A novel model of locking plate and locking spongious screw: a biomechanical in vitro comparison study with classical locking plate.

BACKGROUND: Locking plates are commonly used for the fixation of comminuted, periprosthetic and osteoporotic bone fractures. These plates are secured to the bone with screws, creating a stable connection with fixed angle between the plate and the screws. In this biomechanical in vitro study, our aim is to evaluate and compare the novel locking plate-locking spongious screw model with FDA approved classical locking plate. METHODS: Sawbone PCF-15 osteoporotic bone model was utilized to simulate osteoporotic bone conditions. Two screws were used to attach both the classical locking plate and the novel locking plate-locking spongious screw model to these bone models. The attachment strength of the screws to the bone blocks was measured by pull-out tests. RESULTS: Novel locking plate-locking spongious screw model exhibited an 84.38% stronger attachment to the osteoporotic bone model compared to the current locking plate model. CONCLUSIONS: In conclusion, one of the important problems in the locking plates which is the high Pull-out risk of the locking spongious screws can been resolved with our proposed new model and has a chance of having a better purchase especially in osteoporotic bones.

Comparing two surgical techniques in distal hypospadias repair: Urethral mobilization vs. urethral plate tubularization.

OBJECTIVES: While numerous techniques have been described for hypospadias repair, the Tubularized Incised Plate (TIP) procedure is currently the most frequently used method. On the other hand, urethral mobilization techniques have less frequent preference. In this study, we aimed to compare the outcomes of these two techniques in patients with distal hypospadias, particularly those with the urethral meatus located at the coronal and subcoronal. MATERIAL AND METHODS: A total of 75 patients with distal hypospadias underwent surgery. Patients with glanular and midpenile hypospadias whose meatus was not located at the coronal and subcoronal levels were excluded from the study. 43 patients underwent surgery performing the Eccentric Circummeatal-Based Flap with Limited Urethral Mobilization (ECMB) method, while the remaining 32 patients were treated with the TIP. We analyzed age at surgery, urethral meatus position, ventral or glanular curvature, catheterization duration, and complications. RESULTS: The mean age at operation was 2.6 (±1.1) years. The position of the meatus was coronal in 48 patients (64%), and subcoronal in 27 (36%). Complications consist of fistula in two patients, meatal stenosis in seven patients, and preputial redundancy in two patients. No glans retraction, wound infection or glans dehiscence was observed in any patient. Complication rates were compared and no statistically significant difference was found between the two methods (P>0.05). CONCLUSION: Mobilization techniques may be a preferable and safe option for young surgeons due to their relative ease of perform.

Acetylation of histone H2B marks active enhancers and predicts CBP/p300 target genes.

Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis-regulatory elements, predicting enhancer strength and identifying their target genes is challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a signature of active enhancers. H2BNTac prominently marks candidate active enhancers and a subset of promoters and discriminates them from ubiquitously active promoters. Two mechanisms underlie the distinct H2BNTac specificity: (1) unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300; (2) H2A-H2B, but not H3-H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms current state-of-the-art models in predicting CBP/p300 target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling CBP/p300-dependent gene regulation.

Release of linker histone from the nucleosome driven by polyelectrolyte competition with a disordered protein.

Highly charged intrinsically disordered proteins are essential regulators of chromatin structure and transcriptional activity. Here we identify a surprising mechanism of molecular competition that relies on the pronounced dynamical disorder present in these polyelectrolytes and their complexes. The highly positively charged human linker histone H1.0 (H1) binds to nucleosomes with ultrahigh affinity, implying residence times incompatible with efficient biological regulation. However, we show that the disordered regions of H1 retain their large-amplitude dynamics when bound to the nucleosome, which enables the highly negatively charged and disordered histone chaperone prothymosin α to efficiently invade the H1-nucleosome complex and displace H1 via a competitive substitution mechanism, vastly accelerating H1 dissociation. By integrating experiments and simulations, we establish a molecular model that rationalizes the remarkable kinetics of this process structurally and dynamically. Given the abundance of polyelectrolyte sequences in the nuclear proteome, this mechanism is likely to be widespread in cellular regulation.

SUV39 SET domains mediate crosstalk of heterochromatic histone marks.

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.

Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments.

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability.

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage.

The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play an important role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair.

Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α.

The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions.

Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.

Evaluation of the removal reasons of totally implantable venous devices in children: a retrospective study.

Totally implantable venous access devices (TIVADs) increase the quality of life in children with hematologic and oncologic diseases or organ failures. The aim of this study is to determine the reasons for port removal. The port catheters, implanted and removed in patients between January 2000 and June 2013 were evaluated retrospectively. The patients were divided into two groups, whose port catheters were removed due to completed therapy (completed therapy group, CTG) and whose port catheters were removed because of a port catheter-related complications (complication group, CG). In the CG, the patients whose port catheters are removed for infectious reasons are investigated for whether there is a relationship with age, gender, body mass index (BMI), height and weight at the time of port implantation and removal. In total, 242 patients who underwent port implantation and removal were included in the study. The male to female ratio was 1.32/1 and the mean age of the patients was 9.4±4.9 years (0-24 year). Patients were enrolled in CTG (n=170, 70.2%), and CG (n=72, 29%). There is a positive correlation between BMI and infections (p < 0.05). In the CG, patients under steroid treatment had higher incidence of non-infectious causes than infectious causes (p < 0.05). Oppositely, non infectious complications were higher in steroid free patients (p < 0.05). There was no catheter related mortality in the entire study group. The hematological malignancies and solid tumors are the most common underlying primary disease in patients with port removal because of complications. Infectious complications are most common cause of port removal in children and despite other microorganism, fungi should be considered as a cause of catheter related infections.

Peutz-Jeghers syndrome: an unusual cause of recurrent intussusception in a 7-year-old boy.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant inherited disorder characterized by intestinal hamartomatous polyps in association with mucocutaneous pigmentations. Main symptoms of PJS in childhood are abdominal pain, obstruction, intussusception, and bleeding from hamartomatous polyps. PJS carries a high risk of gastrointestinal cancer with advancing years. Although, intussusception has been reported as a well-known complication of PJS, recurrent intussusception as an alarming finding in a patient with normal gastrointestinal endoscopy is uncommon. A 7-year-old boy who had recurrent intussusception episodes and diagnosed with PJS histopathological confirmation after surgical excision of involved bowel segments is presented to discuss the clinical features and treatment options of recurrent intussusception as a presenting finding of PJS.

Multivalency governs HP1α association dynamics with the silent chromatin state.

Multivalent interactions between effector proteins and histone post-translational modifications are an elementary mechanism of dynamic chromatin signalling. Here we elucidate the mechanism how heterochromatin protein 1α (HP1α), a multivalent effector, is efficiently recruited to the silent chromatin state (marked by trimethylated H3 at Lys9, H3K9me3) while remaining highly dynamic. Employing chemically defined nucleosome arrays together with single-molecule total internal reflection fluorescence microscopy (smTIRFM), we demonstrate that the HP1α residence time on chromatin depends on the density of H3K9me3, as dissociated factors can rapidly rebind at neighbouring sites. Moreover, by chemically controlling HP1α dimerization we find that effector multivalency prolongs chromatin retention and, importantly, accelerates the association rate. This effect results from increased avidity together with strengthened nonspecific chromatin interactions of dimeric HP1α. We propose that accelerated chromatin binding is a key feature of effector multivalency, allowing for fast and efficient competition for binding sites in the crowded nuclear compartment.

Enterobius granuloma: an unusual cause of omental mass in an 11-year-old girl.

Enterobius vermicularis (pinworm) is the only nematode that infects humans. It is one of the most common intestinal parasites. Pinworm commonly infests the terminal ileum and colon, and does not cause severe morbidity unless ectopic infection occurs. However, granulomatous lesions caused by ectopic Enterobius vermicularis infection may lead to unusual clinical symptoms and may be misinterpreted as malignant lesions. Herein, the authors present an 11-year-old girl with pinworm infection who presented with abdominal pain and an omental mass, with special emphasis on the diagnosis and treatment.

Engineering chromatin states: chemical and synthetic biology approaches to investigate histone modification function.

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.

Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a traceless ligation at valine. uH2A could be efficiently incorporated into nucleosomes, thereby opening the way to detailed biochemical and biophysical studies on the function of this PTM. Accordingly, we used uH2A, as well as a previously generated ubiquitylated H2B, in chaperone-coupled nucleosome stability assays to demonstrate that the direct effect of ubiquitylated histones on nucleosomal stability is in fact modest.

Spatial variability of salinity and alkalinity of a field having salination risk in semi-arid climate in northern Turkey.

Spatial variability of salinity and alkalinity is important for site-specific management since they are the most important factors influencing soil quality and agricultural production. The objectives of this study were to analyze spatial variability in salinity and alkalinity and some soil properties affecting salinity and alkalinity, using classical statistics and geostatistical methods, in an irrigated field with low-quality irrigation water diverted from drainage canals. A field of 5 da was divided into 10 m x 10 m grids (5 lines in the east-west direction and 10 lines in the north-south direction). The soil samples were collected from three depths (0-30, 30-60 and 60-90 cm) at each grid corner. The variation coefficients of OM and sand contents were higher than other soil properties. OM had the maximum variability, with a mean of 1.63% at 0-30 cm depth and 0.71% at 30-60 cm depth. Significant correlations occurred between ESP, EC and each of Ca, Mg, K and CaCO(3) contents of the soils (p<0.01). Experimental semivariograms were fitted to spherical and gaussian models. All geostatistical range values were greater than 36 m. The soil properties had spatial variability at small distances at 60-90 cm depth. EC was variable within short distances at 30-60 cm depth. The nugget effect of ESP increased with soil depth. Kriged contour maps revealed that soils had a salinisation and alkalisation tendency at 60-90 cm depth based on spatial variance structure of the EC and ESP values. Spatial variability in EC and ESP can depend on ground water level, quality of irrigation water, and textural differences.