Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general.transcription factors | neural development | retinal development | cell fate determination | gene regulation

Our current study focuses on the expression of two members of the onecut transcription factor family, One-cut1 (Oc1) and Onecut2 (Oc2), in the developing mouse retina. By immunofluorescence staining, we found that Oc1 and Oc2 had very similar expression patterns throughout retinal development. Both factors started to be expressed in the retina at around embryonic day (E) 11.5. At early stages (E11.5 and E12.5), they were expressed in both the neuroblast layer (NBL) and ganglion cell layer (GCL). As development progressed (from E14.5 to postnatal day [P] 0), expression diminished in the retinal progenitor cells and became more restricted to the GCL. By P5, Oc1 and Oc2 were expressed at very low levels in the GCL. By co-labeling with transcription factors known to be involved in retinal ganglion cell (RGC) development, we found that Oc1 and Oc2 had extensive overlap with Math5 in the NBL, and that they completely overlapped with Pou4f2 and Isl1 in the GCL, but only partially in the NBL. Co-labeling of Oc1 with cell cycle markers confirmed that Oc1 was expressed in both proliferating retinal progenitors and postmitotic retinal cells. In addition, we demonstrated that expression of Oc1 and Oc2 did not require Math5, Isl1, or Pou4f2. Thus, Oc1 and Oc2 may regulate the formation of RGCs in a pathway independent of Math5, Pou4f2, and Isl1. Furthermore, we showed that Oc1 and Oc2 were expressed in both developing and mature horizontal cells (HCs). Therefore the two factors may also function in the genesis and maintenance of HCs. J. Comp. Neurol. 520:952–969, 2012.

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.

Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (ϳ80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.

Background: Methicillin resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community infections. Its prevalence varies with country and with hospitals within a country. The current study estimates the prevalence of MRSA strains and investigates their antibiogram in western Nepal. Methodology: A total of 162 S. aureus strains were isolated from various clinical specimens, and antibiotic susceptibility tests were performed using disc diffusion, growth on oxacillin screen agar, and oxacillin minimum inhibitory concentration (MIC). Results: One hundred and twelve (69.1%) strains were found to be MRSA, of which 37 (33.1%) were community acquired and 75 (66.9%) were hospital acquired. Of 112 MRSA strains, 45 (40.1%) were multi-drug resistant. All MRSA strains were found resistant to penicillin, and 91.9%, 87.4%, 77%, and 55.5% were resistant to amoxicillin, ampicillin, trimethoprim/sulfamethoxazole, and cephalexin, respectively. However, low resistance was observed with amikacin (19%), ciprofloxacin (26.5%), and norfloxacin (30.6%). All strains were sensitive to vancomycin. Conclusion: The reported rate of MRSA prevalence is alarming. Given the ability of MRSA to spread from person to person, it is necessary to adhere to rational use of antibiotics and to raise awareness among the concerned communities and tourists who visit this area.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community pathogen. The objectives of this study were to estimate the prevalence of multidrug-resistant MRSA strains in clinical specimens and to investigate the sensitivity pattern of these strains against various antibiotics used for treating hospitalized and out patients. Strains were identified using standard procedures, and their sensitivity pattern was investigated using such techniques as disc diffusion, minimum inhibitory concentration (MIC), and the mecA gene PCR. Among 783 isolates of S. aureus, 301 (38.44%) were methicillin-resistant, of which 217 (72.1%) were found to be multidrug-resistant. Almost all MRSA strains were resistant to penicillin, 95.68% were resistant to cotrimoxazole, 92.36% were resistant to chloramphenicol, 90.7% were resistant to norfloxacin, 76.1% were resistant to tetracycline, and 75.75% were resistant to ciprofloxacin. Vancomycin was the most effective drug, with only 0.33% of MRSA strains being resistant to it. It is concluded that antibiotics other than vancomycin can be used as anti-MRSA agents after a sensitivity test so as to preclude the emergence of resistance to it and that prevailing problems in chemotherapy will escalate unless indiscriminate and irrational usage of antibiotics is checked.

SUMMARY Alternative translation initiation and stop codon readthrough in a few well-studied cases have been shown to allow the same transcript to generate multiple protein variants. Because the brain shows a particularly abundant use of alternative splicing, we sought to study alternative translation in CNS cells. We show that alternative translation is widespread and regulated across brain transcripts. In neural cultures, we identify alternative initiation on hundreds of transcripts, confirm several N-terminal protein variants, and show the modulation of the phenomenon by KCl stimulation. We also detect readthrough in cultures and show differential levels of normal and readthrough versions of AQP4 in gliotic diseases. Finally, we couple translating ribosome affinity purification to ribosome footprinting (TRAP-RF) for cell-type-specific analysis of neuronal and astrocytic translational readthrough in the mouse brain. We demonstrate that this unappreciated mechanism generates numerous and diverse protein isoforms in a cell-type-specific manner in the brain.

Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.