Proximal Urethrostomy Versus Urethroplasty for Complex Urethral Strictures.

Background: The optimal treatment for complex urethral stricture (CUS) is yet to be determined. Comparisons of methods based on validated questionnaires or objective outcome measures are lacking. Objective: To compare proximal urethrostomy and urethroplasty for CUS using objective measures and validated questionnaires, and to evaluate trends in subgroups of patients who underwent proximal urethrostomy as the intended definitive treatment versus first-stage urethroplasty. Design setting and participants: We identified all patients who underwent proximal urethrostomy at our center from 2004 to 2021. The control group comprised patients who underwent urethroplasty for CUS (strictures >6 cm, recurrent posturethroplasty strictures, or CUS due to lichen sclerosus or past hypospadias surgery). Outcome measurements and statistical analysis: The primary outcome was a recurrent stricture at a minimal follow-up of 1 yr. The secondary outcomes included validated questionnaires, uroflowmetry, and residual urine volume. Survival was compared by a Kaplan-Meier analysis. Results and limitations: The study included 57 proximal urethrostomy and 75 urethroplasty patients. Results for these two groups were as follows: the cumulative incidence of stricture recurrence over a median follow-up of 46 mo was 22.6% for proximal urethrostomy versus 36.2% for urethroplasty (p = 0.106); no statistically significant differences were observed between groups in terms of postoperative quality of urination or life, satisfaction with outcome, and erectile function. Both groups had a significant improvement in urinary flow after surgery (19.65 vs 20.29 ml/s), with no difference between the groups (p = 0.796); the proximal urethrostomy group had a significant improvement in postvoid residual after surgery, but there was no difference between the groups in the last follow-up visit (79.16 vs 52.03 ml; p = 0.245). A subgroup analysis of the proximal urethrostomy group showed no significant differences in cumulative primary or secondary outcomes. Limitations included the retrospective design and the relatively small study population. Conclusions: Comparisons of the two groups revealed no significant differences in stricture recurrence, results of validated questionnaires, or objective measures of urination. Patient summary: Proximal urethrostomy is equivalent to urethral reconstruction, and it should be offered as a viable solution for complex urethral stricture.

Effects of high oleic acid peanuts on mice's liver and adipose tissue metabolic parameters and gut microbiota composition.

This study aimed to investigate the effects of two types of peanuts, regular Hanoch (HN) and a new high-oleic cultivar., Hanoch-Oleic (HO), on metabolic parameters and gut microbiota composition. Male C57BL/6 mice were fed with a normal diet (ND) or ND supplemented with HN (NDh) or HO (NDo). Following 18 weeks of diet regimen, the NDo group exhibited reduced body weight and peri-gonadal adipose-to-body weight ratio, paralleled to lesser food consumption. Although blood levels of total cholesterol, HDL-cholesterol, free fatty acids, and liver enzyme levels did not differ between groups, decreased insulin sensitivity was found in the NDh group. Within adipose tissue, the expression of lipolytic and lipogenic enzymes was higher, while those related to lipid oxidation were lower in the NDh group compared to the NDo group. Additionally, HO peanuts consumption promoted the establishment of a healthy microbiota, with an enhanced abundance of Bifidobacterium, Lactobacillus, and Coprococcus genera. In conclusion, the inclusion of the HO peanut cultivar., rather than the conventional peanut cultivar., in a balanced diet was related to better metabolic outcomes and was linked to a favorable microbiota profile.

Chiliadenus iphionoides Reduces Body Weight and Improves Parameters Related to Hepatic Lipid and Glucose Metabolism in a High-Fat-Diet-Induced Mice Model of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has become an epidemic with increasing prevalence. Limited treatment options and poor adherence emphasize the urgent need for novel therapies for the treatment and/or prevention of NAFLD. Bioactive natural compounds found in medicinal plants are promising as novel therapeutic agents for NAFLD. Chiliadenus iphionoides, a medicinal plant with several health-promoting properties, is an encouraging candidate. The current study aimed to elucidate the metabolic effects of C. iphionoides consumption in a high-fat-diet (HFD)-induced model of NAFLD. Male C57BL/6J mice (n = 40, 7-8-week-old) were fed a HFD (60% fat) with/without 0.5 or 2.5 gr C. iphionoides for fifteen weeks. Diet supplementation with C. iphionoides significantly ameliorated HFD-induced weight gain. Likewise, liver and adipose tissue weights were profoundly lower in the C. iphionoides-fed groups. Reduced liver steatosis in those groups was corroborated by histology, plasma liver enzyme levels, and lipid profile, indicating improved liver function and lipid metabolism in addition to enhanced insulin sensitivity. The addition of C. iphionoides to an obesogeneic diet can beneficially alleviate metabolic alterations and may be a practicable strategy for the management of NAFLD.

Metabolic and Microbiome Alterations Following the Enrichment of a High-Fat Diet With High Oleic Acid Peanuts Versus the Traditional Peanuts Cultivar in Mice.

A new Israeli-developed peanut cultivar, "Hanoch-Oleic" (HO), uniquely contains enlarged oleic acid contents and was designed to confer additional beneficial effects over the traditional cultivar, "Hanoch" (HN). This work elucidates metabolic changes and microbiota adaptations elicited by HO addition to a high-fat diet (HFD). Male C57BL/6 mice were fed for 18 weeks with a normal diet or a HFD with/without the addition of HN (HFDh) or HO (HFDo). Body-weight did not differ between HFD-fed mice groups, while liver and adipose weight were elevated in the HFDh and HFD groups, respectively. Insulin-sensitivity (IS) was also decreased in these groups, though to a much greater extent in the traditional peanuts-fed group. Modifications in lipids metabolism were evident by the addition of peanuts to a HFD. Liver inflammation seems to return to normal only in HFDh. Peanuts promoted an increase in α-diversity, with HFDo exhibiting changes in the abundance of microbiota that is primarily associated with ameliorated gut health and barrier capacity. In conclusion, the HO cultivar appears to be metabolically superior to the traditional peanut cultivar and was associated with an improved inflammatory state and microbial profile. Nevertheless, IS-negative effects reinforced by peanuts addition, predominantly NH, need to be comprehensively defined.

Cannabis Extract Effects on Metabolic Parameters and Gut Microbiota Composition in a Mice Model of NAFLD and Obesity.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver abnormalities and has been linked with metabolic syndrome hallmarks. Unfortunately, current treatments are limited. This work aimed to elucidate the effects of three cannabis extracts on metabolic alteration and gut microbiota composition in a mouse model of NAFLD and obesity. Male mice were fed with a high-fat diet (HFD) for 12 weeks. Following the establishment of obesity, the HFD-fed group was subdivided into HFD or HFD that was supplemented with one of three cannabis extracts (CN1, CN2, and CN6) for additional 8 weeks. Metabolic parameters together with intestinal microbiota composition were evaluated. Except for several minor changes in gene expression, no profound metabolic effect was found due to cannabis extracts addition. Nevertheless, marked changes were observed in gut microbiota diversity and composition, with CN1 and CN6 exhibiting microbial abundance patterns that are associated with more beneficial outcomes. Taken together, specific cannabis extracts' addition to an HFD results in more favorable modifications in gut microbiota. Although no marked metabolic effect was disclosed, longer treatments duration and/or higher extracts concentrations may be needed. More research is required to ascertain this conjecture and to establish the influence of various cannabis extracts on host health in general and NAFLD in particular.

Broccoli Florets Supplementation Improves Insulin Sensitivity and Alters Gut Microbiome Population-A Steatosis Mice Model Induced by High-Fat Diet.

Nonalcoholic fatty liver disease (NAFLD) is linked to obesity, type 2 diabetes, hyperlipidemia, and gut dysbiosis. Gut microbiota profoundly affects the host energy homeostasis, which, in turn, is affected by a high-fat diet (HFD) through the liver-gut axis, among others. Broccoli contains beneficial bioactive compounds and may protect against several diseases. This study aimed to determine the effects of broccoli supplementation to an HFD on metabolic parameters and gut microbiome in mice. Male (7-8 weeks old) C57BL/J6 mice were divided into four groups: normal diet (ND), high-fat diet (HFD), high-fat diet+10% broccoli florets (HFD + F), and high-fat diet + 10% broccoli stalks (HFD + S). Liver histology and serum biochemical factors were evaluated. Alterations in protein and gene expression of the key players in lipid and carbohydrate metabolism as well as in gut microbiota alterations were also investigated. Broccoli florets addition to the HFD significantly reduced serum insulin levels, HOMA-IR index, and upregulated adiponectin receptor expression. Conversely, no significant difference was found in the group supplemented with broccoli stalks. Both broccoli stalks and florets did not affect fat accumulation, carbohydrate, or lipid metabolism-related parameters. Modifications in diversity and in microbial structure of proteobacteria strains, Akermansia muciniphila and Mucispirillum schaedleri were observed in the broccoli-supplemented HFD-fed mice. The present study suggests that dietary broccoli alters parameters related to insulin sensitivity and modulates the intestinal environment. More studies are needed to confirm the results of this study and to investigate the mechanisms underlying these beneficial effects.

To Be or Not to Be Eisenmenger: The Different Shades of Blue.

Eisenmenger syndrome refers to any untreated congenital cardiac defect with an intracardiac communication that leads to pulmonary arterial hypertension, reversal of intracardiac shunting, and cyanosis. We describe a 40-year-old cyanotic patient with congenital heart disease with presumed Eisenmenger syndrome who was considered inoperable. Testing revealed a partial atrioventricular septal defect with no evidence of pulmonary arterial hypertension, and the patient underwent successful cardiac repair. (Level of Difficulty: Intermediate.).

Vectorial facial sculpting: A novel sub-SMAS filler injection technique to reverse the impact of the attenuated retaining ligaments.

BACKGROUND: Age-related attenuation of the orbital, zygomatic, and masseteric ligaments contribute to temporal brow ptosis, deepening nasolabial fold, and jowl, respectively. AIMS: To present and assess the vectorial facial sculpting (VFS) technique, a novel nonsurgical panfacial rejuvenation method which focuses on reversal of the impact of attenuated ligaments on the aging face. METHODS: This case series included women who underwent vectorial facial sculpting at a private clinic from June 2018 to January 2019. Sagging tissues in the lateral brow, nasolabial fold, and jowl were repositioned in a directed manner with vectors whose cross-product counteracted the vector of the respective causative attenuated ligament. Filler material was deposited into the supraperiosteal/sub-SMAS plane in right angle vectors at the lateral supra-orbital rim for brow ptosis, pre-auricular and malar region for nasolabial fold and across the jawline region for jowl. Standardized photographs were taken before and at 3 months following treatment. Outcome was assessed by both the patients and an independent investigator using a validated Global Aesthetic Improvement Scale. Adverse outcomes were documented. RESULTS: Forty-five patients of mean age 59.8 ± 3.9 years were included. Mean Global Aesthetic Improvement Scale scores for patients and an independent investigator alike at 3 months following treatment indicated "very much improved" (1.44 ± 0.66 and 1.56 ± 0.78, respectively). The procedure was well tolerated with no adverse events. CONCLUSION: Vectorial facial sculpting is a comprehensive anatomical approach for nonsurgical directional tissue mobilization. Based on physical and mathematical rules to reverse the respective causative attenuated ligament, the technique effectively and safely restores youthful facial contours.

Deep Layer Radiofrequency Thermo-coagulative Technology for Cervicofacial Contouring: Sonographic and Clinical Results.

Radiofrequency energy thermally induces collagen contraction and remodeling. The resultant dermal tightening is well established. However, facial aging encompasses also deeper layers of collagen-containing tissues. We present a deep layer radiofrequency-based thermo-coagulative technique for cervicofacial contouring and evaluate its efficacy. METHODS: This prospective single center study was conducted from June 2017 to June 2018 and included 10 women. Echogenicity and thickness of layers 1-5 of the lower face, lateral neck, and submental regions were sonographically measured at baseline and at 6 weeks postoperatively. Echogenicity analysis was based on the number of high echogenic pixels counted and processed using Matlab-based image application (The Mathworks, Natick, Mass.). Clinical outcome at 12 months postoperatively was evaluated by 2 independent evaluators using a validated 5-point lower face improvement scale and the Merz jawline scale (0-4). Patient satisfaction and adverse effects were recorded. RESULTS: Mean age was 60.2 years (range, 52-76). A statistically significant increase in echogenicity (P ≤ 0.02) and a decrease in thickness (P = 0.01) was noted. Echogenicity increased at 149%, 78%, and 60%, for the lateral neck, lower face, and submental region, respectively. The corresponding decrease in thickness per site was 16%, 6%, and 19%. The average physicians' improvement in lower face contour was 3.8, and the Merz jawline scale was improved from 2.85 at baseline to 1.05 at 12 months postoperatively. Patient satisfaction was high. Side effects were minimal. CONCLUSIONS: Deep layer radiofrequency-based technology thermally induces profound soft tissue tightening and neocollagenesis. It is a safe and effective technique for cervicofacial contouring in selected patients.

Dual-plane hyaluronic acid treatment for atrophic acne scars.

BACKGROUND: Acne is a common condition that affects up to 80% of all adolescents. Scarring may affect some 95% as a function of severity and delay before treatment. The pathogenesis includes enzymatic degradation of collagen fibers and subcutaneous fat. OBJECTIVE: This study aimed to treat atrophic acne scars using the Dual-Plane injection of Hyaluronic Acid. METHODS: A total of 12 patients with moderate-to-severe atrophic acne scars were treated with a novel NAHYCO™ based Hyaluronic Acid filler, using a dual-plane technique for two treatment sessions at a 4-week interval. Results were objectively assessed by two blinded Dermatologists and subjectively evaluated by the patients themselves. RESULTS: A total of 8 out of the 12 patients reported moderate improvement, two indicated marked improvement and two rated minimal improvement. Dermatologists' mean global evaluation score was 2.5 ± 0.43. LIMITATIONS: The small sample size and regional nature of a single-center study. Nevertheless, both the expert dermatologists' and the patients' evaluations of standardized high-resolution medical photographs were consistent, suggesting that this inherent bias was negligible. CONCLUSION: The treatment led to impressive improvement in the depth of the scars, suggesting that this technique can result in safe and rapid amelioration of atrophic acne scars in only two sessions.

Nonsense mutation-dependent reinitiation of translation in mammalian cells.

In-frame stop codons mark the termination of translation. However, post-termination ribosomes can reinitiate translation at downstream AUG codons. In mammals, reinitiation is most efficient when the termination codon is positioned close to the 5'-proximal initiation site and around 78 bases upstream of the reinitiation site. The phenomenon was studied mainly in the context of open reading frames (ORFs) found within the 5'-untranslated region, or polycicstronic viral mRNA. We hypothesized that reinitiation of translation following nonsense mutations within the main ORF of p53 can promote the expression of N-truncated p53 isoforms such as Δ40, Δ133 and Δ160p53. Here, we report that expression of all known N-truncated p53 isoforms by reinitiation is mechanistically feasible, including expression of the previously unidentified variant Δ66p53. Moreover, we found that significant reinitiation of translation can be promoted by nonsense mutations located even 126 codons downstream of the 5'-proximal initiation site, and observed when the reinitiation site is positioned between 6 and 243 bases downstream of the nonsense mutation. We also demonstrate that reinitiation can stabilise p53 mRNA transcripts with a premature termination codon, by allowing such transcripts to evade the nonsense mediated decay pathway. Our data suggest that the expression of N-truncated proteins from alleles carrying a premature termination codon is more prevalent than previously thought.

A 1064-nm Neodymium-doped Yttrium Aluminum Garnet Picosecond Laser for the Treatment of Hyperpigmented Scars.

BACKGROUND: Pigmentation is one of the few major characteristics according to which scars are evaluated. Data on the treatment of the hyperpigmented component of scars are sparse. OBJECTIVE: The authors aimed at evaluating the efficacy of the fractional 1,064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) picosecond laser in the treatment of the hyperpigmented component of scars. METHODS: Sixteen patients with hyperpigmented scars underwent 3 to 8 treatment sessions at 3- to 6-week intervals with the 1,064-nm Nd:YAG picosecond laser (PicoWay, Candela, Resolve handpiece). The treatment response was evaluated by 2 noninvolved dermatologists on a global assessment scale (GAS) of 1 to 4. A Mexameter quantitatively evaluated the melanin content of the scar before and after laser treatments. RESULTS: The average GAS score of the 2 noninvolved dermatologists was 3.31 ± 0.57. The patients assessed their level of tolerance as good or excellent and their satisfaction level as moderate or high. The Mexameter showed that the melanin index decreased considerably (by 39.11 ± 11.58%) in all patients after treatment. CONCLUSION: The fractionated nonablative picosecond Nd:YAG laser was effective for the treatment of the hyperpigmented component of scars.

Radio Peel-Synergism Between Nano-fractional Radiofrequency and 20% Trichloroacetic Acid Chemical Peeling.

BACKGROUND: Microneedling fractional radiofrequency (FRF) and chemical peels are widely used for skin rejuvenation. OBJECTIVE: The authors aimed at evaluating the efficacy and safety of FRF and trichloroacetic acid 20% (TCA20%) peel in different combinations for determining the optimal treatment protocol. METHODS: In this prospective clinical comparison of 4 protocols (FRF alone, TCA20% alone, TCA20% before FRF [TCA→FRF], and TCA20% following FRF [FRF→TCA]), the patients underwent 3.8 ± 1.2 successive treatments of one protocol at 4- to 6-week intervals. The patients and 2 dermatologists evaluated improvement of pigmentation and dyschromia, erythema and blood vessels, laxity and wrinkling, and skin imperfections using a global aesthetic improvement scale (GAIS) and a 1 to 5 scoring system. The patients rated their satisfaction and reported adverse effects and reduced activity. Skin impedance and histological changes following the different protocols were also evaluated on 3 additional volunteers. RESULTS: Sixty-seven patients (age range 22-80 years) were studied. TCA→FRF caused skin impedance to decrease, yielding a more superficial and less-efficient penetration of FRF energy. FRF→TCA produced more significant improvement in overall facial skin appearance (GAIS) and most evaluated skin parameters. Adverse effects and satisfaction rates were similar for all approaches. CONCLUSION: FRF→TCA had the best synergistic effect on skin rejuvenation compared with FRF or TCA20% alone and TCA→FRF.

Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene.

Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine [Formula: see text] pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

Forehead Lift Using Botulinum Toxin.

BACKGROUND: The principle of dynamic muscular activity affecting eyebrow height and shape is well known. We postulate that similarly, dynamics of the fronto-galea-occipital muscles affect forehead height. OBJECTIVES: To present a forehead lift technique using Botulinum toxin injection and evaluate its clinical efficacy and safety. METHODS: Twenty-nine female patients comprised the study group. Forty units of prepared abobutolinumtoxinA (Dysport, 10 U/0.05 mL) were injected into 4 points in the hair-bearing scalp, simulating the points of frontalis origin. The glabella and forehead regions were treated with 50 U each. Standard photographs and measurements were taken before and at 2 weeks following treatment. Forehead height was measured bilaterally drawing a vertical line from mid-pupil to frontal hairline (MPFH) and from medial canthus to frontal hairline (MCFH). We assessed outcome differences in patients with low vs high forehead (cutoff value 5.5 cm forehead height). RESULTS: Mean age was 48 years (range, 29-66 years). Two weeks following treatment, mean frontal height had increased significantly in all measurement points (MCFH right: 4.1 ± 1.8 mm, MCFH left 4.4 ± 1.8 mm, MPFH right 4.4 ± 2.0 mm, MPFH left 4.7 ± 2.3 mm; P <0.001). Low forehead subgroup achieved significantly higher forehead lift compared with high forehead subgroup both in MCFH (6.9% ± 2.0% vs 5.3% ± 2.2%, P = 0.043) and MPFH (8.6% ± 2.5% vs 5.7% ± 2.6%, P = 0.008). No adverse events were documented in any participant. CONCLUSIONS: Botulinum toxin type A injection into frontalis origin can effectively and safely extend forehead height in selected patients. The effect of this technique is greater on patients with low foreheads.

Design of UV-Absorbing Polypropylene Films with Polymeric Benzotriaziole Based Nano- and Microparticle Coatings.

UV-absorbing nanoparticles (NPs) and microparticles (MPs) were prepared by emulsion and dispersion copolymerization of the vinylic monomer 2-(2'-hydroxy-5'-methacryloxyethylphenyl)-2H-benzotriazole (Norbloc (NB)) with the crosslinking monomer divinylbenzene. The effect of the initiator concentration on the size and size distribution of the polyNB (PNB) particles was elucidated. Thin coatings of the formed PNB NPs or MPs of 19 ± 2 and 200 ± 25 nm dry diameter, respectively, onto polypropylene (PP) films were then prepared and characterized. Increasing the concentration or thickness of the PNB NP or MP thin coatings on the PP films decreased their UV transmittance, up to complete UV blocking with just 2 µm of a 4% NP coating. Migration of the UV-absorbing agents from the coated PP films was not observed during three years of storage at room temperature, offering a unique solution to current problems of migration of UV-absorbing additives. The thin coatings obtained by the PNB NPs were superior to those of the PNB MPs, in that no UV transmittance or loss of optical properties of the PP films were observed for the NP coatings, while the coatings produced by the PNB MPs resulted in damaged optical properties, particularly increasing the haze, and achieved incomplete UV blocking.

Synthesis and characterization of crosslinked polyisothiouronium methylstyrene nanoparticles of narrow size distribution for antibacterial and antibiofilm applications.

BACKGROUND: Isothiouronium salts are well known in their variety of antimicrobials activities. The use of polymeric biocides, polymers with antimicrobial activities, is expected to enhance the efficacy of some existing antimicrobial agents, thus minimizing the environmental problems accompanying conventional antimicrobials. METHODS: The current manuscript describes the synthesis and characterization of crosslinked polyisothiouronium methylstyrene (PITMS) nanoparticles (NPs) of narrow size distribution by dispersion co-polymerization of the monomer isothiouronium methylstyrene with the crosslinking monomer ethylene glycol dimetacrylate. RESULTS AND DISCUSSION: The effect of total monomer, crosslinker and initiator concentrations on the size and size distribution of the formed NPs was also elucidated. The bactericidal activity of PITMS NPs of 67 ± 8 nm diameter was illustrated for 4 bacterial pathogens: Listeria innocua, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In order to demonstrate the potential of these PITMS NPs as inhibitor of biofilm formation, polyethylene terephthalate (PET) films were thin-coated with the PITMS NPs. The formed PET/PITMS films reduced the viability of the biofilm of Listeria by 2 orders of magnitude, making the coatings excellent candidates for further development of non-fouling surfaces. In addition, PITMS NP coatings were found to be non-toxic in HaCaT cells. CONCLUSIONS: The high antibacterial activity and effective inhibition of bacterial adsorption indicate the potential of these nanoparticles for development of new types of antibacterial and antibiofilm additives.

Structural Basis for p53 Lys120-Acetylation-Dependent DNA-Binding Mode.

Normal cellular homeostasis depends on tight regulation of gene expression, which requires the modulation of transcription factors' DNA-binding specificity. That said, the mechanisms that allow transcription factors to distinguish between closely related response elements following different cellular signals are not fully understood. In the tumor suppressor protein p53, acetylation of loop L1 residue Lys120 within the DNA-binding domain has been shown to promote the transcription of proapoptotic genes such as bax. Here, we report the crystal structures of Lys120-acetylated p53 DNA-binding domain in complex with a consensus response element and with the natural BAX response element. Our structural analyses reveal that Lys120 acetylation expands the conformational space of loop L1 in the DNA-bound state. Loop L1 flexibility is known to increase p53's DNA-binding specificity, and Lys120-acetylation-dependent conformational changes in loop L1 enable the formation of sequence-dependent DNA-binding modes for p53. Furthermore, binding to the natural BAX response element is accompanied by global conformational changes, deformation of the DNA helical structure, and formation of an asymmetric tetrameric complex. Based on these findings, we suggest a model for p53's Lys120 acetylation-dependent DNA-binding mode.

Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells.

We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents.

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer: Part III: studies on keto esters and acids.

The development of "molecular rulers" would allow one to quantitatively locate the penetration depth of intercalants within lipid bilayers. To this end, an attempt was made to correlate the (13)C NMR chemical shift of polarizable "reporter" carbons (e.g., carbonyls) of intercalants within DMPC liposomal bilayers - with the polarity it experiences, and with its Angstrom distance from the interface. This requires families of molecules with two "reporter carbons" separated by a known distance, residing at various depths/polarities within the bilayer. For this purpose, two homologous series of dicarbonyl compounds, methyl n-oxooctadecanoates and the corresponding n-oxooctadecanoic acids (n=4-16), were synthesized. To assist in assignment and detection several homologs in each system were prepared (13)C-enriched in both carbonyls. Within each family, the number of carbons and functional groups remains the same, with the only difference being the location of the second ketone carbonyl along the fatty acid chain. Surprisingly, the head groups within each family are not anchored near the lipid-water interface, nor are they even all located at the same depth. Nevertheless, using an iterative best fit analysis of the data points enables one to obtain an exponential curve. The latter gives substantial insight into the correlation between polarity (measured in terms of the Reichardt polarity parameter, ET(30)) and penetration depth into the liposomal bilayer. Still missing from this curve are data points in the moderate polarity range.