syndrome.

PURPOSE: To determine the presence of pseudoexfoliative material in the unaffected eyes of patients with clinically unilateral pseudoexfoliation syndrome. DESIGN: Prospective observational case series. PARTICIPANTS: Thirty-two consecutive patients with clinically unilateral pseudoexfoliation syndrome, undergoing routine cataract surgery. METHODS: Transmission electron microscopy (TEM) was used to examine conjunctival and anterior lens capsule specimens in affected and unaffected eyes. MAIN OUTCOME MEASURE: Presence of characteristic pseudoexfoliation syndrome findings on TEM. RESULTS: Transmission electron microscopy demonstrated pseudoexfoliative material on either the anterior capsule or conjunctival sample from the clinically unaffected eye in 26 of the 32 patients with clinically unilateral pseudoexfoliation syndrome (81%; 95% confidence interval, 64%-93%). CONCLUSION: The results suggest that the seemingly uninvolved eye in a patient with clinically unilateral pseudoexfoliation syndrome has an 81% likelihood of being affected ultrastructurally. Several population studies examining conversion rates from unilateral to bilateral disease have found a similar proportion of patients with bilateral pseudoexfoliation syndrome in the later decades of life.

Using aptamers to study protein-protein interactions.

The emerging science of systems biology focuses on the systematic study of complex interactions in whole biological systems. A systemic, or integrative, methodology is employed as the chief means of discovering new properties and understanding the aggregate of processes that occur in a biological system. Accordingly, the Human Genome Project has provided a complete map of genes and resultant proteins corresponding to their function. Protein-protein interactions are important pieces of this biological tapestry, and understanding how they work cooperatively in a cell will result in a better understanding of the whole organism. To accomplish this objective, we report the use of DNA/RNA aptamers as a novel tool for the study and elucidation of protein-protein interactions, both in vivo and in vitro.

Subluxation of suture-fixated posterior chamber intraocular lenses a clinicopathologic study.

PURPOSE: To report the occurrence of subluxation of suture-fixated posterior chamber (PC) intraocular lenses (IOL) and elucidate the mechanisms involved. DESIGN: Prospective clinicopathologic study. PARTICIPANTS: A single 10-0 Prolene suture explanted from a patient who experienced subluxation of his PC-IOL, 11.5 years after placement. Furthermore, multiple 10-0 Prolene sutures and PC-IOLs used for iris fixation were studied as controls. METHODS: Scanning electron microscopy (SEM) was used to analyze the surface of the explanted suture. In addition, randomly selected 10-0 Prolene sutures cut with Vannas scissors and cut with the positioning holes of a randomly selected PC-IOL identical to that implanted in the patient's eye were examined as controls. Finally, the positioning holes of several randomly selected, iris-fixated PC-IOLs were studied using SEM with particular attention to surface quality and edge finish. MAIN OUTCOME MEASURES: Presence of any signs of suture degradation, the character of the cut edge of the suture, as well as the characteristics of the positioning holes of the PC-IOLs. RESULTS: Scanning electron microscopy of the explanted suture revealed sharply cut edges, without significant degradation of the suture, and no intact loop. Scanning electron microscopy of the control suture cut with a PC-IOL demonstrated a similarly cut edge. The positioning holes of the examined PC-IOLs had a sharp edge, and some also had an imperfect finish. CONCLUSION: We conclude that the surface properties of the positioning holes lead to cutting of the suture, and subsequent subluxation of the PC-IOL.

The NOTCH Ligand JAG1 Regulates GDNF Expression in Sertoli Cells.

In the seminiferous epithelium of the testis, Sertoli cells are key niche cells directing proliferation and differentiation of spermatogonial stem cells (SSCs) into spermatozoa. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), which is essential for SSC self-renewal and progenitor expansion. While the role of GDNF in the testis stem cell niche is established, little is known about how this factor is regulated. Our previous studies on NOTCH activity in Sertoli cells demonstrated a role of this pathway in limiting stem/progenitor cell numbers, thus ultimately downregulating sperm cell output. In this study we demonstrate through a double-mutant mouse model that NOTCH signaling in Sertoli cells functions solely through the canonical pathway. Further, we demonstrate through Dual luciferase assay and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis that the NOTCH targets HES1 and HEY1, which are transcriptional repressors, directly downregulate GDNF expression by binding to the Gdnf promoter, thus antagonizing the effects of FSH/cAMP. Finally, we demonstrate that testicular stem/progenitors cells are activating NOTCH signaling in Sertoli cells in vivo and in vitro through the NOTCH ligand JAG1 at their surface, indicating that these cells may ensure their own homeostasis through negative feedback regulation.

Chart documentation by general physicians of the glaucoma medications taken by their patients.

PURPOSE: To estimate the frequency of documentation of glaucoma medications by primary care physicians. DESIGN: Cross-sectional, observational study. METHODS: The general medical records of 100 patients of one glaucoma specialist were reviewed. We recorded whether the mention of eyedrops appeared in the medical record. RESULTS: The median number of glaucoma medications used was 2.5 (range 1 to 5). Fifty-five (55%, 95% confidence interval: 45%-65%) of the medical records of the primary physicians mentioned eyedrops. Alpha-agonists were statistically less frequently documented (13%) in the general medical record than beta-adrenergic blockers (47%) and prostaglandins (44%). CONCLUSION: Almost half of the charts of these primary physicians had no documentation of any eyedrop use by their patients with glaucoma. An important step in reducing drug-induced side effects and interactions with other medications would be better recognition by primary physicians of the ophthalmic drugs used by their patients.

Preferences for eye drop characteristics among glaucoma specialists: a willingness-to-pay analysis.

PURPOSE: To determine the importance that US glaucoma specialists place on attributes of eye drops for lowering intraocular pressure. MATERIALS AND METHODS: We performed a cross-sectional survey by conducting a telephone interview with 113 members of the American Glaucoma Society. We administered a willingness-to-pay (WTP) instrument asking glaucoma specialists how much they would pay to obtain particular characteristics in an eye drop. Demographic data were correlated with the WTP responses. We compared the glaucoma specialists' responses to those previously obtained from patients. The main outcome measure was willingness-to-pay more (in US dollars). RESULTS: Almost all respondents were willing to pay extra to reduce the frequency of administration of eye drops from 3 times a day to once a day, and to avoid blurred vision, drowsiness, or inhibition of sexual performance. Only 54 (48%) were willing to pay more to avoid iris darkening. The mean amount that respondents were willing to pay (relative to US 50 dollars) differed significantly across eye drop characteristics (P < 0.001). The mean amount that the respondents were willing to pay was highest for avoidance of inhibition of sexual performance (US 105 dollars), blurred vision (US 92 dollars), and drowsiness (US 92 dollars). When compared with glaucoma and glaucoma suspect patients, more ophthalmologists were likely to pay extra for desirable eye drop attributes. However, the magnitude of the extra amount was similar between ophthalmologists and patients. CONCLUSIONS: Glaucoma specialists place differing value on various eye drop characteristics. Although the proportion of glaucoma specialists willing to pay more is generally greater than the proportion of patients, the preferences of glaucoma specialists and patients are alike.

Diurnal axial length fluctuations in human eyes.

PURPOSE: This study sought diurnal variations of eye length in human subjects, analogous to those reported in laboratory animals. METHODS: Seventeen subjects, ages 7 to 53 (median 16) years and mean spherical equivalent refractive error -0.68 D (range, -3.00 to +1.00 D), underwent axial length measurements at multiple times during the day between 7 AM and 1 AM the following day, using partial coherence interferometry (PCI), a highly precise, noncontact method. Diurnal axial length measurements were obtained on two or more days in 10 of these subjects. RESULTS: During at least 1 day, 15 subjects showed a statistically significant (ANOVA, P < 0.05) diurnal fluctuation of axial length, with a magnitude generally between 15 and 40 microm. From the diurnal tracings that fit a sine curve using statistical criteria, the mean period of fluctuation was 21.6 +/- 4.33 hours (SD), the mean amplitude was 27.1 +/- 11.9 microm (SD; range, 12.8-41.4 microm), and the maximum axial length tended to occur at midday. Each of the subjects with multiple daily measurements showed axial length fluctuations on at least 1 day, but there were day-to-day differences in the diurnal variations: most notably, four subjects showed axial length fluctuations on each day; in others, the fluctuations were not observed on each testing day. CONCLUSIONS: The human eye undergoes diurnal fluctuations in axial length, with a pattern suggesting maximum axial length at midday. Based on repeated measurements, these daily fluctuations may not appear regularly in all subjects, suggesting the possibility of physiologic influences that must be defined.

Penicillin G acylase-fatty lipid biocomposite films show excellent catalytic activity and long term stability/reusability.

The formation of biocomposite films of the pharmaceutically important enzyme penicillin G acylase (PGA) and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein the enzyme diffuses into the lipid film during immersion in the enzyme solution, thereby leading to the formation of a biocomposite film. The incorporation of the enzyme in both cationic as well as anionic lipids suggests the important role of secondary interactions such as hydrophobic and hydrogen bonding in the enzyme immobilization process. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimentry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by Fourier transform infrared spectroscopy (FTIR) and biocatalytic activity measurements. Whereas the biological activity of the lipid-immobilized enzyme was marginally higher than that of the free enzyme, the biocomposite film exhibited increased thermal/temporal stability. Particularly exciting was the observation that the biocomposite films could be reused in biocatalysis reactions without significant loss in activity, which indicates potentially exciting biomedical/industrial application of these films.

Candida bombicola cells immobilized on patterned lipid films as enzyme sources for the transformation of arachidonic acid to 20-HETE.

Preparation of biocompatible surfaces for immobilization of enzymes and whole cells is an important aspect of biotechnology due to their potential applications in biocatalysis, biosensing, and immunological applications. In this report, patterned thermally evaporated octadecylamine (ODA) films are used for the immobilization of Candida bombicola cells. The attachment of the cells to the ODA film surface occurs possibly through nonspecific interactions such as hydrophobic interactions between the cell walls and the ODA molecules. The enzyme cytochrome P450 present in the immobilized yeast cells on the ODA film surface was used for the transformation of the arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The assembly of cells on the hydrophobic ODA surface was confirmed by quartz crystal microgravimetry (QCM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SEM images confirmed the strong binding of the yeast cells to the ODA film surface after biocatalytic reactions. Moreover, the biocomposite films could be easily separated from the reaction medium and reused.

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers.

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (Kd=1.59nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.

Specific and sensitive tumor imaging using biostable oligonucleotide aptamer probes.

Although several imaging modalities are widely used for tumor imaging, none are tumor type-specific. Different types of cancer exhibit differential therapeutic responses, thus necessitating development of an imaging modality able to detect various tumor types with high specificity. To illustrate this point, CD30-specific oligonucleotide aptamer in vivo imaging probes were conjugated to the near-infrared IRD800CW reporter. Mice bearing xenografted CD30-positive or control CD30-negative lymphoma tumors on contralateral sides of the same mouse were developed. Following a systemic administration of aptamer probes, whole body imaging of tumor-bearing mice was performed. Imaging signal from tumor sites was analyzed and imaging specificity confirmed by tissue immunostaining. The in vivo biodistribution of aptamer probes was also evaluated. Whole body scans revealed that the RNA-based aptamer probes selectively highlighted CD30-expressing lymphoma tumors immediately after systemic administration, but did not react with control tumors in the same mouse. The resultant imaging signal lasted up to 1 hr and the aptamer probes were rapidly eliminated from the body through urinary and lower intestinal tracts. For more sensitive imaging, biostable CD30-specific ssDNA-based aptamer probes were also generated. Systemic administration of these probes also selectively highlighted the CD30-positive lymphoma tumors, with imaging signal detected 4-5 folds higher than that derived from control tumors in the same animal, and lasted for up to 24hr. This study demonstrates that oligonucleotide aptamer probes can provide tumor type-specific imaging with high sensitivity and a long-lasting signal, indicating their potential for clinical applications.