Single-stranded DNA Gap Accumulation is a Functional Biomarker for USP1 Inhibitor Sensitivity.

Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with the drug sensitivity. USP1 inhibition increased monoubiquitinated PCNA at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids therefore represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials.

Protocol for Testing Human Melanoma Exosomes that Shift the Healthy Phenotype of Human Dermal Cells.

Exosomes are small membrane-derived vesicles that transmit DNA constituents, mRNAs, microRNAs, and proteins from donor cells to a receiver cell. Various cells comprising of mesenchymal, immune, and cancer cells discharge exosomes. Cancer cell exosomes form the entry and reprogramming of essentials connected to a tumor environment. Melanoma-derived exosomes transport diverse proteins such as c-MET and RAB27a, which leave a melanoma mark. Increased mesenchymal epithelial transition (MET) expressions in serum exosomes have been considered an indicator of disease progression. Meanwhile, RAB27a has been identified as being involved in exosome discharge and trafficking. Decreased expressions of RAB27a in human melanoma cells have shown to diminish exosome release.We examined the effects of the downregulation and upregulation of RAB27a and c-MET in human dermal fibroblasts by utilizing the isolated exosomes of malignant melanoma cell lines. Melanoma exosomes derived from cancer cells conveyed information to healthy dermal fibroblasts and stem cells while inducing phenotypic change. In this chapter, we show optimized protocols that were used by our group for in vitro analysis with melanoma exosomes.

ΔNp63 Regulates Homeostasis, Stemness, and Suppression of Inflammation in the Adult Epidermis.

The p63 transcription factor is critical for epidermis formation in embryonic development, but its role in the adult epidermis is poorly understood. In this study, we show that acute genetic ablation of ΔNp63, the main p63 isoform, in adult epidermis disrupts keratinocyte proliferation and self-maintenance and, unexpectedly, triggers an inflammatory psoriasis-like condition. Mechanistically, single-cell RNA sequencing revealed the downregulation of cell cycle genes, upregulation of differentiation markers, and induction of several proinflammatory pathways in ΔNp63-ablated keratinocytes. Intriguingly, ΔNp63-ablated cells disappear by 3 weeks after ablation, at the expense of the remaining nonablated cells. This is not associated with active cell death and is likely due to reduced self-maintenance and enhanced differentiation. Indeed, in vivo wound healing, a physiological readout of the epidermal stem cell function, is severely impaired upon ΔNp63 ablation. We found that the Wnt signaling pathway (Wnt10A, Fzd6, Fzd10) and the activator protein 1 (JunB, Fos, FosB) factors are the likely ΔNp63 effectors responsible for keratinocyte proliferation/stemness and suppression of differentiation, respectively, whereas IL-1a, IL-18, IL-24, and IL-36γ are the likely negative effectors responsible for suppression of inflammation. These data establish ΔNp63 as a critical node that coordinates epidermal homeostasis, stemness, and suppression of inflammation, upstream of known regulatory pathways.

A Comprehensive Review: Molecular and Genetic Background of Indirect Inguinal Hernias.

BACKGROUND: The occurrence of indirect inguinal hernias (IIH) is 5 times more prevalent than that of direct inguinal hernias (IH) and it is 7 times more common in males, owing to the attendance of the processus vaginalis (PV) throughout testicular descent. SUMMARY: In children, the immense mainstream of IH is indirect. The progress of IIH development in children is instigated with a patent PV, which is mostly treated by simple herniorrhaphy. Syndromes of the collagen, microfibril, elastin, and glycosaminoglycan constituents of the extracellular matrix may attend to the development of IH. Our recent research showed that the lack of epithelial-mesenchymal transition (EMT) in children contributes to the development of IIH, while the scenario is defined as the opposite in adults. However, there is still a lack of knowledge on all of the genetic and molecular causes of the disease. KEY MESSAGES: Here we aimed to review the published genetic background of IH, the deficiencies of connective tissue causing the disease, recently defined molecular pathways involved including EMT, and possible recurrence reasons. This comprehensive study can deliver an analytic outline aiding to define patients with IH combined with fundamental genetic diseases.

Decellularization Concept in Regenerative Medicine.

Decellularized organs and tissues are effectively utilized in a diversity of regenerative medicine purposes, and the decellularization approaches employed differ as broadly as the tissues/organs of concern. Biological scaffold substances formed by extracellular matrix (ECM) are mostly produced with methods that include decellularization of tissues. Conservation of the multifaceted arrangement and three-dimensional (3D) construction of the ECM is very wanted but it is documented that almost every approach of decellularization cause disturbance of the organization and possible forfeiture of surface organization and conformation. The competence of cell elimination from a tissue is reliant on the basis of the tissue and the precise physical, chemical, and enzymatic approaches that are utilized. Here, the most frequently applied and newly developed decellularization techniques are designated, organ engineering with decellularized scaffolds for different organs, recent knowledge in the field are explained.

Deficiency of epithelial-mesenchymal transition causes child indirect inguinal hernia.

INTRODUCTION: Epithelial-mesenchymal transition (EMT) describes rapid changes in cellular phenotype. During EMT, epithelial cells down-modulate cell-cell adhesion, alter polarity, reorganize cytoskeleton, become isolated, motile, and resistant to anoikis. Epithelial breakdown and epithelial cell migration are the key processes involved in the obliteration of processus vaginalis. The great majority of abnormalities are because of nonobliteration or incomplete fusion of PV. We aimed to analyze the quantitative changes of epithelial genes in adult/child patients and their controls to examine differences of the genesis of these hernias. We also aimed to investigate the potential epigenetic causes of indirect inguinal hernia in adult patients. METHODS: Ten adult, ten child indirect inguinal hernia sacs and ten adult, ten child parietal peritonea were used. Hernial sac samples were obtained from indirect inguinal hernia surgeries. Peritonea of adult patients who underwent open cholecystectomy for cholelithiasis via subcostal incision were included in the study as the healthy control groups. Ages of the children were selected to be between 0 and 5 whereas the age of adults was chosen as 35-55, respectively. Total RNA isolation and cDNA synthesis were made from hernia sacs and peritoneum samples. Relative Keratin 1, Keratin 15, Filaggrin2 and STAT3 expressions were analyzed via qPCR. Indirect inguinal hernia sac cells were seeded and grown in vitro. Child diseased cells were employed in immunocytochemistry (ICC) analysis for Cytokeratin 15, Filaggrin2 and Bcl-2. Adult indirect inguinal hernia cells were examined for H3 modifications through ICC. RESULTS: In child indirect inguinal hernia, Keratin expressions were found higher than their controls. They were meaningfully lower than the healthy group in adult indirect inguinal hernia. Keratin 15, Keratin 1 and Filaggrin2 expressions were all correlating since they are members of related pathways. STAT3 expressions were opposite to Keratin and Filaggrin expressions suggesting that adult cells might have a switch to the mesenchymal state from the epithelial state. Adult indirect inguinal hernia samples have switched to the mesenchymal state whereas child indirect inguinal hernia samples have shown lack of transition. CONCLUSION: Irregular changes of EMT associated genes act in the progression of indirect inguinal hernia. Hence, the information on the epigenetic regulation of EMT in patients with primary inguinal hernia can aid to comprehend the pathogenesis in adults and infers new therapeutic approaches for this disease. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: Level 2.

In vitro artificial skin engineering by decellularized placental scaffold for secondary skin problems of meningomyelocele.

BACKGROUND: Meningomyelocele (MMC) is a condition that is originated by the fusion defect of the neural tube. It is a congenital anomaly and can be characterized by spinal cord defects and impaired skin integrity. It is very important to close the skin openings via three-dimensional artificial skin like construction for preventing infection and maintaining the healthy skin structure. Therefore, we aim to generate artificial skin like structures formed by the own cells of donor for treating the MMC-related skin disorder. METHODS: In this study, waste placental tissues were collected and decellularization process was applied. Decellularized and normal placental tissues were compared by immunohistochemistry (IHC). Donor's own placental stem cells were seeded onto biological scaffold and were differentiated into skin related cell types. Finally, gene expressions were evaluated, and the structural integrity were analyzed with IHC. Tube formation assay was also performed for examining the angiogenesis formation of the tissue in order to evaluate the possibility of a healthy organ development. RESULTS: Characterization experiments proved that the decellularized skin preserved a normal skin 3D construction and vasculature along with significant ECM arrangements. The well-kept placental ECM scaffold was cytocompatible, supportive of mesenchymal cell types. Native organ related scaffold is expected to carry a huge influence in skin tissue engineering via delivering a niche for skin-based cells and even for stem/progenitor cells. Regarding to the data obtained from this study, in vivo investigation the skin-like structure in animal models is thought to be the next step as a future prospect. CONCLUSION: This study is a reference investigation for skin engineering based on placental stem cells and biological scaffolds.

Characterization and Differentiation of Stem Cells Isolated from Human Newborn Foreskin Tissue.

Circumcision is described as a cultural, medical, and religious process which states surgical removal of the foreskin either partly or fully. Cells isolated from the circumcised tissues are referred as foreskin cells. They have been thought as feeder cell lines for embryonic stem cells. Their fibroblastic properties were also utilized for several experiments. The waste tissues that remain after the circumcision thought to have stem cell properties. Therefore, there have been very few attempts to expose their stem cell properties without turning them into induced pluripotent stem cells. Although stem cell isolation from prepuce and their mesenchymal multilineage differentiation potential have been presented many times in the literature, the current study explored hematopoietical phenotype of newborn foreskin stem cells for the first time. According to the results, human newborn foreskin stem cells (hnFSSCs) were identified by their capability to turn into all three germ layer cell types under in vitro conditions. In addition, these cells have exhibited a stable phenotype and have remained as a monolayer in vitro. hnFSSCs suggested to carry different treatment potentials for bone damages, cartilage problems, nerve damages, lesion formations, and other diseases that are derive from mesodermal, endodermal, and ectodermal origins. Owing to the location of the tissue in the body and differentiation capabilities of hnFSSCs, these cells can be considered as easily obtainable and utilizable even better than the other stem cell sources. In addition, hnFSSCs offers a great potential for tissue engineering approaches due to exhibiting embryonic stem cell-like characteristics, not having any ethical issues, and teratoma induction as in embryonic stem cell applications.