Ultra-sensitive, rapid detection of dried bloodstains by surface enhanced Raman scattering on Ag substrates.

A simple water extraction and transfer procedure is found to result in reproducible and highly sensitive 785 nm excited SERS spectra of 24 h dried bloodstains on Ag nanoparticle substrates. This protocol allows confirmatory detection and identification of dried stains of blood that have been diluted by up to 105 in water on Ag substrates. While previous SERS results demonstrated similar performance on Au substrates when a 50% acetic acid extraction and transfer procedure was used, the water/Ag methodology avoids any potential DNA damage when the sample size is extremely small (≤∼1 µL) due to low pH exposure. The water only procedure is not effective on Au SERS substrates. This metal substrate difference results from the efficient red blood cell lysis and hemoglobin denaturation effects of the Ag nanoparticle surfaces as compare to that of Au nanoparticles. Consequently, the 50% acetic acid exposure is required for the acquisition of 785 nm SERS spectra of dried bloodstains on Au substrates.

Surface enhanced Raman scattering specificity for detection and identification of dried bloodstains.

Surface enhanced Raman spectroscopy (SERS) provides highly specific vibrational signatures identifying dried blood for a variety of forensic applications. SERS spectra on Au nanoparticle substrates excited at 785 nm are found to identify dried stains of human and nonhuman blood from seven animals, and distinguish stains due to menstrual and peripheral blood. In addition, the unique SERS bloodstain spectrum is distinct from the SERS spectra of thirty red-brown stains of potential household substances that could be visually mistaken for bloodstains and from food stains that have been shown to give positive results with presumptive colorimetric blood tests. Finally, a SERS swab procedure has been developed and demonstrates that the substrates that a blood sample dried on does not offer any Raman or fluorescence interference for the SERS identification of dried blood. Such bloodstains on porous and nonporous materials are all identical and exclusively due to the heme moiety of hemoglobin. Optimized selection of the extraction solvent is found to control the chemical composition of molecular components appearing in the SERS spectrum of complex, multicomponent biological mixtures, such as body fluids.

Surface enhanced Raman scattering for robust, sensitive detection and confirmatory identification of dried bloodstains.

An optimized procedure is described for the acquisition of 785 nm excited SERS spectra of dried bloodstains and shown to offer great potential for rapid, portable, highly sensitive and specific, confirmatory identification for forensic applications. Following extraction in 1 µL of 50% acetic acid, a robust, highly reproducible SERS spectrum is observed from dried bloodstains resulting from a hematin-like heme moiety (ferric, high spin). As anticipated, this blood signature can be classified with 100% specificity and sensitivity with respect to the SERS spectra of other body fluids. High quality SERS spectra can be observed from stains of blood diluted by as much as 105. Dried blood spectra acquired on Au and Ag SERS active substrates exhibit very different relative intensities at this electronically, non-resonant excitation wavelength (785 nm) indicating that a strong chemical effect contributes to the SERS enhancement of this body fluid. DFT calculations further confirm the vibrational band assignments of the features seen in these SERS spectra of dried blood.

An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles.

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.

Haploinsufficiency of steroidogenic factor-1 in mice disrupts adrenal development leading to an impaired stress response.

Adrenal steroids are essential for homeostasis and survival during severe physiological stress. Analysis of a patient heterozygous for the steroidogenic factor-1 (SF-1) gene suggested that reduced expression of this nuclear receptor leads to adrenal failure. We therefore examined SF-1 heterozygous (+/-) mice as a potential model for delineating mechanisms underlying this disease. Here we show that SF-1 +/- mice exhibit adrenal insufficiency resulting from profound defects in adrenal development and organization. However, compensatory mechanisms, such as cellular hypertrophy and increased expression of the rate-limiting steroidogenic protein StAR, help to maintain adrenal function at near normal capacity under basal conditions. In contrast, adrenal deficits in SF-1 heterozygotes are revealed under stressful conditions, demonstrating that normal gene dosage of SF-1 is required for mounting an adequate stress response. Our findings predict that natural variations leading to reduced SF-1 function may underlie some forms of subclinical adrenal insufficiency, which become life threatening during traumatic stress.

Autocrine and paracrine Müllerian inhibiting substance hormone signaling in reproduction.

Members of the transforming growth factor beta (TGFbeta) superfamily are polypeptide growth factors that exhibit diverse effects on normal cell growth, adhesion, mesenchymal-epithelial interactions, cell differentiation, and programmed cell death. This chapter will discuss the work of ourselves and others on one member of this large superfamily, Müllerian inhibiting substance (MIS, or anti-Müllerian hormone, AMH) and its role in reproductive tract development and the adult gonad. Using recombinant MIS protein, it is possible to begin unraveling the molecular mechanism of duct involution in the embryo. Our recent results suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions. In addition to the developmental effects of MIS in secondary sexual differentiation, expression studies of the MIS ligand and the MIS type II receptor (MISIIR) suggest a potential regulatory role for MIS in adult germ cell maturation and gonadal function. Recent data from others suggest that MIS may act in a paracrine manner to block differentiation of interstitial cells of the adult gonad by repressing all or some steps of steroidogenesis. Our studies are highly suggestive of direct repression of steroidogenic enzyme gene expression by activation of the MIS signaling pathway. Thus, for the first time, an opportunity to define fully target genes and components of the MIS signaling pathway may be possible.

Steroidogenic factor-1: its role in endocrine organ development and differentiation.

The cloning of the first steroid hormone receptor over a decade ago provided vital insight into the mechanisms by which steroid hormones activate gene transcription. When bound by hormone, these receptors function as ligand-dependent transcription factors by binding to unique response elements in the promoter of specific target genes. Over 60 receptors have now been characterized in this superfamily of steroid receptors. Many receptors known as orphan receptors have been cloned by homology and have no known ligands but appear to be mediators of endocrine function in the adult and in many cases are essential developmental regulators in endocrine organogenesis. One such receptor is steroidogenic factor-1 (SF-1). While initially cloned as a transcriptional regulator of the various steroidogenic enzyme genes in the adrenal and gonad, it has become clear through genetic ablation experiments in mice that SF-1 is an essential factor in adrenal and gonadal development and for the proper functioning of the hypothalamic-pituitary-gonadal axis. In addition, these studies have revealed that SF-1 is necessary for the formation of the ventromedial nucleus of the hypothalamus. While we have learned much since the initial cloning of SF-1, the mechanisms by which SF-1 regulates these various developmental programs remain elusive. This article focuses on the characterization of SF-1 and its emerging role in endocrine homeostasis. Specific attention is placed on the mechanisms of action of this unique member of the nuclear receptor superfamily.

Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

Paracrine-mediated apoptosis in reproductive tract development.

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions.

Neuronal expression of the 5HT3 serotonin receptor gene requires nuclear factor 1 complexes.

The 5HT3 receptor (5HT3R) is a serotonin-gated ion channel whose expression is restricted to a subset of cells within the central and peripheral nervous systems. In vitro analysis shows that a small proximal region of the TATA-less 5HT3R promoter is sufficient to direct neuronal-specific reporter gene expression. Three potential regulatory elements conserved between the mouse and human genes were identified within this proximal promoter, two of which are known sites for the ubiquitously expressed factors Sp1 and nuclear factor 1 (NF1). Surprisingly, mutation of the NF1 binding site abolished all reporter activity in cell transfection studies, suggesting that this element is essential for neuronal-specific transcriptional activity of the 5HT3R. Furthermore, a complex of neuronal proteins that includes a member(s) of the NF1 family binds to this site, as shown by gel mobility super shift and DNaseI footprinting analyses. Although NF1 has been proposed to mediate basal transcription of many ubiquitously expressed genes, our data suggest that a member of the NF1 transcription factor family participates in neuronal-specific gene expression by promoting interactions with other regulatory factors found in sensory ganglia.

Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression.

Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. In males, SF-1 participates in sexual development by regulating expression of the polypeptide hormone Müllerian inhibiting substance (MIS). Here, we show that WT1 -KTS isoforms associate and synergize with SF-1 to promote MIS expression. In contrast, WT1 missense mutations, associated with male pseudohermaphroditism in Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked, candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between SF-1 and WT1, most likely through a direct interaction with SF-1. We propose that WT1 and Dax-1 functionally oppose each other in testis development by modulating SF-1-mediated transactivation.

Epibulbar molluscum contagiosum.

PURPOSE: To report a case of epibulbar conjunctival nodules from molluscum contagiosum in a patient with atopic dermatitis. METHODS: A 39-year-old man with atopic dermatitis who was treated with oral prednisone was initially examined with ocular itching, foreign body sensation, and conjunctival injection of the right eye and was found to have three discrete conjunctival nodules. Excision of the nodules led to complete resolution of the signs and symptoms. RESULT: Histopathologic examination of the conjunctival specimen disclosed molluscum contagiosum. CONCLUSION: Patients with defective or suppressed cell-mediated immunity are at increased risk of unusual ocular involvement with molluscum contagiosum.

Suction cup/contact lens complications following penetrating keratoplasty.

PURPOSE: Rigid gas permeable (RGP) contact lenses facilitate visual rehabilitation in cases of high or irregular corneal astigmatism following penetrating keratoplasty. A variety of plunger-like suction cup devices are available to assist in the removal of these lenses. METHODS: We report three patients with serious complications associated with the use of a suction cup device for contact lens removal following penetrating keratoplasty. RESULTS: Two patients suffered corneal wound dehiscence following contact lens removal; one contact lens was removed by the patient's spouse and the other was removed by a trained technician. A third patient triggered a graft rejection, and ultimately, graft failure, after a vigorous attempt at lens removal. CONCLUSIONS: Forces generated by suction cup devices during removal of RGP contact lenses are sufficient to cause significant trauma. Contact lenses with an apical clearance fit may augment these forces, with the potential for complications following penetrating keratoplasty.

The nuclear receptor SF-1 mediates sexually dimorphic expression of Mullerian Inhibiting Substance, in vivo.

Mullerian Inhibiting Substance (MIS) functions to promote regression of the Mullerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.

Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease.

During male gonadal development Müllerian duct regression is mediated by the actions of the hormone Müllerian inhibiting substance (MIS), a member of the transforming growth factor beta superfamily. MIS is considered to be unique among members of this superfamily because bioactivation of MIS via proteolytic processing is hypothesized to occur at its target organ, the Müllerian duct. We find instead that the majority of MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains noncovalently associated with the prodomain. In addition, we have identified two candidate endoproteases that are expressed in the testes and that may be capable of processing MIS in vivo. These kex2/subtilisin-like enzymes, PC5 and furin, are members of the proprotein convertase family that have been implicated in hormone bioactivation via proteolytic processing after dibasic amino acid cleavage recognition sites. Coexpression of PC5 and MIS in transfected mammalian cells results in efficient processing and bioactivation of MIS. Our results suggest that MIS is a natural substrate for PC5, thereby supporting a role for prohormone convertases in the activation of transforming growth factor beta-related hormones during development.

Infectious keratitis with corneal perforation associated with corneal hydrops and contact lens wear in keratoconus.

BACKGROUND: Corneal perforation is an uncommon complication associated with keratoconus. The first cases of infectious keratitis and corneal perforation associated with corneal hydrops and contact lens wear are reported in two keratoconus patients. METHODS: A retrospective chart review and histopathological examination were carried out. RESULTS: Both patients progressed to corneal perforation and emergency penetrating keratoplasty. One patient cultured Fusarium and the second patient Serratia marcesens. Both patients wore contact lenses against medical advice. CONCLUSIONS: The tear in Descement's membrane, stromal oedema, and epithelial bedewing associated with corneal hydrops results in loss of the epithelial-endothelial barrier of the cornea, creating a conduit for infectious organisms through the cornea. Acute hydrops associated with epithelial keratitis, stromal swelling, and a Descement's membrane tear may be a significant risk factor for infectious keratitis and corneal perforation. Contact lenses should not be worn during an active corneal hydrops owing to the increased risk for severe infectious keratitis and corneal perforation.