Improved prediction of DNA and RNA binding proteins with deep learning models.

Nucleic acid-binding proteins (NABPs), including DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in essential biological processes. To facilitate functional annotation and accurate prediction of different types of NABPs, many machine learning-based computational approaches have been developed. However, the datasets used for training and testing as well as the prediction scopes in these studies have limited their applications. In this paper, we developed new strategies to overcome these limitations by generating more accurate and robust datasets and developing deep learning-based methods including both hierarchical and multi-class approaches to predict the types of NABPs for any given protein. The deep learning models employ two layers of convolutional neural network and one layer of long short-term memory. Our approaches outperform existing DBP and RBP predictors with a balanced prediction between DBPs and RBPs, and are more practically useful in identifying novel NABPs. The multi-class approach greatly improves the prediction accuracy of DBPs and RBPs, especially for the DBPs with ~12% improvement. Moreover, we explored the prediction accuracy of single-stranded DNA binding proteins and their effect on the overall prediction accuracy of NABP predictions.

Validated Negative Regions (VNRs) in the VISTA Database might be Truncated Forms of Bona Fide Enhancers.

The VISTA enhancer database is a valuable resource for evaluating predicted enhancers in humans and mice. In addition to thousands of validated positive regions (VPRs) in the human and mouse genomes, the database also contains similar numbers of validated negative regions (VNRs). It is previously shown that the VPRs are on average half as long as predicted overlapping enhancers that are highly conserved and hypothesize that the VPRs may be truncated forms of long bona fide enhancers. Here, it is shown that like the VPRs, the VNRs also are under strong evolutionary constraints and overlap predicted enhancers in the genomes. The VNRs are also on average half as long as predicted overlapping enhancers that are highly conserved. Moreover, the VNRs and the VPRs display similar cell/tissue-specific modification patterns of key epigenetic marks of active enhancers. Furthermore, the VNRs and the VPRs show similar impact score spectra of in silico mutagenesis. These highly similar properties between the VPRs and the VNRs suggest that like the VPRs, the VNRs may also be truncated forms of long bona fide enhancers.

Engineered Bacteriophage-Based In Situ Vaccine Remodels a Tumor Microenvironment and Elicits Potent Antitumor Immunity.

In situ vaccines (ISVs) utilize the localized delivery of chemotherapeutic agents or radiotherapy to stimulate the release of endogenous antigens from tumors, thereby eliciting systemic and persistent immune activation. Recently, a bioinspired ISV strategy has attracted tremendous attention due to its features such as an immune adjuvant effect and genetic plasticity. M13 bacteriophages are natural nanomaterials with intrinsic immunogenicity, genetic flexibility, and cost-effectiveness for large-scale production, demonstrating the potential for application in cancer vaccines. In this study, we propose an ISV based on the engineered M13 bacteriophage targeting CD40 (M13CD40) for dendritic cell (DC)-targeted immune stimulation, named H-GM-M13CD40. We induce immunogenic cell death and release tumor antigens through local delivery of (S)-10-hydroxycamptothecin (HCPT), followed by intratumoral injection of granulocyte-macrophage colony stimulating factor (GM-CSF) and M13CD40 to enhance DC recruitment and activation. We demonstrate that this ISV strategy can result in significant accumulation and activation of DCs at the tumor site, reversing the immunosuppressive tumor microenvironment. In addition, H-GM-M13CD40 can synergize with the PD-1 blockade and induce abscopal effects in cold tumor models. Overall, our study verifies the immunogenicity of the engineered M13CD40 bacteriophage and provides a proof of concept that the engineered M13CD40 phage can function as an adjuvant for ISVs.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

Artificial selection footprints in indigenous and commercial chicken genomes.

BACKGROUND: Although many studies have been done to reveal artificial selection signatures in commercial and indigenous chickens, a limited number of genes have been linked to specific traits. To identify more trait-related artificial selection signatures and genes, we re-sequenced a total of 85 individuals of five indigenous chicken breeds with distinct traits from Yunnan Province, China. RESULTS: We found 30 million non-redundant single nucleotide variants and small indels (< 50 bp) in the indigenous chickens, of which 10 million were not seen in 60 broilers, 56 layers and 35 red jungle fowls (RJFs) that we compared with. The variants in each breed are enriched in non-coding regions, while those in coding regions are largely tolerant, suggesting that most variants might affect cis-regulatory sequences. Based on 27 million bi-allelic single nucleotide polymorphisms identified in the chickens, we found numerous selective sweeps and affected genes in each indigenous chicken breed and substantially larger numbers of selective sweeps and affected genes in the broilers and layers than previously reported using a rigorous statistical model. Consistent with the locations of the variants, the vast majority (~ 98.3%) of the identified selective sweeps overlap known quantitative trait loci (QTLs). Meanwhile, 74.2% known QTLs overlap our identified selective sweeps. We confirmed most of previously identified trait-related genes and identified many novel ones, some of which might be related to body size and high egg production traits. Using RT-qPCR, we validated differential expression of eight genes (GHR, GHRHR, IGF2BP1, OVALX, ELF2, MGARP, NOCT, SLC25A15) that might be related to body size and high egg production traits in relevant tissues of relevant breeds. CONCLUSION: We identify 30 million single nucleotide variants and small indels in the five indigenous chicken breeds, 10 million of which are novel. We predict substantially more selective sweeps and affected genes than previously reported in both indigenous and commercial breeds. These variants and affected genes are good candidates for further experimental investigations of genotype-phenotype relationships and practical applications in chicken breeding programs.

Exosome/antimicrobial peptide laden hydrogel wound dressings promote scarless wound healing through miR-21-5p-mediated multiple functions.

Mesenchymal stem cell (MSC)-based therapy is an effective strategy for regenerative therapy. However, safety and ease of use are still issues to be overcome in clinical applications. Exosomes are naturally derived nanoparticles containing bioactive molecules, which serve as ideal cell-free therapeutic modalities. However, issues such as delivery, long-term preservation and activity maintenance of exosomes are other problems that limit their application. In this study, we proposed the use of rapid freeze-dry-thaw macroporous hydrogels for the encapsulation of HucMSC-derived exosomes (HucMSC-Exos) combined with an antimicrobial peptide coating. This exosome-encapsulated hyaluronic acid macroporous hydrogel HD-DP7/Exo can achieve long-term storage and transport by lyophilization and can be rapidly redissolved for treatment. After comprehensively comparing the therapeutic effects of HucMSC-Exos and HucMSC-loaded hydrogels, we found that HucMSC-Exos could also effectively regulate fibroblasts, vascular endothelial cells, and macrophages and inhibit myofibroblast-mediated fibrosis, thus promoting tissue regeneration and inhibiting scar formation in a mouse model of deep second-degree burn infection healing. These properties of lyophilized storage and whole-process-repair make HD-DP7/Exo have potential application value and application prospects.

High quality assemblies of four indigenous chicken genomes and related functional data resources.

Many lines of evidence indicate that red jungle fowl (RJF) is the primary ancestor of domestic chickens. Although multiple versions of RJF (galgal2-galgal5 and GRCg6a) and commercial chickens (GRCg7b/w and Huxu) genomes have been assembled since 2004, no high-quality indigenous chicken genomes have been assembled, hampering the understanding of chicken domestication and evolution. To fill the gap, we sequenced the genomes of four indigenous chickens with distinct morphological traits in southwest China, using a combination of short, long and Hi-C reads. We assembled each genome (~1.0 Gb) into 42 chromosomes with chromosome N50 90.5-90.9 Mb, amongst the highest quality of chicken genome assemblies. To provide resources for gene annotation and functional analysis, we also sequenced transcriptomes of 10 tissues for each of the four chickens. Moreover, we corrected many mis-assemblies and assembled missing micro-chromosomes 29 and 34-39 for GRCg6a. Our assemblies, sequencing data and the correction of GRCg6a can be valuable resources for studying chicken domestication and evolution.

High-quality genome assembly of a C. crossoptilon and related functional and genetics data resources.

There are four species in the Crossoptilon genus inhibiting at from very low to very high altitudes across China, and they are in varying levels of danger of extinction. To better understand the genetic basis of adaptation to high altitudes and genetic changes due to bottleneck, we assembled the genome (~1.02 Gb) of a white eared pheasant (WT) (Crossoptilon crossoptilon) inhibiting at high altitudes (3,000~7,000 m) in northwest of Yunnan province, China, using a combination of Illumina short reads, PacBio long reads and Hi-C reads, with a contig N50 of 19.63 Mb and only six gaps. To further provide resources for gene annotation as well as functional and population genetics analyses, we sequenced transcriptomes of 20 major tissues of the WT individual and re-sequenced another 10 WT individuals and a blue eared pheasant (Crossoptilon auritum) individual inhabiting at intermediate altitudes (1,500~3,000 m). Our assembled WT genome, transcriptome data, and DNA sequencing data can be valuable resources for studying the biology, evolution and developing conservation strategies of these endangered species.

Whole transcriptome screening for novel genes involved in meiosis and fertility in Drosophila melanogaster.

Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. We identified 94 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction.

Soluble suppression of tumorigenicity 2 associated with atrial fibrillation detected after stroke: A retrospective study.

Background: The soluble suppression of tumorigenicity 2 (sST2) is closely associated with stroke and atrial fibrillation (AF). However, no studies on sST2 and AF detected after stroke (AFDAS) have been reported. This study investigated the correlation between sST2 and AFDAS. Methods: This was a single-center, retrospective, clinical observational study. Patients diagnosed with a transient ischemic attack (TIA) or acute ischemic stroke were enrolled, and all patients underwent sST2 detection and electrocardiogram (ECG) or Holter monitoring for at least 24 h. Results: In total, 970 patients were enrolled, including 72 (7.4 %) with AFDAS. Multivariate analysis showed that age (OR 1.078; 95 % CI, 1.050-1.107; p < 0.001), heart rate (HR) (OR 1.025; 95 % CI, 1.007-1.044; p = 0.007), national institutes of health stroke scale (NIHSS) score (OR 1.089; 95 % CI, 1.029-1.152; p = 0.003), high sensitivity C-reactive protein (hs-CRP) (OR 1.006; 95 % CI, 1.002-1.009; p = 0.001), and sST2 (OR 1.018; 95 % CI, 1.010-1.026; p < 0.001) were independent risk factors of AFDAS. The areas under the curve (AUCs) for age, HR, sST2, hs-CRP, and NIHSS were 0.731, 0.599, 0.815, 0.664, and 0.700, respectively. The conventional model included age, HR, NIHSS score, and hs-CRP level based on multivariate results. After adding sST2 to the model, the model's performance in predicting AFDAS increased significantly. Conclusion: Higher sST2 levels were associated with the occurrence of AFDAS. Thus, sST2 can improve the risk model for AFDAS.

Versatile Hydrogel Dressings That Dynamically Regulate the Healing of Infected Deep Burn Wounds.

Severe burns threaten patient lives due to pain, inflammation, bacterial infection, and scarring. Most burn dressings that are commonly used perform a single function and are not well suited for the management of deep burns. Therefore, a multifunctional antimicrobial peptide- and stem cell-loaded macroporous hydrogel that can fight bacterial infection and regulate wound healing progression by temporally regulating cytokine production by internal stem cells is developed. The macroporous skeletal hydrogel is manufactured via the cryogenic gelation of hyaluronic acid (cryogel). Based on the oxidative polymerization reaction of dopamine, the antimicrobial peptide DP7 is immobilized on the surface of the cryogel (DA7CG). Placental mesenchymal stem cells (PMSCs) are then packaged inside the macroporous hydrogel (DA7CG@C). According to the results of in vitro and in vivo experiments, during the inflammatory phase, DP7 inhibits infection and modulates inflammation; during the proliferative phase, DA7CG@C accelerates the regeneration of skin, blood vessels, and hair follicles via internal stem cells; and during the remodeling phase, DA7CG@C contributes to extracellular matrix remodeling due to the ability of DP7 to regulate the paracrine secretion of PMSCs, synergistically promoting scar-free healing. DA7CG@C can participate in all phases of wound healing; therefore, it is a promising dressing for burn treatment.

Underlying causes for prevalent false positives and false negatives in STARR-seq data.

Self-transcribing active regulatory region sequencing (STARR-seq) and its variants have been widely used to characterize enhancers. However, it has been reported that up to 87% of STARR-seq peaks are located in repressive chromatin and are not functional in the tested cells. While some of the STARR-seq peaks in repressive chromatin might be active in other cell/tissue types, some others might be false positives. Meanwhile, many active enhancers may not be identified by the current STARR-seq methods. Although methods have been proposed to mitigate systematic errors caused by the use of plasmid vectors, the artifacts due to the intrinsic limitations of current STARR-seq methods are still prevalent and the underlying causes are not fully understood. Based on predicted cis-regulatory modules (CRMs) and non-CRMs in the human genome as well as predicted active CRMs and non-active CRMs in a few human cell lines/tissues with STARR-seq data available, we reveal prevalent false positives and false negatives in STARR-seq peaks generated by major variants of STARR-seq methods and possible underlying causes. Our results will help design strategies to improve STARR-seq methods and interpret the results.

Myeloid-derived suppressor cells: key immunosuppressive regulators and therapeutic targets in hematological malignancies.

The immunosuppressive tumor microenvironment (TME) supports the development of tumors and limits tumor immunotherapy, including hematological malignancies. Hematological malignancies remain a major public health issue with high morbidity and mortality worldwide. As an important component of immunosuppressive regulators, the phenotypic characteristics and prognostic value of myeloid-derived suppressor cells (MDSCs) have received much attention. A variety of MDSC-targeting therapeutic approaches have produced encouraging outcomes. However, the use of various MDSC-targeted treatment strategies in hematologic malignancies is still difficult due to the heterogeneity of hematologic malignancies and the complexity of the immune system. In this review, we summarize the biological functions of MDSCs and further provide a summary of the phenotypes and suppressive mechanisms of MDSC populations expanded in various types of hematological malignancy contexts. Moreover, we discussed the clinical correlation between MDSCs and the diagnosis of malignant hematological disease, as well as the drugs targeting MDSCs, and focused on summarizing the therapeutic strategies in combination with other immunotherapies, such as various immune checkpoint inhibitors (ICIs), that are under active investigation. We highlight the new direction of targeting MDSCs to improve the therapeutic efficacy of tumors.

A Dendrimer Peptide (KK2DP7) Delivery System with Dual Functions of Lymph Node Targeting and Immune Adjuvants as a General Strategy for Cancer Immunotherapy.

The clinical efficacy of personalized cancer vaccines still needs to be improved due to their insufficient immune effect. The development of innovative adjuvants and lymph node-targeted delivery systems is the key to improving the clinical efficacy of personalized vaccines. However, there is still a lack of an adjuvant delivery system that is simple in preparation and capable of mass production and integrates adjuvant and lymph node targeted delivery functions. Here, this work reports that a simple dendrimer polypeptide (KK2DP7) nanoparticle enhances the immune efficacy of an OVA/neoantigen-based vaccine. Due to its multiple functions as a delivery vehicle, immune adjuvant, and facilitator of dendritic cell migration, KK2DP7 efficiently increases the efficiency of antigen uptake and cross-presentation by antigen-presenting cells (APCs) and delivers antigens to lymph nodes via APCs. Strikingly, the antitumor effect of KK2DP7/OVA is superior to that of commonly used adjuvants such as poly(I:C), CpG, and aluminum adjuvant combined with OVA. Furthermore, KK2DP7/OVA combined with anti-PD-1 antibody is able to prevent tumor recurrence in a postoperative recurrent tumor model. Thus, KK2DP7-based cancer vaccines alone or in combination with immune checkpoint blockade therapies to treat tumors or postoperative tumor recurrence are a powerful strategy to enhance antitumor immunity.

Cellular Prion Protein Role in Cancer Biology: Is It A Potential Therapeutic Target?

Cancers are worldwide health concerns, whether they are sporadic or hereditary. The fundamental mechanism that causes somatic or oncogenic mutations and ultimately aids cancer development is still unknown. However, mammalian cells with protein-only somatic inheritance may also contribute to cancerous malignancies. Emerging data from a recent study show that prion-like proteins and prions (PrPC) are crucial entities that have a functional role in developing neurological disorders and cancer. Furthermore, excessive PrPC expression profiling has also been detected in non-neuronal tissues, such as the lymphoid cells, kidney, GIT, lung, muscle, and mammary glands. PrPC expression is strongly linked with the proliferation and metastasis of pancreatic, prostate, colorectal, and breast malignancies. Similarly, experimental investigation presented that the PrPC expression, including the prion protein-coding gene (PRNP) and p53 ag are directly associated with tumorigenicity and metastasis (tumor suppressor gene). The ERK2 (extracellular signal-regulated kinase) pathway also confers a robust metastatic capability for PrPC-induced epithelial to mesenchymal transition. Additionally, prions could alter the epigenetic regulation of genes and overactive the mitogen-activated protein kinase (MAPK) signaling pathway, which promotes the development of cancer in humans. Protein overexpression or suppression caused by a prion and prion-like proteins has also been linked to oncogenesis and metastasis. Meanwhile, additional studies have discovered resistance to therapeutic targets, highlighting the significance of protein expression levels as potential diagnostic indicators and therapeutic targets.

Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells.

Circulating tumor cells (CTCs) play a crucial role in tumor recurrence and metastasis, and their early detection has shown remarkable benefits in clinical theranostics. However, CTCs are extremely rare, thus detecting them in the blood is very challenging. New CTC detection techniques are continuously being developed, enabling deeper analysis of CTC biology and potential clinical application. This article reviews current CTC detection techniques and their clinical application. CTCs have provided, and will continue to provide, important insights into the process of metastasis, which could lead to development of new therapies for different cancers.

Codelivery of Shikonin and siTGF-ß for enhanced triple negative breast cancer chemo-immunotherapy.

Although chemoimmunotherapy has achieved considerable success in cancer treatment in recent years, the cure for triple-negative breast cancer (TNBC) remains elusive. The unsatisfied outcomes are likely attributed to deficient tumor immunogenicity, a strong immunosuppressive tumor microenvironment (ITM) and tumor metastasis. To address this issue, we constructed an effective codelivery system, combined with tumor growth factor ß (TGF-ß) small interference RNA (siTGF-ß) and shikonin (SK), to achieve successful chemoimmunotherapy of TNBC. The SK/siTGF-ß NPs (approximately 110 nm) exhibited a uniform structure and good stability. Conjugated FA presented enhanced cellular uptake in 4T1 cells, and siTGF-ß escaped from lysosomes because of the "proton sponge" effect of PEI. Furthermore, SK actually induced satisfactory immunogenic cell death (ICD) and the resulting dendritic cell (DC) maturation facilitated a distinctly enhanced cytotoxic T lymphocyte (CTL) response, generating a positive effect on tumor suppression. Simultaneously, the successful silencing of TGF-ß alleviated the TGF-ß-mediated ITM and inhibited the epithelial-to-mesenchymal transition (EMT), contributing to the infiltration of CTLs, suppression of regulatory T lymphocyte (Treg) proliferation and lung metastasis inhibition. Thus, the SK/siTGF-ß NPs demonstrated the strongest therapeutic effect with delayed tumor growth (TIR = 88.5%) and lung metastasis restraint (77.3%). More importantly, tumor rechallenge assay suggested that the codelivery system produced a long-term immunological memory response to prevent tumor recurrence. Based on boosting the immune response and combating the ITM, SK/siTGF-ß NPs would be a potential approach for TNBC therapy.

Enhanced nose-to-brain delivery of siRNA using hyaluronan-enveloped nanomicelles for glioma therapy.

Gliomas are the most malignant brain tumors, and their treatment is very challenging because of the presence of the blood-brain barrier (BBB). Intranasal administration has been considered a noninvasive strategy for glioma therapy in recent years, but our explorations of the intranasal delivery of siRNA-based therapies are still clearly inadequate. In this study, the cell-penetrating peptide DP7-C was enveloped with hyaluronic acid (HA) to develop the multifunctional core-shell structure nanomicelle HA/DP7-C. In vitro studies of HA/DP7-C revealed low cytotoxicity and a higher cell uptake efficiency, which was associated with the interaction between HA and CD44. In vivo experiments indicated that HA/DP7-C delivered the siRNA to the central nervous system through the trigeminal nerve pathway within hours after intranasal administration and that the interaction between HA and CD44 also increased its accumulation at the tumor site. Successful intracellular delivery of an antiglioma siRNA inhibited tumor growth and ultimately prolonged the survival time and decreased the tumor volume in GL261 tumor-bearing mice. In addition, toxicity tests on rats showed no adverse effects on the nasal mucosa and trigeminal nerves. In conclusion, HA/DP7-C is a potential intranasal delivery system for siRNAs in glioma therapy.

Dextran and peptide-based pH-sensitive hydrogel boosts healing process in multidrug-resistant bacteria-infected wounds.

Traumatic multidrug-resistant (MDR) bacterial infections are deadly threat to the public. To combat MDR bacteria, we developed a dual functional pH-sensitive hydrogel based on peptide DP7 (VQWRIRVAVIRK) and oxidized dextran (DP7-ODEX hydrogel). As an antimicrobial peptide, DP7 can synergize with many antibiotics; thus, we loaded ceftazidime into DP7-ODEX hydrogel, which showed an obvious advantage in MDR P. aeruginosa inhibition. Additionally, due to the interaction between aldehyde groups in oxidized dextran and amine groups from wound tissue, the hydrogel could extend on the irregular surface of skin defects and promote epithelial cells adhesion. DP7 could also be used as a wound-healing peptide and accelerate the healing process. We confirmed that the DP7-ODEX hydrogel exerted formidable therapeutic effects in normal or diabetic wound infection model. According to histomorphology analysis we found that DP7 hydrogel also have a scarless wound healing ability. In summary, we developed a hydrogel fabricated by the dual functional peptide DP7 that can kill multidrug-resistant bacteria colonizing the wound bed and boost scarless wound healing.