Lhx2 promoter to regulate Cre recombinase expression in transgenic mice we have been able to define a distinct progenitor cell population in the forebrain solely committed to eye development. Conditional inactivation of Lhx2 in these progenitor cells causes an arrest in eye development at the stage when the optic vesicle induces lens placode formation in the surface ectoderm. The eye-committed progenitor cell population is present in the −/−Lhx2 embryonic forebrain suggesting that commitment to eye development is Lhx2-independent. However, re-expression of Lhx2 in −/−Lhx2 progenitor cells only promotes development of retinal pigment epithelium cells, indicating that Lhx2 promotes the acquisition of the oligopotent fate of these progenitor cells. This approach also allowed us to identify genes that distinguish Lhx2 function in eye development from that in the forebrain. Thus, we have defined a distinct progenitor cell population in the forebrain committed to eye development and identified genes linked to Lhx2's function in the expansion and patterning of these progenitor cells.Progenitor cells committed to eye development become specified in the prospective forebrain and develop subsequently into the optic vesicle and the optic cup. The optic vesicle induces formation of the lens placode in surface ectoderm from which the lens develops. Numerous transcription factors are involved in this process, including the eye-field transcription factors. However, many of these transcription factors also regulate the patterning of the anterior neural plate and their specific role in eye development is difficult to discern since eye-committed progenitor cells are poorly defined. By using a specific part of the  The vertebrate eye is a complex and highly specialised neurosensory organ that converts light (photons) into electro-chemical pulses that the brain can translate into images. The development of the eye proceeds through co-ordinated interactions between tissues of different embryonic origin. Immediately after initiation of gastrulation, the eye field is specified in the anterior neural plate The development of the eye is regulated by a number of signalling pathways active at different time points during morphogenesis Lhx2, Pax6, Rx, Tlx, Six6, Six3, and ETMitf whereas cells in close proximity to the lens placode, receiving Fgf signalling, start to express Vsx2 . Vsx2 represses Mitf expression which specifies the neural retina Mitf expression specifying the RPE cells Pax2Sox2, Pax6 and Six3These patterning morphogens impose their actions by activating a cascade of transcription factors that establish cellular identity and subsequent interactions with the environment. The expression of a combination of transcription factors in cells in the anterior neural plate defines the eye field. These transcription factors are collectively referred to as the eye field transcription factors and include Lhx2 has been shown to be important in the development of the eyes since eye development is arrested at the optic vesicle stage prior to lens placode induction in −/−Lhx2 mouse embryos Lhx2 in eye development has also been revealed by the observation that over-expression of various combination of eye field transcription factors other than Lhx2 only induce ectopic eyes when endogenous Lhx2 expression is induced Lhx2 is expressed in the anterior neural plate prior to the formation of the optic vesicle and the Lhx2 null embryos have severe defects in other forebrain structures, such as the cerebral cortex and the hippocampus, revealing that Lhx2 is also involved in the patterning of the forebrain Importantly, many of the eye field transcription factors have additional function(s) in the forebrain neuroectoderm as revealed by defective forebrain development in the respective homozygous mutant mouse embryos Lhx2 function(s) at various stages of eye development it is necessary to distinguish its function prior to and after this commitment step. We therefore developed a novel Cre transgenic mouse strain, denoted Lhx2-Cre, where Cre expression was regulated by an 11 kb genomic region of the Lhx2 promoter immediately upstream of the transcriptional start. Cre expression was not detected in all Lhx2 expressing cells; it rather defined a progenitor cells in the optic pit of the prospective forebrain at embryonic day 8.25 (E8.25) committed to generate the neural part of the eye. Thus, the Lhx2-Cre mouse strain will be a very useful tool to examine the function of genes prior to and after commitment of cells during eye development and in the anterior neural plate. Conditional inactivation of Lhx2 in the Cre+ cells led to a developmental arrest just prior to formation of the optic cup and a subsequent deterioration of the optic vesicle. The optic vesicle developed further in these embryos compared with the conventional −/−Lhx2 embryos. Moreover, genes important for lens differentiation were induced in the surface ectoderm leading to the formation of a lens placode. However, further development of the lens placode required maintained Lhx2 expression since the lens placode regressed in mutant embryos. Cre expression was detected in the optic vesicle in −/−Lhx2 embryos suggesting that commitment to eye development in the neural plate is independent of Lhx2 expression. Expression of transgenic Lhx2 in the Cre+ cell in the −/−Lhx2 background could partly rescue eye development as only cells of the RPE layer developed in these animals. Furthermore, by analysing the expression pattern of a number of genes putatively regulated by Lhx2 in a different cellular context Lhx2 function also in eye development.Identification of the cells in the early forebrain solely committed to eye development would be an important step in examining the function of a gene prior to and after commitment to eye development. To elucidate the molecular basis for Lhx2 gene is widely expressed in the prospective forebrain including the optic vesicle/cup, neural retina, optic stalk and RPE cells Lhx2 promoter to drive expression Cre recombinase in transgenic mice. We initially used a 5 kb and an 11 kb genomic region immediately upstream of the Lhx2 transcriptional start site to drive expression of the Cre. We crossed the Lhx2-Cre transgenic mice to the ROSA26 Reporter (ROSA26R) mouse strain to generate Lhx2-Cre:ROSA26R double transgenic mice. These mice enabled the detection of expression and functional activity of Cre recombinase and therefore permitted lineage tracing of Cre expressing cells based on β-Galactosidase  activity Cre in transgenic mice (data not shown). However, when the 11 kb region was used to drive Cre expression in the double transgenic animals we reproducible observed β-Gal+ cells in the neural part of the eye , whereas very few β-Gal+ cells could be detected in the developing forebrain where the endogenous Lhx2 gene is highly expressed -Cre transgenic founder mouse strains  until E12.5  flox/floxLhx2-Cre:Lhx2 mice obtained were anophthalmic and histological sections of newborn mice revealed that all eye structures were lacking in the flox/floxLhx2-Cre:Lhx2 mice  we reproducibly noticed a few cells with an inactivated Lhx2 gene in the optic vesicle (data not shown), and at E9.5 (ss24–28) the Lhx2 gene has been efficiently inactivated in the entire optic vesicle  mutant since the optic vesicle in the conditional mutant comes in close contact with the surface ectoderm and appears to induce a thickening of the surface ectoderm indicative of lens placode formation    . Bmp4 exss18–21) . However vesicle . These rLhx2 mediated function in eye development, we tried to identify novel genes/pathways putatively linked to Lhx2 function. To achieve this we took advantage of a previous global gene expression analysis comparing Lhx2+ stem cells to their Lhx2− progeny in a different cellular context Lhx2 target genes identified in this screen had gene expression pattern in vivo in various organs that overlapped with that of Lhx2, suggesting partly overlapping mechanisms for Lhx2 function in different tissues. Two of these genes, Enc1 and Nuak1, had an expression pattern consistent with this assumption in eye development. Both were expressed in Lhx2 expressing domains, Enc1 was expressed in the prospective neural retina and lens whereas Nuak1 was expressed in the prospective forebrain, but Nuak1 was excluded from the optic cup , further supporting the idea that Fgf15 is mainly a downstream target of BMP4 signalling in the neural retina. In contrast, Nuak1 expression extended into the domain from where it was excluded in the control when Lhx2 was inactivated , where Cre-mediated recombination induces Lhx2-GFP expression  we could observe a bilaterally located small mass of pigmented cells where the eye is normally located  mice, which is a reliable indicator for functional Lhx2 expression in Z/Lhx2-GFP mice after Cre mediated recombination of the double reporter transgene GFP expression in the neural part of the eye at different developmental stages in all Lhx2-Cre:Z/Lhx2-GFP embryos analysed (n = 4) . genotype , and allhthalmic . However located , suggestand Pax6 , whereasdetected , further (n = 4) , suggestLhx2 promoter to regulate the expression of the Cre recombinase we have been able to identify cells in the forebrain solely committed to generate the neural part of the eye, suggesting that these Cre+ cells are the earliest cells committed to eye development. Commitment to this progenitor cell fate is independent of prior Lhx2 expression in the anterior neural plate, and we propose that Lhx2 promote the acquisition of the oligopotent state of this progenitor cell population in addition to its requirement for the subsequent differentiation of the optic vesicle into the optic cup. Eye development progresses further in the conditional mutant compared to the conventional −/−Lhx2 mutant mice since the optic vesicle comes in direct contact with the surface ectoderm and induces a lens placode. However, immediately after the developmental arrest of the optic vesicle in the conditional mutant, both the optic vesicle and the lens placode degenerate leading to a complete lack of these structures. These result reveal that lens development require continuous interactions between the lens placode and the optic vesicle even if the lens placode has acquired many of its molecular characteristics. We have compared the expression pattern of some genes involved in eye development in the optic vesicle and lens placode between the conditional mutant (flox/floxLhx2-Cre-Lhx2 results presented herein) and the conventional mutant  before and after commitment and that the difference is not solely due to the delayed inactivation of the Lhx2 gene in the conditional mutant. We have also identified novel genes putatively coupled to Lhx2 function during eye development that previously have been shown to be linked to Lhx2 function in another stem/progenitor cell system. This approach also allowed us to distinguish the function of Lhx2 in the forebrain to that in eye development.By using a defined part of the Lhx2 mutant has previously shown that Lhx2 is important for the optic vesicle to optic cup transformation Lhx2 is expressed in the entire prospective forebrain prior to optic vesicle formation it is difficult to discern if Lhx2 has a function in the patterning of the forebrain and hence commitment to eye development, or expansion and patterning of the optic vesicle, or both. This is also pertinent to other transcription factors suggested to have a role in eye development such as Pax6, Six3, Six6 and TlxLhx2-Cre transgenic mouse strain that defines the first progenitor cells committed to generate the neural part of the eye in the anterior neural plate it is possible to molecularly define the role of any gene in patterning/commitment to eye development and subsequent expansion/patterning of the eye committed progenitor cells. We could confirm that Lhx2 is required in the optic vesicle to optic cup transformation since development is blocked almost immediately following inactivation of Lhx2 in the optic vesicle. This observation is also in agreement with the finding that induction of eye development by ectopic expression of eye field transcription factors can only occur when endogenous Lhx2 expression is induced Pax2 expression is induced and Mitf expression is transiently induced, whereas this process is completely blocked in the −/−Lhx2 embryos. This observation suggests that Lhx2 is important for both establishing and maintaining the patterning of the optic vesicle. The earliest molecular consequence of inactivation of Lhx2 in the optic vesicle is the down-regulated expression of Six6, whereas expression of most other transcription factors involved in early eye development appears to be unaffected. The down-regulated expression of Six6 is in agreement with the finding that Lhx2 synergises with Pax6 to induce Six6 expression Six6 expression cannot explain the block in eye development since −/−Six6 mice have a relatively mild eye phenotype affecting only the late stages of eye development Lhx2 expression is not required for commitment to the eye-specific progenitor cell since Cre expression is also detected in the distal part of the optic vesicle in −/−Lhx2 embryos. However, expression of transgenic Lhx2 in the eye committed progenitor cells in the −/−Lhx2 background only promote development of RPE cells, suggesting that Lhx2 expression prior to eye commitment is important for the progenitor cell to acquire its oligopotency. Thus, Lhx2 might also regulate the establishment of a fully functional eye progenitor cell.Analysis of the conventional Pax6, which is complicated by the fact that it is expressed in cells both in the optic vesicle and the surface ectoderm/lens placode Pax6 function, at least in mice, have been obtained by performing tissue-specific inactivation of Pax6 in surface ectoderm Pax6 is required cell autonomously in the surface ectoderm for lens development. Furthermore, the developing lens is not necessary for the formation of the neural retina and RPE layer, but is rather required for the correct organisation and localisation of the neuroepithelium of the eye. Our results reveal that lens placode development is also arrested immediately after Lhx2 has been inactivated in the optic vesicle. Thus, continuous interactions between the optic vesicle/cup and the developing lens is required for lens formation although the lens placode has formed and acquired many of its molecular characteristics such as expression of Pax6, Six3 and Sox2There are several hypotheses of how optic vesicle-surface ectoderm/lens placode interactions regulate eye development. Many of these hypotheses are based on the study of Bmp4 and Bmp7 mutant mice have profound eye defects −/−Lhx2 embryos since neither Bmp4 nor Bmp7 are expressed and phosphorylated SMADs, the intracellular mediators of BMP signalling, are not detected in mutant optic neuroepithelium Bmp4 is initiated in the optic vesicle similar to the control animals but is rapidly down-regulated, supporting the notion that Lhx2 function is partially mediated by BMP signalling. This notion is further supported by the down-regulated expression of Fgf15, which is suggested to be a down-stream target of BMP4 signalling Bmp7 expression appears to be unaffected in the conditional mutant and hence lack of Bmp expression cannot solely explain the Lhx2 mutant phenotype. Since Bmp7 has been shown to regulate the expression of Pax2 explains why Pax2 expression is unaffected in the conditional mutant whereas it is absent in the −/−Lhx2 mice Bmps appear therefore to be differently regulated by Lhx2 where both Bmp4 and Bmp7 expression is initiated by Lhx2 but maintained expression of Bmp4 is Lhx2-dependent whereas maintained Bmp7 expression is Lhx2-independent. Moreover, Lhx2 re-expression in the −/−Lhx2 eye committed progenitor cells led to the formation of only RPE cells, which is remarkably similar to the eye phenotype in −/−Bmp7 mice Bmp7 nor Bmp4 is expressed in the −/−Lhx2 optic vesicle Lhx2 in the −/−Lhx2 eye progenitor cells might be partly due to suboptimal expression of the Bmps when Lhx2 expression is turned on in the optic vesicle. Alternatively, the level of transgenic Lhx2 expression might not be sufficient to induce enough BMP expression at the correct time in the optic vesicle.BMP signalling has been shown to be important for eye development since both Lhx2 function by comparing global gene expression in Lhx2+ progenitor cells to their Lhx2− progeny Lhx2 in various tissues and progenitor cell populations, suggesting that mediators of Lhx2 function partly overlap in different tissues/progenitor cell populations. In this study we identified a number of genes that also overlap with Lhx2 expression during eye development. The gene Enc1  encodes a Kelch-related protein suggested to be important in the organisation and function of the cytoskeleton Enc1 was expressed in the prospective neural retina and the lens placode, and was not detected in these tissues in the conditional Lhx2 mutant. Since Lhx2 is expressed in the neural retina but not expressed in the lens placode it suggests that Lhx2 regulate Enc1 in neural retina by a cell autonomous mechanism and in the lens placode by a cell nonautonomus mechanism. Putative mediators of the cell nonautonomous regulation of Enc1 in the lens placode remains to be elucidated, but could partly include mediators of BMP4 signalling since this signalling pathway has been linked to Lhx2 function in eye development and is important for lens development Lhx2 in eye development is further emphasised by the expression pattern of Nuak1, an adenosine monophosphate-activated protein kinase (AMPK)-related kinase  suggested to be involved in the regulation of ploidy and senescence Nuak1 is normally expressed by the neural ectoderm in the forebrain where Lhx2 is also expressed, but its expression is excluded in the optic vesicle and its derivatives, e.g. prospective neural retina, RPE cells, and optic stalk. However, in the conditional Lhx2 mutant the expression domain of Nuak1 extends into these domains of the developing eye. The most simplistic explanation for this phenotype is that Lhx2 promotes Nuak1 expression in the forebrain neural ectoderm whereas it suppresses Nuak1 expression in eye committed neural ectoderm. Thus, the combined effects of down-regulated expression of Six6, Enc1, Fgf15 and Bmp4 in the optic vesicle, misexpression of Nuak1 in the optic vesicle and down-regulated expression of Enc1 in the lens placode, might partly explain the developmental arrest and degeneration of the eye in the conditional Lhx2 mutants. Lhx2 has been suggested to regulate key determinants of both dorsal and ventral identity Lhx2 function presented here starts to explain how Lhx2 accomplishes that.We have previously identified genes putatively linked to Cre expression is regulated by the promoters of Crx, Rx, Pax6 and Six3 genes directing Cre expression to the developing eye Rx-Cre and the Six3-Cre transgenic mice also reveal Cre expression in neural tissue outside of the eye domain in the forebrain, and the Pax6-Cre and Crx-Cre mouse strains show restricted expression within the developing eye. To our knowledge the Lhx2-Cre mouse strain is the first mouse model where progenitor cells solely committed to generate the neural part of the eye can be identified in the anterior neural ectoderm. Lineage tracing of these cells revealed that they do not contribute to any other cells in the prospective forebrain. The conditional inactivation of Lhx2 in these eye committed progenitor cells also confirmed this assumption since the forebrain appears to be intact, which is in contrast to the −/−Lhx2 embryos that lack several forebrain structures Lhx2-Cre mouse strain will be a very useful tool to elucidate the specific role(s) of any gene in the patterning/commitment of anterior neural plate into eye committed progenitor cells, and the subsequent expansion/pattering of the optic vesicle. Moreover, the role of specific genes in the ability of the optic vesicle to communicate with surface ectoderm and hence induce and promote lens development can also be studied in detail by using the Lhx2-Cre mouse strain.Transgenic mice have previously been generated where The mice were maintained at the animal facility at Umeå University and all experiments involving animals were approved by the local Animal Review Board .Lhx2-Cre transgenic construct was generated by using an 11 kb DNA fragment of the Lhx2 promoter, which included the first 36 bp of the Lhx2 coding sequence. The Lhx2 promoter fragment was fused in-frame with Cre recombinase cDNA and a SV40 polyadenylation signal was added. Pronuclear injection of the DNA construct generated two founder lines of which one was chosen for further studies. Generation of ROSA26R mice, −/−Lhx2 mice, flox/floxLhx2 mice and Z/Lhx2-GFP transgenic mice has been described previously floxLhx2 allele were: LOX 5′-GCCAGACTAGCAGACGCTGC-3′ and SDL2 5′-CCACCGGTACTCCTCTTCAGAG-3′. Primers used to identify the Z/Lhx2-GFP transgene were GFPforward 5′-TTCCACCATATTGCCGTC-3′ and GFPreverse 5′-AGAACTTGCCGCTGTTCA -3′. Primers used to identify the Lhx2-Cre transgene were: 1084: 5′-GCGGTCTGGCAGTAAAAACTATC-3′ and 1085: 5′-GTGAAACAGCATTGCTGTCACTT-3′. Primers used to genotype the ROSA26R mice were: Lac3 5′-GGT TGT TAC TCG CTC ACA-3′ and Lac4 5′- CGT TAA AGT TGT TCT GCT TC-3′. The morning of the vaginal plug was considered as E0.5.The 2, 5 mM EGTA, 0.02% NP40 and 0.01% sodium deoxycholate) and subsequently incubated in X-gal buffer  , 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide) over night at room temperature. The reaction was stopped with 3×5 minutes washes with PBS, and sections were mounted in 87% glycerol. Whole-mount in situ hybridisation and in situ hybridisation using DIG labelled probes were performed essentially as previously described Lhx2 , Bmp4 , Pax6 , Bmp7 , Pax2 (IMAGE clone: 40142573), Cre , Rx (IMAGE clone: 5366450), Sox2 (IMAGE clone: 6413283), Otx2 , Six6 , Six3 , Vsx2 (IMAGE clone: 6492679), Mitf (IMAGE clone: 40047440), Enc1  and Nuak1 .Embryos were isolated and fixed in 4% paraformaldehyde (PFA) in PBS at 4°C. Embryos used for β-Gal staining were fixed for 30 minutes and embryos used for in situ hybridisation were fixed for 1–2 hours. After fixation the embryos were transferred to 30% sucrose in PBS for 24 hours at 4°C, mounted in Tissue-tek (Sakura) and stored at −80°C. Sectioning (8–10 µm) was performed on a cryostat (Microm HM505E) and collected on superfrost plus slides (Menzel-Gläser). For hematoxylin-eosin staining, tissue sections were incubated in Mayer's hematoxylin solution for 2 minutes, in water for 15 minutes, in eosin solution for 2 minutes, in 95% ethanol for 2×1 minutes, in 99% ethanol for 2×1 minutes and in xylene for 5 minutes. The slides were mounted with DPX mounting media (VWR). For β-Gal staining, tissue sections were washed for 3×20 minutes in wash buffer , E9.5 (D) and E10.5 (F). β-Gal staining of sections of a head from Lhx2-Cre:ROSA26R double transgenic embryo at E12.5 derived from two different Lhx2-Cre transgenic founder mouse strains revealing that all neural parts of the eye are β-Gal+ in both founder mice . β-Gal staining of a sagittal section of a whole Lhx2-Cre:ROSA26R double transgenic embryo at E9.5 (E) and a transversal section of a head at E10.5 (G). β-Gal+ cells can be detected in the midbrain at E9.5 , in the olfactory placode (OE) at E10.5  and in cells of the hindbrain at E10.5 . NR, neural retina. RPE, retinal pigment epithelium. OS, optic stalk. Scale bar: A–E and F–G 500 µm.(TIF)Click here for additional data file.Figure S2Conditional inactivation of Lhx2 in the eye committed progenitor cell population cause anophthalmia. All adult flox/floxLhx2-Cre:Lhx2 animals are anophthalmic (C) whereas the flox/floxLhx2 mice develop normal eyes (A). This phenotype is already manifested at postnatal day 1 since no eye structures can be detected on sections of the head of flox/floxLhx2-Cre:Lhx2 animals (D) whereas flox/floxLhx2 animals develop normal eyes (B). Scale bar: 400 µm.(TIF)Click here for additional data file.Figure S3Increased number of apoptotic cells in the mutant optic vesicle. Immunohistochemical analysis of coronal sections of control optic vesicle (A) and mutant (flox/floxLhx2-Cre:Lhx2) optic vesicle (B) at E9.5 to detect the presence of activated caspase-3. Scale bar: 100 µm.(TIF)Click here for additional data file.Figure S4Fgf15 expression is significantly down-regulated in the optic vesicle in the conditional mutant. In situ hybridisation analyses of coronal sections of the optic vesicles in control (A) and mutant (flox/floxLhx2-Cre:Lhx2) embryos (B) at E9.5 to detect Fgf15 expression.(TIF)Click here for additional data file.Figure S5Lhx2 expression is induced following Cre-mediated recombination of the Z/Lhx2-GFP transgene. Schematic representation of the vector used to generate the Z/Lhx2-GFP transgenic mouse strain (upper panel) and the organisation of this vector after Cre-mediated recombination (lower panel). The blue arrows correspond to the mRNA that is generated before and after Cre-mediated recombination of this vector. We utilised an expression system based on the Z/AP double reporter vector developed by Lobe and co-workers β-Geo  is followed by an expression cassette consisting of the Lhx2 cDNA, an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) cDNA. Thus, cells expressing Cre recombinase will delete the β-Geo gene and initiate expression of Lhx2 and GFP since the Lhx2-GFP part is placed immediately downstream of the promoter/enhancer. Supplementary reference. 1. Lobe, C., Koop, K., Kreppner, W., Lomeli, H., Gertsenstein, M. and Nagy, A. (1999). Z/AP, a double reporter for cre-mediated recombination. Develop. Biol. 208:281–292.(TIF)Click here for additional data file.


Unconstrained rigid docking, flexible side chain docking and protein crystal structure determinations reveal a water-mediated hinge binding mode for a series of benzimidazole ligands of the protein kinase CHK2. This binding mode is different from those previously postulated in the literature and may provide a useful approach to selective small molecule inhibitor design. Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature. It has been postulated that selective inhibition of CHK2 could increase the efficacy of genotoxic cancer therapies in a p53 mutant background by modulating resistance pathways and may also be radioprotective to normal p53 wild-type tissues.As part of our in-house drug discovery project, we were particularly intrigued by a published series of benzimidazole CHK2 inhibitors for which two closely related binding modes have been proposed, neither of which appeared entirely consistent with the reported SAR (vide infra).1, 2–6, 7, 8 and 9,10, 11 and 12, Literature SAR includes analogues where the benzimidazole scaffold has been modified or replaced  and has In silico docking of small molecules into protein binding sites has become a powerful tool for medicinal chemistry design22.1We divided the 19 publicly available CHK2 protein–ligand crystal structures into two groups. The first comprises crystal structures in which the ligand mimics ADP and interacts with the hinge region of CHK2 through one or two hydrogen bonds between the ligand and residues Glu302 and Met304 A.42 The 2.22CN5) and to NSC1095555 (PDB ID: 2W0J) as representative high resolution parent structures for docking . To validate the suitability of these structures for docking, ligands were removed and re-docked into the empty structures using GOLD.We carried out best practice unconstrained rigid docking using a selected set of 50 potent compounds from the published benzimidazole series .15–17 We8–12, 2CN5), hydrogen bond interactions with both Glu302 and Met304 were observed for the lowest energy poses of 14 active compounds ; the other 36 compounds did not make these interactions. However, even for the 14 ligands that did bind to the hinge, no additional hydrogen bonds with the protein were identified from the Protein–Ligand Interaction Fingerprint  that form a high number of hydrogen bonds (3 out of a possible 4) (13-induced conformation are the two hydrogen bonds to the hinge (Glu302 and Met304) and an interaction between the carboxylate side chain of Asp368 and the N3 atom of the benzimidazole scaffold  that form the highest possible number of hydrogen bonds (4 out of a possible 4)  A. The thscaffold A. For thsible 4) B. The lie Lys249 B. Dockin8 to 12, into the30-induced conformation provides the optimal compromise of a high number of hydrogen bonds (4 out of a possible 4) adopted by a high number of biochemically active ligands (50%) docked into this ligand-induced protein conformation. Thus, the introduction of side chain flexibility into the docking protocol delivers an optimal binding mode mediated by a conserved water molecule to the hinge region. This binding mode is consistent with the observed experimental SAR (vide infra) and is significantly different from the two closely related binding modes previously postulated in the literature.Considering all possible solutions, these results indicate that the ligand 30-induced conformation with the crystal structure 2W0J  representing different ligand binding modes were obtained from the Protein databank. To optimize the positioning of hydrogen atoms in the ATP binding pocket, each crystal structure was subject to Protonate3D as implemented in MOECHK2 crystal structures  to those on the protein surface (lowest priority) until the limit of 10 was achieved.Crystal and Library in GOLD as described by Lovell et al.The permitted amount of flexibility for the 10 selected residues was defined by the parameters 4.6To identify the protein residues interacting with a docked inhibitor, the resulting protein-inhibitor complexes were analysed using the protein–ligand interaction fingerprint (PLIF) implemented in MOE, a method similar to the SIFt.4.7Each ligand-induced protein conformation was analysed by plotting the trade-off between the number of polar interactions formed by the benzimidazole-5-carboxamide scaffold in the ligand-induced binding mode and the number of docked ligands adopting that particular binding mode . The sel4.8The CHK2 kinase domain  was produced as a GST-fusion protein and purified as previously described.4.919, 30, 44 and 47 for protein–ligand crystallography are described in Compound structures have previously been disclosed although preparative methods have not been described for all compounds.4.1019, 4A9S; 30, 4A9R; 44, 4A9T and 47, 4A9U.Atomic coordinates and structure factors for the crystal structures of ligand-bound CHK2 can be accessed using the following PDB codes:


GST is a family of enzymes that are important in protection of the body against oxidative stress.Investigate the association between GSTT1 and GSTM1 polymorphism and hypertension.GSTT1 and GSTM1 genotypes were detected by PCR. The fragments were then analyzed by agarose gel electrophoresis.There is no significant association between GSTT1 & GSTM1 polymorphism and hypertension GSTT1 & GSTM1 polymorphism can be considered a risk factor for hypertension. Essential hypertension is a complex multi-factorial disorder with many genetic, environmental and demographic factors contributing to this disorder. Several experimental and clinical studies have highlighted the role of oxidative stress in development of hypertension 2]3]4][3][4]2][[4][3][4][3][4]2]. GST actThe present study aimed to test the hypothesis that the loss of activity of the enzyme due to a deletion polymorphism in the GSTT1 and GSTM1 may affect the risk of developing hypertension. We have also studied other risk factors for hypertension.The control group consisted of 33 non-hypertensive individuals with mean age 41.7 ± 13.6 years.The case group consisted of 30 hypertensive patients with mean age 40.1 ± 14 years. The study was conducted in the Biochemistry department in Dubai Medical College for girls. All subjects signed an informed consent to participate in the study. The hypertensive patients underwent a standardized evaluation consisting of a questionnaire, physical examination, and laboratory tests. Weight, height, waist and hip circumferences were measured. Body mass index (BMI) and Waist Hip Ratio (WHR) were calculated. Hypertension was defined as blood pressure ≥ 140/90 mmHg or current use of anti-hypertensive medication.Subjects with body mass index (BMI) ≥ 25 kg/m2 were considered positive for obesity as defined by World Health organization. Those with waist circumference (WC) > 88 cm for women and > 95 cm for men or with waist hip ratio (WHR) > 0.8 for women and > 0.95 for men were considered positive for abdominal obesity .Lipids and lipid fraction measurements were performed using routine enzymatic tests (DiaSys Kits) as previously described 9]9]DNA was extracted from white blood cells by a salting-out method .For genotype analysis, the GSTT1 and GSTM1 were amplified by using multiplex polymerase chain reaction (PCR) protocol 12]13].[13].12][.[13].12]Statistical analysis was done with SPSS software version 11.0 . Difference in genotype prevalence and association between case and control group were assessed by the Chi-square test. Correlation coefficient, Odds Ratio (OR) and 95% CI were used to describe the strength of association.The distribution of genotypes of GSTM1 and GSTT1 in cases and control are shown in The results of anthropometric measurements & lipid profile among the cases and control are displayed in Oxidative stress may contribute to the generation and/or maintenanceof hypertension via a number of possible mechanisms 15]16].[16].15][.[16].15]


Immunogen development for HIV-1 vaccines can be based on epitope identification of naturally occurring neutralizing antibodies in HIV-1 infected patients. A neutralizing monoclonal antibody, HJ16, was obtained at IRB from a patient recruited at ITM which recognized a new epitope in the CD4bs and neutralized mostly tier 2 strains that were not neutralized by b12[HJ16 resistance was induced by culturing the sensitive replication competent VI1090 (CRFO2_AG) strain (IC50 0.10 µg/ ml) in increasing amounts of HJ16 on freshly isolated PBMC until a resistant strain was obtained that was able to replicate in almost 200 µg/ml HJ16. Neutralizing activity was measured using TZMbl and PBMC neutralization assays. Site-directed mutagenesis was carried out to induce the observed mutations into the VI1090 expressing vector.Sequencing of the HJ16 sensitive versus the resistant strain revealed a distinct point mutation where the neutral asparagine was replaced by the negative charged aspartic acid. This N276D point mutation is only seen in 0.69% of the group M strains described in the Los Alamos database . Introduction of this mutation into the HJ16 neutralization sensitive construct rendered the mutated strain resistant to neutralization by at least 100 fold higher HJ16 concentration.Results show that the mutation in the N-linked glycosylation site at N276 has a distinct influence on sensitivity to the HJ16 CD4bs antibody. Although it is disappointing that a single mutation seems to induce resistance it is obvious that this mutation is quite unique and occurs rarely in natural infection rendering this epitope very suitable for further vaccine development.


In Figure 1, not all of the data is included. The incorrect version of the figure represents miR-26a expression analysis in 20 breast cancer specimens and 15 normal breast tissues. The revised version includes 52 breast cancer specimens and 29 normal breast tissues. Please see the revised version of Figure 1 here:


Tightly controlled concentration gradients of morphogens provide positional information and thus regulate tissue differentiation and morphogenesis in multicellular organisms. However, how such morphogenetic fields are formed and maintained remains debated. Here we show that fibroblast growth factor 8 (Fgf8) morphogen gradients in zebrafish embryos are established and maintained by two essential mechanisms. Firstly, Fgf8 is taken up into the cell by clathrin-mediated endocytosis. The speed of the uptake rate defines the range of the morphogenetic gradient of Fgf8. Secondly, our data demonstrate that after endocytosis the routing of Fgf8 from the early endosome to the late endosome shuts down signaling. Therefore, intracellular endocytic transport regulates the intensity and duration of Fgf8 signaling. We show that internalization of Fgf8 into the early endosome and subsequent transport towards the late endosome are two independent processes. Therefore, we hypothesize that Fgf8 receiving cells control both, the propagation width and the signal strength of the morphogen. The family of fibroblast growth factors (Fgfs) is currently believed to consist of 23 structurally related polypeptides controlling a wide range of biological functions Fgf8 is one member of the Fgf family with key inductive functions during development of neural ectoderm, mesoderm and limb formation R447H mutant fruit flies, it has been shown, that depletion in uncoating activity correlates with decreased endocytosis One internalization route for growth factors such as Fgfs is via Clathrin mediated endocytosis (CME) a major mechanism for controlled cargo uptake which can be subdivided into several steps The identity of the uptake mechanism for Fgf8 has been a matter of some controversy Although some aspects of Fgf8 internalization route are understood, the factors that execute key functions during specific steps of Fgf8 endocytosis are still unclear. Furthermore, it is not fully understood how endocytosis regulates Fgf8 signaling and it is also unclear whether the signals emanate from the membrane and/or from internalized ligand-receptor complexes. Therefore, localization of the internalization route and subsequent identification of the signaling compartments is crucial.Here the molecular machinery involved in CME of Fgf8 was characterized and the influences of endocytosis on Fgf8 signaling were examined. Therefore, different steps in internalization – the clathrin mediated uptake and the transport from early to late endosomes- were analyzed. Firstly, we analyzed whether Hsc70 controls the endocytosis of Fgf8 by regulating CME. We show that knock-down of Hsc70 leads to a reduction in Fgf8 internalization and subsequently to an increase in long-range signaling of Fgf8 in zebrafish. Consistently, overexpression of Hsc70 leads to enhanced internalization of Fgf8 into early endosomes. Furthermore, we find that Fgf8 displays a decreased signaling activity in the early endosome compared to the plasma membrane. Finally we show that Fgf8 signaling is terminated by transport of the ligand to the late endosome. Therefore, we conclude that the receiving cells control Fgf8 signaling at two independent levels: firstly the dynamic of CME is important for regulating the activity range of Fgf8, and secondly the rate of transport from early endosomes to late endosomes determines Fgf8 signaling strength.All zebrafish husbandry and experimental procedures were performed in accordance with the German law on Animal Protection and were approved by Local Animal-Protection Committee  and the Karlsruhe Institute of Technology (KIT).Danio rerio) were maintained at 28°C on a 14-h light/10-h dark cycle 2O2) and the transgenic zebrafish line Tg(Dusp6:EGFP)pt6, which was received from Michael Tsang.Breeding zebrafish (Hsc70 full length coding sequence was amplified with primer pairs (forward/reverse); 5′-TGG TGG CAC TTT TGA TGT GT-3′/5′-TCC CTC TCT GCA GTC TGG TT-3′ from a zebrafish cDNA library at stage 24 h. The Hsc70 full-length cDNA was cloned in the expression vector pCS2+. The plasmid was then also linearized with NOT1 and transcribed using SP6 Message Machine Kit (Ambion).pCS2+Fgf8 GFP plasmid hsc70 MO1 (5′ATAAAACAGAGATGGATGAAGATGC 3′) and hsc70 MO2 (5′ AGCTGGTCCCTTGGACATTGTGTCA 3′) were used at a concentration of 0.5 mM. A volume of 1–2 nl was injected per embryo. The injection of MO oligomers was performed in the yolk cell close to the blastomeres at one to two-cell stage.For transient knock-down of gene expression, Morpholino-antisense oligomers , For transient knock-down, Hsc70 MO at a concentration 0.5 mM, was injected at the one- to two-cell stages. Samples of 30 embryos were collected at 28hpf, homogenized and dissolved in 1% triton lysis buffer. Protein solution was then eluted with Lammli buffer and heated to 95°C for 5 min and subjected to SDS-PAGE.For immunoblot, a Hsc70 polyclonal antibody  was used at a dilution 1∶50,000. This was followed by incubation with anti-mouse IgG-HRP  at a dilution 1∶10,000. Chemiluminescence detection was performed using ECL Western Blotting Substrate Kit (Pierce).6 cells in a 10cm plate. After 24 hours cells were transfected according to the manufacturer’s protocol using Promofectin (Promocell). Briefly two mixtures containing DNA/serum free DMEM and Promofectin/serum-free DMEM were combined incubated for 20 min at RT and added to the cells. 24 hrs after transfection the culture medium was replaced and Bafilomycin A1 was added. 24 hrs later the cells were lysed in sodium dodecyl sulfate (SDS)–sample buffer containing 100 mM dithiothreitol (DTT) and subjected to Western blot analysis. Activated Erk was monitored using an antibody against phosphorylated Erk . For the loading control the membrane was stripped  and reprobed with an Erk antibody (Erk 1 (K-23) Santa Cruz). Blots were stained using the enhanced chemiluminescence system (Thermo Fisher Scientific). The quantification of proteins bands in Western blot analysis was performed using the program ImageJ.HEK 293 cells were cultivated to 70–80% confluency before transfection. For transfection of Fgf8 and Hsc70 cells were seeded at 2×10The statistical analysis was performed on three independent experiments. All quantifications are given as mean plus standard deviation.in situ hybridization was carried out as described previously fgf8, erm and pea3, using an RNA labeling and detection kit (Roche). Stained embryos were dissected and mounted in glycerol.Whole-mount mRNA After ISH staining, embryos were then incubated in 25 µM SYTOX nucleic acid stain (Invitrogen) overnight. After washing in 1×PBS embryos were mounted laterally for confocal analysis.in situ hybridization experiments. Inhibition of molecular transport from the early to the late endosomes was performed by treatment of embryos with 100 nM bafilomycin A1 (Sigma-Aldrich) with 1% DMSO or 1% DMSO only for a period of 1 hour at 30% epiboly stage and subjected to live imaging or fixed at 75% epiboly stage for in situ hybridization experiments. Texas red Transferrin was purchased from Invitrogen.Inhibition of Fgf receptor-mediated signaling was performed by incubating live embryos in 16 µM of SU5402  in 1% DMSO or with 1% DMSO only at 30% epiboly stage for a period of 3 hours and fixed at 75% epiboly stage for Zebrafish PAC2 cells were used to study the intracellular localization of Fgf8. The following endosomal markers were used: RFP-Rab5a (human) from Ari Helenius, pCS2+mCherry-dmRab7 from Jim Smith, mRFP-Clathrin from Ernst Ungewickell, dsRed-LAMP1 from Erez Raz. Cells were transfected separately with Fgf8-GFP at a concentration of 1 µg and Hsc70 transfected along with mRFP-Clathrin, RFP-Rab5a, mCherry-Rab7 at a concentration of 600 ng in DMEM solution. 24 hours post transfection, Fgf8-GFP transfected cells were co-cultured with cells transfected with Hsc70 and the respective endosomal markers.Prior to imaging, embryos were mounted in 70% glycerol. Images were taken with the help of an Olympus SZX16 microscope equipped with a DP71 digital camera using the imaging software Cell A. For confocal analysis, embryos were embedded for live imaging in 1.5% low melting point agarose (Sigma-Aldrich) dissolved in 1x E3 solution at 50% epiboly stage. Live embryos as well as cells were images using 20x and 63x water immersion objective. Confocal image stacks were then obtained using the Leica TCS SP5 X confocal laser-scanning microscope. Images were further processed using Imaris 6 (Bitplane AG).erm and pea3, were then measured using Olympus Cell A software. The area of expression was calculated with respect to the area of the entire embryo. Confocal experiments were quantified using Imaris 6 (Bitplane AG).The areas for Fgf target gene expression erm and pea3 by in-situ hybridization  and PAC2 cells transfected with endocytic markers , late endosomes (72%) and lysosomes 78%; . ActivatWe next analyze the consequence of Fgf8 endocytosis for signaling. We used a cell cultivation assay in which we analyzed effective Fgf8 signaling by quantification of Erk1 and Erk2 phosphorylation levels. Transfection of Fgf8 led to a 12-fold stronger level of Erk1/2 phosphorylation compared to the unstimulated control cell culture . SimultaBased on the data from the cell culture experiments we tested our hypothesis if the transport of Fgf8 along the endocytic route influences the signaling range. First we analyzed the endocytic route of Fgf8 in zebrafish embryos. We found that Fgf8 was taken up into Rab5 positive early endosomes, however, co-localization of Rab5 and Fgf8 was a rare event  supportiAlthough Fgf-Fgfr interaction has been extensively studied, the internalization We hypothesized that the rate of CME alters the intracellular uptake of Fgf8 and therefore determines the extracellular availability of Fgf8 and, hence the signaling range  & 2. Preerm and pea3 positive cells rather than Fgf8 spreading. An increased rate of endocytosis uncoupled from cell migration leads to a decrease of the spreading range In parallel, our experiments suggest that Fgf8 signaling can occur at the cell membrane and to a lesser extend in Rab5 positive early endosomes, which contrasts with other growth factor signaling pathways. As a result of impaired CME by expression of Dynamin2 K44A, activated Egfr shows a prolonged residence at the plasma membrane, which leads to a reduced activity of downstream signaling components such as MAPKs Erk1/2 or the p85 subunit of phosphatidylinositol 3 kinase, PI3 kinase After CME, Fgf8 localizes to early endosomes and late endosomes. Blockage of Fgf8 transport to late endosomes increases signaling. This suggests that - in addition to signaling at the membrane - signaling occurs also from early endosomes. Indeed the hypothesis that Fgf signaling can occur through the entire endocytic route has been postulated already in the 80s Here we conclude that the dynamics of Clathrin-mediated endocytosis of Fgf8-Fgfr complexes regulates the signaling range of Fgf8, whereas the retention time of the activated signaling complex at the plasma membrane and in the early endosome defines signaling strength.Figure S1A. Immunoblot analysis of the expression of Hsc70 protein during knockdown and over expression of Hsc70. Embryos were injected with Hsc70 morpholinos as well as Hsc70 mRNA and samples at 28hpf were subjected to immunoblot analysis. Embryos injected with Hsc70 MOs showed a reduction in the expression of Hsc70 when compared to the expression in wild type samples. Embryos overexpressing Hsc70 showed an increase in the expression of Hsc70 when compared to the expression in the wild type. ISH for Fgf8 expression. (B) Shows the expression of Fgf8 at 75% epiboly stage. Fgf8 expression remains unaltered during overexpression (C), knock-down of Hsc70 (D), and injection of Hsc70 MO and mRNA (E).(TIF)Click here for additional data file.Figure S2Endocytosis of Transferrin. Transferrin is taken up into the endosomes in fish fibroblasts (A–A”). Activation of Hsc70 leads to the formation of clusters of Transferrion, most likely in early endosomes (B–B”).(TIF)Click here for additional data file.Figure S3Injection procedure to measure the distance of Fgf8 distribution. Figure shows the schematic diagram of the experiment performed to analyze the spreading of Fgf8 from its source. (A) Fgf8 GFP DNA was injected into live zebrafish embryos at the one cell stage. (B) Zebrafish embryo at 50% epiboly stage show a mosaic expression of Fgf8. (C) Confocal microscopy analysis of live embryos show Fgf8 expressed in specific cells creating local sources from which the distance travelled by Fgf8 from the source to receiving cells is measured .(TIF)Click here for additional data file.Figure S4Quantification of co-localization studies of Fgf8 with endosomal markers. Transfection of Hsc70 led to the formation of Fgf8 clusters with a diameters over 0.8 µm. These clusters co-localize similarly strongly in Rab5 positive early endosomes. Three independent experiments were quantified.(TIF)Click here for additional data file.


PAX6) on human chromosome 11p13 is an essential transcription factor for eye formation in animals. Mutations in PAX6 can lead to varieties of autosomal-dominant ocular malformations with aniridia as the major clinical signs. Known genetic alterations causing haplo-insufficiency of PAX6 include nonsense mutations, frame-shift mutations, splicing errors, or genomic deletions. The purpose of this study was to identify genetic defects as the underlying cause of familial aniridia in a large Chinese family.The paired box gene 6 . Quantitative real-time PCR was applied to verify the abnormal aCGH findings in the proband and to test five other family members.All exons of PAX6 in the proband. The aCGH analysis showed two copies of PAX6 but revealed a 566 kb hemizygous deletion of chromosome 11p13, including four annotated genes doublecortin domain containing 1 (DCDC1), DnaJ homolog subfamily C member 24 (DNAJC24), IMP1 inner mitochondrial membrane, IMMP1Landelongation factor protein 4 (ELP4) downstream of PAX6. Quantitative real-time PCR verified the deletion in the proband and further identified the deletion in a blind fashion in four affected family members but not in the one with a normal phenotype.There were no detectable pathogenic mutations in the exons of PAX6 should be the cause of the familial aniridia in this Chinese family, although two copies of PAX6 are intact. aCGH evaluation should be applied if there is a negative result for the mutation detection of PAX6 in patients with aniridia.The 566 kb hemizygous deletion of chromosome 11p13 downstream of  Eight-five percent of individuals with aniridia inherit this disorder as an autosomal-dominant trait, 13% occur as part of the autosomal-dominant WAGR syndrome , and the remaining 2% occur as part of other disorders, including Peters’ anomaly and Gillespie’s syndrome, in either autosomal-dominant or autosomal-recessive inheritances [Aniridia OMIM 06210 is PAX6) is located on chromosome 11p13 and contains 14 exons encoding a protein (PAX6) with 422 amino acids. PAX6 is a transcriptional factor controlling development of a diversity of organs and tissues  by recognizing specific DNA sequences of its downstream target genes [PAX6 gene primarily cause aniridia due to its haplo-insufficiency [PAX6 represent the major causes of aniridia, genomic rearrangements involving the downstream region of PAX6 were identified in patients with aniridia although both copies of PAX6 are intact in these patients [The paired box gene 6  techniques. To our knowledge this is the first case found in an Asian population and is one of few similar cases with this kind of genetic mechanism [In the present study, we report a genomic microdeletion in the downstream region of echanism ,11,12.A five-generation Chinese family with familial aniridia was recruited from Heilongjiang province, northeastern China. There were 15 affected individuals in this family from which five affected members  and one unaffected individual (IV:4) participated in this study . Ocular PAX6 in the proband (III:4) were amplified and sequenced with the ABI BigDye Terminator Cycle Sequencing kit v3.1,  according to the described protocol [(ELP4) gene within the deleted genomic region, primers within the PAX6 gene, and reference primers within the Oral-facial-digital syndrome 1 protein (OFD1) gene on the human X chromosome. Both ELP4 and PAX6 were assayed as test genes compared with OFD1, and both ELP4 and PAX6 were assayed as test/controls.All exons of protocol . The supprotocol . The digprotocol . IndividThe clinical findings are summarized in PAX6 from the six participants of the family were amplified and sequenced using standard techniques. No intragenic point mutation or deletion could be identified (data not shown).All exons of PAX6  according to HG18 , DnaJ homolog subfamily C member 24 (DNAJC24), IMP1 inner mitochondrial membrane (IMMP1L), and elongation factor protein 4 (ELP4). The remaining 34 CNVs were considered to be benign because these CNVs were identified in healthy individuals documented in the Database of Genomic Variation or in recent publications [The aCGH analysis detected 35 copy number variations (CNVs) in the proband’s genome . One of ications ,18.ELP within the deleted region, which showed one threshold cycle difference between the patient and reference DNA samples for the amplification of the test gene . We used the qPCR method for testing the other five family members in a blind fashion, four affected family members with eye anomalies, and one healthy individual. All four patients were confirmed to carry the deletion, including ELP, but not PAX6. The phenotypic normal individual showed two copies for both ELP and PAX6 (data not shown).The 566 kb hemizygous deletion of chromosome 11p13 in the proband was verified by qPCR using primers targeted to est gene . Two copDCDC1, DNAJC24, IMMP1L, and ELP4. The proximal breakpoint of this deletion is approximately 123 kb from the 3′ end of PAX6. We postulate that this 566 kb heterozygous deletion is the underlying cause of the familial aniridia and acts by disrupting the transcription in one of the two PAX6 alleles, even though the two copies of PAX6 were intact in all individuals investigated in this study.The major finding in this Chinese family with familial aniridia is the presence of a 566 kb heterozygous deletion containing four annotated genes: PAX6 controlling the expression of this gene, if disrupted, leading to aniridia and other eye anomalies. To our knowledge, this is the first case found in Asian patients with aniridia and one of few similar cases with this kind of genetic mechanism.Our finding provides further evidence of the existence of the remote 3′ regulatory elements in the downstream region of PAX6 are apparently free of mutations. 2) Several publications reported similar observations in patients with aniridia, but the chromosomal breakpoint from the 3′ end of PAX6 and the fragment of deletion were different. For example, two aniridia pedigrees have been characterized in which the disease segregates with chromosomal re-arrangements that involve 11p13 but do not disrupt the PAX6 gene since the chromosomal breakpoint is at least 85 kb away from the 3′ end of PAX6 [ELP4 gene region, not involving PAX6, was present in all subjects with aniridia but not in the investigated normal relatives [: metallophosphoesterase domain containing 2 (MPPED2), doublecortin domain containing 5 (DCDC5), DCDC1, DNAJC24, IMMP1L, zinc finger CLS domain containing 3 (DPH4), and ELP4) has been characterized that starts 35 kb from the 3′ end of PAX6 in a patient with aniridia, autism, and mental retardation [DCDC1, DNAJC24, IMMP1L, and ELP4) was found in patients with aniridia [PAX6. Deletions in this region have been shown to abolish PAX6 expression and cause aniridia and other eye anomalies due to loss of enhancers and a downstream regulatory region [Our postulation is based on following evidence: 1) The heterozygous deletion segregated with aniridia in the five affected individuals but not in the unaffected individual, while the exons and splicing regions of elatives . A 1.3 My region ,19,20.DCDC1 gene encodes a member of the doublecortin family, which is highly expressed in testis and fetal brain [DNAJC24 is one of several enzymes involved in synthesis of diphthamide, which is a unique posttranslationally modified histidine found only in translation elongation factor 2 [IMMP1L encodes a peptidase similar to mitochondrial inner membrane peptidase (IMP1), which is one of the catalytic subunits of the IMP complex proteolytically removing the mitochondrial targeting presequence of nuclear-encoded proteins [ELP4 encodes a component of the six subunit elongator complex, a histone acetyltransferase complex that associates directly with RNA polymerase II during transcriptional elongation. Two recent reports indicate that ELP4 is possibly associated with the centrotemporal sharp wave electroencephalogram (EEG) trait in rolandic epilepsy and speech sound disorder [Little information is known about the four annotated genes within the 566 kb deletion in this study. It is also unknown whether these genes are involved in any of the phenotypic features found in these individuals carrying the deletion. The al brain . DNAJC24factor 2 . IMMP1L proteins . ELP4 endisorder .PAX6 exons, copy number variation should be investigated in the ﬂanking regions of PAX6. We suggest that patients, such as the subjects reported here, should be investigated using high resolution aCGH techniques in a clinical setting if sequencing analyses for PAX6 in patients with aniridia is negative.Submicroscopic copy number variations may play a role in human diseases either by loss of gene expression regulatory elements or by disrupting coding sequences. As well as the point mutations in


Inherited factors contribute to the burden of prostate cancer, however the identification of susceptibility genes for prostate caner has been challenging. To establish the contribution of eight founder alleles in three DNA damage repair genes  to prostate cancer in Poland, and to measure the impact of these variants on survival among patients, 3750 men with prostate cancer and 3956 cancer-free controls were genotyped for 3 founder alleles in BRCA1 , 4 alleles in CHEK2 , and 1 allele in NBS1 (657del5). Strong associations were seen for both CHEK2 and NBS1. BRCA1 was not associated with the risk of prostate cancer, NBS1 mutation was associated with poor survival - mortality was significantly worse for carriers of a NBS1 mutation than for non-carriers . We conclude that a founder mutation in NBS1 predisposes to aggressive prostate cancer.


C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [The GGGGCC G4C2) intronic repeat expansion within ia (FTD) ,2. The m intronicbe toxic .in situ hybridisation probes for G4C2 to detect RNA foci in transfected cells and human brain tissue form C9ORF72 mutation carriers. We assessed the toxicity by cell counting, activated caspase, PARP cleavage and Annexin V expression transfected cells and by TUNEL assay in zebrafish embryos. We probed transfected cells with antibodies to 30 RNA binding proteins and then tested the best candidates by immunohistochemistry in human brain tissues. We used biotinylated G4C2 x72 probes to pull down repeat binding proteins and confirmed binding by western blot.In order to determine whether expanded G4C2 transcripts might be toxic and sequester RNA binding proteins we generated G4C2 repeats with 8x, 38x and 72x and expressed them in a range of neuronal and non-neuronal cell lines, primary neurons and zebrafish embryos. We used fluorescent Cellular toxicity was associated with the nuclear retention of transcripts containing 38x and 72x repeats and the appearance of RNA foci. By placing the repeats 3’ to EGFP we were able to monitor nuclear export and show that transfected neuroblastoma cells, but not HEK cells, declined in number over time due to apoptotic cell death. This was most marked in those showing the greatest nuclear retention and foci, indicating cell type and neuron-specific toxicity of longer G4C2 repeats.In order to explore the sequestration hypothesis we screened antibodies to 30 RNA binding proteins on 72x transfected neuroblastoma cells and demonstrated colocalization of G4C2 RNA foci with three proteins. Using biotinylated 72x RNA we were only able to pull down one of these indicating that it is able to bind G4C2 RNA directly. On probing human cerebellar ALS and FTD tissues we detected a striking colocalization of this protein with 70% of RNA foci.Our observation that longer stretches of G4C2 RNA form neurotoxic foci and bind specific RNA binding proteins is similar to other intronic microsatellite expansion disorders where CUG (DM1) and CCUG (DM2) intronic repeats generate RNA foci in cells, animal models and in patients with myotonic dystrophy.


Plasmodium falciparum infection, in particular the central role of natural killer (NK) cell-derived interferon gamma (IFN-γ), is becoming increasingly recognised. Recently, it has been shown that IFN-γ production in response to P. falciparum antigens is in part regulated by killer-cell immunoglobulin-like receptor (KIR) genes, and a study from malaria-exposed Melanesians suggested an association between KIR genotypes and susceptibility to infection. This prompted us to determine and compare the frequencies of 15 KIR genes in Gambian children presenting with either severe malaria (n = 133) or uncomplicated malaria (n = 188) and in cord-blood population control samples (n = 314) collected from the same area. While no significant differences were observed between severe and uncomplicated cases, proportions of individuals with KIR2DS2+C1 and KIR2DL2+C1 were significantly higher among malaria cases overall than in population control samples. In an exploratory analysis, activating KIR genes KIR2DS2, KIR3DS1 and KIR2DS5 were slightly higher in children in disease subgroups associated with the highest mortality. In addition, our data suggest that homozygosity for KIR genotype A might be associated with different malaria outcomes including protection from infection and higher blood parasitaemia levels in those that do get infected. These findings are consistent with a probable role of KIR genes in determining susceptibility to malaria, and further studies are warranted in different populations.The relevance of innate immune responses to  Plasmodium falciparum can result in asymptomatic parasite carriage, an uncomplicated febrile disease or a potentially life-threatening illness. Apart from clinical immunity that gradually develops with repeated exposure Infection with the malaria parasite Natural killer (NK) cells are a key component of innate immunity. They kill their targets (diseased cells) by means of cytotoxic activity P. falciparum displays considerable heterogeneity  rather than cytotoxicity is the major contribution of NK cells to host defence ogeneity , 12 and ogeneity , 14. Thubright and CD56dim expressing cells bright cells produce more IFN-γ than CD56dim cells, CD56dim cells represent about 80% of the NK cell population dim population bright cells are KIR negative, whereas the majority of CD56dim cells express KIRs that are highly polymorphic  and 2 pseudogenes (KIR2DP1 and KIR3DP1). The expression and function of each of these genes influence the expression and function of other members of the gene family KIR molecules are glycoproteins encoded by a diverse and compact set of genes on chromosome 19. They are expressed on specialised lymphoid cells mainly NK cells and a subpopulation of γδ T cells and some memory αβ T cells KIRs and their interactions regulate NK cell activity in modulating disease outcomes  and KIR systems , 22 encooutcomes , 26–33.  alleles , 35, andP. falciparum infection, only a few studies have investigated the role of KIRs in malaria. A study that compared KIR genotypes in Melanesians with and without malaria parasitaemia found evidence for an increased number of activating KIR genes in parasitaemic individuals KIR genes among Gambian children with severe or uncomplicated forms of malaria, and cord-blood population control samples to assess whether individual KIR genes or genotypes are associated with the occurrence of disease or parasitaemia levels. Given that epidemiological findings have in the past implicated individual HLA class I alleles (e.g. HLA-B*53) Whilst there is accumulating evidence favouring an important role of NK cells in DNA samples were obtained from buccal swabs collected from Gambian children with uncomplicated malaria  and severe malaria .The typing technique used in this study (PCR-SSP) allowed us to detect the presence of inhibitory and activating KIR genes including those found in genotype A. The number of specific genes found at the centromeric end of B genotypes is variable including KIR2DS2, 2DL2, 2DL5, 2DS3 and 2DS5, in addition to the framework genes 3DL3 and 3DP1. However, telomeric B motifs are specifically identified with the presence of KIR3DS1 and 2DS1 genes in addition to 2DL4 and 3DL2 present in most motifs.In contrast to the conserved nature of genotype A in terms of gene content, KIR genotype B is highly polymorphic in gene content. Genotype B is a combination of activating and inhibitory KIR genes at the telomeric end all belong to KIR haplotype A . An opposite effect was observed for heterozygote (c-A/B) carriers . No such differences were seen with telomeric genotypes.We adopted the recent techniques and terminologies used by Cooley et al. lotype A . On the  KIR2DS4 . Analysi KIR2DS4  did not  KIR2DS4 . Howevercarriers , with a vs 12.5%, respectively, P = 0.004; adjusted for ethnicity and multiple comparisons P = 0.012), suggesting that having two copies of haplotype A may be protective against malaria infection. An interesting observation was that none of the 14 children with CM+SRD, considered to be the most critically ill patients, was homozygous for KIR genotype A, while a substantial proportion of children with less severe forms of malaria were homozygous for genotype A . Given the small number of individuals per group, this should be considered an exploratory analysis that encourages further investigations in other populations with larger sample sizes.The proportion of individuals homozygous for KIR genotype A varied significantly amongst the three groups . Whilst HLA-Cw*16:01 frequency  to be lowest amongst children with severe malaria . This observation, however, should be taken with caution given the small number of individuals used in this comparison. None of the HLA-B alleles was found to have any impact on malaria in this population. The frequencies of HLA-B or -C dimorphic groups and subgroups  were similar between groups ]. The frequencies of these two compound genotypes were significantly higher in cases compared with population control , suggesting that carriers of KIR2DL2+C1 and/or KIR2DS2+C1 were more at risk of being infected with malaria parasites than those without any of these genotypes. However, these two genes are in strong linkage disequilibrium (LD) even in African populations (It has been shown that KIR and HLA interact in an epistatic manner to modulate human immunodeficiency virus (HIV) disease outcome ulations , 48 makiKIR genes (I/A ratio) or the carriage of a particular KIR genotype is associated with parasite load measured on admission. When the ratios were grouped into four bins containing similar numbers of individuals, the level of parasitaemia was significantly different (PANOVA = 0.03) showing a significant increase with increasing I/A ratio , and parasitaemia levels declined significantly with increasing B content of the KIR genotype  and in a population control group. HLA class I (B and C) genotyping was performed on a proportion of samples with enough genomic DNA left after KIR typing. Possible KIR–HLA interactions were explored as compound genotypes in individuals expressing KIR molecules together with their putative ligand(s). Consistent with previous studies in other West African populations  compared with the population control group. Amongst the participants with available HLA type individuals carrying either 2DL2 or 2DS2 together with their corresponding ligand (HLA-C group 1) were significantly more frequent in the infected group. Taken together, these observations suggest that these genes could be involved in increased susceptibility to malaria infection caused by P. falciparum. Although a few of our study participants have one without the other, KIR2DL2 and 2DS2 genes are in strong LD in most populations worldwide including those of African origin (KIR gene (2DL2) and its corresponding activating counterpart (KIR2DS2) have been associated with other diseases. Absence of these genes has been associated with resistance to infection with herpes simplex virus type-1 (HSV-1) KIR2DS2 in the absence of its specific ligands has been associated with increased susceptibility to psoriatic arthritis In this study, we determined the presence or absence of 15 ulations , 48, we n origin , 48, 50.KIR genes did not differ significantly between disease subgroups, it is worth noting that prevalence of three activating KIR genes, KIR2DS2, KR2DS5 and KIR3DS1 were highest in the most critically ill children presenting with CM+SRD  contributes to exacerbated inflammatory responses that have been implicated repeatedly in the pathogenesis of severe malaria (Although the proportion of subjects with individual h CM+SRD . While t malaria , 56. ButKIR3DS1 was the least frequent KIR gene in the Gambian population, in line with previous data from other African populations (KIR gene evolution amongst different populations worldwide has suggested that KIR3DS1/L1 has been under positive selection in modern Sub-Saharan African populations, with KIR3DS1 being rare and KIR3DL1 allotypes predominating KIR3DS1, such as reduced susceptibility to malaria or protection from severe forms of malaria, for which our data provide some support. In a study that compared KIR genotypes in Melanesian individuals with and without malaria parasitaemia living in a malaria hyper-endemic area KIR3DS1 positivity was equally distributed in both groups, but KIR3DS1/L1 heterozygosity was significantly associated with being parasite positive.Present in 6.4% and 12.5% of our population control samples and malaria cases respectively, ulations , 50, 55.KIR genes influence susceptibility to malaria. The involvement of genes located at the centromeric part of the KIR locus in modulating the outcome of malaria and in particular the role of KIR2DL2 and KIR2DS2 in the susceptibility to P. falciparum malaria warrant further investigations, and we recommend that larger studies should be performed across different populations to test this hypothesis with additional power. Such studies are needed to complement genome-wide approaches KIR haplotypic diversity.Taken together, our data provide some support for the hypothesis that