Sea ice is a crucial component of the Arctic climate system, yet the tools to document the evolution of sea ice conditions on historical and geological time scales are few and have limitations. Such records are essential for documenting and understanding the natural variations in Arctic sea ice extent. Here we explore sedimentary ancient DNA (aDNA), as a novel tool that unlocks and exploits the genetic (eukaryote) biodiversity preserved in marine sediments specifically for past sea ice reconstructions. Although use of sedimentary aDNA in paleoceanographic and paleoclimatic studies is still in its infancy, we use here metabarcoding and single-species quantitative DNA detection methods to document the sea ice conditions in a Greenland Sea marine sediment core. Metabarcoding has allowed identifying biodiversity changes in the geological record back to almost ~100,000 years ago that were related to changing sea ice conditions. Detailed bioinformatic analyses on the metabarcoding data revealed several sea-ice-associated taxa, most of which previously unknown from the fossil record. Finally, we quantitatively traced one known sea ice dinoflagellate in the sediment core. We show that aDNA can be recovered from deep-ocean sediments with generally oxic bottom waters and that past sea ice conditions can be documented beyond instrumental time scales. Our results corroborate sea ice reconstructions made by traditional tools, and thus demonstrate the potential of sedimentary aDNA, focusing primarily on microbial eukaryotes, as a new tool to better understand sea ice evolution in the climate system. While these methods have advantages such as availability of large datasets, rapid measurement, seasonal sea ice reconstructions, potential for quantitative reconstructions, they do have some immanent limitations such as poor preservation, indirect relation with sea ice, absence under permanent sea ice or a limited regional application ) lication , 4.Here we demonstrate the potential of sedimentary aDNA metabarcoding for sea ice reconstructions and of specific sea ice organisms as palaeo-sea ice indicators, focusing mainly on DNA originating from the microbial eukaryotes. Compared to the traditional proxies, our approach has a strong advantage through a more direct link to sea ice via the broader eukaryote (sea ice) community and/or individual sea ice organisms, whose genetic signatures  have been preserved in sediments and can be used to characterize past biodiversity . Molecul25 (PIP25) index were calculated following ref. [A multicore (MC) and a 19.6-m long Calypso core (CC) were recovered from the East Greenland Sea  and a 5-cm resolution N. pachyderma sinistral isotope stratigraphy [The age model for the core is based on linear interpolation between 10 tie points determined via AMS tigraphy  from 345Full details about the methods, protocols and bioinformatical pipelines are available in the The raw data were quality-trimmed and error-corrected using published bioinformatics tools . This waPolarella glacialis in sedimentary aDNA. Primers amplifying the ribosomal ITS1 region of P. glacialis, Polarella-ITS-44F (5′-CGACTGGGTGGAGATGGTTG-3′) and Polarella-ITS-138R (5′-CCCAGGTGTTTAAGCCAGGT-3′), were designed and tested for efficiency and specificity . All clones (N = 10) from ddPCR amplification of P. glacialis ITS1 from a mixture of all six DNA subsamples from the core surface sediment gave best hit to P. glacialis when compared to GenBank using the blastn algorithm. PCR reactions were performed in C1000 Touch thermocycler with deep-well module (Bio-Rad). PCR products were cloned using a standard cloning kit and Sanger sequenced for verification purposes. Quantitative amplification of P. glacialis ITS1 gene fragments from aDNA was followed by droplet generation and post-PCR enumeration. The ddPCR results were normalised to P. glacialis ITS1 copies per g sediment.Droplet digital PCR (ddPCR) analysis was conducted to quantify the abundance of the sympagic dinoflagellate Protoceratium reticulatum. Nematosphaeropsis labyrinthus and Impagidinium pallidum were abundant and the sample also contained heterotrophic taxa . The samples at 24 and 249 cm contained a characteristic low-diversity assemblage in higher concentrations (respectively 286 and 187 cysts/g sed). Sample 24 cm recorded an autotrophic dinoflagellate cyst assemblage dominated by Spiniferites (including Spiniferites elongatus) and the common presence of Nematosphaeropsis labyrinthus and cysts of Protoceratium reticulatum. Such assemblage is not typically associated with sea ice. In contrast, sample 249 cm was dominated by heterotrophic taxa like Islandinium minutum, Brigantedinium and Round Brown Cysts. This sample also recorded cysts of the sea ice dinoflagellate Polarella glacialis (n = 2). Together, the dinoflagellate cyst assemblage indicated a nutrient-rich environment, associated with  sea ice. Fresh water algae Halodinium and Pediastrum were also recorded.Most samples recorded very few dinoflagellate cysts  was recorded in sample 249 cm, where also high values of HBI-III (0.06 µg/g TOC), brassicasterol (27.46 µg/g TOC) and dinosterol (3.24 µg/g TOC) were recorded  and dinosterol (<2.37 µg/g TOC) showed low values. IP25 only indicates presence or absence of seasonal sea ice, but can be used in combination with phytoplankton biomarkers  to calculate the PIP25 index, which allows to reconstruct sea ice and sea surface conditions, respectively [BIP25 and PDIP25 indices (0.65–0.66), our surface sample and sample 249 cm indicate seasonal sea ice conditions. In all samples where IP25 is (near) zero, this can be interpreted as either sea ice free or permanent sea ice conditions due to limitations of the PIP index [The mono-unsaturated highly branched isoprenoid IP diatoms . In surfnditions , 27. Itsde novo chimera removal resulted in 143,750 reads that clustered into 977 aDNA operational taxonomic units (OTUs) with a 97% similarity cut-off. See within each sediment layer, or α–diversity [between sediment samples, is represented as the unique fraction distance  varied from undetectable (limit of detection 200 pg per assayed sample volume) to 1795 ng DNA per g sediment, with a rapid drop in recoverable double-stranded DNA (dsDNA) from surface to downcore sediments. After sequencing the 18S rDNA gene, quality-filtering, merging, clustering with singleton removal, and iversity , was calac, ref. ). Princiac, ref. . Most noac, ref. .Fig. 3DiConstrained correspondence analysis (CCA) of metabarcoding results (OTUs) using dinoflagellate cyst and biomarker concentrations could explain only 14.4% of the observed genetic diversity in the metabarcoding results   on the downcore samples and identified 348 OTUs with significant discriminatory power  with IP25 and PBIP25 for four putative cercozoan OTUs , one OTU resembling a polar centric diatom (OTU_5051) and a Gymnodinium-like OTU (OTU_333) . The environmental variables employed were concentrations of dinocysts (measure for productivity), brassicasterol and dinosterol , and the sea ice diatom biomarker IPPolarella glacialis. We chose to target this species because it is a known sea-ice associated dinoflagellate that was identified during the microscope analysis. Using PCR primers designed to specifically amplify the P. glacialis ribosomal RNA ITS1 region  contained an approximately 10- to 100-fold higher abundance of detectable P. glacialis ITS1 gene copies compared to the other downcore samples (<40 to 266 copies g/sediment).In addition to qualitative investigation of sedimentary aDNA using metabarcoding, we employed a quantitative approach  to specifically quantify DNA sequences from 1 region , we obseent Fig. . HighestIn our attempt to explore the applicability of using sedimentary aDNA to reconstruct Arctic sea ice on Late Quaternary time scales we demonstrate that DNA from Arctic sediments of ca. 100,000 years old is well preserved, even from a region with generally oxic bottom waters, and that it can be used to describe the sea ice history. We recorded aDNA in all samples of our sediment core in the Greenland Sea Fig. . Our lowWith our generalist approach, using a moderately short fragment of ~260 base pairs targeting a wide diversity of eukaryotic organisms, of which we focus specifically on the micro-sized ones that compare best with traditional sea ice proxies, we gathered the broad molecular signature of Late Quaternary sediments in the East Greenland Sea Fig. . The conCryothecomonas lineage of the Cryomonadida consists of heterotrophic grazers that forage on sea-ice brine communities [25 and low dinoflagellate cyst concentrations (low productivity) likely reflects a permanent sea ice cover between ~17.5 (24 cm) to 51 cal kyr BP (390 cm), except at ~33.7 cal ka BP . IP25 is usually absent in sediments underlying open water and under permanent sea ice conditions, but the low concentration of phytoplankton productivity biomarkers  and dinoflagellate cysts indicate a limited productivity that is most consistent with a permanent or extended sea ice cover. The metabarcode signature between ~17.5 to 51 cal kyr BP may thus be reflecting a permanent sea ice cover. The elevated dinocyst concentrations of mainly Spiniferites at 17.5 cal kyr BP suggests productivity, possibly related to a return to seasonally sea ice free conditions in the region.In the downcore samples (24 to 590 cm), the metabarcoding results show a remarkably strong agreement with the pattern derived from traditional sea ice proxies . In samples 24 to 390 cm, a consistent metabarcoding signature with abundant marine stramenopile and Cercozoa sequences occur Fig. . The marmunities . The mosmunities . Also womunities , 46. In 25, while also brassicasterol, dinosterol and dinocyst concentrations (productivity) increased. The dominance of the sea ice associated dinoflagellate Islandinium minutum and presence of the sea-ice dinoflagellate Polarella glacialis, both detected using microscopy (cysts) and genetic tools (see below) indicate seasonal sea ice in this sample  to have a different signature compared to the other samples Fig. , indicat25 biomarker (and its derived indices PBIP25), we identified significant correlations  were found to be significantly correlated to measured concentrations of IP25. These latter two are discriminatory OTUs for the sample 249 cm, in which the IP25 concentration was highest. OTUs classified as known sea-ice associated taxa, such as dinoflagellates in the Suessiaceae family , were also positively associated with IP25, however this association was not found to be statistically significant  are unclike OTU , 51, 52)like OTU  may be pm Arctic , 55 and m Arctic  surface m Arctic , unfavorP. glacialis cysts in our slides, and previous successful identification of P. glacialis in paleoenvironmental genomics work in Antarctica [P. glacialis in all samples and demonstrated increased abundances in the surface sample (sample 1 cm) and sample 249 cm   fragments are preferential for targeted or quantitative studies [We employed a broad taxonomic characterization of the sedimentary aDNA to reconstruct sea ice in the geological past. To achieve the necessary balance between high phylogenetic resolution and DNA detection in geological samples dating back ~100,000 years, we chose a moderate target amplicon length (~260 base pairs). One of the key challenges of a broad sedimentary aDNA metabarcoding detection approach is that the generated sequence information represents an amalgamation of taxa of diverse origins, not only from sea ice. DNA present in marine sediments can reflect biological diversity present in the sediment biome , 59, or majority . For thi studies , 69, par studies .N = 8) and for DNA extraction (N = 10) were routinely checked for contamination, and where PCR products were visible on an agarose gel (N = 2), purified and sequenced as separate samples. These sequences were bioinformatically removed from all sample data prior to statistical analysis  and sea ice in the geological record is demonstrated indirectly via other sea ice proxies. The relationship between metabarcode signatures, both on microbial eukaryotic community and individual protist species level, in marine sediments and sea ice environments needs to be established and calibrated in the modern environment to demonstrate the link between the sedimentary aDNA data and sea ice environments in the modern ocean. That will allow to fully develop sedimentary aDNA as an independent sea ice proxy and exploit its potential for understanding the evolution of the Arctic cryosphere. We demonstrate here that this approach has a tremendous and untapped potential, even in regions with oxygen-rich bottom waters [In summary, we have shown how past microbial ecology can be used in climate research by demonstrating that universal metabarcoding and single-species quantitative DNA approaches can characterize sea ice evolution on a Late Quaternary timescale and both corroborate and complement sea ice reconstructions using traditional paleo-sea ice proxies. The major shifts in the Greenland Sea sedimentary aDNA profiles determined from metabarcoding co-occur with changes in palynology and sea ice biomarkers, all demonstrating a shift from a permanent to seasonal sea ice regime. Furthermore, detailed bioinformatic analyses revealed previously unknown OTUs  in samples where traditional proxies indicate sea ice presence. Subsequently, the sea ice dinoflagellate m waters .www.pangaea.de at doi:10.1594/PANGAEA.900724. Metabarcoding sequence data is freely available from the public databases as a Sequence Read Archive with accession ID PRJEB27691.Palynological, biomarker and ddPCR concentration data are freely available from the Bjerknes Centre Data Centre (BCDC) and Supplementary informationSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Table 4Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Captions


These findings are further supported by experimental evidence of a substantial reduction in phosphorylation of DARPP32 and GLUR1, two PKA targets in CTIP2-deficient MSNs. Moreover, we show that CTIP2-dependent dysregulation of protein phosphorylation is shared by HD hPSC-derived MSNs and striatal tissues of two HD mouse models. This study therefore establishes an essential role for CTIP2 in human MSN homeostasis and provides mechanistic and potential therapeutic insight into striatal neurodegeneration.The mechanisms underlying the selective degeneration of medium spiny neurons (MSNs) in Huntington disease (HD) remain largely unknown. CTIP2, a transcription factor expressed by all MSNs, is implicated in HD pathogenesis because of its interactions with mutant huntingtin. Here, we report a key role for CTIP2 in protein phosphorylation via governing protein kinase A (PKA) signaling in human striatal neurons. Transcriptomic analysis of CTIP2-deficient MSNs implicates CTIP2 target genes at the heart of cAMP-Ca •CTIP2 protects human MSNs against oxidative stress-induced cell death•PKA-regulated DARPP32 and GLUR1 phosphorylation is reduced in CTIP2-deficient MSNs•HD MSNs share CTIP2-dependent downregulation of protein phosphorylation by PKA In this article, Fjodorova and colleagues provide mechanistic and potential therapeutic insight into striatal neurodegeneration caused by CTIP2 hypofunction. CTIP2 plays an essential role in human MSN homeostasis via regulating PKA-dependent DARPP32 phosphorylation and protecting MSNs from oxidative stress-induced cell death. Protein phosphorylation deficits occur in human and mouse Huntington disease MSNs due to CTIP2- and mHTT-co-regulated molecular signaling abnormalities. Inhibitory γ-amino butyric acid (GABA)-releasing medium spiny neurons (MSNs) are the principal projection neurons of the basal ganglia, receiving inputs from both glutamatergic cortical neurons and midbrain dopaminergic neurons. DARPP32 is a class-defining protein marker for striatal MSNs and a central mediator of dopaminergic and other first-messenger signaling in these cells. Specifically, phosphorylation of DARPP32 at threonine 34 (pDARPP32-Thr34) by protein kinase A (PKA), following dopamine D1 receptor activation, is critically involved in regulating electrophysiological, transcriptional and behavioral responses of MSNs to physiological and pharmacological stimuli, including antidepressants, neuroleptics, and drugs of abuse . Thus, tCTIP2  is a transcription factor expressed by all MSNs and is required for MSN development and transcriptional regulation of striatal genes . CTIP2 dUsing CRISPR/Cas9 genome-edited human embryonic stem cells (hESCs) as a model, we uncover a role for CTIP2 in regulating protein kinase and phosphatase levels and activity and subsequently phosphorylation of DARPP32-Thr34 in MSNs. We show for the first time that deficits in PKA-dependent protein phosphorylation occur in human and mouse HD MSNs, potentially owing to CTIP2- and mHTT-co-regulated molecular signaling abnormalities as suggested by transcriptomic analysis. This study provides evidence of a central role for CTIP2 in human MSN homeostasis and supports the hypothesis that CTIP2 may mediate regional specificity of HD pathogenesis.CTIP2 gene (in vitro (DIV), both control and CTIP2KO hESCs gave rise to high yields of cells expressing lateral ganglionic eminence (LGE) markers GSX2, FOXP2, ISL1, and MEIS2, subpallial marker ASCL1, with very few TBR1+ cortical cells  hESC lines were generated using the CRISPR/Cas9-assisted genome editing technology with guide RNAs targeting exon 2 of the IP2 gene A–S1D. ToIP2 gene . At 20 dy period C–1F. TakS-nitroso-N-acetylpenicillamine (SNAP), which was previously shown to generate reactive oxygen species and induce oxidative stress  analysis of CTIP2KO no. 4 and control MSN cultures at 20 and 40 DIV . Global principal component analysis showed clear sample segregation based on genotype and developmental stage A. Analys2+ transport, and cation channel activity were significantly dysregulated in our CTIP2KO datasets  appeared in the PKA gene set , is known to dephosphorylate DARPP32-Thr34 in a Cat manner . Westernt manner D and 3E.t manner F and 3G,t manner H, providFinally, we demonstrate that reduction in pDARPP32-Thr34 levels is directly caused by the loss of CTIP2 in MSNs. To this extent we re-introduced CTIP2 into CTIP2KO no. 4 MSNs via viral delivery of CTIP2 transgene in postmitotic MSN neurons and determined levels of DARPP32 phosphorylation I and S1EBecause CTIP2 level is reduced in several HD models that share some of the deficits with CTIP2KO MSNs, we investigated potential overlap between CTIP2- and mHTT-mediated transcriptomic changes by comparing our MSN20 and MSN40 DEGs with four publicly available datasets of human and mouse HD models . This anTo strengthen this observation and provide the first experimental support, we examined DARPP32 and Glur1 phosphorylation, and Ppp3ca and Ppp3cc phosphatase levels, in animal and human cell models of HD. More than a 50% decrease in pDarpp32-Thr34 levels was observed in striatal homogenates of R6/1 and Q175 mice compared with wild-type (WT) littermates A and 4B.Ctip2-deficient mice exhibited normal expression of immature MSN markers (Meis2 and Nolz1) in the developing LGE at embryonic day 14.5. However, the number of Darpp32+ neurons was significantly reduced in the newborn striatum. It remained unclear therefore whether Darpp32+ MSNs could initially be generated in Ctip2−/− mice. Using a panel of LGE and postmitotic MSN markers, our study provides the first evidence that CTIP2 deficiency does not compromise the initial birth of MSNs from hESCs.We have developed an efficient and versatile platform for investigating the role of transcription factor CTIP2 in human MSN development and homeostasis. CTIP2-driven DEGs identified in the current study are significantly enriched in striatal genes associated with both direct and indirect pathways in the basal ganglia, which is in line with previous findings in mice . These C2+ signaling and kinase activity. ADD3 and CNGA3 regulate synaptic plasticity, Ca2+ transport, and activation of cAMP-dependent PKA, and were significantly downregulated in CTIP2-deficient MSNs , HD hESCs (46Q and 51Q), and HD hiPSCs 71Q; see .imagej.net) from >5,000 cells/sample blind to the experimental condition.Cells were incubated with primary antibodies overnight at 4°C followed by Alexa Fluor secondary antibodies for 1 h. Staining quantification was acquired manually in ImageJ .ad libitum.Animal work was done under UK Home Office personal and project licenses in accordance with the requirements of the UK Animals (Scientific Procedures) Act 1986. Animals were group-housed and received water and food Equal amounts of proteins for each sample were separated on 4%–12% Bolt Bis-Tris Plus gels (Thermo Fisher Scientific) and transferred via electro-blotting to a polyvinylidene difluoride membrane . Membranes were incubated with primary antibodies overnight at 4°C followed by secondary antibodies for 1 h. All blot images were quantified in ImageJ blind to the experimental condition.1, n2, and n3, where n1 and n2 are the number of control and CTIP2KO (or HD) samples, respectively, from n3 independent experiments.Statistical analysis was performed using one- or two-way ANOVA tests and considered statistically significant at p < 0.05. Sample sizes for each test are indicated in the figure legends as n = nM.F. and M.L. conceived the study and designed the experiments. M.F. carried out and analyzed the CTIP2 hESC and HD animal experiments with support from Z.L. and E.D. D.C.L.F. contributed to the RNA-seq data analysis and interpretation. M.L. (Paris) and A.L.P. contributed HD hPSC data and S.P.B. supplied HD animals. M.F. and M.L. wrote the manuscript and S.P.B. edited the manuscript.


Diffraction of flexural-gravity waves in an ice cover by a bottom mounted structure with vertical walls is studied. The problem is solved by using the so-called vertical modes corresponding to the roots of the dispersion relation for flexural-gravity waves. These modes reduce the original three-dimensional problem to a set of two-dimensional diffraction problems with non-homogeneous boundary conditions on the rigid walls. Two unknown functions presenting in the boundary conditions for each mode are determined using the conditions at the contact line between the ice cover and the vertical walls. The clamped conditions at the contact line, where the ice cover is frozen to the wall, are considered in this study. The solution of the problem is obtained for a single vertical circular cylinder frozen in the ice cover. A general approach to the problem for vertical cylinders of any shapes is presented. The diffraction problems with vertical walls extended to infinity are discussed.This article is part of the theme issue ‘Modelling of sea-ice phenomena’. The water depth H is taken as the length scale, 1/ω as the time scale, A is the scale of the deflections and AHω is the scale of the velocity potential of the flow. The ice deflection, w, and the velocity potential, ϕ, are periodic in time,Φ, in ∈D. The plane z = − 1 corresponds to the flat rigid bottom, and the plane z = 0 corresponds to the ice–fluid interface. The potential Φ also satisfies the following boundary conditions,N is the normal unit vector to the vertical wall Γ and ∂Φ/∂N is the normal derivative of the potential on the wall. The equation of thin ice plate can be written in the form, see  for an elastic plate of constant thickness, Ei is the Young modulus of the ice, ν is the Poisson ratio, ρ is the water density and g is the gravitational acceleration. The condition at infinity follows from ∈Γ, between the ice cover and the surface of the cylinder can be complicated in practical problems. The present method of vertical modes is not sensitive to the types of these conditions. The method is demonstrated here for the ice cover being frozen to the vertical cylinder, which is modelled by the clamped conditions,The fluid flow and ice deflection are caused by an incident hydroelastic wave,irection , where Aorm, see ,2.5wherce sheet , ω0 =  = Wnfn(z) satisfies Laplace's equation (fn(z) is a non-trivial solution of the following spectral problem:n is a root of the dispersion relation  is a solution of the equation0 and −ϰ0, where ϰ0 > 0, infinite number of pure imaginary roots, ϰn = iμn and −ϰn, where n≥1 and μn+1≥μn > 0, and four complex roots, ϰ−2 = − a + ib, ϰ−1 = a + ib, −ϰ−2 and −ϰ−1, where a > 0 and b > 0 /(ϰnsinh[ϰn]), normalized by the condition f′n(0) = 1, are known as the vertical modes. They are orthogonal, 〈fj, fn〉 = 0, 〈fn, fn〉 = Qn, where j≠n and the scalar product of two functions F(z) and G(z), which are bounded together with their derivatives up to the third order in the interval −1 ≤ z ≤ 0, is defined byQn = (ϰ2n(ϰ4n + δ)2 + q(5ϰ4n + δ − q))/(2ϰ2nq2). For the imaginary roots of the dispersion relation, ϰn = iμn, where μn > 0 and n≥1, we have μn = πn − q(πn)−5 + O(n−6) as n → ∞. Therefore, Qn = O(n8) as n → ∞. The conditions at infinity for equation  =equation , n≥ − 2,Γ for equation , where N = 0 on the vertical wall Γ and S is a curvilinear coordinate along the wall. Using (1) we can calculate the derivative ∂Φ/∂N near the wall, where N > 0, by differentiating the series (1) term by term. Then, to evaluate this normal derivative on the wall, we take the limit as N → 0. In this way, we satisfy the wall boundary condition (2) as the limit where the boundary Γ is approached from the flow region D. This procedure of satisfying the boundary condition (2) at the vertical wall Γ is needed here because the vertical mode method requires the derivatives up to the third order, see the definition of the scalar product (Φ/∂N)]/∂z is equal to zero on the vertical wall, ∈Γ, −1 < z < 0, including the limit as z → 0. On the other hand, the derivative ∂[(∂Φ/∂z)]/∂N = ∂W/∂N, is equal to zero as N → 0 only for the clamped conditions at the contact line. The boundary condition on the vertical wall (2) is understood here as the limit where N → 0. In addition, the first and third derivatives in z at z = 0 in the definition of the scalar product (F′′′(z), G′′′(z), F′(z) and G′(z) where z → 0−.The boundary conditions on equation  are deril. Using 1 we can e series 1 term byondition 2 as the ondition 2 at the  product . Howevercal wall 2 is unde product  are undeΓ for equation (2) on the vertical modes fk(z), k≥ − 2. By definition,Wk/∂N)(S) is the normal derivative of the function Wk on Γ. On the other hand, by using , and f′k(0) = 1, f′′′k(0) = ϰ2k, see . Taking the limit as z → 0− in these equations and using the kinematic condition (3), we findN > 0. Finally taking the limit in (N → 0 and equating the results of (α(S) = (∂W/∂N)(S) is the slope of the ice plate at the vertical wall Γ and Q(S) = − (∂/∂N)(∇2W) is the non-dimensional shear force on Γ with the scale DiAH−3.Here the limit of the integral is zero, see 2, and f′ϰ2k, see  and the equation  gives ∂3ondition 3, we finlimit in  as N → 0sults of  and 3.72, and f′k≥ − 2, provides the required boundary conditions at Γ for equation  and α(S) are unknown in advance and should be determined by using the conditions at the contact line. Note that these functions do not depend on the index of the vertical mode k, see  can be decomposed asn≥ − 2 and n≠0. The diffraction potential, WD), is the solution of equation (0/∂N)(S) = 0 on Γ. The radiation potentials n≥ − 2, are the solutions of equation   3.10) anent wave .There are two difficulties with the derived solution. First, it should be explained how the solution with the edge conditions  can be u4.Γ between the vertical wall and the ice plate. Notations (R(S) is the radius of curvature of the curve Γ. The boundary conditions (α(S) = 0 and Q(S) = 0 are known as conditions of sliding edge, where the plate edge is free to move vertically but its rotation is not allowed /n and the Laplace equation  in , O(n−7) and O(n−3) correspondingly. Therefore, condition (1) and the kinematic condition (3) on the ice–fluid interface are satisfied term by term. The far-field condition (2).Differentiating the series for y, z) in  twice innd using  and  in  five timondition , we findondition  convergeondition  is satisondition . The bouondition 1 and theondition 3 on the ondition  is not rondition , see equondition  for detaondition –2.7) wi wiΦ at Γ, we find2) is satisfied if both series on the right-hand side of  is the function on the left-hand side of the dispersion relation  over CM tends to zero as M → ∞. On the other hand, the contour integral can be evaluated by the residue theorem, it is equal to the first series in  and α(S). This result indicates that the series , n≥ − 2, are not independent and at least two of them can be presented as linear combinations of other functions, see [fn(z), n≥ − 2 is complete on the interval −1 < z < 0. Moreover, each vertical mode corresponds to a certain root of the dispersion relation  = 0 and (∂R/∂ϰ) = 0 with respect to δ and q for ϰ = iμ, μ > 0. The solutions of the linear system are shown in δ and q at the cusp point provide a triple root. If the characteristics of the ice cover are given but the frequency of the incident wave varies, then the non-dimensional parameters δ and q are related by δ + q(m/ρH) = (H/Lc)4. For the characteristics of the ice cover and the water depth from the calculations for δ = 0.3844 − 0.009q, see dotted line in −1.The result that both series in  are equarelation . For som6.The method of vertical modes in the three-dimensional problems of flexural-gravity waves and their interaction with vertical walls has been described and applied to the problems with different conditions at the contact line between the ice cover and the vertical wall. The method can only be used for a constant water depth. In this study, the ice deflection is caused by an incident wave and the vertical wall represents a bottom-mounted vertical cylinder. It has been shown that the problem requires a general solution of an auxiliary problem with unknown in advance shear and deflection slope along the contact line. This general solution is obtained by using the vertical modes. Then the two unknown functions at the contact line are obtained using the physical conditions at the contact line. By using a set of orthogonal functions defined at the contact line, the latter problem can be reduced to a system of algebraic equations for any shape of the wall. This approach was applied to the problem of flexural-gravity wave interaction with a vertical circular cylinder frozen in the ice cover.There are several open questions left after this study. It is not clear how to generalize the approach to problems with waves generated by a periodic external pressure near the walls. How can this method be used in problems with vertical walls extended to infinity? In particular, for an ice channel. It is not clear how to apply the method for conditions where the dispersion relation for the flexural-gravity waves has multiple roots. How can problems be solved with mixed conditions at the contact line? On the other hand, the method of vertical modes is very suitable for radiation problems with oscillating structures or parts of them.


Screw fixation is a typical technique for the isolated subtalar joint. However, no consensus has been reached on how to select the most suitable insertion position and direction. This study aims to find the ideal screw insertion and then explore its influence on the clinical efficacy of subtalar fusion by analyzing the effects of different cannulated screw insertions on the stress distribution, anti-rotary strength, and anti-inversion/eversion strength of the subtalar joint.In this study, we investigated three cannulated screw insertions for subtalar fusion: screw insertion with the most uniform stress distribution (group A), lateral-medial parallel screw insertion (group B), and traditional longitudinally parallel screw insertion (group C). The effects of these three insertions on the loading stress of the subtalar joint  were comparatively analyzed with the three-dimensional finite element method to screen the ideal screw insertion. Moreover, a prospective study was conducted to analyze the influence of the ideal screw insertion on subtalar fusion, including the fusion rate, fusion time, and clinical efficacy .P < 0.05), and the AOFAS score increased from 57.0 to 85.6 (P < 0.05), with a good and excellent rate of 95.8%.Group B was worse than group A with respect to the stress distribution uniformity, but slightly better than group C, and better than both groups A and C in terms of the anti-rotary strength and anti-inversion/eversion strength. The screw insertion based on the most uniform stress distribution is not feasible in surgery. Therefore, the lateral-medial antiparallel screw insertion is the ideal insertion. From January 2012 to June 2016, 48 cases were treated by subtalar fusion with the ideal screw insertion, and then followed up for 30.6 months (12–48 months). The fusion was proved in all 48 cases with a fusion rate of 100% by X-ray or CT scan. The mean time of fusion was 12.8 weeks (12–16 weeks). The VAS score decreased from 6.00 before operation to 1.03 on the last visit (The lateral-medial parallel screw insertion not only demonstrates a good stress distribution profile of the subtalar joint but also has advantages such as easy localization and operation during surgery, as well as a high fusion rate and few complications after surgery. Therefore, it is a safe, accurate, and effective fixation mode that is worthy of being popularized clinically. Subtalar fusion is the gold standard therapy for severe subtalar arthritis  to mainlBased on the above issues and with the purpose of providing a good, feasible screw insertion approach for subtalar fusion and thus obtaining a satisfactory fusion rate, this study was planned to first identify the screw insertion with the most uniform stress distribution of the subtalar joint by 3D finite element analysis. Then, this study comparatively analyzes the differences of three screw insertions in terms of the stress distribution, anti-rotary strength, and anti-inversion/eversion strength to screen the ideal screw insertion and finally further analyzes the effects of the ideal screw insertion on the efficacy of subtalar fusion in a prospective form.A male patient was selected who was 28 years old with a height of 160 cm and a body weight of 52 kg. X-ray examination: No improper lower limb alignment and abnormal ankle bony structure were detected by ankle X-ray posterior-anterior and lateral projection scans; foot tumor, deformation, and other lesions were excluded.CT examination: The scan range was from 5 cm above the ankle to the planta pedis of the whole feet. The scan parameters were as follows: tube current of 100 mA, tube voltage of 120 kV, and slice thickness of 0.75 mm. The up-down plain scan was performed to obtain the 2D CT images of 527 slices. The 2D CT imaging data were processed in the PACS image processing workstation system, and all data were saved in the DICOM format.The raw data in the DICOM format were read by Menu bar > File > import image in the Mimics17.0 software, and the coronal and axial continuous 2D images were constructed with the CT sagittal data.Via Menu bar > Segmentation > Thresholding, the CT threshold of the bony structures in the whole feet was automatically segmented and extracted as 226-2710HU, and the CT value of the bony structures was read.The 3D model was calculated by Segmentation > Calculate 3D, and the talus and calcaneus entities were segmented separately by Segmentation > CT bone segmentation.The talus and calcaneus entities were duplicated and reversely translated 50 mm along the reference axis. Then, the talus and calcaneus models were migrated opposite along the reference axis, and Boolean operations were performed on the talus and calcaneus entities to delete the original talus and calcaneus entities separately and save the intersection community. Finally, the whole 3D entity models of the talus and calcaneus were generated and output in a format of “x_t” for use in the subsequent finite element analysis.This study uses a simplified model and hardly considers the screw threads. Therefore, the details of the screw threads were neglected, and the screws were simplified as 7.3 mm × 8 cm glossy cylinders.Group A: The screw insertion with the most uniform stress distribution of the subtalar joint was analyzed.The ranges for the screw insertion were set as follows: (1) two screws penetrated the subtalar joint; (2) the bony areas for the screw entry were the bone surface of the talar neck  and areas of posterior and posterosuperior calcaneal tubercles; and (3) the screw insertion with the most uniform stress distribution of the subtalar joint was found by changing the distance between two screws and the angle between the screw and the subtalar joint and loading a stress of 50 Mpa on the calcaneus along the screw.Group B: The effects of lateral-medial parallel screw insertion on the stress distribution of the subtalar joint were analyzed.Procedures of the lateral-medial parallel screw insertion: First, the talar neck and posterior calcaneal tubercle were divided in triplicate transversally. Second, two screws were inserted separately from the lateral-medial equal division points of the talar neck into the corresponding lateral-medial equal division points of the posterior calcaneal tubercle. Finally, a stress of 50 Mpa was loaded on the calcaneus along the screw, and thereafter, the stress distribution of the subtalar joint was observed.Group C: The effects of the traditional anteroposterior longitudinally parallel screw insertion on the stress distribution of the subtalar joint were analyzed.Procedures of the traditional anteroposterior longitudinally parallel screw insertion: First, the posterior calcaneal tubercle was divided in triplicate longitudinally. Second, two screws were inserted separately toward the talus via the subtalar joint from the equal division points of the posterior calcaneal tubercle. Finally, a stress of 50 Mpa was loaded on the calcaneus along the screw, and thereafter, the stress distribution of the subtalar joint was observed.The stress distribution nephogram, uniform stress, and maximum uniform area were used as indicators to evaluate the stress distribution. A bigger uniform stress and maximum uniform area indicated a more uniform stress distribution.Comparison of anti-inversion/eversion strength: The subtalar joint inversion was simulated by confining the calcaneal body and then exerting an upward constant traction force on the body laterally after two screws were inserted. The distance of the talus from the subtalar joint was compared, and a smaller distance suggested a higher anti-inversion/eversion strength.Comparison of anti-rotary strength: Similarly, the subtalar joint rotation was imitated by exerting a horizontally constant traction force on the talar neck laterally. The distance of the talus from the lateral border of the subtalar joint (which served as the reference) was compared, and a smaller distance also reflected a higher anti-rotary strength.The horizontal and vertical displacements between the talus and calcaneus were evaluated with an indicator of “mm.” A smaller maximum displacement corresponded to a higher anti-inversion/anti-rotary strength.This was a prospective study approved by the Ethics Committee of Southwest Hospital. Before operation, X-ray, CT, and MRI scans, as well as the AOFAS scoring, VAS scoring, and conventional admission evaluation, were routinely performed.The inclusion criteria are as follows: (1) severe subtalar arthritis, i.e., peripheral pain of the subtalar joint, aggravated during walking, seriously influencing daily life and (2) no efficacy of drug, local block, physical rehabilitation, and other conservative treatments for over 6 months. The exclusion criteria are as follows: (1) severe internal diseases, (2) severe subtalar infection, and (3) severe osteoporosis.From January 2012 to June 2016, a total of 48 cases of severe subtalar arthritis were treated by subtalar fusion with the ideal screw insertion. Before operation, the course of the disease was 32 months (1–360 months), and the age was 43.7 years (19–74 years).After satisfactory anesthesia, an incision was made from the lower part of the fibular apex to the basilar part of the fourth metatarsus, and the complete cartilage was removed to approximately 2 mm of the subchondral bone with osteotome and osteotribe, so that the joint surfaces were completely coupled. The subtalar joint was fixed with the ideal screw insertion . The talar neck and posterior calcaneal tubercle were divided in triplicate transversally, the foot was kept in a neutral position, two 2.0 mm guide wires were inserted separately from the lateral-medial equal division points of the talar neck into the corresponding lateral-medial equal division points of the posterior calcaneal tubercle Fig. , and twoAt 6 weeks, 3 months, 6 months, and on the last visit after operation, the functional evaluation and satisfaction evaluation were performed separately with the VAS pain scoring and AOFAS hindfoot function scoring. The ankle-hindfoot function scoring results were classified into four levels: level 1, 90–100, excellent; level 2, 72–89, good; level 3, 41–71, medium; and level 4, 1–40, poor. The excellent and good results were satisfactory, and the medium and poor results were unsatisfactory. At 1 day, 6 weeks, 3 months, 6 months, 1 year, and thereafter every half a year after operation, X-ray examination was performed; CT examination were performed if the fusion could not be assessed by X-ray examination. The postoperative complications were observed.t test. P < 0.05 suggested that a difference was statistically significant.The measurement data were presented as The finite element models for the subtalar bony structure and double-screw fixation and three different screw insertion models were effectively established. There were 120,000 grids of information generated for the subtalus, 70,000 grids of information for the calcaneus, and 20,000 grids of information for the screws. The established subtalar joint model had a good morphology and good coupling; thus, it could be used to analyze the stress distribution of the subtalar joint.2, the overall stress distribution was not uniform, the maximum peak stress was 40 MPa, the areas with a higher stress were distributed in a band shape, and the stress was 23~27 MPa; so, there was a wide range of stress concentration  was 7.076 × 10−3 mm, 6.257 × 10−3 mm, and 7.145 × 10−3 mm, respectively ; X-ray or CT scan showed bony fusion and good bony suitability of the subtalar joint in 48 cases , expanded to the medical life science field from the structural engineering field, has become a practical, efficient, and digital analysis approach. FEA can be applied to effectively simulate the true geometry of the ankle and the stress distribution of various ankle parts . The subThe screw insertion with the most uniform stress distribution of the subtalar joint was found by 3D FEA. Combining the anatomic characteristics of the talus and calcaneus, the lateral screw was near the boundary of the talar neck and the medial screw was close to the talar surface of the ankle joint in the area of the talar neck. The former was located at the boundary of the posterior calcaneal tubercle and the latter was adjacent to the Achilles’ tendon terminal in the area of the posterior calcaneal tubercle. Therefore, inserting the lateral screw easily caused the split of the lateral talar and calcaneal bones, while inserting the medial screw easily injured the articular capsule, talar cartilage, and Achilles’ tendon terminal during the surgical procedures. It was seen that the screw insertion had disadvantages such as difficult localization and insertion and a big injury to adjacent tissues in the insertion area. In terms of the stress distribution uniformity, anti-rotary strength, and anti-inversion/eversion strength, the traditional longitudinally parallel screw insertion was inferior to the lateral-medial parallel screw insertion. When the screw was inserted toward the talus via the posterior calcaneal tubercle, the projection point of the screw was located on the talar surface of the ankle joint. Since the screw was inserted from the posterior calcaneal tubercle, the screw insertion position in the talar neck was not considered. The advantages were simple operation and easy localization, and the disadvantage was the possibility of injuring the talar surface if the selected screw length was unsuitable. The lateral-medial parallel screw insertion was worse than the screw insertion with the most uniform stress distribution in terms of the stress distribution uniformity, but comparable to the latter and superior to the traditional longitudinally parallel screw insertion in terms of the anti-rotary strength and anti-inversion/eversion strength. The advantages were that the talar neck and posterior calcaneal tubercle were divided in triplicate and the screws were inserted from the equal division points; the localization was simple, the insertion was convenient, and the insertion position was reliable and stable. The major movement of the subtalar joint was the inversion/eversion; the lateral-medial parallel screw insertion was vertical to its movement axis while the longitudinally parallel screw insertion was parallel to this axis. Thus, our independently designed lateral-medial parallel screw insertion demonstrated a stronger anti-rotary strength and anti-inversion/eversion strength in biomechanics than the traditional anteroposterior longitudinally parallel screw insertion. It was further confirmed by 3D finite element analysis.Numerous clinical and biomechanical experiments have shown that the firm internal fixation is beneficial for improving the joint fusion rate, decreasing the infection rate, and promoting early functional training. Our follow-up results suggested that after operation, the AOFAS score was markedly increased, and the VAS scoring results indicated a significant pain relief in all patients. Muñoz  treated The lateral-medial parallel screw insertion not only demonstrates a good stress distribution profile of the subtalar joint but also has advantages such as easy localization and operation during surgery, as well as a high fusion rate and few complications after surgery. Therefore, it is a safe, accurate, and effective fixation mode which is worthy of being popularized clinically.


Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response. The study of the mechanisms of DNA repair is a major challenge in the fight against cancer. Indeed, these mechanisms are often exacerbated in cancer cells. Thus, they provide radio- and chemotherapy resistance properties. Understanding DNA repair pathways allows us to consider new methods to limit this phenomenon in order to reduce tumor progression.Homologous recombination (HR) is one of the mechanisms of DNA repair. Very well preserved during the evolution, it allows faithful repair. It is a precise and highly complex mechanism that is finely regulated. It involves many proteins whose central actor is the RAD51 recombinase. RAD51 is distributed between the cytoplasm and the nucleus but its nuclear translocation is increased in response to DNA damage with the help of the BRCA2 protein ,4. This The activity of RAD51 is precisely regulated by post-translational modifications (PTM) as well as by its association with other protein partners such as BRCA2 [Indeed, a sequential phosphorylation of RAD51 on S14 by CK2 and then on T13 by Plk1 has been proven demonstrated . This doSimilarly, RAD51 phosphorylation on T309 by Chk1 seems to be required for the formation of RAD51 nuclear foci on DNA damage site. Interestingly, the absence of this phosphorylation increases cell sensitivity to DNA damage suggesting the importance of these PTM in the DDR biological process .Actually, only one tyrosine kinase family has been described to phosphorylate RAD51 and it concerns c-ABL or its oncogenic fusion protein BCR/ABL which are able to phosphorylate RAD51 on Y315 and Y54 residues. Thus, the phosphorylation of RAD51 by c-ABL modulates its recruitment on DSB sites and its strand exchange activity . In cellSeveral studies suggest that the HR pathway is modulated by receptor tyrosine kinases (RTK). For example, the inhibition of EGFR, overexpressed in many cancers, leads to a decrease in homologous recombinant repair by acting on the Ras/Raf or IRS1 . AnotherHGFR is expressed in a large number of cell types  and plays a role mainly in embryonic development but also in carcinogenesis. It is activated by its HGF ligand, which is mainly expressed in the placenta. Ligand binding allows dimerization of the receptor and leads to its autophosphorylation on intracellular tyrosines of the receptor. This autophosphorylation triggers the tyrosine kinase activity of the receptor. Then, many signaling channels are activated in the cell. They affect mostly cell proliferation and survival.The overexpression or the presence of constitutive mutations of the c-MET receptor, but also the overexpression of the ligand in many human cancers such as carcinomas, sarcomas and glioblastomas is related to significant morbidity ,16,17,18Our study shows, for the first time, the existence of a direct link between RAD51, the key protein for DNA repair by HR, and a membrane receptor with tyrosine kinase activity, c-MET. By combining in vitro and in silico approaches, we show that c-MET is able to phosphorylate RAD51 on several tyrosine residues. These phosphorylations are closely related to the oligomeric state of RAD51 and to its interaction with the BRC motif of BRCA2. Our results suggest that c-MET-mediated RAD51 phosphorylation could play a potential role in the BRCA2-dependent regulation of RAD51.E. coli) that ensures the production of non-phosphorylated RAD51.To evaluate the phosphorylation of RAD51 in vitro, we used purified RAD51-WT recombinant proteins and the C-terminal catalytic domain of c-MET. This catalytic domain is indeed constitutively active even in the absence of the HGF ligand because tyrosines of the catalytic domain are phosphorylated . InteresImmunoblot analysis showed a significant increase in the phosphorylation state of a protein at 37 kDa, revealed by both the anti-phosphotyrosine antibody and the antibody directed against the RAD51 protein A. This sA specific c-MET kinase inhibitor, PHA665752, was also used to test the specificity of this phosphorylation. PHA665752 is a competitive ATP inhibitor that fits into the ATP pocket of the c-MET receptor at residues I1084-K1110 . A signiWe first used several phosphorylation prediction softwares . The results suggest that among all RAD51 tyrosines, six are potentially phosphorylatable by c-MET: Y159, Y178, Y191, Y205, Y301 and Y315.To verify and validate these bioinformatic prediction results, two experimental approaches were used, the first by site-directed mutagenesis of tyrosine residues of RAD51 and the second by mass spectrometry.Using site-directed mutagenesis, each tyrosine residue of interest was replaced by a phenylalanine residue. This leads to the production and purification of non-phosphorylatable RAD51 mutants with a purity of over 95% . Indeed,E. coli BL21-DE3 bacteria and then purified on a NiNTA column. After the production of 11 proteins (mutants and wild type), in vitro phosphorylation assays using the c-MET kinase were performed . A migration of RAD51 phosphorylated by c-MET was carried out on SDS-PAGE gel. Staining with Coomassie blue G-250 allowed the identification of the bands which were then stored in a H2O/Acetic acid 10% medium. MS analysis confirmed the phosphorylation of 3 of the 4 previously obtained sites: Y159, Y191 and Y205.By combining all the results obtained by these different methods of biostatistics, biochemistry and biophysics, we concluded that the in vitro phosphorylation of RAD51 by the c-MET kinase occurs at four tyrosines: Y159, Y191, Y205 and Y315.The crystallographic data of the RAD51 nucleofilament obtained by Brouwer et al. , led us The visualization on a trimer of RAD51 allowed us to position the four residues in the central monomer  increase in the phosphorylation of the two mutants compared to the RAD51-WT protein .The RAD51 protein is normally present in monomer-oligomer equilibrium . We usedWe also performed the same type of experiment with the no-polymerizable mutants F86E B and A19This convergence of phospho/non-phospho ratios demonstrates that the phosphorylation of RAD51 by c-MET is increased with the monomeric forms and emphasizes the importance of the polymerization state on the phosphorylation level of RAD51 by c-MET.There is a tight relationship between the oligomerization and phosphorylation state of RAD51. To find out whether the level of oligomerization of RAD51 changes when the protein is phosphorylated, we carried out a crosslinking assay of the oligomer forms of RAD51 before and after phosphorylation. The proteins were then analyzed by SDS-PAGE and immunoblotting. Revelation with an anti-RAD51 antibody shows that RAD51 phosphorylation by c-MET leads to an increase in multimer forms of RAD51 A. IndeedIs the structural equilibrium of RAD51 closely related to its phosphorylation, what could be the effect of phosphorylation on the RAD51 nucleofilament? Nucleofilament formation, which is highly dependent on the self-association capacity of RAD51, is indeed an essential step in HR.®). A biotinylated single-stranded DNA was immobilized on a streptavidin tip, and then incubated in a solution containing phosphorylated or non-phosphorylated RAD51 protein. This approach enabled us not only to evaluate the association of the phosphorylated RAD51 protein with the DNA but also to follow the dissociation of the DNA with or without the BRC4 peptide. Concerning the association phase of RAD51 with DNA, we observed that there is no difference between the phosphorylated and non-phosphorylated form , a significant increase of about 20% of recombination frequency is observed . SimilarIn the last decade, DNA repair pathways have been described as promising targets in cancer therapy . UnderstPrevious results highlight the possible connection between c-MET receptor and RAD51 since kinase inhibition reduces the protein level of RAD51  and induThe activity of RAD51 involves many protein-protein interactions which determine the function and the role of RAD51 in the DNA repair context. The RAD51 protein presents different oligomeric forms in buffer solution ,36. StruNucleotide cofactors are also important elements to consider as they modulate the equilibrium and configuration of RAD51 oligomers. Schay et al.  demonstrThe self-association of RAD51 involves a polymerization motif located in its N-terminal domain. Mutations in this region such as F86E, A89E lead exclusively to monomeric forms and thus to the abrogation of the self-association of RAD51 ,26.By using non-polymerizable mutants, we have shown that the level of RAD51 phosphorylation by c-MET is strongly related to the oligomeric state of RAD51 .This RAD51 monomer-monomer self-association property holds a prominent place in the formation of the RAD51 filament on single-stranded DNA (ssDNA). Disturbance in the monomer-monomer interface may impact nucleofilament formation and thus recombinase activity.By combining in silico prediction, mass spectroscopy (MS) and biochemical approaches, we identified four phosphorylatable tyrosines  by c-MET kinase.It is interesting to note that the Y315 phosphorylation detection failed by MS method. In our previous work, we proposed an explanation of this absence of detection. After proteolysis of RAD51 by trypsin, the length (28 amino acids) of Tyr315-containing peptide and its negative charge (pHi = 4.03) could interfere with its detection by MS , Y315 phThe four phosphorylatable tyrosine residues that we identified in this study are all located in these monomer-monomer interfaces N-1/N or N/N+1 or in their vicinity . The Y19Although our findings show that the phosphorylation of tyrosine residues seems to strengthen monomer/monomer interactions by promoting the oligomeric state of RAD51, the nucleofilament formation does not seem to be affected by these phosphorylations. Indeed phosphorylated RAD51 binds to single stranded DNA and exhibits a dissociation constant (KD = 140 nM) similar to the binding affinity of non-phosphorylated RAD51 (KD = 128 nM). Theses KD values are in the same order of magnitude and are consistent with the data in the literature .The BRC4-28 peptide inhibitor is able to destabilize the multimeric forms of RAD51. It is derived from the BRCA2 protein and it is capable of mimicking monomer-monomer interfaces and causes also the dissociation of the nucleofilament of RAD51 . Our resIt is noteworthy that this peptide corresponds to the BRC interaction motif of the BRCA2 protein, which has a central role during HR through controlling the RAD51 recombinase. BRCA2 contains eight BRC motifs (from BRC1 to BRC8) which are able to bind directly to RAD51 with varying affinity . TherefoThe low sensitivity of the phosphorylated RAD51 forms to the BRC4-28 inhibitor can be explained by the localization of the RAD51 interaction site and by the formation of new bonds generated by phosphate groups on tyrosine residues. Molecular modelling of RAD51-BRC4  shows thThis study shows that the recombinase activity of RAD51 of presynaptic filaments containing phosphorylated RAD51 was less sensitive to the inhibitory effect of the BRC4 peptide. These results highlight the importance of the BRCA2-RAD51 interaction in cells and can explain the impact of c-MET activation on homologous recombination repair where BRCA2 and RAD51 are confidentially connected.On the other hand, it is difficult to assess the contribution and effects of each of the four phosphorylatable sites on RAD51 activity. According to the literature Y315 phosphorylation has little effect on the in vitro RAD51 activities  suggestiOur preliminary cellular result  shows thThe FASTA Sequence of RAD51 was uploaded on 3 web sites  to predict six RAD51 phosphorylation sites induced by c-MET kinase activity after data compilation.PyMOL 2.0 software was used for modelling of RAD51 and its mutation or phosphorylation. We used PDB Structure 5nwl  and 1n0wEscherichia coli BL21- DE3 strain at 37 °C. The bacteria are then lysed in buffer . His-tagged RAD51-proteins were purified on a NiNTA resin  . Imidazole used for purification was removed by dialysis . Protein concentrations were determined by the Bradford assay. The purity of proteins was analyzed by SDS- PAGE followed by Coomassie staining. All His-tagged RAD51 proteins were kept at −80 °C prior to use.The cDNA of His-tagged RAD51 was cloned in pET15b vector . All RAD51 Y/F, RAD51 F86E and RAD51 A190/192L were generated by site-directed mutagenesis using overlapping oligo-nucleotides. The DNA sequences of the mutant clones were checked and validated. RAD51-WT and RAD51 mutants were then over-expressed in RAD51-Y54F-for: ggaggctgttgcctttgcgccaaagaaggRAD51-Y54F-rev: ccttctttggcgcaaaggcaacagcctccRAD51-Y159F-for: gaaggaaaggccatgttcattgacactgagggRAD51-Y159F-rev: ccctcagtgtcaatgaacatggcctttccttcRAD51-Y178F-for: ggcagtggctgagaggtttggtctctctggcagtgRAD51-Y178F-rev: cactgccagagagaccaaacctctcagccactgccRAD51-Y191F-for: cctggataatgtagcatttgctcgagcgttcaacacRAD51-Y191F-rev: gtgttgaacgctcgagcaaatgctacattatccaggRAD51-Y205F-for: cagacccagctcctttttcaagcatcagccatgRAD51-Y205F-rev: catggctgatgcttgaaaaaggagctgggtctgRAD51-Y216F-for: gccatgatggtagaatctaggtttgcactgcttattgtagactgRAD51-Y216F-rev: cagtctacaataagcagtgcaaacctagattctaccatcatggcRAD51-Y228F-for: gtgccaccgcccttttcagaacagactactcgRAD51-Y228F-rev: ccgagtagtctgttctgaaaagggcggtggcacRAD51-Y232F-for: ccctttacagaacagacttctcgggtcgaggtgagcRAD51-Y232F-rev: gctcacctcgacccgagaagtctgttctgtaaagggRAD51-Y301F-for: cccatgcatcaacaaccagattgtttctgaggaaaggaagaggggRAD51-Y301F-rev: cccctcttcctttcctcagaaacaatctggttgttgatgcatgggRAD51-Y315F-for: cagaatctgccaaatcttcgactctccctgtcttcRAD51-Y315F-rev: gaagacagggagagtcgaagatttggcagattctgRAD51-F86E-for: ctcgtgcccatgggcgagaccacggcgacggagRAD51-F86E-rev: ctccgtcgccgtggtctcgcccatgggcacgagRAD51-A190/192L-for: cctggataatgtactgtatctgcgagcgttcaacacRAD51-A190/192L-rev: gtgttgaacgctcgcagatacagtacattatccaggYAPKKELINIKGISEAKADKILAEAAKLVPMGFTTATEFHQRRSEIIQITTGSKELDKLLQGGIETGSITEMFGEFRTGKTQICHTLAVTCQLPIDRGGGEGKAMYIDTEGTFRPERLLAVAERYGLSGSDVLDNVAYARAFNTDHQTQLLYQASAMMVESRYALLIVDSATALYRTDYSGRGELSARQMHLARFLRMLLRLADEFGVAVVITNQVVAQVDGAAMFAADPKKPIGGNIIAHASTTRLYLRKGRG ETRICKIYDSPCLPEAEAMFAINADGVGDAKD.MAMQMQLEANADTSVEEESFGPQPISRLEQCGINANDVKKLEEAGFHTVEAVAThe circular dichroism (CD) spectra of purified RAD51 protein were measured using a J-810 spectropolarimeter  and a mini-quartz cell with a path length of 0.2 cm. The spectra were recorded in the range from 210 to 260 nm and averaged over five scans to increase the signal-to-noise ratio . The final concentration of each RAD51 protein was 4 µM in phosphate buffer saline (PBS), pH 7.4.2; 3 mM MnCl2; 3 μM Na-orthovanadate; 1.2 mM DTT; 50 μg/mL PEG20.000). Reactions were incubated at 30 °C for 1 h and terminated by addition of Laemmli SDS sample dilution buffer. Proteins were separated by SDS-PAGE 12% gel, and phosphorylation was visualized by immunoblot. The c-MET inhibitor, PHA665752  was used during the c-MET kinase Assay at 1 µM.Recombinant active c-MET (0.1 µM)  was incubated with 2 mM of ATP and 10 µM of recombinant RAD51 (WT or mutant) in 25 µL kinase buffer  was cross-linked by incubation with 20 mM BS3  at room temperature for 30 min. Cross-linking was terminated by adding Tris 1M pH 8.0 for 15 min and analyzed by SDS-PAGE 10% gel and western blot.Samples were separated by electrophoresis on 12% SDS–polyacrylamide gels and transferred to nitrocellulose membranes . Membranes were blocked for 1 h in 0.1% TBS-Tween20 containing 5% BSA. Primary antibodies were rabbit anti-RAD51  or mouse anti-pTyr . Membranes were washed three times with 0.1% TBS-Tween20 for 10 min and incubated for 1 h with anti-mouse Alexa-Fluor680-conjugated or anti-rabbit Alexa-Fluor800-conjugated secondary antibodies . Membranes were scanned at 700 nm and 800 nm, respectively, using the Odyssey infrared imaging system . Images were treated and proteins were quantified with the Odyssey software. The amounts of phospho-RAD51 on the blot were normalized to the amount of RAD51 and the ratio of phospho-RAD51 to total RAD51 was determined from three independent experiments.Biotinylated DNA polydT (100 mM) was bound on a streptavidin coated biosensor (tip). RAD51 proteins (4 µM) were prepared in reaction buffer made of PBS buffer 1× and ATP 2 mM. Kinetics were divided in three steps. Firstly, baseline of the buffer was measured for 10 s, then the association step between ssDNA and RAD51 at 4 µM was monitored for 200 s and finally the dissociation step of the bound RAD51 was monitored for 200 s in buffer with or without BRC4. Tip was regenerated in NaOH (50 mM) twice for 40 s before reuse.2 at 37 °C for 20 min. The reaction was initiated by adding supercoiled pPB4.3 DNA (200 μM in bp). After incubation of 30 min at 37 °C, the reactions were stopped and deproteinized by a stop solution . The reaction mixtures were further incubated for 15 min at 37 °C. After adding 5-fold loading dye , the reaction products were separated by electrophoresis on 1% agarose gel. The electrophoresis was carried out in 0.5× TAE buffer  at 100 V for 2 h. The labeled products (100-ssDNA and D-loop) were visualized and quantified by the detection of the IRD-700 dye with the 700 nm infrared fluorescent detection channel of an Odyssey Infrared Imager (LI-COR Biosciences) [100-ssDNA labeled IRD700 (1 μM)  was incubated with 0.5 μM RAD51 in the presence or absence of the indicated amounts of the corresponding molecule in 10 μL of standard reaction buffer containing 20 mM Tris–HCl (pH 8), 1 mM ATP, 1 mM DTT, 1 mM CaClciences) .RG37 cells stably expressing the direct-repeat (DR)-GFP construct were generously given by Dr LOPEZ B. . RG37 cells were plated into 12-well plates, 1 day later pretreated by HGF (50 ng/mL) and subsequently transiently transfected with pCBA-HA-I-SceI  using Lipofectamine2000 . Transient expression of I-SceI endonuclease generated a DNA double strand break at the integrated GFP gene sequences and stimulated HR. GFP signal was assayed 72-hr post-transfection on a CytoFLEX flow cytometer . For each experiment, 10,000 cells were scored per treatment group, and the recombination percentage was calculated in comparison with the number of GFP-positive cells of the control condition RG37 I-SceI.t-test. Statistical significance was assumed at * p < 0.05, ** p < 0.01, *** p < 0.001 or Non-Significant NS for p > 0.05. Each error bars represents s.d. Statistical analysis and signal values representations were performed using the Microsoft Excel 2013 software.Statistical analysis was performed using paired Student’s This study demonstrates the direct link between RAD51 and c-MET wherein the phosphorylation of RAD51 impact its oligomeric state but also its interaction with the BRC motif of BRCA2. Our results suggest that c-MET-mediated RAD51 phosphorylation could play a potential role in the BRCA2-dependent regulation of RAD51. Given our encouraging in vitro findings, the next step should be to expand our investigations in cells to assess the RAD51 phosphorylation level according to c-MET inhibition in the DNA damage response pathway. The expected results could pave an attractive therapeutic strategy to overcome resistance by combining the modulation of c-MET and RAD51 activity with anticancer treatments.


To identify the optimal viewing angles for every proximal screw in PHILOS plate-fixed proximal humeral fractures.Three fresh-frozen human cadaveric bodies with six intact shoulders were studied. All three bodies were put in the beach chair position and PHILOS plates were placed on the proximal humerus. Head screws penetrating 1 mm into the joint were fitted one by one. Fluoroscopy was conducted in the 180° horizontal plane and the 120° coronal plane to analyze each screw’s penetration in every shoulder. Images were taken every 5°, then all images were analyzed to identify the sensitive angles.The range of optimal viewing angles to visualize penetration of every head screw was identified. In the coronal plane, the angles in the range between 0° and 10° were sensitive to all screws except No. 8 and No. 9. Furthermore, penetration of screws No. 8 and 9 could not be identified on any axillary view, but could be identified in the horizontal plane from − 30° to − 10° and from 10° to 35° respectively.We recommend a 0°–10° axillary view with 30° arm abduction combined with two horizontal angles in the range of − 30° to − 10° and 10° to 35° for routine fluoroscopy during surgery. Our results will be helpful in avoiding primary screw penetration. Proximal humeral fracture accounts for approximately 5% of all bone fractures, and the rate is rising with the increasing age of the population –3. AlthoThe aim of our study therefore was to explore the optimal viewing angles of intraoperative fluoroscopy for every proximal screw in PHILOS plate-fixed proximal humeral fractures, and then suggesting some intra-operative fluoroscopy angles for avoiding screw penetration.This study was approved by ethics of committee of Shanghai sixth people’s hospital, the approval number is 2016-ky-005, and all donors had signed agreement about further cadaveric research and education before body donation. Three fresh-frozen human cadaveric bodies with six intact shoulders were obtained, which comprised 2 females and 1 male, with a mean age of 72.2 years (range 64–82). All the specimens included the entire scapula, clavicle, humerus and intact associated soft tissues. Gross examination was performed and clinical histories were reviewed to exclude any history of bone diseases.All three bodies were put in a beach chair position to simulate the intraoperative situation. The PHILOS plates were placed on the proximal humerus, 5 mm lateral to the bicipital groove and 8 mm distal to the tip of the greater tuberosity, in all shoulders through the standard deltopectoral approach. Two non-locking screws were initially placed in the shaft holes to fix the plates in position. Second, we simulated the methods used in the study by Spross et al. ; the subHead screws were numbered to facilitate identification and recording Fig. . The No.In order to simulate the axillary view, the c-arm was placed on the coronal plane of the body and the arm was placed at 30° of abduction Fig. . Taking Overall, a total of 62 images were obtained for the penetration of No. 1 screw. The same method was then applied for the imaging investigation of penetration of the next 8 screws, to give a total of 558 images from one shoulder. All 6 shoulders underwent the same process and 558 × 6 images were collected.All images were numbered and were independently and randomly reviewed for screw penetration (yes/no) by two experienced attending doctors. The two doctors were blinded to the experimental design and process. All discordant cases were reevaluated by a third examiner, and the results were assigned by consensus of the three examiners. Based on these results, for each shoulder, we were able to draw a viewing angle range to detect penetration of every head screw.Statistical analysis was performed using the statistical program SPSS version 20.0 . The optimal viewing angle range of every screw was set between the average minimum and maximum of the six shoulders. In this study each screw has an angle range in each shoulder, so the average of the six angle ranges was calculated for each screw, with the lower boundary set as the average of the lower boundary of the six angle ranges and the higher boundary as the average of the higher boundary of the six angle ranges. In order to simplify clinical application, all the angles were measured to the nearest multiple of five greater than the average lower value for the lower boundary, for example if the average value was 8 we chose 10; and we chose the first multiple of five less than the average higher value for the higher boundary, for example if the average value was 8 we chose 5  show the same high sensitivity. In the horizontal plane, at least two different projections should be used to detect screws No. 8 and 9, one in the range between − 30° and − 10°, another between 10° and 35°, which equate to internal rotation of the arm 10–30° and external rotation of 10–35°. In addition, we found that the standard lateral view was not able to identify any screw penetration, which may be because 1 mm is too short to be detected. We recommend that our results should be used intraoperatively to reduce the rate of primary screw penetration rather than at follow-up. This is because, on the one hand, a large proportion of secondary screw penetrations are secondary to severe humeral head deformities, such as varus and necrosis, so that the optimal angles for these heads would be different from our data, while on the other hand, it may be impractical to perform fluoroscopy using such accurate angles at each follow-up.In the process of carrying out this study, we chose to rotate the c-arm rather than rotate the arm , because it is easier and more precise to control the angle according to the angle indexes on the c-arm, and it is also more practical during surgery.Based on our results, we can conclude that the sensitivity of every radiographic projection, for anteroposterior and outlet radiographs, means that penetration of three screws may be missed, while the result of a standard axillary view is similar to that found in the study by Spross et al., showing that it is sensitive to all screws but No. 8 and 9. So we recommend 0°–10° axillary view with the arm at 30° abduction combined with two horizontal angles in the range of − 30° to − 10° and 10° to 35° as a good combination; however, this is not the only one which can achieve 100% sensitivity, because the standard 0° axillary view with the arm at 30° abduction is used in many places and is easy to remember.There are some limitations to this research. Firstly, the protocol was designed based on healthy shoulders with no history of bone diseases, so we simulated an anatomical reduction model. Although for some fractures it is difficult to obtain the absolute anatomical reduction , we beliIn conclusion, after studying the viewing angles of every proximal screw on PHILOS plate-fixed proximal humeral fractures, we obtained an optimal viewing angle range for every screw. Consequently, we recommend 0°–10° axillary view with the arm at 30° abduction, while another two horizontal angles in the range of − 30° to − 10° and 10° to 35° should be included. Our results will be helpful for avoiding screw penetration during surgery.


The transition of rod precursor cells to post-mitotic rod photoreceptors can be promoted by extrinsic factors such as insulin-like growth factor 1 (IGF-1), which regulates phosphatidylinositide concentration, and consequently the 3-phosphoinositide-dependent protein kinase-1 (PDPK-1). PDPK-1 is a 63 kDa cytoplasmic kinase that controls cell proliferation and differentiation. In the mouse retina, PDPK-1 and its phosphorylated derivative p-PDPK-1 (Ser241), showed peak expression during the first postnatal (PN) day with a substantial decline by PN7 and in the adult retina. Though initially widely distributed among cell types, PDPK-1 expression decreased first in the inner retina and later in the outer retina. When PDPK-1 is inhibited in neonatal retinal explants by BX795, there is a robust increase in rod photoreceptor numbers. The increase in rods depended on the activity of PKC, as BX795 had no effect when PKC is inhibited. Inhibition of PDPK-1-dependent kinases, such as P70-S6K, but not others, such as mTORC-1, stimulated rod development. The P70-S6K-dependent increase in rods appears to be correlated with phosphorylation of Thr252 and not at Thr389, a substrate of mTORC-1. This pathway is also inactive while PKC activity is inhibited. We also found that inhibition of the kinase mTORC-2, also stimulated by insulin activity, similarly increased rod formation, and this effect appears to be independent of PKC activity. This may represent a novel intracellular signaling pathway that also stimulates photoreceptor development. Consistent with previous studies, stimulation of STAT3 activity is sufficient to prevent any PDPK-1, P70-S6K, or mTORC2-dependent increase in rods. Together the data indicate that PDPK-1 and other intrinsic kinases downstream of IGF-1 are key regulators of rod photoreceptor formation. Cells within the retina develop in stereotyped order that is well conserved across vertebrates . In the 3K) activity, increasing phosphorylation of the 3′-hydroxyl on intracellular phosphatidylinositol (PIP3) .Extrinsic insulin-like growth factor 1 (IGF-1) signaling is one such signal that promotes the transition of rod precursor cells to post-mitotic photoreceptors . Canonicl (PIP3) . PIP3 is3 binding to the PDPK-1 pleckstrin homology domain targets it to the plasma membrane and allows it to phosphorylate protein kinase B (AKT1/PKB) at Thr308  phosphorylation. This alteration in IGF-1 signaling is correlated with decreased phosphorylation of the PI3K regulatory subunit P55 (PIK3R3). This shift in PI3K regulation likely decreases PDPK-1 activity and signaling to it’s downstream effectors  are important signals in rod photoreceptor development. In this study we show that inhibiting PDPK-1 greatly increases the development of rod photoreceptors in retinal explants, in part through PKC-STAT3 signaling and in part through other pathways.As a key intermediate between PIThis study was carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition). The protocol was approved by the Pennsylvania State University College of Medicine Institutional Animal Care and Use Committee.ad libitum access to rodent chow. All ex vivo explant culture experiments used retinal tissue from early postnatal day 1 (PN1) mice.C57B1/6J mice were purchased from Jackson laboratory  and housed in a room with an ambient temperature of 25°C, 30–70% humidity, a 12-h light–dark cycle, and ex vivo retinal explant cultured system . Semi-quantitative analysis of band intensity was performed using ImageJ1.Western blots were performed as previously described . BrieflyImmunolabeling of retinal tissue was performed as previously described . BrieflyTable 2 for dilutions) overnight at 4 °C. The following day, sections were washed and incubated in secondary antibody for 3 h, followed by incubation in 1 μg/mL Hochest-33258 for 20 min. After three washes, slides were mounted with 0.5% n-propyl gallate in 1:1 glycerol: PBS. Sections were imaged using an Olympus FLUOVIEW FV1000 confocal microscope. In each set of experiments, acquisition parameters for each antibody were held constant. Fluorescent intensity measurements were derived using the ImageJ measurement tool.Prior to immunohistochemical labeling of deparaffinized or frozen tissue section, we unmasked antigens in a sodium citrate buffer (pH 6.0). Sections were permeabilized in 0.2% Triton-X-100 in PBS, blocked in 5% non-fat milk, and incubated in primary antibodies  as a reference gene.RNA was isolated from PN1 retinas using TRIzol (Invitrogen) and purified using RNeasy Mini Kits (Qiagen) as per the manufacturer’s instructions. Final RNA concentration was measured on a Gene Spect III (Hitachi) spectrophotometer. 2 μg RNA samples were reverse transcribed in a total reaction volume of 50 μL using SuperScript III reverse transcriptase (Invitrogen). Quantitative PCR annealing temperatures were set at 60°C for 40 cycles on a Bio-Rad iCycler, followed by a melt curve analysis to confirm primer specificity. Primer sequences are listed in t-test. For the statistical analysis of more than two groups, we used a one-way ANOVA with a Newman–Keuls post hoc test.All statistical analyses were performed using GraphPad Prism. To compare the means of two groups, we used a two-tailed, unpaired p < 0.01; Figure 1A). Similarly, p-PDPK-1 (Ser241) levels declined to 72 ± 7% at PN7 (p < 0.05) and 65 ± 5% at PN28 . To determine whether PDPK-1 protein was expressed in specific retinal cell populations we labeled retinas fixed at PN1, PN7, or PN28 with antibodies recognizing PDPK-1 or p-PDPK-1 (Ser241). During PN1, both forms of PDPK-1 were widely distributed across the retina, from the outer neuroblast layer to inner neuroblast layer. At PN7, much of the labeling had disappeared and the heaviest labeling of PDPK-1 (but not pPDPK-1) was on cell bodies at the outer edge of the inner nuclear layer and suggests that PDPK-1 is present in horizontal cells . At PN28, outer plexiform labeling remained strong, and the inner plexiform layer was diffusely labeled. We tested whether the p-PDPK-1-expressing population of cells of the outer neuroblast layer were dividing or post-mitotic by labeling fixed PN1 retinas for both proliferating cell nuclear antigen , and p-PDPK-1 (Ser241) . Though many cells were only labeled with a single marker, there was co-localization (yellow) of PDPK-1 and PCNA in cells proximal to the outer limiting membrane . This shows that pPDPK-1 is present in a portion of dividing cells of the early postnatal retina.In the mouse retina, the protein levels of PDPK-1 and its active form, phosphorylated at Ser241, significantly declined between postnatal days (PN) 1, 7, and 28. Relative to PN1, total PDPK-1 protein decreased to 82 ± 15% at PN7 and 31.4% at PN28 (50: 6 nM) at concentrations ranging from 10 nM to 20 μM for 96 h. BX795 significantly increased rhodopsin labeling, with a peak effect on rhodopsin (340 ± 25% of control) observed at 1 μM . Concentrations of BX795 greater than 1 μM decreased rhodopsin labeling, suggesting off-target or non-specific effects that negatively impact rod development. Notably, we see the same pattern of rhodopsin labeling in explats treated with increasing concentrations of an alternative PDPK-1 inhibitor, GSK2334470 . To determine whether the action of BX795 was on rod photoreceptors, rather than just on opsin, we tested the expression of a variety of other markers. Though some rod genes (Crx and Sag) showed little change, the expression of others  significantly increased, suggesting that the action of BX795 was on more than a single rod gene , and that in our experiments, rhodopsin is a valid proxy for overall photoreceptor development.To determine the role of PDPK-1 in photoreceptor development, we treated retinal explants with the competitive inhibitor, BX795 (ICp < 0.0001) relative to untreated control explants. Treatment of explants with 50 nM of the PKC inhibitor Go7874 or the combination of IGF-1 and Go7874 decreased rhodopsin labeling to 69 ± 7% (n.s.), and 54 ± 16% (n.s.) of control, respectively .Our previous studies have documented the stimulatory effects of IGF-1 on rod photoreceptor development via a pathway involving activation of PKC , 2011. Wp < 0.05) . When Go7874 was co-applied with BX795 there was a decrease in rhodopsin labeling (51 ± 40%) relative to controls, similar to the level of labeling seen with Go7874 alone (48 ± 49%). These data show that the BX795-mediated increase in rhodopsin levels requires PKC activity.BX795 also significantly increased rhodopsin levels relative to controls , suggesting that mTORC-1 does not normally regulate photoreceptor development. To confirm that the inhibitory effects of rapamycin were still active over similar time periods, we tested the effect of 30 min and 48 h rapamycin treatment on phosphorylation of the mTORC1 target p-P70-S6K (Thr389). Our data suggest that rapamycin is even more effective at decreasing Thr389 levels over this time period than at 30 min, confirming our result .3-Phosphoinositide-dependent protein kinase-1 lies upstream of AKT1 and thus mTORC-1 signaling . To test50 = 160 nM) , though to a lesser extent than with 100 nM BX795. Concurrent inhibition of PDPK-1 and P70-S6K  greatly increased rhodopsin levels, but the increase in rhodopsin was not statistically different from inhibition of PDPK-1 alone , suggesting that PDPK-1 and P70-S6K may work in the same pathway to regulate photoreceptor development.P70-S6K is major substrate of both PDPK-1 and mTORC-1. Using the P70-S6K inhibitor PF4708671 (IC 160 nM) , we testFigures 5A,C). As shown earlier, rhodopsin levels were reduced in Go7874-treated retinas . Similarly, rhodopsin was elevated in explants treated with PF4708671 . When explants were treated with Go7874 and PF4708671, opsin levels increased to a lower extent than with PF4708671 alone . These data suggest that while much of the action of P70-S6K is dependent on PKC, the remaining increase in rhodopsin in Go7874 plus PF4708671-treated retinas suggests that part of P70-S6K’s effect on rhodopsin is independent of PKC.To test more directly if P70-S6K is upstream of PKC, we treated explants with 200 nM PF4708671, 50 nM Go7874, or a combination of these inhibitors for 96 h (p < 0.05) but not at Thr252 . On the other hand, BX795 significantly decreased both Thr389 phosphorylation  and Thr252 phosphorylation . This suggests that the effects of P70-S6K on rod photoreceptor development are particularly dependent on its phosphorylation at Thr252.mTORC-1 and PDPK-1 activate P70-S6K kinase activity on separate amino acid residues  . Using r50 = 10 nM). Treatment of explants with 100 nM KU0063794 enhanced rod photoreceptor development by 236 ± 15% relative to untreated control explants . The combined use of KU0063794 and BX795 also stimulated rod development  approximately to the same extent as BX795 alone . These data show that mTORC-2 inhibition is likely a component of PDPK-1-inhibition-dependent photoreceptor development.The mTORC2 complex is a regulator of cell growth and metabolism, and the mTORC2 component rictor is a downstream target of P70-S6K . We hypoFigure 6B, co-application of Go7874 with KU0063794 to retinal explants led to a 210 ± 21% increase in rhodopsin above control, not significantly different from the increase seen with KU0063794 alone . These data suggest that the action of mTORC-2 on rod photoreceptor development is independent of PKC.To determine whether mTORC-2 requires PKC to inhibit rod development, we tested the ability of Go7874 to block the KU0063794 induced increase in rhodopsin levels. As shown in Figures 7A,B). We also tested whether these inhibitors would be sufficient to overcome levels of STAT3 activation stimulated by cytokines such as leukemia inhibitory factor (LIF). We tested the effect of 20 ng/mL LIF on rod photoreceptor development induced by 100 nM BX795, 100 nM KU0063794, or 200 nM PF4708671, and found that LIF-induced STAT3 activity completely halts photoreceptor development, regardless of the presence of BX795, KU0063794, or PF4708671 . We also confirmed that the actions of LIF are STAT3 dependent, since while no rods develop in explants treated with 20 ng/mL LIF and 100 nM BX795, rhodopsin labeling is rescued in explants treated with a cocktail of 100 nM BX795, 20 ng/mL LIF, and 50 μM of the STAT3 inhibitor Stattic . These data suggest STAT3 activity is a prominent regulator of cell fate in the developing retina, perhaps more so than the factors we have studied that stimulate rod development.Activation of STAT3 by phosphorylation of Tyr705 blocks rod photoreceptor development. We hypothesized that inhibitors which increase rhodopsin levels would do so through a pathway that ultimately decrease p-STAT3 Tyr705 levels. 100 nM BX795, but not 100 nM KU0063794 or 200 nM PF4708671, significantly decreased resting p-STAT3 Tyr705 . In the early postnatal retina when PDPK-1 levels are greatest, both active and total PDPK-1 are evenly distributed across the tissue , suggesting that PDPK-1 may play a role in the terminal differentiation of multiple retinal cell types. Developing retinal cells extend radial processes across much of the retina. This makes it difficult to measure the co-localization of the cytoplasmic PDPK-1 enzyme and the nuclear proliferation marker PCNA. Nevertheless, we did find some double-labeled cells at the outer margin of the retina, where developing cells undergo mitosis. Interestingly PCNA is cytosolic in these cells, which in granulocytes correlates with differentiation in to neutrophils , suggesting that it may have a unique function within this cell type. However, the overall decline in PDPK-1 levels suggests that the developmental role of PDPK-1 has concluded by PN7. We did not find any significant perinuclear localization of the active or inactive form of the enzyme, as has been reported by others . Our data showed that this effect is dose dependent up to 1 μM BX795, but the decrease in rhodopsin labeling at greater concentrations suggest that it can affect other proteins at higher concentrations . We used 100 nM BX795 in our studies, as it has high specificity for PDPK-1 and a minimal effect on downstream factors such as AKT-1 . While this concentration lessened the magnitude of effect on rhodopsin, we feel that the increased specificity make our results easier to interpret. Additionally, this dose of BX795 was sufficient to increase the expression of multiple rod genes , and we would expect an even greater effect at concentrations of BX795 closer to 1 μM.A previously characterized developmental shift in PIter ages . As a mater ages . PDPK-1 Figure 3).Our previous work had shown that the basal or IGF-1 stimulated formation of photoreceptors is dependent on PKC activity , 2011. IFigure 4). This was not completely unexpected, as direct inhibition of AKT/PKB also does not appear to regulate rod development . One such target that is key in mediating PDPK-1 signaling is P70-S6K, which increases translational activity within a cell by phosphorylating the S6 ribosomal subunit . This finding, while significant, initially seemed to conflict with the lack of effect we observed in rapamycin-treated retinas, as both mTORC-1 and PDPK-1 increase P70-S6K activity. However, these kinases target different activating residues on P70-S6K (Thr389 for mTORC-1 and both Thr389 and Thr252 for PDPK-1). While rapamycin strongly inhibited phosphorylation of Thr389 (without affecting Thr252), BX795 decreased phosphorylation at both sites . Comparing these effects, and the ability of P70-S6K inhibition to increase rod photoreceptor development, we suggest that PDPK-1 suppresses photoreceptor development by activating Thr252 of P70-S6K. To our knowledge, this is the first demonstration of the differential effects of phosphorylating these amino acid residues on P70-S6K.3-Phosphoinositide-dependent protein kinase-1 activity leads to the phosphorylation of a number of targets including AKT-1/PKB, mTORC-1, P70-S6K, and mTORC-2. Canonically, PDPK-1 phosphorylates and activates AKT-1/PKB at Thr308, which then indirectly activates mTORC-1. mTORC-1 is has been implicated in the regulation of many diverse processes within cells, from increasing survival to regulating energy homeostasis and cell growth . Despiteelopment . However subunit . We inhiFigure 6). Like mTORC1, mTORC2 is activated in response to PI3K signaling .The protein mTOR can be a component of two multi-protein complexes, mTORC-1 and mTORC-2. mTORC-2 is a rapamycin-insensitive protein complex that is responsive to growth factor signaling cascades, and regulates cell proliferation, survival, and metabolism . mTORC-2ignaling , and is ignaling , though ignaling , and thuAdditionally, it may be possible that P70-S6K stimulates rod development through interactions with both PKC- and mTORC2-dependent developmental pathways. If this is the case, it would explain why the rod-stimulating effects of PF4708671 do not appear to be fully silenced by PKC inhibition, though rhodopsin levels in explants treated with PF4708671 were not statistically distinct from those in explants treated with PF4708671 and Go7874. Regardless, it is still remains to be seen specific role this alternate pathway plays in the developmental cellular physiology of photoreceptors.One central characteristic of rod photoreceptor development is that it is fully inhibited by STAT3 activity . PKC actIt will also be important to fully define the developmental role of PDPK-1. There is strong evidence that both its activity and its subcellular localization are highly regulated. Further knowledge concerning the identity of the PDPK-1 regulators, their impact on PDPK-1 localization, and the consequences of PDPK-1 localization on downstream signaling will substantially expand our understanding of how multiple signals trigger or inhibit rod photoreceptor development.TX and DH performed the statistical analysis and wrote the sections of the manuscript. TX wrote the first draft of the manuscript. All authors contributed to conception and design of the study and manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.