The function of math5 and myocilin in mammalian eye development and disease

Complex molecular events underlie vertebrate eye development and disease. The eye is composed of two major tissue types: the anterior and posterior segments. During development, the retinal progenitor cells differentiate into six neuronal and one non-neuronal cell types. These cell types later organize into the distinct laminar structure of the mature retina which occupies the posterior segment. In the developed anterior segment, both the ciliary body and trabecular meshwork regulate intraocular pressure created by the aqueous humor. The disruption in intraocular pressure can lead to a blinding condition called glaucoma. To characterize molecular mechanisms governing retinal development and glaucoma, two separate mouse knockout lines carrying mutations in math5 and myocilin were subjected to a series of in vivo analyses. ^ Math5 is a murine homologue of Drosophila atonal , a bHLH proneural gene essential for the formation of photoreceptor cells. The expression of math5 coincides with the onset of retinal ganglion cell differentiation. The targeted deletion of mouse math5 revealed that a null mutation inhibits the formation of a majority of the retinal ganglion cells. The mutation also interferes with the normal development of other retinal cell types such as amacrine, bipolar and photoreceptor cells. These results suggest that math5 is a proneural gene responsible for differentiation of retinal ganglion cells and may also have a role in normal development of other neuronal cell types within the retina. ^ Myocilin has two unique protein coding regions bearing homology to non-muscle myosin of Dictyostelium discoideum and to olfactomedin, an extracellular matrix molecule first described in the olfactory epithelium of the bullfrog. Recently, autosomal dominant forms of myocilin mutations have been found in individuals with primary open-angle glaucoma. The genetic linkage to glaucoma suggests a role of myocilin in normal intraocular pressure and ocular function. However, the analysis of mice heterozygous and homozygous for a targeted null mutation in myocilin indicates that it is dispensable for normal intraocular pressure or ocular function. Additionally, the lack of a discernable phenotype in both heterozygous and null mice suggests that haploinsufficiency is not a critical mechanism for MYOC-associated glaucoma in humans. Instead, disease-causing mutations likely act by gain of function. ^ In summary, these studies provide novel insights into the embryonic development of the vertebrate retina, and also begin to uncover the molecular mechanisms responsible for the pathogenesis of glaucoma. ^

THE MULTIPLE LOCATION TIME WEIGHTED INDEX: USING PATIENT ACTIVITY SPACES TO CALCULATE PRIMARY CARE SERVICE AREAS

Geographic health planning analyses, such as service area calculations, are hampered by a lack of patient-specific geographic data. Using the limited patient address information in patient management systems, planners analyze patient origin based on home address. But activity space research done sparingly in public health and extensively in non-health related arenas uses multiple addresses per person when analyzing accessibility. Also, health care access research has shown that there are many non-geographic factors that influence choice of provider. Most planning methods, however, overlook non-geographic factors influencing choice of provider, and the limited data mean the analyses can only be related to home address. This research attempted to determine to what extent geography plays a part in patient choice of provider and to determine if activity space data can be used to calculate service areas for primary care providers. During Spring 2008, a convenience sample of 384 patients of a locally-funded Community Health Center in Houston, Texas, completed a survey that asked about what factors are important when he or she selects a health care provider. A subset of this group (336) also completed an activity space log that captured location and time data on the places where the patient regularly goes. Survey results indicate that for this patient population, geography plays a role in their choice of health care provider, but it is not the most important reason for choosing a provider. Other factors for choosing a health care provider such as the provider offering “free or low cost visits”, meeting “all of the patient’s health care needs”, and seeing “the patient quickly” were all ranked higher than geographic reasons. Analysis of the patient activity locations shows that activity spaces can be used to create service areas for a single primary care provider. Weighted activity-space-based service areas have the potential to include more patients in the service area since more than one location per patient is used. Further analysis of the logs shows that a reduced set of locations by time and type could be used for this methodology, facilitating ongoing data collection for activity-space-based planning efforts.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

An analysis of the function of region 1.2 of the primary sigma factor, sigma 70, from Escherichia coli

The sigma (σ) subunit of eubacterial RNA polymerase is required for recognition of and transcription initiation from promoter DNA sequences. One family of sigma factors includes those related to the primary sigma factor from E. coli, σ70. Members of the σ70 family have four highly conserved domains, of which regions 2 through 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by σ 70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter specific binding by holoenzyme. This study primarily investigates the functional role of region 1.2 during transcription initiation. In vivo and in vitro characterization of thirty-two single amino acid substitutions targeted to region 1.2 of E. coli σ70 as well as a deletion of region 1.2, revealed that mutations in region 1.2 can affect promoter binding, open complex formation, initiated complex formation, and the transition from abortive transcription to elongation. The relative degree of solvent exposure of several positions in region 1.2 has been determined, with positions 116 and 122 likely to be located near the surface of σ70. ^ During the course of this study, the existence of two “wild type” variants of E. coli σ70 was discovered. The identity of amino acid 149 has been reported variably as either arginine or aspartic acid in published articles and in online databases. In vivo and in vitro characterization of the two reported variations of E. coli σ70 (N149 and D149) has determined that the two variants are functionally equivalent. However, in vivo and in vitro characterization of single amino acid substitutions and a region 1.2 deletion in the context of each variant background revealed that the behavior of some mutations are greatly affected by the identity of amino acid 149. ^

The Open Door Mission: Measuring and predicting outcomes of one community-based substance abuse treatment program

The objectives of this study were to identify and measure the average outcomes of the Open Door Mission's nine-month community-based substance abuse treatment program, identify predictors of successful outcomes, and make recommendations to the Open Door Mission for improving its treatment program.^ The Mission's program is exclusive to adult men who have limited financial resources: most of which were homeless or dependent on parents or other family members for basic living needs. Many, but not all, of these men are either chemically dependent or have a history of substance abuse.^ This study tracked a cohort of the Mission's graduates throughout this one-year study and identified various indicators of success at short-term intervals, which may be predictive of longer-term outcomes. We tracked various levels of 12-step program involvement, as well as other social and spiritual activities, such as church affiliation and recovery support.^ Twenty-four of the 66 subjects, or 36% met the Mission's requirements for success. Specific to this success criteria; Fifty-four, or 82% reported affiliation with a home church; Twenty-six, or 39% reported full-time employment; Sixty-one, or 92% did not report or were not identified as having any post-treatment arrests or incarceration, and; Forty, or 61% reported continuous abstinence from both drugs and alcohol.^ Five research-based hypotheses were developed and tested. The primary analysis tool was the web-based non-parametric dependency modeling tool, B-Course, which revealed some strong associations with certain variables, and helped the researchers generate and test several data-driven hypotheses. Full-time employment is the greatest predictor of abstinence: 95% of those who reported full time employment also reported continuous post-treatment abstinence, while 50% of those working part-time were abstinent and 29% of those with no employment were abstinent. Working with a 12-step sponsor, attending aftercare, and service with others were identified as predictors of abstinence.^ This study demonstrates that associations with abstinence and the ODM success criteria are not simply based on one social or behavioral factor. Rather, these relationships are interdependent, and show that abstinence is achieved and maintained through a combination of several 12-step recovery activities. This study used a simple assessment methodology, which demonstrated strong associations across variables and outcomes, which have practical applicability to the Open Door Mission for improving its treatment program. By leveraging the predictive capability of the various success determination methodologies discussed and developed throughout this study, we can identify accurate outcomes with both validity and reliability. This assessment instrument can also be used as an intervention that, if operationalized to the Mission’s clients during the primary treatment program, may measurably improve the effectiveness and outcomes of the Open Door Mission.^