Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.

Experimental Support for Human Papillomavirus Genome Amplification Early after Infectious Delivery.

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate-early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using 5-ethynyl-2'-deoxyuridine (EdU) pulse-labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1- and E2-dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number, confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In the absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, a lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number. IMPORTANCE It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846), we demonstrate herein that viral genome is indeed amplified in an E1- and E2-dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV life cycle using mutational approaches.

Decellularized skin pretreatment by monophosphoryl lipid A and lactobacillus casei supernatant accelerate skin recellularization.

BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

Extracellular vesicles derived from human foreskin cells (hFS-Exo) accelerate cell migration and angiogenesis through MAPK pathway: an in vitro study.

BACKGROUND: Wound healing is one of the important processes in the body. Attempts to create new drugs are of interest due to the side effects of natural and chemical wound healing compounds. To overcome this obstacle, stem cells have been used as healing agents. However, both difficulties in collection and risks such as rejection and teratoma in the recipient body have limited the use of stem cells, directly. Since the potential content of the stem cells can be transferred to the recipient cells by vesicles, small extracellular vesicles have recently become prominent agents. METHODS AND RESULTS: The wound-healing effect of extracellular vesicles derived from foreskin cells was investigated in both keratinocyte and endothelial cells. Migration assay, RT-PCR, Col1a1 ELISA and Western Blot experiments were utilized to reveal healing effect of EVs and its possible molecular pathways. EV-treated groups exhibited more proliferative, invasive, and migrative characteristics. When comparing to the control group, new vessel formation was induced in EV groups. An increase in gene levels of growth factors related to wound healing and change in the mitogen-activated protein kinase (MAPK) signaling pathway proteins in EV-treated groups were determined. Possible molecular mechanisms underlying cell movements were associated with the MAPK pathway. It was found that human foreskin cell EVs (hFS-Exo) may have a potential to heal wounds in a short period of time by triggering the MAPK pathway. CONCLUSIONS: hFS-Exo could be a new promising wound healing agent in the future.

Genital calcinosis cutis: Microscopic evidence supporting a dystrophic origin.

Calcinosis cutis (CC) is characterized by the deposition of calcium salts in the skin and subcutaneous tissues. CC involving the vulva or foreskin (prepuce) is uncommon. We present a 9-year-old female with vulvar CC and a 15-year-old male with preputial CC. Microscopic review of excisional specimens revealed calcification associated with follicular cysts in the vulvar case and lichen sclerosus in the preputial case, suggesting a dystrophic origin to a subset of cases of genital CC that might otherwise be classified as idiopathic. The clinical implication of these findings is the need for close histopathologic scrutiny and ongoing clinical surveillance of patients with genital CC initially deemed idiopathic.

Mono-2-ethylhexyl phthalate-induced downregulation of MMP11 in foreskin fibroblasts contributes to the pathogenesis of hypospadias.

Hypospadias is one of the most common congenital anomalies of the male urogenital system, and di(2-ethylhexyl) phthalate (DEHP), a widely used endocrine-disrupting chemical (EDC), is considered a significant risk factor for this condition. Mono-2-ethylhexyl phthalate (MEHP), the toxic active metabolite of DEHP, has been proven to affect penile development and ultimately result in the hypospadias phenotype. However, while it is acknowledged that hypospadias arises from the aberrant development of multiple penile tissues, the specific impact of MEHP on human foreskin tissue development and its underlying molecular mechanisms of action remain unclear. In this study, we constructed an in vitro toxicity assay for MEHP using human foreskin fibroblasts and employed high-throughput RNA sequencing to investigate the molecular mechanisms subserving the defects in cellular function. We subsequently conducted multi-omics data analysis using public databases to analyze key target genes, and identified MMP11 as a chief downstream gene responsible for the effects of MEHP on HFF-1 cell migration. Through molecular docking analysis and molecular biology experiments, we further demonstrated that the nuclear receptor PPAR-gamma was activated upon binding with MEHP, leading to the suppression of MMP11 expression. Additionally, we found that epigenetic modifications induced by MEHP were also involved in its pathogenic effects on hypospadias. Our research highlights the crucial role of impaired cellular proliferation and migration in MEHP-induced hypospadias. We identified the MEHP/PPAR-gamma/MMP11 pathway as a novel pathogenic mechanism, providing important potential targets for future preventive strategies with respect to hypospadias.

Improvising on the Fly: Comparison of a Novel Technique for Emergent Zipper Release to a Well-Established Technique in a Simulated Setting.

BACKGROUND: Penile skin zipper entrapment is an emergent medical condition in which the penile skin, scrotal skin, or foreskin gets caught within the teeth of a zipper or the slider itself. This can lead to complications such as urethral involvement, skin loss, or tissue necrosis. We propose a novel technique to aid in the release of entrapped skin utilizing wire cutters directed at the inferior portion of the zipper pull. OBJECTIVES: To describe a novel technique to free entrapped penile skin and compare its performance to the well-established median bar technique in a simulated setting. METHODS: A randomized cross-over design was used to compare techniques on successful release, time to release and tissue injury using an animal model of raw chicken skin entrapped in a zipper. Statistical significance was assessed at p < 0.05. RESULTS: Twenty-two participants were included. There was no statistically significant difference between the novel technique and the median bar technique regarding successful release (100% vs 95.5%, respectively), median time to release (29.1 vs 26.4 seconds, respectively), or frequency of tissue injury (22.7% vs 27.3%). CONCLUSION: Performance using our novel technique for removal of penile skin from a zipper is similar to the median bar release technique regarding. Our novel technique may be a valid treatment option for the release of entrapped penile skin in a zipper mechanism in the emergency department setting.

Reverse circumcision foreskin advancement flap for reconstructing penile shaft skin defects in adults with burn injuries in the perineal region.

AIM: Genital burns are rare injuries. Reconstruction of penile skin defects should consider cosmetic and functional outcomes. Skin grafts can develop scar contractures and carry hair follicles, causing unwanted results. These downsides remain unsolved issues. This work aimed to describe a new foreskin advancement flap method for completely reconstructing penile shaft skin defects in severely burned patients. MATERIALS AND METHODS: From 2021 to 2023, four patients with third-degree burns in the genital area were enrolled in this investigation. We describe a series of cases with deep burns to the penile shaft and surrounding area that needed debridement and reconstruction using a novel technique called "reverse circumcision," which consists of tangential excision of the penis and a foreskin advancement flap without longitudinal cuts with less morbidity, preservation of function, and a better aesthetic appearance. The patients had an average follow-up of nine months. RESULTS: The reverse circumcision technique was established for patients with severe burns in the genital area. The four patients were satisfied with the postoperative results and the aesthetic results of the procedure without reporting any complications. No scarring or contractures were observed on the glans or penile shaft after surgery. CONCLUSIONS: Compared with other flap methods, the use of a reverse circumcision foreskin advancement flap was more straightforward, feasible, and effective. In adults, the foreskin tissue completely covers the penile shaft skin defect. It is a viable reconstructive surgical technique that is easily reproducible and has excellent aesthetic and functional results. For this surgical technique, tissue transfers, bulky regional flaps, or skin grafts were not needed.

hsa_circ_0000417 downregulation suppresses androgen receptor expression and apoptotic signals in human foreskin fibroblasts via sponging miR-6756-5p.

BACKGROUND: Dysregulated apoptosis of penile mesenchymal cells during male urethragenesis has been previously demonstrated to underly hypospadiac urethral closure failure, and androgen receptor (AR) has been shown to play a central role in regulating penile mesenchyme cell proliferation and survival. However, the regulatory mechanisms upstream and downstream of AR remain poorly understood. Our clinical data and bioinformatics analysis previously indicated that hsa_circ_0000417, a circRNA significantly downregulated in hypospadias preputial specimens, may act as a ceRNA for AR via sequestering hsa_miR-6756-5p, and that the biological functions of hsa_circ_0000417 may significantly involve the PI3K/AKT pathway. In this study, we employed human foreskin fibroblasts (HFF-1) to experimentally validate this putative hsa_circ_0000417/miR-6756-5p/AR axis and its impact on penile mesenchymal cell proliferation and apoptosis. METHOD AND RESULTS: We showed that hsa_circ_0000417 knockdown significantly promoted proliferation and suppressed apoptosis of HFF-1 cells. Mechanistically, hsa_circ_0000417 functioned as a molecular sponge for miR-6756-5p in HFF-1 cells and relieved the latter's translational repression on AR mRNA, leading to decreased AKT activation and increased expression of pro-apoptotic proteins BAX and cleaved-caspase 9. Conversely, elevated levels of miR-6756-5p resulted in diminished AR expression concomitant with enhanced AKT activation and HFF-1 cell proliferation. CONCLUSIONS: Collectively, our data describe for the first time a circRNA-mediated post-transcriptional regulatory mechanism of AR and its functional consequences in penile mesenchymal cells in the context of hypospadias. These findings may contribute to advancing our current understanding of the roles of AR and mesenchymal cell fate decisions during penile morphogenesis.

Crosstalk of moderate ROS and PARP-1 contributes to sustainable proliferation of conditionally reprogrammed keratinocytes.

Conditionally reprogrammed cell (CRC) technique is a promising model for biomedical and toxicological research. In the present study, our data first demonstrated an increased level of PARP-1 in conditionally reprogrammed human foreskin keratinocytes (CR-HFKs). We then found that PARP inhibitor ABT-888 (ABT), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC), or combination (ABT + NAC) were able to inhibit cell proliferation, ROS, PARP-1, and ROS related protein, NRF2, and NOX1. Interestingly, knockdown of endogenous PARP-1 significantly inhibited cell proliferation, indicating that the increased PARP-1 expression was critical for CR. Importantly, we found that a moderate level of ROS contributed the cell proliferation and increased PARP-1 since knockdown of PARP-1 also inhibited the ROS. The similar inhibition of cell proliferation, ROS, and expression of PARP-1 and NRF2 proteins was observed when CR-HFKs were treated with hydroquinone (HQ), a key component from skin-lightening products. Moreover, the treatment of HQ plus treatment of ABT, NAC, or combination can further inhibit cell proliferation, ROS, expression of PARP-1, and NRF2 proteins. PARP-1 knockdown inhibited the population doubling (PDL) and treatment of HQ inhibited the PDL further, as well as the change of ROS. Finally, we discovered that pathways including cyclin D1, NRF2, Rb and pRb, CHK2, and p53, were involved in cell proliferation inhibition with HQ. Taken together, our findings demonstrated that crosstalk between ROS and PARP-1 involves in the cell proliferation in CR-HFKs, and that inhibition of CR-HFK proliferation with HQ is through modulating G1 cell cycle arrest.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

[Balanoposthitis in the clinical practice of a urologist and dermatovenereologist: an interdisciplinary problem].

Balanoposthitis is a common inflammatory disease of the male genitals, which occurs more often in uncircumcised men. The cause of balanoposthitis can be an infectious, inflammatory or autoimmune process, as well as traumatization. In most cases, after proper intimate hygiene and the use of neutral moisturizers, the symptoms of balanoposthitis are completely stopped. In the case of torpid course of balanoposthitis and in the absence of improvement after drug therapy, it is necessary to exclude the malignant process. In the review article, the authors present the data of modern scientific research on the qualitative and quantitative composition of the microbiome in balanoposthitis. Differences in the composition of the microbiome were revealed in patients with balanoposthitis and healthy patients from the control group with excess foreskin. It was found that in patients with balanoposthitis, a impaired in hydration of the skin of the glans penis was revealed. Staphylococcus warneri and Prevotella bivia are the most common species associated with balanoposthitis and positively correlate with the severity of the disease. Candida infection, as an etiological factor of balanoposthitis, often occurs in children and may be associated with diaper rash. The prevalence of Gardnerella vaginalis as a pathogen in the male urogenital tract has not been fully studied. Currently, there are no reliable scientific studies that make it possible to attribute G. vaginalis to the etiological factor of balanoposthitis in men. However, it should be borne in mind that balanoposthitis may have a polymicrobial and synergistic etiology with the participation of G. vaginalis and anaerobic bacteria in the lower genital tract of men. The review article is clearly illustrated with clinical examples of the disease from the personal practice of the authors.

Repression of Mafb promotes foreskin fibroblast proliferation through upregulation of CDK2, cyclin E and PCNA.

Mafb plays a significant role in the development and differentiation of various organs, tissues and cells. Nevertheless, its role in the control of external genital cell proliferation and function in the mechanism of hypospadias remains unknown. In this study, the expression of Mafb in foreskin fibroblasts was inhibited by siRNA. The Cell Count Kit-8 (CCK-8) assay showed cell proliferation increased after transfection, and the number of cells entered the S phase significantly increased via flow cytometry. Both mRNA and protein levels of cyclin E, cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) were significantly upregulated in the siRNA group. Meanwhile, twenty-five prepuce tissue samples were collected from hypospadias repair surgery. These samples were divided into two groups: the severe and mild groups. Normal prepuce tissue specimens were obtained during circumcision as the normal control. The upregulated expression of cyclin E, CDK2 and PCNA and downregulated Mafb expression were observed in the hypospadias group. This study reveals for the first time that the reduction in Mafb promotes the foreskin fibroblast proliferation. Thus, downregulated Mafb expression may cause hypospadias by upregulating CDK2, cyclin E and PCNA. These findings can shed new light on the embryonic development of the urethra.

Comparative analysis on the outcomes in circumcising children using modified Chinese ShangRing and conventional surgical circumcision.

OBJECTIVE: To compare the differences and outcomes of surgical procedures, clinical effect, complications and patients' satisfaction between disposable oval-shaped circumcision device (Modified Chinese ShangRing series, Kiddie love®) and conventional circumcision in the treatment of children with phimosis or redundant prepuce. METHODS: The clinical data were retrospectively analyzed in 114 children with phimosis or redundant foreskin undergone circumcision using a disposable oval-shaped circumcision device, a modified Chinese ShangRing series, Kiddie Love® (Kiddie Love group) in our hospital between January 2018 and February 2020, and another 114 children with similar conditions circumcised by conventional surgical procedure before January 2018 (conventional group). The two groups were compared regarding the operative time, intraoperative blood loss, postoperative pain scores, healing time, the incidence of complications and guardian's satisfaction. RESULTS: Circumcision was successfully completed in children in both groups. The operative time, intraoperative blood loss, postoperative pain scoring in 24 h by VAS, pain at the removal of the device or stitches and wound healing were (6.4 ± 1.6) min, (34.1 ± 6.4) min; (0.7 ± 0.2) ml, (2.6 ± 0.6) ml; (2.2 ± 1.0) points, (1.3 ± 0.5) points; (23.7 ± 3.9)day, (15.9 ± 2.8)day, respectively for Kiddie Love group and conventional group(either P < 0.05 or P > 0.05). The two groups were significantly different in the incidence of hematoma, edema and incision dehiscenceyet were insignificant in incision infection. Children in both groups were followed up from 6 to 31 months (mean: 23 months), and the satisfaction rate was 94.7% (108/114) in parents of the children circumcised by the ShangRing and 83.3% (95/114) in those of children treated by conventional circumcision (P < 0.05). CONCLUSION: Modified Chinese ShangRing, Kiddie Love®, has superiorities, including simpler procedure, shorter operative time, less blood loss, fewer complications, better cosmetic results and higher satisfaction of patients over conventional circumcision in the treatment of children with phimosis or redundant foreskin, and worthy of wider clinical recommendation.

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury.

PURPOSE: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. METHODS: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). RESULTS: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already non-irradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. CONCLUSIONS: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

[Functional phimosis: prevalence, diagnosis and treatment in outpatient practice].

AIM: To study the prevalence of functional phimosis determined during erection in patients over 18 years of age, as well as the features of its diagnosis and treatment in outpatient practice. MATERIALS AND METHODS: A retrospective study that included 201 patients who underwent circumcision at a mean age of 42.7 years, was carried out. Complaints, history, initial examination and autophotography of the penis during erection were evaluated. The subjects were divided into 2 groups. The group I (n=38) included patients complaining of the inability to reveal the glans penis during erection, while in group II (n=163) men with similar complaints in a f laccid state of the penis were included. All patients underwent circumcision under local anesthesia. RESULTS: The proportion of functional phimosis was 18.9%. The mean age in groups I and II was significantly different (29.47+/-8.82 and 45.6+/-19.4 years, respectively, p<0.01). In 14 (36.8%) patients of group I, a short frenulum was also diagnosed. Primary phimosis was detected in 26.3% and 14.1% of patients in groups I and II (p<0.05), respectively. The acquired phimosis was diagnosed in 73.7% and 85.9% (p<0.05) of cases, respectively. There were no concomitant diseases in patients with "functional" phimosis, while in men with "pathological" phimosis, 22.7% of patients had various comorbidities, such as diabetes mellitus, coronary heart disease, hypertension, etc. CONCLUSIONS: Among patients who visit a urologist with a diagnosis of phimosis, almost every fifth man has functional form (18.9%). For the diagnosis of the phimosis, the history taking and autophotography of the penis during erection have an important role. In this category of patients, surgical treatment can be performed on an outpatient basis.

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model.

BACKGROUND: Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. As a widely used material in clinic, there is little information on the biobanking of ECSs and its repair effect after storage. RESULTS: Two methods for biobanking foreskin ECSs were compared in a short term (7 days): 4-degree storage and programmed cryopreservation. Cell sheet integrity, viability, apoptosis, immunogenicity, mechanical properties and function were evaluated. In vivo, ECSs were directly transplanted to skin defect models and histological examination was performed at 1 week postoperatively. We successfully extracted human foreskin-derived primary epithelial cells and fabricated them into ECSs. Compared with 4-degree storage, programmed cryopreservation preserved the ECS structural integrity, enhanced the mechanical properties, decreased HLA-I expression, and increased cell viability and survival. An increased proportion of melanocytes with proliferative capacity remained in the cryopreserved sheets, and the undifferentiated epithelial cells were comparable to those of the fresh sheets. In vivo, cryopreserved ECSs could reduce inflammatory cell infiltration and promote connective tissue remodeling, epithelial cell proliferation and vascular regeneration. CONCLUSIONS: Programmed cryopreservation of ECSs was superior and more feasible than 4-degree storage and the cryopreserved ECSs achieved satisfying skin wound healing in vivo. We anticipate that the off-the-shelf ECSs could be quickly used, such as, to repair human epithelial defect in future.

Genome-Wide Transcriptome Analysis of Human Papillomavirus 16-Infected Primary Keratinocytes Reveals Subtle Perturbations Mostly due to E7 Protein Expression.

It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.

Is it safe to use a thermocautery device for circumcision? Examination of the histopathological changes emerging after thermocautery-assisted circumcisions.

Multiple number of techniques and devices have been described concerning circumcision method so far. One of them is thermocautery device, and it has been widely used. Although there is controversy that the penis may be damaged due to the heat generated during the use of the device, not enough histopathological studies have been conducted. We aimed to determine the histopathological changes in human foreskin caused by heat after circumcision with thermocautery and to demonstrate the safety of the use of a thermocautery-assisted circumcision. Forty-one patients were divided into two groups according to the thermal energy used during cutting with thermocautery as follows: high temperature (Group 1, n = 22) and low temperature (Group 2, n = 19). The effect of the heat intensity and depth of the coagulation necrosis produced with thermocautery-assisted circumcisions performed at low and high temperatures were evaluated. The difference between the groups was not statistically significant. Tissue damage is extremely limited in thermocautery-assisted circumcisions, even when it is used at high temperatures. The thermocautery device can be used for effective and safe circumcisions.