Regions Of High Homology Research Articles

Evaluation of pathogenic variants detected in high homology regions of the PMS2 gene. How effective is long-range PCR?

Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, most laboratories do not clearly report the origin of this molecular finding. The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the PMS2 gene in a Brazilian sample. An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with PMS2. In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Comparative genomics of the Leukocyte Receptor Complex in carnivores.

The mammalian Leukocyte Receptor Complex (LRC) chromosomal region may contain gene families for the killer cell immunoglobulin-like receptor (KIR) and/or leukocyte immunoglobulin-like receptor (LILR) collections as well as various framing genes. This complex region is well described in humans, mice, and some domestic animals. Although single KIR genes are known in some Carnivora, their complements of LILR genes remain largely unknown due to obstacles in the assembly of regions of high homology in short-read based genomes. As part of the analysis of felid immunogenomes, this study focuses on the search for LRC genes in reference genomes and the annotation of LILR genes in Felidae. Chromosome-level genomes based on single-molecule long-read sequencing were preferentially sought and compared to representatives of the Carnivora. Seven putatively functional LILR genes were found across the Felidae and in the Californian sea lion, four to five genes in Canidae, and four to nine genes in Mustelidae. They form two lineages, as seen in the Bovidae. The ratio of functional genes for activating LILRs to inhibitory LILRs is slightly in favor of inhibitory genes in the Felidae and the Canidae; the reverse is seen in the Californian sea lion. This ratio is even in all of the Mustelidae except the Eurasian otter, which has a predominance of activating LILRs. Various numbers of LILR pseudogenes were identified. The structure of the LRC is rather conservative in felids and the other Carnivora studied. The LILR sub-region is conserved within the Felidae and has slight differences in the Canidae, but it has taken various evolutionary paths in the Mustelidae. Overall, the process of pseudogenization of LILR genes seems to be more frequent for activating receptors. Phylogenetic analysis found no direct orthologues across the Carnivora which corroborate the rapid evolution of LILRs seen in mammals.

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1.

The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.

The impact of pre-existing cross-reactive immunity on SARS-CoV-2 infection and vaccine responses.

Pre-existing cross-reactive immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in infection-naive subjects have been described by several studies. In particular, regions of high homology between SARS-CoV-2 and common cold coronaviruses have been highlighted as a likely source of this cross-reactivity. However, the role of such cross-reactive responses in the outcome of SARS-CoV-2 infection and vaccination is currently unclear. Here, we review evidence regarding the impact of pre-existing humoral and T cell immune responses to outcomes of SARS-CoV-2 infection and vaccination. Furthermore, we discuss the importance of conserved coronavirus epitopes for the rational design of pan-coronavirus vaccines and consider cross-reactivity of immune responses to ancestral SARS-CoV-2 and SARS-CoV-2 variants, as well as their impact on COVID-19 vaccination.

Abstract 2924: Target identification, selectivity profiling and mechanistic insights of a Cdk9 inhibitor using complementary proteomics methods

Abstract In an effort to map the selectivity and understand the mode of action of a CDK9 inhibitor (compound 1), we employed several orthogonal proteomics methods. Our results show a clear selectivity against the CDK family of kinases, with the highest specific affinity towards CDK9. In addition, our multi-method approach allows us to also map the protein binding sites of the inhibitor, identify non-kinase (off-) targets as well as detect the cellular molecular responses to the added inhibitor. Here we describe the methods used, their strengths and weaknesses, how they can be used in the drug discovery pipeline and how they synergize to provide mechanistic insights of compounds of interest. We have used chemoproteomics, kinase affinity tools (kinobeads), Cellular Thermal Shift Assay (CETSA) and Limited Proteolysis (LiP). The results obtained clearly show that CDK9 is the primary target of Compound 1, with affinity curves highly correlated between the different target deconvolution techniques. The chemoproteomic approach rely on a compound derivate, able to bind to a sepharose bead. Subsequently, the binding competition assays are performed on lysed cell material. The choice of a mild lysis buffer allowed us to identify, not only CDK9, but also it’s molecular partners in the p-Tefb complex (Cyclin T1, Cyclin T2 and Aff4) with similar concentration response behavior. The results for the kinases identified in the study were strikingly similar when also profiling the compound without the chemical modification using the kinobeads assay. In the CETSA experiments, where both lysed cells and intact cells were profiled, the lysate experiment most closely resembles that of the previous pull-downs. Here, only direct binders of Compound 1 show a thermal shift, for example several of the pulled down kinases but not the p-Tefb complex partners that were co-competed previously. In the intact cell version of CETSA, not only the direct binders of the compound show stability shifts, but also downstream events and other secondary modulatory effects leave thermal traces in the cell. For example, Compound 1 also binds to GSK3A/B, causing their melting temperature to increase. Inhibition of GSK3 further affects the phosphorylation state and cellular location of FOXK1, which in turn is identified as a destabilized protein. Finally, Limited Proteolysis was used to identify target protein and using the LiP-Quant approach their LiP scores were assigned. Further, out of the identified CDK targets, mapping of peptide cleavage pattern was performed for the members of the CDK family for which structural data is published. The result identified the peptides to be directly adjacent to the ATP binding pocket of CDK9 or regions of high homology. The use of complementary techniques, based on unique biological and biochemical processes, allow robust and confident characterization of inhibitor compounds. Citation Format: Adam Hendricks, Nigel Beaton, Alexey Chernobrovkin, Eric Miele, Ghaith Hamza, Piero Ricchiuto, Ronald Tomlinson, Tomas Friman, Cassandra Borenstein, Bernard Barlaam, Sudhir Hande, Chris De Savi, Rick Davies, Martin Main, Joakim Hellner, Kristina Beeler, Yuehan Feng, Roland Bruderer, Lukas Reiter, Daniel Martinez Molina, Maria Paola Castaldi. Target identification, selectivity profiling and mechanistic insights of a Cdk9 inhibitor using complementary proteomics methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2924.

High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing.

Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.

Next-generation sequencing of newborn screening genes: the accuracy of short-read mapping

Newborn screening programs are an integral part of public health systems aiming to save lives and improve the quality of life for infants with treatable disorders. Technological advancements have driven the expansion of newborn screening programs in the last two decades and the development of fast, accurate next-generation sequencing technology has opened the door to a range of possibilities in the field. However, technological challenges with short-read next-generation sequencing technologies remain significant in highly homologous genomic regions such as pseudogenes or paralogous genes and need to be considered when implemented in screening programs. Here, we simulate 50 genomes from populations around the world to test the extent to which high homology regions affect short-read mapping of genes related to newborn screening disorders and the impact of differential read lengths and ethnic backgrounds. We examine a 158 gene screening panel directly relevant to newborn screening and identify gene regions where read mapping is affected by homologous genomic regions at different read lengths. We also determine that the patient’s ethnic background does not have a widespread impact on mapping accuracy or coverage. Additionally, we identify newborn screening genes where alternative forms of sequencing or variant calling pipelines should be considered and demonstrate that alterations to standard variant calling can retrieve some formerly uncalled variants.

Metadata Analysis of mcr-1-Bearing Plasmids Inspired by the Sequencing Evidence for Horizontal Transfer of Antibiotic Resistance Genes Between Polluted River and Wild Birds.

We sequenced the whole genomes of three mcr-1-positive multidrug-resistant E. coli strains, which were previously isolated from the environment of egret habitat (polluted river) and egret feces. The results exhibit high correlation between antibiotic-resistant phenotype and genotype among the three strains. Most of the mobilized antibiotic resistance genes (ARGs) are distributed on plasmids in the forms of transposons or integrons. Multidrug-resistant (MDR) regions of high homology are detected on plasmids of different E. coli isolates. Therefore, horizontal transfer of resistance genes has facilitated the transmission of antibiotic resistance between the environmental and avian bacteria, and the transfer of ARGs have involved multiple embedded genetic levels (transposons, integrons, plasmids, and bacterial lineages). Inspired by this, systematic metadata analysis was performed for the available sequences of mcr-1-bearing plasmids. Among these plasmids, IncHI2 plasmids carry the most additional ARGs. The composition of these additional ARGs varies according to their geographical distribution. The phylogenetic reconstruction of IncI2 and IncX4 plasmids provides the evidence for their multiregional evolution. Phylogenetic analysis at the level of mobile genetic element (plasmid) provides important epidemiological information for the global dissemination of mcr-1 gene. Highly homologous mcr-1-bearing IncI2 plasmids have been isolated from different regions along the East Asian-Australasian Flyway, suggesting that migratory birds may mediate the intercontinental transportation of ARGs.

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials.

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.

Chromosomal inversions associated with environmental adaptation in honeybees.

Chromosomal inversions can facilitate local adaptation in the presence of gene flow by suppressing recombination between well-adapted native haplotypes and poorly adapted migrant haplotypes. East African mountain populations of the honeybee Apis mellifera are highly divergent from neighbouring lowland populations at two extended regions in the genome, despite high similarity in the rest of the genome, suggesting that these genomic regions harbour inversions governing local adaptation. Here, we utilize a new highly contiguous assembly of the honeybee genome to characterize these regions. Using whole-genome sequencing data from 55 highland and lowland bees, we find that the highland haplotypes at both regions are present at high frequencies in three independent highland populations but extremely rare elsewhere. The boundaries of both divergent regions are characterized by regions of high homology with each other positioned in opposite orientations and contain highly repetitive, long inverted repeats with homology to transposable elements. These regions are likely to represent inversion breakpoints that participate in nonallelic homologous recombination. Using long-read data, we confirm that the lowland samples are contiguous across breakpoint regions. We do not find evidence for disruption of functional sequence by these breakpoints, which suggests that the inversions are likely maintained due to their allelic content conferring local adaptation in highland environments. Finally, we identify a third divergent genomic region, which contains highly divergent segregating haplotypes that also may contain inversion variants under selection. The results add to a growing body of evidence indicating the importance of chromosomal inversions in local adaptation.

The Development and Validation of Clinical Exome-Based Panels Using ExomeSlicer: Considerations and Proof of Concept Using an Epilepsy Panel

Exome-based panels are becoming the preferred diagnostic strategy in clinical laboratories. This approach enables dynamic gene content update and, if needed, cost-effective reflex to whole-exome sequencing. Currently, no guidelines or appropriate resources are available to support the clinical implementation of exome-based panels. Here, we highlight principles and important considerations for the clinical development and validation of exome-based panels. In addition, we developed ExomeSlicer, a novel, web-based resource, which uses empirical exon-level next-generation sequencing quality metrics to predict and visualize technically challenging exome-wide regions in any gene or genes of interest. Exome sequencing data from 100 clinical epilepsy cases were used to illustrate the clinical utility of ExomeSlicer in predicting poor-quality regions and its impact on streamlining the ad hoc Sanger sequencing fill in burden. With the use of ExomeSlicer, >2100 low complexity and/or high-homology regions affecting >1615 genes across the exome were also characterized. These regions can be a source of false-positive or false-negative variant calls, which can lead to misdiagnoses in tested patients and/or inaccurate functional annotations. We provide important considerations and a novel resource for the clinical development of exome-based panels.

Role of Homology and DNA Double‐Strand Breaks in Initiation of Intrachromosomal Recombination in Mammalian Cells

As part of our continuing investigations into the mechanisms of intrachromosomal recombination in mammalian cells, we stably transfected thymidine kinase deficient mouse fibroblasts with a DNA construct, pKM1, harboring a herpes thymidine kinase (tk) gene rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I‐SceI. We refer to this latter tk sequence as the “recipient.” The construct also contained a closely linked truncated “donor” tk sequence. The recipient and donor were positioned as direct repeats with respect to transcriptional orientation. The donor could potentially restore function to the recipient gene via recombination with the recipient, and such events could be provoked by induction of a double‐strand break (DSB) at the I‐SceI site in the recipient. Such DSB repair events were recoverable by selection for tk‐positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient in pKM1, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Our earlier work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In work presented here, we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest that the search for high homology prior to recombination is not mediated by an invading DNA terminus and may actually be independent of DSB formation. Further, the homology that enabled the recovered recombination events was located at a discrete distance from the actual invading DNA terminus. In an extension of our studies, we designed two additional constructs, pCW1 and pCW2. These constructs were similar to pKM1 except the recipient sequence in each was disrupted by an 8bp XhoI linker insertion and each construct contained a I‐SceI recognition site positioned within vector sequences at a location outside of either the recipient or donor sequence. Cells transfected with constructs pCW1 and pCW2 are being used to further explore whether a chromosomal DSB can induce and/or influence the nature of genetic exchanges occurring at a discrete distance from the DSB when the genetic exchanges themselves do not directly repair the DSB. Progress addressing this latter issue is presented.Support or Funding InformationThis work was supported by grant MCB 1157416 from the National Science Foundation to ASW and BCW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High homology is not required at the site of strand invasion during recombinational double-strand break repair in mammalian chromosomes

We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the “recipient”) rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated “donor” tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient. Repair events were recoverable by selection for tk-positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Previous work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In the current study we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest a search for high homology prior to recombination that is not mediated by an invading DNA terminus.

Mismatch Tolerance During Homologous Recombination in Mammalian Cells

We investigated the homology dependency of recombination in cultured thymidine kinase-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) which was rendered non-functional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a closely linked truncated “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via recombinational repair provoked by induction of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. We used three different donor sequences that contained 16, 25, or 33 mismatches relative to the recipient. The mismatches within these “homeologous” sequences were clustered and were surrounded by regions of high homology. Previous work indicated that mammalian cells fastidiously avoid recombination between homeologous sequences. We show that when homeologous sequences are surrounded by high homology, mismatches are frequently included in gene conversion events. Gene conversion tracts from spontaneous recombination revealed that in each event either all or none of the mismatches were transferred from donor to recipient, suggesting that recombination must begin and end in high homology. This requirement was somewhat relaxed for DSB-induced events. Knock-down of mismatch repair provided evidence that incorporation of mismatches into gene conversion tracts involved repair of mismatched heteroduplex intermediates. Our results indicate that mismatch repair of multiple mispaired bases does not function to impede exchange between homeologous sequences. Supported by grant MCB: 1157416 from the NSF to ASW and BCW.

Cloning and expression analysis of Drosophila extracellular Cu Zn superoxide dismutase.

In the present study, we cloned and sequenced the mRNAs of the Sod3 [extracellular Cu Zn SOD (superoxide dismutase)] gene in Drosophila and identified two mRNA products formed by alternative splicing. These products code for a long and short protein derived from the four transcripts found in global expression studies (Flybase numbers Dmel\\CG9027, FBgn0033631). Both mRNA process variants contain an extracellular signalling sequence, a region of high homology to the Sod1 (cytoplasmic Cu Zn SOD) including a conserved AUG start, with the longer form also containing a hydrophobic tail. The two fully processed transcripts are homologous to Caenorhabditis elegans Sod3 mRNA showing the same processing pattern. Using an established KG p-element+ insertion line (KG06029), we demonstrate that the Sod3 codes for an active Cu Zn SOD. We found differing expression patterns across sex with higher levels of expression of Sod3 in females. There is a correlation of Sod1 and Sod3 gene expression and activity that can explain why Sod3 was not seen in earlier studies of Sod1. Finally, we found no effect on lifespan with the Sod3 hypomorph mutation (Sod3KG06029) but did observe a significant increase in resistance to paraquat and H2O2 (hydrogen peroxide).

Molecular Markers of Spermatogonial Stem Cells in the Domestic Cat

Spermatogonial stem cells (SSCs) represent an exciting new avenue for assisted reproduction in endangered and genetically valuable species. Before this technology can be applied to wildlife, species-specific markers are required to evaluate SSC enrichment strategies and monitor subsequent in vitro culture. This study was designed to evaluate six conserved SSC markers (THY1, GPR125, GFRalpha1, PLZF, UCHL1 and OCT4) in the cat. Testes from three juveniles and three adults were obtained following routine castrations and processed for mRNA extraction. RT-PCR of whole testis and cell suspensions enriched for SSCs by differential plating confirmed that all six SSC markers are expressed in both the whole testis and SSC-enriched cell fractions. The expression of all six putative SSC marker genes in the cat testis suggests conservation of SSC markers, and perhaps self-renewal mechanisms, in felids.

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha-keratins and keratin-associated beta-proteins (formerly beta-keratins) has allowed the production of epitope-specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha-keratin range (40-70 kDa) or beta-protein range (10-30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha-proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin-associated beta-proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin-associated beta-proteins is typical for keratin-associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine-rich or sulfur-rich proteins). In turtles and crocodilians epidermis, keratin-associated beta-proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine-rich versus cysteine-glycine-rich keratin-associated beta-proteins in cells sequentially produced from the basal layer and not from the alternation of beta- with alpha-keratins. The process gives rise to Oberhäutchen, beta-, mesos-, and alpha-layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin-associated proteins (KAPs) of the epidermis, the keratin-associated beta-proteins of sauropsids are capable to form filaments of 3-4 nm which give rise to an X-ray beta-pattern as a consequence of the presence of a beta-pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates.

Blocking of Frizzled Signaling With a Homologous Peptide Fragment of Wnt3a/Wnt5a Reduces Infarct Expansion and Prevents the Development of Heart Failure After Myocardial Infarction

The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.

CDNA Cloning of Goat DNA Methyltransferase 1, Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1 (DNMT1) gene, to screen an effective shRNA-producing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells. In this study, PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions. Next, 3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector, acquiring 3 new vectors, respectively termed pRNAD1, pRNAD2 and pRNAD3. Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing. Finally, the developmental rates of nuclear transfer (NT) embryos generated using donor cells with and without the effective shRNA vector respectively, as well as in vitro fertilization (IVF) embryos were observed and recorded. The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned (GenBank no. FJ617538). Furthermore, an effective interfering shRNA (pRNAD2) was obtained, with its interference effect being 47.88%. Finally, NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls ( P < 0.05), reaching the similar rates to IVF embryos ( P > 0.05). In conclusion, goat DNA methyltransferase 1 gene cDNA was cloned and sequenced, an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved, which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.

New aspects of sphingosine 1-phosphate signaling in mammalian cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine by the enzyme, sphingosine kinase. S1P regulates cell function via a family of five closely related G-protein-coupled receptors (GPCR) termed S1P1-5 which bind S1P with high affinity (Chun et al., 2002). The S1P receptors relay the effects of S1P through signaling networks that regulate cell proliferation, survival, migration etc. A number of growth factors also activate sphingosine kinase to produce intracellular S1P which binds to putative intracellular proteins to produce cellular responses. There are two isoforms of sphingosine kinase termed SK1 and SK2. Sphingosine kinase 1 (SK1) was purified as a 49 kDa protein from rat kidney (Olivera et al., 1998) and subsequently two variants termed SK1a and SK1b were cloned from mouse (Kohama et al., 1998). The identification of SK2 was by BLAST searches of EST data using the mouse SK1 sequence. SK2 has regions of high homology with SK1, but contains an additional 240 amino acids located at the N-terminus and in the centre of the protein (Liu et al., 2000). Multiple spliced variant forms of SK1 and SK2 have subsequently been cloned (reviewed by Alemany et al., 2007). SK1 and SK2 are partially redundant since SK1−/− or SK2−/− mice are normal whereas elimination of both genes is embryonic lethal ([Allende et al., 2004] and [Mizugishi et al., 2005]). However, these enzymes may also have different sub-cellular localisations and antagonistic roles. For instance, SK1 enhances growth and survival whereas SK2 has been shown to be involved in the regulation of apoptosis (Spiegel and Milstien, 2003)