Temporal and Spatial Epigenome Editing Allows Precise Gene Regulation in Mammalian Cells.

Cell-type specific gene expression programs are tightly linked to epigenetic modifications on DNA and histone proteins. Here, we used a novel CRISPR-based epigenome editing approach to control gene expression spatially and temporally. We show that targeting dCas9-p300 complex to distal non-regulatory genomic regions reprograms the chromatin state of these regions into enhancer-like elements. Notably, through controlling the spatial distance of these induced enhancers (i-Enhancer) to the promoter, the gene expression amplitude can be tightly regulated. To better control the temporal persistence of induced gene expression, we integrated the auxin-inducible degron technology with CRISPR tools. This approach allows rapid depletion of the dCas9-fused epigenome modifier complex from the target site and enables temporal control over gene expression regulation. Using this tool, we investigated the temporal persistence of a locally edited epigenetic mark and its functional consequences. The tools and approaches presented here will allow novel insights into the mechanism of epigenetic memory and gene regulation from distal regulatory sites.

CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

Angiogenic Heparin-Mimetic Peptide Nanofiber Gel Improves Regenerative Healing of Acute Wounds.

Wound repair in adult mammals typically ends with the formation of a scar, which prevents full restoration of the function of the healthy tissue, although most of the wounded skin heals. Rapid and functional recovery of major wound injuries requires therapeutic approaches that can enhance the healing process via overcoming mechanical and biochemical problems. In this study, we showed that self-assembled heparin-mimetic peptide nanofiber gel was an effective bioactive wound dressing for the rapid and functional repair of full-thickness excisional wounds in the rat model. The bioactive gel-treated wounds exhibited increased angiogenesis (p < 0.05), re-epithelization (p < 0.05), skin appendage formation, and granulation tissue organization (p < 0.05) compared to sucrose-treated samples. Increased blood vessel numbers in the gel-treated wounds on day 7 suggest that angiogenesis played a key role in improvement of tissue healing in bioactive gel-treated wounds. Overall, the angiogenic heparin-mimetic peptide nanofiber gel is a promising platform for enhancing the scar-free recovery of acute wounds.

Virus-like nanostructures for tuning immune response.

Synthetic vaccines utilize viral signatures to trigger immune responses. Although the immune responses raised against the biochemical signatures of viruses are well characterized, the mechanism of how they affect immune response in the context of physical signatures is not well studied. In this work, we investigated the ability of zero- and one-dimensional self-assembled peptide nanostructures carrying unmethylated CpG motifs (signature of viral DNA) for tuning immune response. These nanostructures represent the two most common viral shapes, spheres and rods. The nanofibrous structures were found to direct immune response towards Th1 phenotype, which is responsible for acting against intracellular pathogens such as viruses, to a greater extent than nanospheres and CpG ODN alone. In addition, nanofibers exhibited enhanced uptake into dendritic cells compared to nanospheres or the ODN itself. The chemical stability of the ODN against nuclease-mediated degradation was also observed to be enhanced when complexed with the peptide nanostructures. In vivo studies showed that nanofibers promoted antigen-specific IgG production over 10-fold better than CpG ODN alone. To the best of our knowledge, this is the first report showing the modulation of the nature of an immune response through the shape of the carrier system.

Relationship between erectile dysfunction and silent coronary artery disease: detection with multidetector computed tomography coronary angiography.

AIM: Our aim was to determine the relationship between erectile dysfunction (ED) and silent coronary artery disease (CAD) by multidetector computed tomography (MDCT) coronary angiography. METHODS: Thirty consecutive men with nonhormonal and nonpsychogenic ED and with no cardiac symptoms were evaluated. Medical history, physical examination and laboratory investigation were performed. The five-item brief form of the International Index of Erectile Function (IIEF-5) was performed for evaluation of ED. The Agatston score (AS) was determined from MDCT images under beta blockade to induce bradycardia. The MDCT coronary angiography findings were evaluated by two radiologists blinded to the clinical findings. Patients were classified into three categories (mild, moderate and severe ED) according to IIEF-5 scores and into five categories (very low, low, moderate, moderately high and high CAD risk) according to the AS. RESULTS: Mean age was 58.3 ± 8.7 years (46-79). 6 patients had hypertriglyceridemia, 4 had hypercholesterolemia and 4 had hyperglycemia. All patients had normal early morning testosterone levels. Regarding IIEF-5 scores, none of them had mild ED, 14 had moderate ED and 16 had severe ED. Of the 14 patients with moderate ED, 21.4% had low and 28.5% had moderate CAD risk regarding AS. Of the 16 patients with severe ED, 25% had moderate, 31.2% had moderately high and 25% had high CAD risk regarding AS. Increasing age was a risk factor for high AS (p = 0.045). There was a significant correlation between AS and ED severity (p = 0.01). CONCLUSIONS: ED and CAD often coexist. MDCT coronary angiography can detect coronary lesions and allow appropriate medical intervention.

Generation of Chimeric "ABS Nanohemostat" Complex and Comparing Its Histomorphological In Vivo Effects to the Traditional Ankaferd Hemostat in Controlled Experimental Partial Nephrectomy Model.

Purpose. Using the classical Ankaferd Blood Stopper (ABS) solution to create active hemostasis during partial nephrectomy (PN) may not be so effective due to insufficient contact surface between the ABS hemostatic liquid agent and the bleeding area. In order to broaden the contact surface, we generated a chimeric hemostatic agent, ABS nanohemostat, via combining a self-assembling peptide amphiphile molecule with the traditional Ankaferd hemostat. Materials and Methods. In order to generate ABS nanohemostat, a positively charged Peptide Amphiphile (PA) molecule was synthesized by using solid phase peptide synthesis. For animal experiments, 24 Wistar rats were divided into the following 4 groups: Group 1: control; Group 2: conventional PN with only 0.5 ml Ankaferd hemostat; Group 3: conventional PN with ABS + peptide gel; Group 4: conventional PN with only 0.5 ml peptide solution. Results. Mean warm ischemia times (WITs) were 232.8 ± 56.3, 65.6 ± 11.4, 75.5 ± 17.2, and 58.1 ± 17.6 seconds in Group 1 to Group 4, respectively. Fibrosis was not different among the groups, while inflammation was detected to be significantly different in G3 and G4. Conclusions. ABS nanohemostat has comparable hemostatic efficacy to the traditional Ankaferd hemostat in the partial nephrectomy experimental model. Elucidation of the cellular and tissue effects of this chimeric compound may establish a catalytic spark and open new avenues for novel experimental and clinical studies in the battlefield of hemostasis.

Growth factor binding on heparin mimetic peptide nanofibers.

Immobilization of growth factors in scaffolds is important for controlling their dose and bioactivity for regenerative medicine applications. Although numerous covalent and noncovalent immobilization strategies have been proposed, better growth factor loading and dose control inside the scaffold is necessary. Nature of the binding site on the growth factor interacting with scaffold is critical for preserving and achieving maximal growth factor functionality, which has been a relatively less emphasized issue in previous studies. We recently reported heparin mimetic peptide nanofibers, which mimic chemistry of heparan sulfates. Heparin mimetic nanofibers were shown to bind to vascular endothelial growth factor (VEGF) and direct endothelial cells to angiogenesis. Here, we further investigated interactions between heparin mimetic peptide nanofibers and growth factors. We tested bioactivity of the nanofiber bound growth factors in order to understand the potential use of these peptide nanofiber scaffolds as analogues of heparan sulfates. We observed that heparin mimetic peptide nanofibers demonstrate better binding profiles to VEGF, hepatocyte growth factor (HGF), and fibroblast growth factor-2 (FGF-2) than control peptide nanofibers. We also identified that the heparin-binding domain of VEGF is critical for its interaction with these nanofibers. However, the heparin-binding site is not indispensable for binding of all growth factors to nanofibers. We also showed that binding of growth factors to nanofibers does not cause any loss in bioactivity through in vitro cell culture assays with PC-12 cells. These results reveal that heparin mimetic peptide nanofibers can effectively mimic heparan sulfates in extracellular matrix and provide an optimal milieu for spatial presentation of important growth factors. These properties make peptide nanofiber scaffolds promising materials for regenerative medicine applications through efficient and precisely controlled growth factor delivery.

Cooperative effect of heparan sulfate and laminin mimetic peptide nanofibers on the promotion of neurite outgrowth.

Extracellular matrix contains an abundant variety of signals that are received by cell surface receptors contributing to cell fate, via regulation of cellular activities such as proliferation, migration and differentiation. Cues from extracellular matrix can be used for the development of materials to direct cells into their desired fate. Neural extracellular matrix (ECM) is rich in axonal growth inducer proteins, and by mimicking these permissive elements in the cellular environment, neural differentiation as well as neurite outgrowth can be induced. In this paper, we used a synthetic peptide nanofiber system that can mimic not only the activity of laminin, an axonal growth-promoting constituent of the neural ECM, but also the activity of heparan sulfate proteoglycans in order to induce neuritogenesis. Heparan sulfate mimetic groups that were utilized in our system have an affinity to growth factors and induce the neuroregenerative effect of laminin mimetic peptide nanofibers. The self-assembled peptide nanofibers with heparan sulfate mimetic and laminin-derived epitopes significantly promoted neurite outgrowth by PC-12 cells. In addition, these scaffolds were even effective in the presence of chondroitin sulfate proteoglycans (CSPGs), which are the major inhibitory components of the central nervous system. In the presence of these nanofibers, cells could overcome CSPG inhibitory effect and extend neurites on peptide nanofiber scaffolds.

Microscopic characterization of peptide nanostructures.

Peptide-based nanomaterials have been utilized for various applications from regenerative medicine to electronics since they provide several advantages including easy synthesis methods, numerous routes for functionalization and biomimicry of secondary structures of proteins which leads to design of self-assembling peptide molecules to form nanostructures. Microscopic characterization at nanoscale is critical to understand processes directing peptide molecules to self-assemble and identify structure-function relationship of the nanostructures. Here, fundamental studies in microscopic characterization of peptide nanostructures are discussed to provide insights in widely used microscopy tools. In this review, we will encompass characterization studies of peptide nanostructures with modern microscopes, such as TEM, SEM, AFM, and advanced optical microscopy techniques. We will also mention specimen preparation methods and describe interpretation of the images.

Heparin mimetic peptide nanofibers promote angiogenesis.

New blood vessel formation (angiogenesis) is one of the most important processes required for functional tissue formation. Induction of angiogenesis is usually triggered by growth factors released by cells. Glycosaminoglycans (e.g., heparan sulphates) in the extracellular matrix aid in proper functioning of these growth factors. Therefore, exogeneous heparin or growth factors were required for promoting angiogenesis in previous regenerative medicine studies. Here we report for the first time induction of angiogenesis by a synthetic nanofibrous peptide scaffold without the addition of any exogenous growth factors or heparin. We designed and synthesized a self-assembling peptide amphiphile molecule that is functionalized with biologically active groups to mimic heparin. Like heparin, this molecule has the ability to interact with growth factors and effectively enhance their bioactivity. The nanofibers formed by these molecules were shown to form a 3D network mimicking the structural proteins in the extracellular matrix. Because of heparin mimicking capabilities of the peptide nanofibers, angiogenesis was induced without the addition of exogenous growth factors in vitro. Bioactive interactions between the nanofibers and the growth factors enabled robust vascularization in vivo as well. Heparin mimetic peptide nanofibers presented here provide new opportunities for angiogenesis and tissue regeneration by avoiding the use of heparin and exogenous growth factors. The synthetic peptide nanofiber scaffolds enriched with proper chemical functional groups shown in this study can be used to induce various desired physiological responses for tissue regeneration.

Biochemical recurrence after radical prostatectomy: is the disease or the surgeon to blame?

PURPOSE: The PSA recurrence develops in 27 to 53% within ten years after radical prostatectomy (RP). We investigated the factors (disease grade and stage or the surgeon's expertise,) more likely to influence biochemical recurrence in men post-radical prostatectomy for organ-confined prostate cancer by different surgeons in the same institution. MATERIALS AND METHODS: A total of 510 patients that underwent radical prostatectomy were investigated retrospectively. Biochemical recurrence was defined as detection of a PSA level of ≥ 0.20 ng/mL by two subsequent measurements. The causes, which are likely to influence the development of PSA recurrence, were separated into two groups as those related to the disease and those related to the surgical technique. RESULTS: Biochemical recurrence was detected in 23.5% (120 cases) of 510 cases. The parameters most likely to influence biochemical recurrence were: PSA level (p < 0.0001), T stage (p < 0.0001), the presence of extracapsular invasion prostate (p < 0.0001), Gleason scores (p = 0.042, p < 0.0001) and the presence of biopsy with perineural invasion (p = 0.03). The only surgical factor that demonstrated relevance was inadvertent capsular incision during the surgery that influenced the PSA recurrence (p < 0.0001). CONCLUSION: The PSA recurrence was detected in 21.6% of patients who had been treated with radical prostatectomy within 5 years, which indicates that the parameters related to the disease and the patient have a pivotal role in the PSA recurrence.

Biochemical recurrence after radical prostatectomy: is the disease or the surgeon to blame?

PURPOSE: The PSA recurrence develops in 27 to 53 percent within ten years after radical prostatectomy (RP). We investigated the factors (disease grade and stage or the surgeon's expertise,) more likely to influence biochemical recurrence in men post-radical prostatectomy for organ-confined prostate cancer by different surgeons in the same institution. MATERIALS AND METHODS: A total of 510 patients that underwent radical prostatectomy were investigated retrospectively. Biochemical recurrence was defined as detection of a PSA level of > 0.20 ng/mL by two subsequent measurements. The causes, which are likely to influence the development of PSA recurrence, were separated into two groups as those related to the disease and those related to the surgical technique. RESULTS: Biochemical recurrence was detected in 23.5 percent (120 cases) of 510 cases. The parameters most likely to influence biochemical recurrence were: PSA level (p < 0.0001), T stage (p < 0.0001), the presence of extracapsular invasion prostate (p < 0.0001), Gleason scores (p = 0.042, p < 0.0001) and the presence of biopsy with perineural invasion (p = 0.03). The only surgical factor that demonstrated relevance was inadvertent capsular incision during the surgery that influenced the PSA recurrence (p < 0.0001). CONCLUSION: The PSA recurrence was detected in 21.6 percent of patients who had been treated with radical prostatectomy within 5 years, which indicates that the parameters related to the disease and the patient have a pivotal role in the PSA recurrence.

The effect of testosterone treatment on urodynamic findings and histopathomorphology of pelvic floor muscles in female rats with experimentally induced stress urinary incontinence.

OBJECTIVE: In recent studies, it has been observed that androgen receptors are densely located in pelvic floor muscles. We aimed to investigate the effect of testosterone on urodynamic findings and histopathomorphology of pelvic floor muscles in rats with experimentally induced stress urinary incontinence. MATERIALS AND METHODS: Twenty-eight adult female rats were randomized into four groups. Group I: rats in which SUI was induced and single-dose testosterone was administered 30 days later, group II: rats in which SUI was induced and single-dose testosterone was administered within the same session, group III: rats in which SUI was induced and saline was injected intramuscularly 30 days later, and group IV: the sham group. In order to demonstrate objectively the curative and preventive role of testosterone in experimental model of SUI, urodynamic examination and histopathomorphological evaluation of levator ani muscle were performed. RESULTS: Myofiber areas in groups I and II were detected to be significantly larger than those of the control group (P < 0.001). Another parameter was leak point pressure value by urodynamy. Regarding this parameter, LPP values in groups 1, 2 and 4 were observed to be significantly higher than those of group 3 (P < 0.001). The results of the comparison among groups 1, 2 and 4 revealed no significance (P > 0.05), which indicates that testosterone provides continence in a similar way to the group in which sciatic nerve section was not performed. CONCLUSIONS: In the present study, it has been demonstrated that testosterone has both preventive and curative effects on rat models of experimental SUI.

Electrostatic effects on nanofiber formation of self-assembling peptide amphiphiles.

Self-assembling peptide amphiphile molecules have been of interest to various tissue engineering studies. These molecules self-assemble into nanofibers which organize into three-dimensional networks to form hydrocolloid systems mimicking the extracellular matrix. The formation of nanofibers is affected by the electrostatic interactions among the peptides. In this work, we studied the effect of charged groups on the peptides on nanofiber formation. The self-assembly process was studied by pH and zeta potential measurements, FT-IR, circular dichroism, rheology, atomic force microscopy, scanning electron microscopy and transmission electron microscopy. The aggregation of the peptides was triggered upon neutralization of the charged residues by pH change or addition of electrolyte or biomacromolecules. Understanding the controlled formation of the hydrocolloid gels composed of peptide amphiphile nanofibers can lead us to develop in situ gel forming bioactive collagen mimetic nanofibers for various tissue engineering studies including bioactive surface coatings.

Is it possible to predict sepsis, the most serious complication in prostate biopsy?

OBJECTIVE: Prostate biopsy for the diagnosis of prostate cancer by transrectal ultrasonography (TRUS) is a common procedure used in daily urology practice with a low complication rate and easy applicability. In this study, the precipitating factors and prophylaxis for sepsis, the worst complication of the procedure, were assessed. PATIENTS AND METHODS: 2,023 Patients with suspected prostate cancer who underwent biopsy by TRUS in one center were assessed retrospectively. The relationship between sepsis and age, serum total prostate-specific antigen (PSA) level, PSA density, prostate volume, number of biopsies, number of repeated biopsies, accompanying diagnosis of prostatitis, presence of urethral catheter, and presence of diabetes mellitus was assessed. Data were analyzed using the t test and logistic regression analysis. RESULTS: Of the 2,023 patients, 62 (3.06%) developed sepsis within 5 days after biopsy. There was no significant relationship between the biopsy and the above parameters using the logistic regression analysis. Using the t test, it was found that the number of biopsy cores (p < 0.001), presence of urethral catheter (p < 0.0001), and presence of diabetes mellitus (p < 0.0001) were predictive factors for sepsis. CONCLUSION: Sepsis is a rare but life-threatening complication after prostate biopsy by TRUS. Although preoperative prophylactic oral antibiotics and enema before biopsy have proven to be effective in decreasing urinary tract infection rates, patients with urethral catheter, diabetes mellitus or those to undergo biopsy from more sites than ten cores should be closely monitored after biopsy.

Percutaneous nephrolithotomy: variables that influence hemorrhage.

OBJECTIVES: Renal hemorrhage is one of the most common and worrisome complications of percutaneous nephrolithotomy (PCNL). This study attempted to identify variables that might influence hemorrhage during PCNL to help urologists establish preventative and treatment strategies for bleeding during PCNL procedures. METHODS: The data of 193 patients (193 PCNL procedures) were retrospectively analyzed. Hemorrhage was estimated by the postoperative decrease in hematocrit factored by the quantity of any blood transfusion. Various preoperative and operative factors were assessed for their association with blood loss using univariate, forward multivariate regression and correlation analysis. RESULTS: The mean patient age was 45.7 +/- 14.4 years (range 5 to 74). The overall stone-free rate was 85.4%. The average hematocrit decrease was 8.7% +/- 5.39% (range 0.3 to 24.7). Forward multivariate regression analysis identified five significant variables that influenced PCNL-related hemorrhage: stone type (P = 0.003), number of tracts (P = 0.010), method of dilation (P = 0.010), diabetes (P = 0.022), and stone surface area (P = 0.049). A statistically significant difference was found in relation to the occurrence of hemorrhage between patients with caliceal stones and partial staghorn stones (P = 0.008) and complete staghorn stones (P = 0.006), single tracts and multiple tracts (P = 0.038), balloon dilators and Amplatz dilators (P = 0.007), patients with small stones (1000 mm2 or smaller) and large stones (greater than 1000 mm2; P = 0.018) on univariate analysis. Also, the stone surface area (P = 0.019) and number of tracts (P = 0.024) showed a positive correlation with the mean hematocrit decrease. CONCLUSIONS: Staghorn stones, multiple tracts, the presence of diabetes, and large stones were associated with increased renal hemorrhage during PCNL on multivariate analysis. However, balloon dilation was associated with decreased hemorrhage.