S. aureus virulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disrupt S. aureus manganese transport and thus abrogate to its virulence.Work outlined in this report demonstrated that MntC-dependent manganese transport is required for  Staphylococcus aureus is a human pathogen that has developed several approaches to evade the immune system, including a strategy to resist oxidative killing by phagocytes. This resistance is mediated by production of superoxide dismutase (SOD) enzymes which use manganese as a cofactor. S. aureus encodes two manganese ion transporters, MntABC and MntH, and a possible Nramp family manganese transporter, exemplified by S. aureus N315 SA1432. Their relative contributions to manganese transport have not been well defined in clinically relevant isolates. For this purpose, insertional inactivation mutations were introduced into mntC, mntH, and SA1432 individually and in combination. mntC was necessary for full resistance to methyl viologen, a compound that generates intracellular free radicals. In contrast, strains with an intact mntH gene had a minimal increase in resistance that was revealed only in mntC strains, and no change was observed upon mutation of SA1432 in strains lacking both mntC and mntH. Similarly, MntC alone was required for high cellular SOD activity. In addition, mntC strains were attenuated in a murine sepsis model. To further link these observations to manganese transport, an S. aureus MntC protein lacking manganese binding activity was designed, expressed, and purified. While circular dichroism experiments demonstrated that the secondary and tertiary structures of this protein were unaltered, a defect in manganese binding was confirmed by isothermal titration calorimetry. Unlike complementation with wild-type mntC, introduction of the manganese-binding defective allele into the chromosome of an mntC strain did not restore resistance to oxidative stress or virulence. Collectively, these results underscore the importance of MntC-dependent manganese transport in S. aureus oxidative stress resistance and virulence.IMPORTANCE Work outlined in this report demonstrated that MntC-dependent manganese transport is required for S. aureus virulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disrupt S. aureus manganese transport and thus abrogate to its virulence. The acquisition of transition metal ions is essential for all forms of life. It has been estimated that up to a third of all proteins  and almoEscherichia coli, Streptococcus pyogenes, and Streptococcus pneumoniae, encode only one type of Mn2+ transport system, while others, including Bacillus subtilis, Salmonella enterica serovar Typhimurium, Yersinia pestis, and Staphylococcus aureus, encode a member of each transporter family  has been shown to increase sensitivity to methyl viologen S. aureus N315 SA1432 revealed sequences that are highly similar to consensus motif I  and motif II  sequences from the family II subclass of Nramp proteins . Consistent with our previous results  conditions. The cells were harvested during the early exponential phase of growth, and SOD activity was assessed for both wild-type and mutant strains of PFESA0179. For these experiments, bacterial cells were cultured in medium without supplemental manganese or in medium supplemented with 0.4 µM, 2 µM, or 10 µM MnSO4. Similar low levels of SOD activity were observed across all PFESA0179 strains in medium lacking supplemental manganese . To confirm that the reduced virulence observed with PFESA0179 mntC is not unique to the PFESA0179 strain background, the virulence of another clinical isolate, PFESA0186, and that of its isogenic mntC mutant were compared in the mouse sepsis model. In agreement with observations made with PFESA0179 mntC, PFESA0186 mntC was significantly attenuated compared to its wild-type counterpart  or PFESA0186 mntC  was monitored for 5 days postchallenge. The figure depicts results from a meta-analysis of three independent experiments. Download FIG S1, PDF file, 0.3 MB.Virulence of PFESA0186 and PFESA0186 Copyright © 2018 Handke et al.2018Handke et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mntC mutant in the mouse sepsis model could be directly attributed to defects in manganese transport, a manganese binding-deficient allele of mntC was designed for use in complementation studies. The crystal structure of MntC has been solved independently by two groups  spectroscopy, providing information on the secondary structure and tertiary structure of the protein, respectively  can be explained by the presence of an irreversibly bound divalent metal ion. This observation has been made previously with S. aureus MntC, where bound metal ions could not even be extracted by EDTA . Similarly, compared to the strain encoding wild-type MntC, complementation with MntC H50K H123K did not restore virulence to PFESA0179 mntC (P < 0.0001). These results confirm that MntC-dependent manganese transport is critical for S. aureus pathogenesis.To determine whether the virulence defects observed with S. aureusin vitro and to enhance its sensitivity to oxidative stress via a reduction in bacterial SOD activity , both mntC and mntH were required for full SOD activity  by those investigators, all strains exhibited low SOD activity. These results can be interpreted to mean that MntABC is of greater importance than MntH for SOD activity upon increasing the level of manganese limitation. Results from the SOD assay presented in our work agree with these prior studies, although the approach used was different. In contrast to increasing the level of manganese limitation by calprotectin sequestration, for the current work, known concentrations of manganese were added to the manganese-depleted culture medium prior to SOD activity assessment. Supplementation of the medium with submicromolar manganese concentrations was sufficient to elevate SOD activity but did so only in strains where mntC was intact. The notion of MntC, as part of the tripartite MntABC transporter, acting as the main contributor to SOD activity under conditions of manganese limitation is consistent with its reported high affinity for manganese (dissociation constant [Kd] in the nanomolar range)   51). In. InS. aumntH nonsense mutation in the 8325-4 strain background .A similar set of observations was made when the mutant set was assayed for methyl viologen resistance, where MntC was the primary contributor to oxidative stress resistance, and a minimal contribution and no contribution to resistance could be attributed to MntH and SA1432, respectively. It should be noted that this small contribution of MntH was observed only in the PFESA0179 strain background. This observed difference between PFESA0179 and 8325-4 can be explained by the presence of a preexisting ckground , a lesioS. aureus  were randomly selected from the internal strain collection of Pfizer, Inc., and aligned against the N315 SA1432 sequence, SA1432 homologs were found in each of the strains . The level of nucleotide identity of SA1432 homologs in these isolates was very high: ST5 strains had an average nucleotide identity of 100%, ST8 strains had an average nucleotide identity of 99.9% , ST22 strains had an average nucleotide identity of 97.5% , ST30 strains had an average nucleotide identity of 97.6% , and ST45 strains had an average nucleotide identity of 96.3% . In addition, all 100 isolates were predicted to encode a fully intact open reading frame (ORF) . Additional work will be required to define the function of this highly conserved protein in S. aureus.Although the initial characterization work whose results are shown in S. aureusmntC mutant strains are significantly attenuated in animal models of infection. This observation is in good agreement with those of Diep et al., who demonstrated that an S. aureus USA300 mntC mutant was significantly attenuated in a mouse lethal challenge model . MntC is attractive as a vaccine candidate because of its high level of conservation , FabC1, was recently identified by phage display methods  and MAb 305-101-8 (interference group 3), significantly reduced the S. aureus bacterial burden in an infant rat model of infection  or on Difco tryptic soy agar . To make TSB-c medium , 10 µg/ml (erythromycin), and 7 µg/ml (chloramphenicol).Primers, strains, and plasmids used in the current study are listed in c medium , TSB wasc medium . The med10.1128/mSphere.00336-18.4TABLE S1 TABLE S1, DOCX file, 0.03 MB.Strains and reagents used in the study. Download Copyright © 2018 Handke et al.2018Handke et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the http://www.cbs.dtu.dk/services/TMHMM/). TransportDB 2.0 (http://www.membranetransport.org/transportDB2/index.html) was used to classify SA1432 as a member of the Nramp family of transporters. Alignment of amino acid motifs was performed with AlignX .Prediction of transmembrane helices was performed with TMHMM v. 2.0 on the CBS web server (mntC::aacA-aphD knockout cassette consisting of two fragments homologous to the S. aureus chromosome (mntC-1 and mntC-2), flanking gentamicin resistance genes aacA to aphD, was constructed as follows. The primer sets (DNA templates) used to amplify each of the fragments were as follows: (i) for mntC-1, oLH81/oLH342 (S. aureus strain COL genomic DNA); (ii) for aacA to aphD, oLH343/oLH344 (pGO1); (iii) for mntC-2, oLH345/oLH314 (COL genomic DNA). All PCRs were performed with iProof polymerase , and all cloning and molecular biology techniques were performed with enzymes from New England Biolabs  and as described previously , flanking the erythromycin resistance gene (ermC), was constructed as follows. The primer sets (DNA templates) used to amplify each of the fragments were as follows: (i) for mntH-1, oLH315/oLH316 (Newman genomic DNA); (ii) for ermC, oLH317/oLH318 (pE194); (iii) for mntH-2, oLH319/oLH320 (Newman genomic DNA). After purification, the three PCR products were spliced together by amplification with oLH315 and oLH320, and the knockout cassette was cloned into pSPT181 at the BamHI/XmaI sites to give pLH58. After electroporation into S. aureus RN4220, the plasmid was integrated at mntH, and the final mntH::ermC mutant was obtained after plasmid excision by secondary recombination. The mutation was introduced into other S. aureus isolates by transduction performed with protocols described previously (mntH was confirmed by PCR.An eviously . In the cat knockout cassette consisting of two fragments homologous to the S. aureus chromosome (SA1432-1 and SA1432-2), flanking the chloramphenicol resistance gene (cat), was constructed as follows. The primer sets (DNA templates) used to amplify each of the fragments were as follows: (i) for SA1432-1, oLH321/oLH322 (Newman genomic DNA); (ii) for cat, oLH323/oLH324 (pC194); (iii) for SA1432-2, oLH325/oLH326 (Newman genomic DNA). After purification, the three PCR products were spliced together by amplification with oLH355 and oLH356, and the knockout cassette was cloned into pSPT181 at the BamHI/XmaI sites to give pLH59. After electroporation into S. aureus RN4220, the plasmid was integrated at SA1432, and the final SA1432::cat mutant was obtained after plasmid excision by secondary recombination. The mutation was introduced into other S. aureus isolates by transduction with protocols described previously (A SA1432::eviously . In the 4 was determined according to standard CLSI guidelines (The MIC of methyl viologen (Sigma-Aldrich) in TSB-c and TSB-c supplemented with 10 µM MnSOidelines  as descridelines . The stoS. aureus strains were inoculated into 5 ml TSB-c medium and incubated overnight at 37°C with shaking (225 rpm). A volume of 150 µl of overnight cultures was inoculated into baffled 125-ml flasks containing 25 ml of TSB-c and, where indicated, supplemental concentrations of MnSO4. Flasks were incubated at 37°C with shaking (225 rpm). Cultures were allowed to grow until they reached an optical density at 600 nm (OD600) of 0.6 to 0.9, corresponding to the exponential phase of growth. At that point, 5 ml from each culture was decanted into three 15-ml conical tubes (for triplicate assessment of SOD activity). Cells were pelleted by centrifugation at 2,400 × g for 10 min at 4°C. From that point onward, cell pellets were kept on ice. The culture supernatant was removed by aspiration, the pellet was resuspended in 500 µl of ice-cold 1× phosphate-buffered saline , and the cells were transferred to microcentrifuge tubes. The cells were pelleted by centrifugation for 2 min at 16,000 × g in a microcentrifuge. Following aspiration of the supernatant, the cells were resuspended in 500 µl of ice-cold 1× PBS. Cell suspensions were added to a lysing matrix B tube , and samples were processed in a Qiagen Retsch MM300 TissueLyser instrument  for 60 s at a frequency setting of 30. Tubes were chilled on ice for 2 min. The cell samples were again processed using a TissueLyser instrument as described above. Samples were spun at 16,000 × g for 1 min in a microcentrifuge to precipitate cellular debris. A volume of 200 µl of supernatant was extracted and placed in a clean tube, and protein samples were placed on ice. Protein concentrations were determined for each sample in triplicate with a Quick Start Bradford protein assay kit (Bio-Rad). SOD activity was assessed with a SOD assay kit (Sigma-Aldrich) and was determined in triplicate for each strain during at least three independent experiments. Statistically significant differences in the levels of SOD activity in the wild-type and mutant strains were determined with a t test performed using GraphPad Prism version 7.04 for Windows .E. coli and purified as described previously  has been deleted, has been described previously . H50K H1eviously ; howevermolar represents the calculated molar ellipticity (in millidegrees per decimole per square centimeter), Θmeasured represents the experimentally measured CD signal, l represents the cuvette path length (1 cm), and C represents the molar concentration of the protein, andMRE represents the calculated mean residue ellipticity, MRW represents the mean residue molecular weight (113 g/mol), l represents the cuvette path length (0.1 cm), and c represents the protein concentration in milligrams per milliliter.All CD experiments were done on a Jasco J-810 automated recording spectropolarimeter  equipped with a Jasco Peltier-type PTC-423S 6-position cell holder. Temperature was maintained at 20°C. All spectra were recorded with a data pitch of 0.1 nm, a spectral bandwidth of 3 nm, and a scanning speed of 50 nm/min. Data corresponding to five accumulations were collected and averaged for each spectrum. Near-UV CD spectra of the samples at 0.8 to 0.9 mg/ml in a mixture of 50 mM Na cacodylate and 150 mM NaCl (pH 7.0) were recorded between 320 and 250 nm using 1-cm-path-length rectangular quartz cuvettes. Far-UV CD spectra of the samples at 0.12 to 0.13 mg/ml in 1× PBS (pH 7.4) were recorded between 260 and 200 nm using 1-mm-path-length rectangular quartz cuvettes. Far- and near-UV CD spectra of the corresponding buffers were recorded with the same parameters and subtracted from the protein spectra to provide baseline correction. Baseline corrected spectra were smoothed using adjacent-neighbor averaging of 21 points and normalized to either molar (near-UV CD) or mean residue (far-UV CD) ellipticity, using 2 dissolved in the same buffer. The experimental temperature was 37°C. An initial 2-µl injection was followed by 8-µl injections to reach saturation. Heat flows of MnCl2 dilution were taken into account by performing buffer titration with the same ligand solution and subtracting the result from the protein titration data. Wild-type MntC titration data were fitted to the data corresponding to the “single class of binding sites” using Origin 5.0 software provided by the ITC manufacturer. No fitting was possible in the case of MntC H50K H123K, since no binding was taking place.All ITC experiments were done using a VP-ITC isothermal titrating calorimeter  in a mixture of 50 mM Na cacodylate and 150 mM NaCl (pH 7.0). Proteins were extensively dialyzed against experimental buffer. Wild-type MntC (32.5 µM) or MntC H50K H123K (49.2 µM) samples were titrated with 0.33 mM MnClmntC  was amplified from S. aureus COL genomic DNA with primers oLH646 and oLH647. The 3′ portion of mntC was amplified from either pLP1215 or pLH89 with primers oLH648 and oLH615. After purification, the mntC PCR products were spliced together by amplification with oLH646 and oLH615. Following addition of 3′ A overhangs, the spliced mntC PCR products were cloned into pCR2.1-TOPO by the use of a TOPO TA cloning kit (Thermo Fisher Scientific). The wild-type mntC allele and mntC allele encoding H50K H123K substitutions were amplified from these clones with primers oLH651 and oLH652, and a cassette containing the native mntABC promoter preceded by a transcriptional terminator (TT-PmntABC) was amplified from pLH76 (mntABC and mntC PCR products were joined and cloned using Gibson assembly master mix (New England Biolabs) to generate pLH110 (expressing wild-type mntC) and pLH107 . An integrative null vector (containing only TT-PmntABC) was also constructed by amplification of TT-PmntABC from pLH76 with primers oLH671 and oLH672. The resulting fragment was cloned into pLH71 as described above, and the clone was named pLH112. Integrative complementation plasmids were purified from E. coli DC10B . Kaplan-Meier survival curves were plotted, and statistical significance was assessed with log rank (Mantel-Cox) tests. All animal work was performed in strict accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols at an American Association of Laboratory Animal Science (AALAS)-accredited facility .Female 9-to-12-week-old CD1 mice  were used for virulence studies. 10.1128/mSphere.00336-18.1TEXT S1 TEXT S1, DOCX file, 0.02 MB.Supplemental Materials and Methods. Download Copyright © 2018 Handke et al.2018Handke et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


The tumor suppressor p53 (TP53) is the most frequently mutated human gene. Mutations in TP53 not only disrupt its tumor suppressor function, but also endow oncogenic gain-of-function (GOF) activities in a manner independent of wild-type TP53 (wtp53). Mutant TP53 (mutp53) GOF is mainly mediated by its binding with other tumor suppressive or oncogenic proteins. Increasing evidence indicates that stabilization of mutp53 is crucial for its GOF activity. However, little is known about factors that alter mutp53 stability and its oncogenic GOF activities. In this review article, we primarily summarize key regulators of mutp53 stability/activities, including genotoxic stress, post-translational modifications, ubiquitin ligases, and molecular chaperones, as well as a single nucleotide polymorphism (SNP) and dimer-forming mutations in mutp53. TP53 has the highest mutation rate, the majority of which are missense mutations with amino acid changes mainly in the DNA binding domain. There are hotspot mutations including R175, G245, R248, R249, R273, and R282, accounting for ~30% of p53 mutations [TP53-null Saos2 cells enhanced soft agar colony formation and tumor development in mice. Since then, considerable evidence has solidified that mutp53 not only loses wild-type TP53 (wtp53) function, but also enhances tumor malignancy in vitro and in vivo independent of wtp53. The primary role of the tumor suppressor p53 (TP53) is to control cell cycle progression, senescence, DNA repair, cell death, and cell metabolism, leading to inhibition of tumorigenesis ,2. Compautations . Such onThe oncogenic function of mutp53 is mainly caused by altered structure and properties of mutp53 to bind with other oncogenic  or tumor suppressive  proteins ,13,14. MGenerally, mutp53 can be roughly divided into two subtypes, DNA contact and conformational (or misfolded/unfolded) mutants. DNA contact mutants have mutations in amino acids where TP53 directly contacts with DNA, leading to impaired TP53’s binding activity while sparing the intact TP53 structure. On the other hand, conformational mutants robustly alter the TP53 structure and disrupt the DNA binding activity ,27. As mLike wtp53, mutp53 stability is regulated by various genotoxic stresses, nutrient depletion, and oncogenic or tumor suppressive proteins ,25, whicMutations in TP53 impair the transcriptional activity (loss of function: LOF). The majority of TP53 mutations are missense mutations. These dysfunctional TP53 mutant proteins are present in the cells and retain or gain abilities to bind with other proteins, resulting in altered function of these binding partners to enhance tumor malignancy, referred to as GOF ,12,13,14−/−TP53 mice [R172HTP53-knockin mice, it remains unclear whether levels of other TP53 mutants also increase by these environmental stresses in mice. Due to mutp53’s inability to transactivate downstream targets including its ubiquitin ligase MDM2, it was considered that mutp53 could be accumulated in both cancer and normal tissues. However, studies using mutp53 knockin mice have demonstrated that mutp53 levels are low in most normal tissues, while mutp53 is accumulated in many cancer tissues ,23,31. T−/− mice ,31. Addi−/− mice ,32,33,34Mutp53 can be degraded by cellular conditions associated with autophagy . SuppresBy mainly focusing on the involvement of PTMs, specific chaperones, and ubiquitin ligases in mutp53 stability/activity, we briefly discuss chemical compounds that can alter mutp53 stability/activity. These include mutp53 accumulators , degraders/depleters , and reactivators  ,41,42. DNA damage agents result in increased levels of mutp53 to alter function of TP53-binding partners, which contributes to chemoresistance ,44,45,46https://clinicaltrials.gov/). Cancer cells are frequently addicted to mutp53, since knockdown of TP53 mutants attenuates malignant properties of cancer cells and tumor development in mice . Tumor shttps://clinicaltrials.gov/). Many TP53 mutants are structurally altered, especially in the DNA binding domain, thereby losing the DNA binding potential . ResearcAccumulated studies demonstrate efficacy of mutp53 degraders/depleters and reactivators to inhibit tumor progression in vitro and in vivo; however, it remains unclear whether these compounds cause PTMs of mutp53 and if the PTMs induced by them play crucial roles in mutp53 degradation or reactivation. Wtp53 is post-translationally modified following a variety of genotoxic and cellular stresses at approximately 15% of amino acid residues, including serine, threonine, and lysine residues, through phosphorylation, acetylation, and ubiquitination ,60,61. MPhosphorylation of proteins is one of the major mechanisms for regulating protein stability or function and transmitting signals in the cell. Following various stresses, wtp53 is phosphorylated at several serine/threonine residues, including serine 15 (S15), threonine 18 (T18), serine 20 (S20), serine 46 (S46), and serine 392 (S392) . IntriguUllrich et al.  report tThe N-terminal serine residues of mutp53 are phosphorylated at multiple sites. Adorno et al.  show thaAnother well-studied phosphorylated serine residue at the N-terminus of mutp53 is S15, a key serine residue to activate wtp53 following genotoxic stress . MelnikoAdditionally, stathmin 1  which plays a role in microtubule destabilization and dynamics contributes to mutp53 stability with phosphorylation at S15 and S37 . KnockdoBesides S15, T18 and S46 are phosphorylated on wtp53, which is enhanced by PML ,113,114.R172HTP53 mutations, concomitant homozygous deletion of Pin1 attenuates tumor progression [S46 of wtp53 is known to be required for TP53-mediated apoptosis ,116,117.gression . Pin1 algression . Thus, iC-terminal serine residues of mutp53 are also phosphorylated. Polo-like kinase 2 (PLK2), which is activated by DNA damage, phosphorylates mutp53  at T377, leading to enhanced binding of mutp53 with p300, increased acetylation of mutp53, and induction of the GOF activity including increase in cell proliferation, NF-Y’s transcriptional activity on cyclin A, cyclin B, cdk1, and cdc25C, and Adriamycin resistance .Additionally, Matsumoto et al.  reveal SOverall, phosphorylation of wtp53 stabilizes and enhances the function of wtp53 as a transcription factor and a tumor suppressor. On the other hand, phosphorylation of mutp53 results in context-dependent changes in the stability and oncogenic GOF. Further detailed studies are needed to clarify the role of each phosphorylation site in mutp53 GOF.CBP/p300, P/CAF (p300/CBP-associated factor), and Tip60 are well-documented acetyltransferases for wtp53 which enhance wtp53 transcriptional activity , while HThirteen lysine (K) residues on wtp53 are reported to be acetylated. Three  are in the DBD, two  are in the domain containing a nuclear localization signal (NLS), and eight  are located in the C-terminal regulatory domain including a tetramerization/oligomerization domain, a nuclear export signal (NES), and a negative regulatory domain  124,125125. NotaMinamoto et al.  report tp21 and PUMA promoters. Additionally, Knowell et al. [Intriguingly, Perez et al.  observe l et al.  find thal et al. .As mentioned in the previous section, glucose restriction triggers mutp53 degradation via autophagic cell death in several mutp53-carrying cancer cell lines, including TOV112D (R175H), MDA-MB-231 (R280K), T47D (L194F), BT-20 (K132Q), and PANC1 (R280T) . IntriguPUMA, NOXA), suggesting reactivation of wtp53 activity through deacetylation. In support of their findings, Zhang et al. [Moreover, Yi et al.  observe g et al.  demonstrThus, some lysine residues contribute to stabilization and activation of GOF mutp53, while other residues can alter the structure of mutp53 to restore wtp53 activity upon acetylation or deacetylation. Detailed analyses for each lysine residue in mutp53 are required to understand the exact role of mutp53 acetylation in its stabilization, GOF activity, and restoration of wtp53 activity.Ubiquitination of mutp53 plays a crucial role in the stability and subcellular localization of mutp53, which impacts its GOF activities. Polyubiquitination of mutp53 leads to its degradation, while its monoubiquitination may alter subcellular localization of mutp53 likewise wtp53 ,131. The−/−TP53−/−MDM2 cells, whereas R273H mutp53 which is not ubiquitinated in the same cells is present in the nucleus [Lukashchuk and Vousden  reveal t nucleus . Similar nucleus  observe Recently, Frum et al.  report tR248W/−TP53 HCT116 cells, they also show that MDM2-B enhances mutp53 GOF including tumor growth and metastasis in vivo. Additionally, MDM2-B is overexpressed in R172H/R172HTP53 mouse lymphomas. Thus, it is postulated that any proteins which inhibit MDM2 activity could stabilize mutp53 to increase the GOF activities.MDM2 has more than 40 splice variants, but it is unclear whether all variants are translated and have functional effects on TP53 . SeveralCHIP is a cofactor that interacts with HSC70 and accelerates ubiquitin-dependent degradation of chaperone substrates through its carboxyl-terminal U-box . Mutp53 Another ubiquitin ligase which can ubiquitinate and degrade wtp53 is Pirh2. Intriguingly, arsenic trioxide (ATO) induces Pirh2-mediated degradation of multiple TP53 mutants in SW480 (R273H), MiaPaCa-2 (R248W), HaCaT (H179Y/R282W) cells, as well as HCT116 cells expressing R175H and R273H .In summary, mutp53 is ubiquitinated by MDM2 and other ubiquitin ligases. Unfolded/misfolded mutp53 may be ubiquitinated by multiple ubiquitin ligases such as MDM2 and CHIP, while ubiquitination of DNA contact mutp53 is mainly executed by MDM2. Additionally, ubiquitinated mutp53 appears be exported to the cytoplasm for degradation likewise wtp53. Thus, ubiquitination and degradation of mutp53 may be dependent on its binding to molecular chaperones and their associated ubiquitin ligases.Deubiquitinases (DUBs) are a group of proteases that cleave ubiquitin from proteins. USP10 is a cytoplasmic ubiquitin-specific protease and is shown to deubiquitinate wtp53 to reverse MDM2-induced wtp53 ubiquitination, nuclear export, and degradation . KnockdoUSP15 is another deubiquitinase that is reported to increase the stability of MDM2 and hence reduce wtp53 activity to inhibit apoptosis in cancer cells . HoweverThus, several ubiquitin ligases and deubiquitinases can regulate ubiquitination and degradation of both wtp53 and mutp53; however, it remains unclear whether a ubiquitin ligase which ubiquitinates both wtp53 and mutp53 modifies the same lysine residues, if different ubiquitin ligases modify the same lysine residues, and which lysine residues are involved in subcellular localization of mutp53. It is also unknown if other ubiquitin-like modifications such as neddylation and SUMOylation occur in mutp53 and whether these modifications alter the stability, GOF activities, and subcellular localization of mutp53.Endogenous and environmental stresses, such as heat shock, oxidative stress, inflammation, infection, chemicals, and irradiation, significantly impact behavior of the cell. Heat shock proteins (HSPs) play central roles in responding to these stresses as molecular chaperones. Molecular chaperones interact with a variety of proteins to promote their proper folding in order to prevent generation of unfolded/misfolded or damaged proteins, causing them to be refolded or degraded .HSP family proteins, including HSP90, HSP70 (DnaK homologue), and HSP40 (DnaJ homologue), play major roles in protein folding, protein maturation, protein localization, proteolysis, and cell signaling . There aR172H/R172HTP53 and R248Q/−TP53 mice with minimal effects on TP53-null mice. Recently, Ingallina et al. [HSP90 plays a crucial role in stabilizing mutp53. Geldanamycin, a HSP90 inhibitor, alters the physical association of mutp53 with the HSP90 complex, leading to reduced protein half-life of several TP53 mutants via ubiquitination and proteasomal degradation, in multiple cancer cell lines, including T47D L194F), MDA-MB-468 (R273H), and SKBR3 (R175H) ,150,151.F, MDA-MBHSP70 is another chaperone which is involved in mutp53 stabilization or degradation. Muller et al.  show thaMortalin is a member of mitochondrial HSP70 and is also known as mtHSP70/Grp75 (mot-2). Mortalin is involved in mitochondrial biogenesis regulating mitochondrial import of nuclear-encoded proteins . Mortalip21, PUMA, and MDM2, as well as mutp53’s binding to the TP53-resposible elements of these genes. Moreover, CTM restores MDM2’s ability to induce mutp53 degradation. Additionally, Tracz-Gaszewska et al. [HSP40 is another chaperone that binds with conformational or misfolded mutp53 (R175H) . Mass spa et al.  observe a et al. . Intrigua et al. .R248W/−TP53 HCT116 cells as well as H1299 and Saos2 cells exogenously expressing R175H, R248W, and R273H mutp53. They observe that BAG2 inhibits MDM2’s activity on mutp53 and accumulate the protein levels of mutp53. Furthermore, BAG2 knockdown enhances sensitivity of mutp53-expressing cells to 5-Fluorouracil, a chemotherapy drug, and reduces migration, tumor growth, and lung colonization of R248W/−TP53 HCT116 cells. Moreover, BAG2 is elevated in a diverse range of human cancers, including colorectal cancers, lung cancers, breast cancers and sarcomas, while high levels of BAG2 are associated with increased mutp53 levels and poor prognosis in these cancer patients [BCL2-associated athanogene (BAG) family proteins are co-chaperones that interact with mutp53. Through mass spectrometry analyses, two BAG family proteins, BAG2 and BAG5, are found to bind with mutp53 (mouse R172H) and promote its GOF activity ,102. Yuepatients . Later, Thus, multiple chaperone and co-chaperone proteins play crucial roles in mutp53 stability and GOF activities as well as restoration of wtp53 structure and activity. Currently, only HSP90 inhibitors are in clinical trials for cancer therapy. Development of inhibitors for other HSPs and chaperones/co-chaperones would greatly help understand mechanisms behind restoration of wtp53 activity and mutp53 stabilization/GOF and may also advance current therapeutic strategies for cancers carrying mutp53.A SNP in wtp53 at codon 72 alters localization and function of wtp53, where arginine 72 (72R)-wtp53 has greater ability to localize to the mitochondria and induce apoptosis more efficiently than proline 72 (72P)-wtp53 . EvidencTP53-null H1299 or PC3 cells, as well as by comparing results between two colon cancer cell lines, HT29 (72P-R273H) and SW620 (72R-R273H). They reveal that 72P-mutp53 more efficiently binds to and inhibits activity of PCG-1α, a master regulator of mitochondrial function, as compared with 72R-mutp53. As a result, cancer cells carrying 72R-mutp53 have increased mitochondrial function, leading to enhanced metastatic capacity. Moreover, 72R-mutp53 is correlated with poorer outcomes in human breast cancer, as compared with 72P-mutp53 [Recently, Basu et al.  reportedP-mutp53 . FurtherP-mutp53 ,164.Oligomerization of TP53 has been implicated in TP53’s DNA binding/transcriptional activity, ubiquitination/degradation, and interactions with TP53 binding partners . RecentlThis article has summarized multiple factors that impact levels, subcellular localization, and GOF activities of mutp53, as well as those inducing reactivation of wtp53 or depletion of mutp53. These include the upstream regulators of mutp53, such as genotoxic stress, PTMs, ubiquitin ligases, and molecular chaperones, as well as different sequences in mutp53 including codon 72 polymorphism and mutations in the oligomerization domain. Although studies examining upstream regulators of mutp53 GOF activities are limited, unlike wtp53, many upstream regulators of wtp53 also appear to impact mutp53 stability and GOF. However, the biological consequences are distinct between wtp53 and mutp53, which can be dependent on types of TP53 mutations, cellular context, and experimental conditions. To clarify these differences, detailed studies using various TP53 mutants in a well-controlled experimental setting should be performed in the future.In conclusion, stability and activity of mutp53 are altered by multiple factors, some of which regulate wtp53 activity. However, the biological consequences are distinct. More studies are required to obtain clear-cut conclusion of the roles of mutp53 PTMs and SNPs in the GOF activity and cancer progression.


Six groups were prepared having different wt% concentrations of ZrO2 nanoparticles: 0% (control), 1.5%, 3%, 5%, 7%, and 10%, respectively. Flexural strength and flexural modulus were measured using a three-point bending test and surface hardness was evaluated using the Vickers hardness test. Fracture toughness and impact strength were evaluated using a single edge bending test and Charpy impact instrument. The fractured surfaces of impact test specimens were also observed using a scanning electron microscope (SEM). Statistical analyses were conducted on the data obtained from the experiments. The mean flexural strength of ZrO2/PMMA nanocomposites (84 ± 6 MPa) at 3 wt% zirconia was significantly greater than that of the control group (72 ± 9 MPa) (p < 0.05). The mean flexural modulus was also significantly improved with different concentrations of zirconia when compared to the control group, with 5 wt% zirconia demonstrating the largest (23%) improvement. The mean fracture toughness increased in the group containing 5 wt% zirconia compared to the control group, but it was not significant. However, the median impact strength for all groups containing zirconia generally decreased when compared to the control group. Vickers hardness (HV) values significantly increased with an increase in ZrO2 content, with the highest values obtained at 10 wt%, at 0 day (22.9 HV0.05) in dry conditions when compared to the values obtained after immersing the specimens for seven days (18.4 HV0.05) and 45 days (16.3 HV0.05) in distilled water. Incorporation of ZrO2 nanoparticles into high impact PMMA resin significantly improved flexural strength, flexural modulus, fracture toughness and surface hardness, with an optimum concentration of 3–5 wt% zirconia. However, the impact strength of the nanocomposites decreased, apart from the 5 wt% zirconia group.Acrylic resin PMMA (poly-methyl methacrylate) is used in the manufacture of denture bases but its mechanical properties can be deficient in this role. This study investigated the mechanical properties  and fracture behavior of a commercial, high impact (HI), heat-cured denture base acrylic resin impregnated with different concentrations of yttria-stabilized zirconia (ZrO In practical applications, denture base materials experiences different types of stresses, such as compressive, tensile and shear, which can lead to premature failure. Intra-orally, repeated mastication over a period of time can lead to denture base fatigue failure. Extra-orally, denture bases can also experience high impact forces when dropped by accident ,2. ImpacHigh impact (HI) denture base resins are widely used in prosthetic dentistry. These materials are provided in either powder or liquid forms and are processed in the same manner as other heat-cured, poly-methyl methacrylate (PMMA) resins. HI resins are reinforced with butadiene-styrene rubber, with the rubber particles grafted to the poly-methyl methacrylate so that the particles are covalently bonded into the polymerized acrylic matrix in order to better absorb mechanical loads ,8,9,10. Many attempts have been made to improve the strength of denture base resins, including the addition of metal wires and plates made of either Co-Cr alloy or stainless steel. However, these materials present limitations contrary to the standard requirements, including poor adhesion between the acrylic resin and reinforcing metal. This separation can result in a reduction in overall mechanical strength within the prosthesis, as well as poor aesthetics. Additionally, metal-reinforced denture bases can become noticeably heavier ,14. Othe2) is a bio-ceramic material that has been widely used for various dental applications, such as crowns and bridges, implant fixture “screws” and abutments, and orthodontic brackets [1/2) [In recent years, several investigations have focused on improving the mechanical properties of PMMA acrylic resins by adding nanomaterials, such as bio-ceramic nanoparticles, due to their special characteristics . Zirconibrackets . Zirconits [1/2) . Furtherts [1/2) ,24. A nuts [1/2) ,25. HoweThe purpose of this study is to evaluate the effects of zirconia nanoparticle addition at low concentrations (up to 10%) to a commercially available, high-impact, PMMA denture base resin on selected mechanical properties such as flexural strength, impact strength, fracture toughness, hardness and fracture behaviour. 2) nanoparticles  were chosen as the inorganic filler agent for fabricating the nanocomposite denture base specimens.A commercially available, Metrocryl HI denture base powder,  and Metrocryl HI (X-linked) denture base liquid   were selected as the denture base material. Yttria-stabilized zirconia , and mixed at 1500 rpm for 20 min. Following the initial mixing, 7 wt% silane coupling agent  was added slowly over a period of 20 s. The mixture was then placed in the speed mixer at 1500 rpm for 10 min and divided equally into two tubes and spun in a centrifuge at 23 °C at 4000 rpm for 20 min. The supernatant (separated toluene) was removed, and the remaining silanized nanoparticles were transferred into a personal solvent evaporator  for 3 h of drying at 60 °C.To determine the most appropriate weight percentages of zirconia nanoparticles for the current study, preliminary investigations were undertaken using 1.5 wt%, 10 wt% and 15 wt% mixtures. Based on these results and knowledge from relevant literature, a decision was made to utilize the following weight percentages of silanized zirconia nanoparticles in the denture base formulation: 0.0% (control), 1.5 wt%, 3.0 wt%, 5.0 wt%, 7.0 wt%, and 10.0 wt%. The composition details of the specimen groups used in this study are described in The silane-treated zirconia and acrylic resin powders were weighed according to Flexural strength of the nanocomposite specimens was evaluated using a 3-point bend test in a universal testing machine  in accordance with British International Standard for Denture Base Polymers (2487: 1989) . The dimIC) in MPa m1/2 according to Equation (2) [Fracture toughness tests were conducted using a single edge span notch bending test on the Zwick universal testing machine in accordance with the British International Standard Organization (BS EN ISO 20795-1:2008) ,29. The 2) utilized a universal pendulum impact testing machine (Zwick/Roell Z020 Leominster). Specimen dimensions were 80 mm (l) × 10 ± 0.01 mm (w) × 4 ± 0.01 mm (h), in accordance with the European International Standard Organization (EN ISO 179-1:2000) [2) was calculated using Equation (4) [c is the breaking energy in joules absorbed by breaking, h is the thickness in mm, and bN is the remaining width in mm after notching.The Charpy V-notch impact test (kJ/m-1:2000) . The spetion (4) : (4)aiN=0.05) of the specimens was measured using a micro-hardness testing machine . Specimens were 65 mm length × 10 mm width × 2.50 mm thickness, and the test load was fixed at 50 g for 30 s. The Vickers hardness was calculated by measuring the diagonals of the pyramid-shaped indentation impressed on the specimen. A total of three indentations were taken at different points in each specimen one side, and then a mean value was calculated. The mean hardness values for all the specimens were determined demonstrative of the materials in the dry condition at day 0. The specimens were then stored individually in 37 ± 1 °C distilled water for 7 d ± 2 h, and were then re-immersed for a total of 45 d ± 2 h. From the raw data, the mean hardness values for each sample group were calculated [The Vickers hardness  . The fractured surface was also studied to identify failure mechanism. Specimens were mounted onto aluminium stubs and sputter-coated with gold after which SEM visualization was performed using a secondary electron detector at an acceleration voltage of 2.0 kV. p < 0.05).Flexural strength, modulus, impact strength, fracture toughness and Vickers hardness data were analysed using a statistical software . Non-significant Shapiro–Wilk and Levene tests showed that the data of flexural and fracture toughness were normally distributed and there was homogeneity of variance. The flexural and fracture toughness data were analysed using a one-way analysis of variance (ANOVA) with the Tukey honestly significant difference post-hoc test at a pre-set alpha of 0.05. Impact strength and hardness data demonstrated nonparametric distributions as evidenced by significant Shapiro–Wilk test results for two groups, and therefore the Kruskal–Wallis test was used to analyse the results as well as to compare the differences among the test groups at a pre-set alpha of 0.05. In addition, the Friedman’s two-way analysis test was applied to identify any significant difference between the three immersion time groups  for the specimen group containing 3 wt% zirconia. However, the mean values of flexural modulus showed a significant increase (p < 0.05) for all specimens, except that containing 7 wt% zirconia, which was not significantly different (p > 0.05) from the control group. The flexural strength data in the table demonstrates that an addition of zirconia nanoparticles to the HI PMMA gradually increased the strength up to 3 wt% and then gradually decreased for other compositions when compared to the control group (0 wt%). The highest value of flexural strength was recorded for the group containing 3 wt% zirconia (83.5 MPa) in comparison with the control group (72.4 MPa), representing a 15% increase in the flexural strength. However, a higher percentage of zirconia nanoparticles (7 wt% to 10 wt%) in the specimens reduced the strength, which was comparable to the control group. A similar behaviour was also found for the flexural modulus of the nanocomposites with increasing zirconia content  of flexural strength values presented in  content . Howeverp < 0.05). Furthermore, after the initial decrease of fracture toughness at 1.5 wt% zirconia, the values slightly increased in the groups containing 3 wt% and 5 wt% zirconia, but they were not statistically significant increases (p > 0.05). The mean values of the fracture toughness  of the n(p > 0.05), as shown in The values of the impact strength for all nanocomposite groups were not statistically significant p < 0.05) for the specimen groups containing 7 wt% and 10 wt% zirconia in both dry (0 day) and wet (7 days) conditions. From the graphical presentation of the hardness results  to create reactive functional groups. This could be responsible for improving the flexural properties of the nanocomposites at lower concentrations of zirconia nanoparticles. Moreover, the improvement in flexural strength and flexural modulus could be a result of the improved dispersion of the ZrO2 nanoparticles when mixing with the speed mixer machine during the preparation stage. This improvement would decrease the agglomeration tendency in the composites. Additionally, the large interfacial area of the nanoparticles contributes to more contact points between the ZrO2 and PMMA, thus enhancing mechanical interlocking and offers additional flexibility in the nanocomposites [The inorganic reinforcing nano-fillers have a large surface area that provides high surface energy, and this produces nanoparticles with a strong tendency to aggregate. This characteristic may decrease the chemical interaction between the nanoparticles and the base PMMA . In thismposites .2 nanoparticles in HI heat-cured denture base acrylic resin are available in the literature. In contrast, investigators have worked on improving the mechanical properties of conventional heat-cured denture base acrylic resin by incorporating different types of fillers [2O3 and ZrO2 with a filler concentration of 5 wt%. Moreover, the flexural modulus increased with an increase in Al2O3/ZrO2 nanoparticle concentration [2O3 particles to PMMA denture bases on flexural strength. They found that a 6% increase in flexural strength value with 2.5 wt% Al2O3 compared to a control group could be obtained. Zhang et al. [2 nanoparticles and micro-particles of aluminium borate whiskers (ABWS) at concentrations of 1 wt%, 2 wt%, 3 wt%, and 4 wt% on the flexural strength of PMMA denture base resin. They found that 2 wt% nano-ZrO2 with a ZrO2/ABWS ratio of 1:2 improved flexural strength by 32% when compared to a control group. These previous studies in the literature were in agreement with the results obtained in this study, which revealed that zirconia positively influenced the flexural properties of HI PMMA with an optimum zirconia concentration between 3 wt% and 5 wt%.Only a few studies on the effect of adding ZrO fillers . Alhareb fillers  showed antration . The grentration , and thig et al.  investigIC) is a critical stress intensity factor that provides information on crack formation [2 nanocomposites with increasing filler content could be due to a number of reasons, such as particle distribution in the polymer matrix, the type and size of the particles, the concentration of the added particles, and chemical reactions between the particles and polymer [2 nanoparticles to the PMMA matrix causes agglomeration, which acts as a stress raiser in the centre of the matrix and reduces the mechanical properties of the polymer material with increasing concentrations of the TiO2 nanoparticles. Fangqiang et al. [2 particles in PMMA matrix using two strategies during mixing: Physical method and chemical method. The physical method was conducted by melt blending, high-energy ball milling or ultrasonic vibration. In the chemical method, when mixing nanoparticles with an MMA monomer, the inorganic ZrO2 nanoparticles acted as a core, and the monomer as a shell structure by in situ polymerization of the monomers, known as grafting. The chemically modified nanoparticle surfaces with MMA enhanced the dispersion stability of the nanoparticles in the polymer matrix. Owing to a combined physical and chemical preparation, it was observed that the dispersion of ZrO2 nanoparticles in the polymer matrix was enhanced and particle aggregation and phase separation decreased to a demonstrable extent. The results of the present study on fracture toughness are consistent with those reported in the study of Alhareb et al. [2O3/ZrO2) showed an improvement in fracture toughness but an increase in zirconia concentration decreased toughness.Fracture toughness  to PMMA denture bases on impact strength. The results showed that the impact strength decreased with an increase in ZrO2 nanoparticle concentration. The finding of the impact strength in the present study is in agreement with that of the previous study, with the exception of the 5 wt% ZrO2/PMMA nanocomposite results. This result can be explained by the fact that a concentration of 5 wt% might be the optimum quantity to improve particle distribution and reduce amalgamation. Asar et al. [2, TiO3, and Al2O3, with 1% and 2% by volume on the impact strength of the PMMA acrylic resin. In contrast to the current study, the findings showed a slight increase in the values of impact strength with 2% ZrO2 addition.The incorporation of hard ZrOgatively ,36. A stgatively  evaluater et al.  investigDenture base materials should also have adequate abrasion resistance to prevent high wear of the material by abrasive denture cleansers, food or general functional forces . Greater2 nanocomposites with different ZrO2 concentrations  using indentation and pendulum hardness tests. They found that the hardness values were increased with an increase in the ratio of ZrO2 to PMMA, with the highest value being 15 wt%. Zhang et al. [2/ABW content, and the optimum hardness was achieved at 3 wt% ZrO2 nanoparticles. They suggested that the decrease in surface hardness with higher filler loading was caused by poor adhesion of the particles to the resin matrix and filler clustering within the matrix. In another study, the incorporation of aluminium oxide (Al2O3) with percentages of 0.5 wt%, 1 wt%, 2.5 wt% and 5 wt% to PMMA acrylic resin exhibited an improvement in Vickers hardness with an increase of Al2O3 filler concentrations [The finding of the present study is in agreement with a study by Yiqing et al. , who evag et al.  investig2 nanocomposite matrix. At low magnification, the fractured surfaces of the nanocomposite specimens exhibited less ductile fracture compared to the control group with a large amount of fragment crack deformation, which formed an irregular surface. Furthermore, the distribution of ZrO2 nanoparticles in the polymer matrix was not homogeneous with evidence of agglomerations, which could reduce the impact strength, particularly at high ZrO2 concentrations (10 wt%).The lower impact strength in the nanocomposites can be related to the presence of voids and clustering of the nanoparticles ,36. At hThe flexural strength of the high impact (HI) heat-cured PMMA denture base was significantly enhanced by the addition of zirconia nanoparticles with 3 wt% when compared to the pure acrylic material (control group).The flexural modulus of the high impact (HI) heat-cured PMMA denture base was significantly enhanced compared to the control group by addition of zirconia nanoparticles with 1.5 wt%, 3 wt%, 5 wt% and 10 wt%. The 7 wt% of zirconia showed a non-significant enhancement compared to the control group.2 concentration. The fracture toughness was slightly increased at 5 wt%, but this was not significantly different compared to the control group.The fracture toughness of the zirconia-reinforced PMMA was significantly decreased, particularly at 10 wt% ZrOFor all zirconia contents, the impact strength of the nanocomposites was significantly lower than that of the control group. However, at 5 wt% and 3 wt% zirconia content, the proportion of reduction in impact strength was not significantly different from that of the control group.Surface hardness continuously increased with increase of zirconia content, in the dry condition at day 0. However, in the wet condition after seven days, and 45 days surface hardness was decreased with all groups.Addition of zirconia in PMMA between 3 wt% and 5 wt% zirconia would provide the optimum mechanical properties suitable for denture base applications.With consideration to the limitations of this study, the following conclusions can be drawn:


Proceedings of the 15th International Congress on Circumpolar Health August 5-10, 2012, Fairbanks, Alaska, USA. This extensive publication includes nearly 100 full length papers, 90 extended abstracts and nearly 100 short abstracts. The full publication is freely available through the journal website.


Purpose: In severe hypospadias staged repair is commonly used and it is regarded as feasible, safe, and durable. In this article we want to describe the results of a modification of the staged repair: a midline incision of the graft during the second stage.Materials and Methods: This is a consecutive single team (2 surgeons) retrospective series. Between 2014 and 2017, 250 patients underwent hypospadias repair, among them 35 patients that had primary staged hypospadias surgery with completed first and second stage repair. 24 (68.6%) cases received a preputial skin graft and 11 (31.4%) buccal mucosa graft. Median age at first stage was 1.5 (0.5–22.1) years, mean time between first and second stage operation was 0.72 (0.4–1.76) years. Follow up rate was 100%, mean follow up period was 1.50 (0.4–3.8) years.Results: The total complication rate was 22.9%. In buccal mucosa repair the complication rate was 36.4% and in preputial graft repair the complication rate was 16.7%, respectively. In 23 patients (65.7%) during second stage urethroplasty a midline incision was performed . Complication rate in non-incised urethroplasty was 8.3%, in incision at glandular level 37.5%, in incision at penile level 13.3% and in incision at urethral opening 16.7%, respectively.Conclusions: Two stage repair is the method of choice in the correction of severe hypospadias. In selected cases a midline incision of the graft is feasible and can be applied if needed. Randomized studies will be needed to evaluate the true benefit of incising the graft. Correction of severe hypospadias is a difficult task. Although there are many techniques available, today the two stage repair is the preferred method for many surgeons . In a fiThis is a consecutive single team (2 surgeons), single hospital retrospective series. Between 2014 and 2017, 250 patients underwent hypospadias repair. 48 patients had staged repair. Out of them we could identify 35 patients that had simple primary staged hypospadias surgery with completed first and second stage repair. 24 (68.6%) cases received a preputial skin graft and 11 (31.4%) received a buccal mucosa graft. Patient characteristics are shown in A first stage hypospadias repair is performed a described previously . PreputiIn our institution a staged repair is performed when there is severe hypospadias [according to the SIU classification ]. The toideal width of the future neo-urethra during second stage urethroplasty. We screened the literature but objective parameters that allow to predict successful tubularization are still unclear . In our practice the margins of the neo-urethra are defined by the surgeon's experience. However, during second stage we sometimes are faced by the three following problems: (a) the glandular graft/urethral plate is not wide enough to create a nice glandular urethra (or the glandular groove is not deep enough), (b) the graft/urethral plate has shrunken and/or is not wide enough on the penile shaft and/or the scarred edges of the graft do not allow wider incision lines, (c) urethral opening is tight. Even in good graft take, buccal mucosa graft sometimes are bulky, thick, and not easily tubularizable. To address these issues a deep midline incision (like the TIP incision) has been introduced in our clinical practice.Repair of severe hypospadias is difficult and there is a persistent high complication rate . Since tLongitudinal incision of the tissue that should become the neo-urethra has been applied for many years . In 1994From hypospadias surgery point of view there are major differences between an untouched urethral plate and a well-taken preputial skin graft or buccal mucosa graft. Grafted tissues are thick and usually well-fixed to the underlying tissue  . Often iAlthough not widely reported, in severe hypospadias a coronal meatus may be cosmetically acceptable. However, most surgeons would at least try to have nice glandular urethra and glanduloplasty. A situation where a midline incision can be applied is when the glandular graft/urethral plate is not wide enough to create a nice glandular urethra. The graft might be bulky or the glans can have a shallow appearance or may be small in size. In our series in 22.9% of all cases a glandular incision was performed. No urethral stricture or meatal stenosis occurred. However, there was one glandular breakdown.In the majority of staged hypospadias repairs there is nice graft take and after 6 months or so the tissues are healthy, well-vascularized and subtle. Urethroplasty is performed easily and the outcome is favorable. However, in our series there are cases where the graft/urethral plate has contracted and primarily is not wide enough for safe urethroplasty. Moreover, we observed the scarred edges of the graft that do not allow wider incision lines. These things happen more in buccal mucosa than in preputial grafts. In buccal mucosa sometimes is bulky, thick, and not easily to be tubularized. Incision of the penile part of the graft is now routinely performed. In our series there had been 1 fistula, 1 breakdown, and 1 case of lichen sclerosus of the entire urethroplasty. Again, we did not encounter any stricture or problem of longitudinal scarring which might be expected following midline incision of the graft in the first place.To avoid meatal stenosis in the first stage the urethra is incised ventrally. However, in some cases there is scarring or fibrosis where the graft meets the original urethral tissue resulting in a tight urethral opening, sometimes so tight that an 8 Fr. catheter could not be inserted. This problem was addressed by a deep midline incision. In our series we could not identify any problem related to such an incision. There was no stricture recorded at the level proximal anastomosis. There was one distal fistula which can hardly be related to a very proximal incision.Even in the largest series worldwide our modification of incising the graft was not used or not reported [Shukla et al. 700 cases , Bracka This is a single center retrospective short to mid-term study with a limited number of patients and longterm results are still pending. There is no control group and the technique of incising a graft has not been applied in a randomized setting but by surgeon's subjective judgment. Therefore, many facts remain unclear: for example: is the complication rate influenced by the incision or has the incision been applied in more difficult cases? Moreover, we do not routinely perform cystoscopy in hypospadias patients, therefore endoscopic findings following midline incision can not be reported.Two stage repair is the method of choice in the correction of severe hypospadias. In selected cases a midline incision of the graft is feasible and can be applied if needed. Randomized studies will be needed to evaluate the true benefit of incising the graft.The datasets generated for this study are available on request to the corresponding author.There is Medical University Vienna ethical committee approval for this study - number 2265/2017.AS, UT, MH, MM, and DH contributed to study concept, design, and drafting of the manuscript. AS, UT, and DH contributed to acquisition of data. AS, UT, MH, and MM contributed to analysis and interpretation of data. AS contributed to critical revision of the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently we comment in International Brazilian Journal of Urology a important paper about the use of penile skin fap in anterior penile urethral stricture , 2. In t


Lithium disilicate rods (n = 50) were surface etched by hydrofluoric acid (HF). Five different dual cure resin cements were used for cementing rods to bovine dentin. Ten rods of each test group were cemented with each cement. Test specimens were thermocycled before tensile bond strength testing. Fracture morphology was visualized by light microscope. Mean surface roughness  was calculated for randomly selected rods. Cohesive fracture in cement was the most frequent observed fracture morphology. Combination of adhesive and cohesive fractures were second most common. Fracture characterized as an adhesive between rod and cement was not observed for KHF2 etched zirconia. Highest mean tensile bond strength was observed when cementing air borne particle abraded zirconia with Variolink Esthetic (Ivoclar Vivadent). All surface treatments resulted in Sa values that were significant different from each other. The number of cohesive cement fractures observed suggested that the cement was the weakest link in bonding of zirconia and lithium disilicate.To evaluate debonding mechanism of zirconia and lithium disilicate cemented to dentin mimicking what could occur in a clinical setting. A null hypothesis of no difference in tensile bond strength between groups of zirconia and lithium disilicate cemented with resin cements was also tested. Zirconia rods ( Zirconia has become one of the most used ceramic in prosthetic dentistry the last decades .The material has a high flexural strength  due to iDespite excellent mechanical properties of zirconia there are complications related to clinical use. Loss of retention of tooth supported crowns is reported as one of the most frequent technical complication. Many approaches have been studied with the aim to increase bond strength between resin cement and zirconia . TribochAir borne particle abrasion using particles of aluminum oxide, diamond or boron nitride is the most used surface treatment . This teHigh crystallinity of zirconia and lack of glass phase makes the material resistant to etching by hydrofluoric acid (HF). This is in contrast to lithium disilicate, where etching by HF establish micromechanical and chemical bond to silanoles and resin cement .An alternative method of surface etching of zirconia was studied by Ruyter et al. . High shWhen cementing ceramic restorations using resin cement, the tooth substance is often pretreated by acidic etch and adhesive components, either as multiple or single step. The pretreatment creates mechanical interlocking and chemical bond between tooth substance and adhesive .In a clinical setting, loosening of the restoration may occur in the weakest part, which represents the bond strength. This could be the cement – restoration bond, in the cement – tooth structure bond or in the cement itself.The aim of the present study was to evaluate debonding mechanism of zirconia and lithium disilicate cemented to dentin mimicking what could occur in a clinical setting, and to test the null hypothesis that no difference in tensile bond strength between groups of zirconia and lithium disilicate cemented with resin cements would be found.n = 150) were extracted , cut 2 cm length and embedded in epoxy resin  with buccal surface exposed. Embedded teeth were ground at DP-U2 with rotating 500-grit silicon carbide paper  under water until 5 × 5 mm dentin surface was obtained and further stored in distilled water.Bovine mandibular incisors  and lithium disilicate  rods with diameter of 5 mm and length of 11.5 mm were produced by CAD/CAM technique. Rods were produced with a notch in the circumference , lithium disilicate rods (n = 50) formed one group. The groups were:2O3, Korox)Zir-A: zirconia, air borne particle abraded, 50 µm aluminum oxide (Al2)Zir-E: zirconia, etched by potassium hydrogen difluoride (KHFLDS: lithium disilicate etched by 4.5% hydrofluoric acid (HF)Zirconia rods were randomly assigned to two different surface treatment groups (n = 10 of each group with each cement).Five resin based cements were used for cementation of the rods  for 10 min at 280 °C for the KHF2 to melt. After cooling, rods were thoroughly steam cleaned and ultrasonically cleaned in distilled water for 15 min. Finally, they were air-dried.KHFZirconia rods were air borne particle abraded at 2.5 bar for 10 s. The nozzle was kept perpendicular to the zirconia surface at 10 mm distance. Rods were air steamed and ultrasonically cleaned in distilled water for 15 min before thoroughly air-dried.The bonding surface of lithium disilicate glass ceramic rods were etched with hydrofluoric acid  for 20 s, cleaned by running water >20 s and thoroughly air-dried.The surface on randomly selected rods was studies in scanning electron microscope , with energy dispersive spectroscopy, EDS. Surface roughness was measured using a confocal microscope (Sensofar S neox). Mean surface roughness  was calculated for randomly selected rods .Five different dual cure resin cements were used for cementing rods to bovine dentin; Multilink Automix (Ivoclar Vivadent), Variolink Esthetic (Ivoclar Vivadent), Panavia F2.0 , Duo-Link (Bisco) RelyX Unicem (3 M ESPE) .Cementation was performed according to producers’ manual and primer was applied when recommended .2 etched zirconia, air borne particle abraded zirconia and HF etched lithium disilicate, were cemented by each cement.Ten rods from each of the three groups; KHFDentine was cleaned using pumice powder dispensed in water prior to cementation.After placing the rods onto dentin, a standardized 882 g seating load was applied by a cementation jig. Excess cement was removed using quick stick micro-brush before light curing 20 s each from 4 directions.All specimens were kept dry at room temperature for 15 min following cementation and thereafter immersed in 37°C distilled water for 24 h.2O3 to remove cement remnant outside the rods and evaluated by light microscopy. Test units were thermocycled 5000 cycles in 5 °C and 55 °C water baths.Specimens were sandblasted using AlSpecimens were mounted in a universal mechanical test machine . Tensile force was applied until break using a centered wire with a cross head speed of 1 mm/min. Rods and dentin were studied in light microscope  for visualizing fracture morphology.Fractures were classified in to 5 different types: (1) adhesive failure between cement and rod, (2) adhesive failure between cement and dentin, (3) cohesive in cement (4) cohesive failure in dentin, (5) combination of adhesive and cohesive failure.p < .05 was regarded as statistical significant different.Microsoft Excel (version 14.2.3) was used for calculating mean tensile bond strength and standard deviation. Komogorov–Smirnov was used for calculation of normality and differences among groups were evaluated using ANOVA tests followed by Tukey’s HSD test. Evaluations were done (1) among rod materials for each cement and (2) among cements for each rod material. 2 etched zirconia.Cohesive fracture in cement was the most common fracture morphology visualized by light microscope, as presented in Adhesive fracture between cement and dentin and cohesive fracture in dentin was observed for Multilink Automix, Variolink Esthetic and RelyX Unicem. This was also the cements with the highest tensile bond strength, as shown in Mean tensile bond strength and standard deviation for the three test groups cemented with different dual cure resin cements are illustrated in Sa value after surface treatment of randomly selected rods were measured using a confocal microscope. As presented in The aim of the present study was to evaluate debonding mechanism of zirconia and lithium disilicate cemented to dentin to mimic what can occur in a clinical setting. A null hypothesis of no difference in tensile bond strength between groups of zirconia and lithium disilicate cemented with resin cements was also tested.2 etched and air borne particle abraded) and lithium disilicate (HF etched).Five dual cure resin cements were used for cementing rods of zirconia  [In a clinical setting loosening of restorations might be because of debonding between cement and dentin, between cement and ceramic or cohesive fracture in cement. Main focus of previous studies on zirconia has been on increasing bond strength between the ceramic and resin cement . Ruyter ps (OH−) . Adhesiv1 value is 2.2 [The 10-MDP containing cement, Panavia F2.0, had the lowest tensile bond strength and was the cement with the highest number of cohesive fractures. The low value is in contrast to the previous study on 10-MDP containing cement . 10-MDP e is 2.2 . Primarye is 2.2 .Multilink Automix was the only cement showing adhesive debonding to lithium disilicate. This was somewhat unexpected finding. Lithium disilicate etched with hydrofluoric acid and primed with silane containing primer is known for establishing micromechanical and chemical bond between resin cement and ceramic . AdhesivCombination of adhesive and cohesive fractures in dentin were also common for the three cements with the highest tensile bond strength, this specially applies for zirconia rods regardless of surface treatment. The bonding seemed to be nearly as strong as the inherent strength of the dentin, which must be regarded as the maximum bond strength.All cements had cohesive fractures to different degree. These observations indicate that cements have different cohesive bond strength that will affect the retention of adhesive cemented restorations.A thin and uniform cement layer is recommended to reduce shrinkage stresses during polymerization and loading failure of the ceramic . HoweverCements used today are mainly in the form of automix to ensure equal amount and even mix of components in the cement system. In the present study one cement, Panavia F2.0, was mixed by hand as recommended by the manufacturer. Panavia F2.0 showed the lowest mean tensile bond strength for all three ceramics. The mixing procedure could result in non-homogenous cement and contributed to the weak bond strength.Micro roughness in the bonding surface of zirconia and lithium disilicate is necessary to establish good bond to resin cement . In thisTo obtain a clinical perspective in the present study, dentin was chosen as substrate for which zirconia and lithium disilicate were cemented to. Individual differences in dentin are detected in several studies  and can The number of cohesive cement fractures observed in the present study suggested that the cement was the weakest link in bonding of ceramics. The null hypothesis of no difference in tensile bond strength between groups of zirconia and lithium disilicate cemented with resin cements was rejected for some combinations.


Deteriorated arterial function and high incidence of cardiovascular events characterise diabetes mellitus. Metformin and recent antidiabetic drugs, SGLT2 inhibitors, reduce cardiovascular events. We explored the possible effects of empagliflozin’s effect on top of metformin treatment on endothelial function and arterial stiffness parameters in type 1 diabetes mellitus (T1DM) patients.Forty T1DM patients were randomised into three treatment groups: (1) empagliflozin (25 mg daily), (2) metformin (2000 mg daily) and (3) empagliflozin/metformin . The fourth group received placebo. Arterial function was assessed at inclusion and after 12 weeks treatment by: endothelial function , arterial stiffness [pulse wave velocity (PWV) and common carotid artery stiffness (β-stiffness)]. For statistical analysis one-way analysis of variance with Bonferroni post-test was used.Empagliflozin on top of metformin treatment significantly improved endothelial function as did metformin after 12 weeks of treatment: FMD [2.6-fold (P < 0.001) vs. 1.8-fold (P < 0.05)] and RHI [1.4-fold (P < 0.01) vs. 1.3-fold (P < 0.05)]. Empagliflozin on top of metformin treatment was superior to metformin in improving arterial stiffness parameters; it significantly improved PWV and β-stiffness compared to metformin . Metformin alone did not influence arterial stiffness.Empagliflozin on top of metformin treatment significantly improved arterial stiffness compared to metformin in T1DM patients. Endothelial function was similarly improved in all treatment groups. Empagliflozin seems to possess a specific capacity to decrease arterial stiffness, which could support its cardioprotective effects observed in large clinical studies.Trial registration Clinical trial registration: NCT03639545 Diabetes mellitus is a systemic, chronic metabolic disease that affects all organs of the body. Of particular importance is the effect of diabetes on the cardiovascular system, where it makes the arteries prone to microvascular as well as macrovascular complications. It is well-known that cardiovascular diseases represent the most common factor for diabetes mellitus-related morbidity and mortality . The mecIn recent years, the treatment goals of diabetes patients have been broadened from merely glycaemia control to cardiovascular protection. The newest drugs in the antihyperglycemic group are sodium glucose co-transporter type 2 (SGLT2) inhibitors, which are currently registered only for treatment of type 2 diabetes mellitus (T2DM) patients. Basically, these drugs increase glucose excretion through the urine and consequently decrease blood glucose values through the inhibition of SGLT2 channels in the kidneys . During The beneficial effects of SGLT2 inhibitors on arterial function have been established in prior studies –13. Howehttp://clinicaltrials.gov (NCT03639545).In this 12-week, prospective, double-blind randomised clinical study forty male T1DM patients from Diabetes outpatient clinic Ljubljana were recruited. All patients were treated with insulin, either through intensive insulin therapy or insulin pump therapy. The patients were equally randomised into three treatment groups, that received the treatment in addition to insulin. The groups were as follows: (1) empagliflozin group (receiving 25 mg daily), (2) metformin group (receiving 2000 mg daily) and (3) empagliflozin/metformin group (receiving empagliflozin 25 mg daily and metformin 2000 mg daily). The fourth group received placebo. The duration of the study period was 12 weeks. All subjects voluntarily participated in this study and signed an informed consent. The study was approved by the National Medical Ethics Committee of Slovenia. The study was registered at 2) or liver failure, benign prostatic hyperplasia, prostatic carcinoma, frequent urinary tract infections or body mass index below 23 kg/m2.For inclusion in the study, patients needed to have a confirmed T1DM diagnosis and be between 30 and 65 years of age. Exclusion criteria included diagnosed advanced heart failure , kidney  measurements of endothelial function ; and (ii) measurements of arterial stiffness . Additionally, venous blood samples were obtained at the beginning and at the end of the study. Glycated haemoglobin (HbA1c) was assessed using the VITRO 5.1FS Chemistry System . An automated sphygmomanometer was used for blood pressure measurements. Arterial function assessment was performed in standardised conditions, in a quiet and temperature-controlled environment. All the patients were subject to prior 8 h fasting . Alcohol and caffeine abstinence were also required. Each of the measurements were performed at the same time and by the same examiner, both at the beginning and the end of study. Ultrasound measurements were obtained using an Aloka ProSound α7 machine with an integrated high resolution eTracking system. Reactive hyperaemia index was measured using an Endopat 2000 device , while cfPWV was obtained using a SphygmoCor device  with SphygmoCor CvMS software (version 9). Continuous electrocardiogram recording was also performed during all measurements.Endothelial function was assessed in accordance with current guidelines  through Plethysmography measurement of finger arterial pulse wave amplitude was measured using an Endopat 2000 device . PatientBoth measurements were performed on the right common carotid artery. Patients laid in a supine position, their head additionally elevated by 45º and tilted to the side by 30º. Using the device’s software, stiffness was measured through analysis of the pulse waves obtained. The tracker pair was fixed at the anterior and posterior wall of the common carotid artery. The machine obtained the pressure waveforms from the changing arterial diameters that were calibrated based on systolic and diastolic blood pressure. Both cPWV and β-stiffness were then calculated automatically as a mean of 12 beats.A Sphygmocor with a CvMS software device was used in accordance to the Consensus document on arterial stiffness for measurement of cfPWV . The latThe values were expressed as mean ± SEM. Their differences were assessed by one-way analysis of variance (ANOVA). For significant interactions, the Bonferroni post-test was performed. A P-value of less than 0.05 was considered statistically significant. Statistical analyses were processed using the GraphPad Prism 5.0 software. Due to a small number of participants, eligible arterial stiffness parameters, i.e. cPWV and cfPWV, were combined into one (PWV), thus increasing the results’ value.Patient characteristics are presented in Table Brachial artery FMD significantly improved after 12-weeks treatment with empagliflozin , metformin  and with empagliflozin/metformin  compared to basal values in separate groups. On the other hand, there were no significant differences in FMD improvements between the treatment groups  significantly improved with empagliflozin , metformin  and empagliflozin/metformin combination . There were no significant differences in RHI improvements between the treatment groups   empagliflozin and metformin have similar effects on endothelial function and divergent effects on arterial stiffness parameters, and (ii) empagliflozin seems to have a unique capacity  to improve arterial stiffness.This study explored the effects of empagliflozin alone and its effects on top of metformin on structural and functional arterial properties. Metformin, as an optimal comparator, was chosen due to its previously proven beneficial effects on arterial function and reduction of cardiovascular events –19. T1DMEmpagliflozin/metformin also significantly decreased HbA1c values, proving their synergistic effects on glycaemia control, whereas separate agents did not significantly affect HbA1c.The translation of these results to clinical practice allows for interesting interpretations. The majority of T2DM patients are treated with metformin and addition of SGLT2 inhibitors is one of therapeutic options. Based on our results, metformin alone significantly improves endothelial function and the addition of empagliflozin would not result in significant benefits. Therefore, the cardioprotective effects of empagliflozin cannot be attributed solely to endothelial function improvement. In contrast, adding empagliflozin to metformin therapy would importantly improve arterial stiffness. The degree of expected decrease of arterial stiffness (14% to 18%) would result in an important predicted reduction of cardiovascular risk in the range of 10–20% . InteresSeveral prior studies examined the effects of SGLT2 inhibitors on arterial characteristics. Empagliflozin 25 mg daily significantly decreased systolic blood pressure and several arterial stiffness parameters after 8 weeks of treatment in young T1DM patients, i.e. augmentation indexes at the radial, carotid and aortic positions, carotid-radial PWV . In anotThe present study is the first of its kind to explore the effect of empagliflozin on arterial function by extensively studying numerous vascular parameters and using a comparator. We observed an improvement in macrovascular endothelial function, which was also found after acute treatment with dapagliflozin in prior studies . The autOur study is the first to reveal that empagliflozin, even on top of metformin, seems to have a unique capacity to improve arterial stiffness that is not shared by metformin and seems not to be related to improvement of endothelial function. Consequently, it could be deduced that the improvement of arterial stiffness by empagliflozin is not associated to nitric oxide, inflammation and oxidative stress, which are all well-known factors involved in endothelial function improvement . Taken aEmpagliflozin on top of meformin treatment significantly improved arterial stiffness compared to metformin in T1DM patients. Endothelial function was similarly improved in all treatment groups. Arterial stiffness improvement by empagliflozin was substantial and in ranges that could be of clinical relevance. The effect was independent from endothelial function improvement, suggesting the specific, unique capacity of empagliflozin to improve arterial stiffness. That could support its cardioprotective effects and that of other SGLT2 inhibitors observed in large clinical studies.


While capillary forces are negligible relative to gravity at the macroscale, they provide adequate force to effectively manipulate millimeter to micro meter objects. The fluidic actuation can be accomplished using droplets that also act as bearings. While rotary droplet bearings have been previously demonstrated, this paper addresses the positioning accuracy of a droplet-based bearing consisting of a droplet between a moving plate and a stationary substrate with constrained wetting region under a normal load. Key wetting cases are analyzed using both closed form analytical approximations and numerical simulations. The vertical force and stiffness characteristics are analyzed in relation to the wetting boundaries of the supporting surface. Case studies of different wetting boundaries are presented and summarized. Design strategies are presented for maximizing load carrying capability and stiffness. These results show that controlled wetting and opposing droplet configurations can create much higher stiffness fluidic bearings than simple droplets. Due to scaling relations, capillary force becomes dominant as the length scale decreases below the millimeter scale and can dominate from micrometer to millimeter scales . The relA new class of bearing based on the capillary bridge was proposed by several groups and rotating micromachines were demonstrated in references ,12,13,14The same principle could be further extended to use droplets to support vertical loads in linear translational motion. With electrowetting actuation, the droplets can serve as both bearing and actuator. Moon and Kim demonstrated this concept by using electrowetting to move droplets which carried a solid platform for biochemical analysis . In theiFor linear bearings, the stiffness and load carrying capacity are critical parameters. The vertical stiffness determines the precision of the bearing in the transverse direction under varying loads. For rotational bearings, it was noted in reference  that theγ is the surface tension, ρ the density difference between the droplet and the ambient, and g the acceleration due to gravity), the effect of gravity on the fluid can be neglected. For water in an air ambient, the capillary length is approximately 2.7 mm.In its simplest form, a droplet-based bearing consists of a capillary bridge formed between two parallel surfaces. The interfacial tensions act at the triple point where the liquid, solid, and ambient fluid meet. The angle between the liquid and solid is the contact angle. Lambert and co-workers have shown that both the Laplace approach and interfacial energy approach are equivalent . In hereh) is known. The forces acting on the top plate are the surface tension force and the pressure force. The vertical component of the surface tension (TF) acting on the top plate is found by integrating around the interface. For the case of a circular droplet with constant wetting angle:γ is the interfacial tension of the liquid-ambient, topR is the radius of the contact area and θ is the contact angle of the liquid and top plate. The pressure force, which originates from the pressure jump across the liquid/ambient interface can be calculated using the Laplace pressure equation:R1 and R2 are the principle radii of the curved interface. Giving the separation distance (h) between the plates, one principle radius can be found by geometry:θ and R2 >> h), the second principle radius can be approximated by the radius of the plate contact (R2 = topR). The top radius is used as it will be assumed to be prescribed while the other may vary with load. Then the normal force (NF) on the top plate is as follows:As shown in D = 2topR) to get the following:The equation is nondimensionalized by normalizing the height  [Once the equilibrium shape of the surface was found, a small displacement in the z direction 50 µm) was applied and the energy change was calculated. The normal force was found using the principle of virtual work (= dE/dz) ,21. Resu0 µm was An effective fluid bearing must be able to support the normal loads applied and should also have a high stiffness to minimize the displacement caused by changes in applied forces. While the force and stiffness are linearly dependent on surface tension, the droplet diameter, the substrate/plate gap, and contact line constraints will nonlinearly impact the performance of the fluid bearing. This paper considers the impact of the droplet aspect ratio (gap height/diameter) for three different wetting arrangements in a simple fluid bearing for different contact line constraint conditions.θ = Φ), Equation . The remainder of the top surface and the entire bottom were non-wetting (contact angle = 165°). Droplet volume was fixed at 50 µL. The vertical forces were extracted and then normalized by the top contact diameter and compared to Type 1 performance for various droplet heights. The result is presented in In a practical bearing, the droplet contact line must be constrained relative to one of the plates while sliding across the other. This could be done by defining a specific wetting region on one plate while the other plate remains hydrophobic—breaking the symmetry of the droplet wetting. This non-symmetric wetting was simulated in Surface Evolver using a hydrophilic circular region on the top (moving) plate (contact angle = 10°) with different wetting radius , the force drops dramatically relative to the Type 1 case as the bottom plate contact area decreases while the top contact line remains stationary. While the lower normal force in this region is undesirable, the increased stiffness in the transition region is favorable for reducing variation in plate positioning with applied normal forces. For a given wetting area, this also allows the bearing to operate at a higher gap height with favorable stiffness. Larger gaps and smaller aspect ratios should reduce the drag introduced by the fluids in a linear actuator.Physical constraints can also be added to the edge of the wetting region to reliably constrain the wetting boundary even beyond 180° . This could be accomplished using a protruding surface as illustrated in h/D) to at least 0.25. This would be the most favorable design for droplet based bearings. High stiffness and large force could be achieved with larger gap height when the contact line is constrained by both geometry and wetting. As discussed earlier, the viscous drag introduced by the droplet could impose a speed limit on the bearing. By operating at larger height, the drag would be reduced and the maximum speed of the bearing could be increased.The Type 3 design can increase the working range of the bearing from 0.1 aspect ratio .This analysis provides insight into droplet and wetting arrangements that will provide the most accurate fluidic bearing in electrowetting and other applications. To achieve precise translational motion in the For a typical application the size and weight of the plate being carried is fixed. Equation  indicateSome case studies are used to demonstrate the tradeoffs when designing such bearing. All the calculations are based on Equation  Althoug does notIn this first case study , the nor3) with dimension of 10 × 10 × 0.5 mm, the minimal force needed to support the weight of the plate is 1078 µN. Assuming an air ambient and water droplets with surface tension 0.072 N/m, and 110° contact angle on a Cytop coating. At 1 mm gap height, a single 6.8 mm diameter droplet would provide sufficient force to support the plate. However, due to stability issues mentioned above, multiple droplets are preferred. h/D < 0.3, CA = 110°). As the droplets are parallel springs, the total stiffness is the number of droplets multiplied by the stiffness of a single droplet.For a square glass plate  as above. Instead of fixing the gap height, the bearings are designed for a maximum displacement of the top plate of ±10 µm at 150% overloading. This can be translated to a design requirement of fixed stiffness . In the prior cases, the preload is applied by gravity which makes the plate position sensitive to orientation. Alternatively, we propose a symmetrical droplet bearing which uses opposing droplets to apply a preload to the plate, the resulting force/stiffness could be symmetrical around the middle point which could further increase the reliability of the device—either orientation around the x—y plane is possible. The simplified case with no wetting contrast (Type 1) is analyzed below .D’) times πγ and the change in height with respect to the middle point (dz) was divided by the equilibrium diameter (D’). The results are presented in Toph = Bottomh). The droplet volume was 50 µL each.To illustrate the force-displacement relation, calculations based on Equation  were perz-direction could be fine-tuned by using either the volume of the droplet, the wetting pattern or the gap height, to change the preload on the bearing and further improve stiffness as seen in As seen in The normal force of a capillary bridge between two parallel surfaces was analyzed using both Laplace-based calculation and simulation. The force-deflection response of three wetting conditions—uniform wetting, defined wetting region and constrained top-wetting were analyzed using numerical simulation and compared to a simplified closed form approximation. This approximation is shown to provide useful reference information for predicting key aspects of each wetting type. For all cases, smaller gaps create higher stiffness and larger forces. The peak force and stiffness are achieved with geometrical constraints on the top plate that enable effective contact angles above 180° at the edge of the wetting boundary.For ultimate precision, an opposing droplet configuration was proposed. Simplified force analysis showed a symmetrical stiffness response due to the preloading effect. The stiffness could be further fine-tuned by changing the total gap height. For linear droplet bearing actuated by electrowetting, a series of design cases were presented to demonstrate the advantage of using multiple droplets to support the same load. The larger actuation force capability of multiple droplets could improve the speed of the actuator. When designing such actuators, the total area and the gap height should be the driving parameters to meet required load and stiffness performance levels.