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Metabolic changes in an organism often occur much earlier than macroscopic manifestations of disease, such as invasive tumors. Therefore, noninvasive tools to monitor metabolism are fundamental as they provide insights into in vivo biochemistry. NMR represents one of the gold standards for such insights by observing metabolites. Using nuclear spin hyperpolarization greatly increases the NMR sensitivity, enabling μmol/L sensitivity with a time resolution of about one second. However, a metabolic phantom with reproducible, rapid, and human-like metabolism is needed to progress research in this area. Using baker's yeast as a convenient metabolic factory, we demonstrated in a single study that yeast cells provide a robust and rapidly metabolizing phantom for pyruvate and fumarate, including substrates with a natural abundance of 13 C: we observed the production of ethanol, carbon dioxide, bicarbonate, lactate, alanine from pyruvate, malate, and oxaloacetate from fumarate. For observation, we hyperpolarized pyruvate and fumarate via the dissolution dynamic nuclear polarization (dDNP) technique to about 30% 13 C polarization that is equivalent to 360,000 signal enhancement at 1 T and 310 K. Major metabolic pathways were observed using tracers at a natural abundance of 13 C, demonstrating that isotope labeling is not always essential in vitro. Enriched [1- 13 C]pyruvate revealed minor lactate production, presumably via the D-lactate dehydrogenase (DLD) enzyme pathway, demonstrating the sensitivity gain using a dense yeast solution. We foresee that yeast as a metabolic factory can find application as an abundant MRI phantom standard to calibrate and optimize molecular MRI protocols. Our study highlights the potential of using hyperpolarization to probe the metabolism of yeast and other microorganisms even with naturally abundant substrates, offering valuable insights into their response to various stimuli such as drugs, treatment, nourishment, and genetic modification, thereby advancing drug development and our understanding of biochemical processes.

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Dissolution dynamic nuclear polarization (dDNP) increases the sensitivity of magnetic resonance imaging by more than 10,000 times, enabling in vivo metabolic imaging to be performed noninvasively in real time. Here, we are developing a group of dDNP polarized tracers based on nicotinamide (NAM). We synthesized 1- 15 N-NAM and 1- 15 N nicotinic acid and hyperpolarized them with dDNP, reaching (13.0 ± 1.9)% 15 N polarization. We found that the lifetime of hyperpolarized 1- 15 N-NAM is strongly field- and pH-dependent, with T 1 being as long as 41 s at a pH of 12 and 1 T while as short as a few seconds at neutral pH and fields below 1 T. The remarkably short 1- 15 N lifetime at low magnetic fields and neutral pH drove us to establish a unique pH neutralization procedure. Using 15 N dDNP and an inexpensive rodent imaging probe designed in-house, we acquired a 15 N MRI of 1- 15 N-NAM (previously hyperpolarized for more than an hour) in less than 1 s.

Clinical Issue: Despite being one of the main pillars of modern diagnostics, magnetic resonance imaging (MRI) uses only a tiny fraction of its potential: no more than a millionth of all nuclear spins contribute to the MRI signal. In order to increase this fraction, called polarization, MRI scanners with stronger magnetic fields are being developed. However, even the most modern scanners do not exploit the potential of MRI., Methodological Innovations: To make full use of this potential, hyperpolarized MRI (HP-MRI) is an excellent tool: quantum mechanical tricks can be used to generate contrast agents whose nuclear spins can deliver a MRI signal that is up to a 100,000 times stronger. This signal enhancement allows imaging of in vivo processes that would be otherwise impossible to measure. It is particularly interesting to introduce these magnetically labeled nuclei into metabolic processes so that the metabolism can be investigated non-invasively and in vivo., Performance: Small but diagnostically important changes in metabolism could be found before macroscopic tissue changes were otherwise visible. High-resolution images can be acquired within a few 100 ms, enabling metabolic monitoring in real-time. Heart, brain, and prostate are among the organs that have already been investigated in over 90 clinical trials using this emerging technology., Achievements: So far, displaying tissue in a similar manner was only possible using nuclear medicine, e.g., positron emission tomography (PET) utilizing radionuclides and without resolution of various metabolic steps. A change in tumor metabolism following treatment was shown within hours in HP-MRI. These applications coupled with background information about the technology are the subject of this review., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

There are over 29,000 children and young people (CYP) with Type 1 diabetes mellitus (T1DM) in England and Wales and another 726 with Type 2 diabetes mellitus (T2DM). There is little effect of deprivation on the prevalence of T1DM whereas the association of deprivation on the percentage of CYP with T2DM is striking with 45% of cases drawn from the most deprived backgrounds. A number that has not changed over the last 4 years. Data from the UK and USA as well as other countries demonstrate the impact of deprivation on outcomes in diabetes mellitus with clear effects on measures of long-term control and complications. In the UK black CYP had higher glycosylated haemoglobin (HbA1c) values compared to other groups. Within the black group, CYP from a Caribbean background had a higher mean HbA1c (77.0 mmol/mol (9.2%)) than those from Africa (70.4 mmol/mol (8.6%)). Treatment regimen (multiple daily injections or insulin pump therapy) explained the largest proportion of the variability in HbA1c followed by deprivation. Those in the least deprived areas had an average HbA1c 5.88 mmol/mol (0.5%) lower than those living in the most deprived areas. The picture is complex as UK data also show that deprivation and ethnicity is associated with less use of technology that is likely to improve diabetes control. Increased usage of pump therapy and continuous glucose monitoring was associated with a younger age of patient (less than 10 years of age), living in the least deprived areas and white ethnicity. This gap between pump usage amongst CYP with T1DM living in the most and least deprived areas has widened with time. In 2014/15 the gap was 7.9% and by 2018/19 had increased to 13.5%. To attain an equitable service for CYP with diabetes mellitus we need to consider interventions at the patient, health care professional, community, and health care system levels., Competing Interests: CJP, RV and PCH have no conflicts of interest to declare and the review was undertaken with no supporting funding source., (© 2021 The Authors.)

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A-tracts are sequences of repeated adenine bases that, under the proper conditions, are capable of mediating DNA curvature. A-tracts occur naturally in the regulatory regions of many organisms, yet their biological functions are not fully understood. Orienting multiple A-tracts together constructively or destructively in a phase has the potential to create different shapes in the DNA helix axis. One means of detecting these molecular shape differences is from altered DNA mobilities measured using electrophoresis. The small molecule netropsin binds the minor groove of DNA, particularly at AT-rich sequences including A-tracts. Here, we systematically test the hypothesis that netropsin binding eliminates the curvature of A-tracts by measuring the electrophoretic mobilities of seven 98-base pair DNA samples containing different numbers and arrangements of centrally located A-tracts under varying conditions with netropsin. We find that netropsin binding eliminates the mobility difference between the DNA fragments with different A-tract arrangements in a concentration-dependent manner. This work provides evidence for the straightening of A-tracts upon netropsin binding and illustrates an artificial approach to re-sculpt DNA shape.

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant., Competing Interests: None.

Background: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients., Methods: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis., Results: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054)., Conclusions: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.

The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are 'captured' by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to ∼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to ∼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a-DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 μs-1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: To evaluate, in rats, the open field videothermometry in real time while performing left pneumonectomy for early diagnosis of cardiopulmonary changes., Methods: Twelve non-specific pathogen-free Wistar rats were randomly allocated into two groups; pneumectomy group (GP) and sham surgery group (GS). Mean arterial pressure, videothermometry in real time, of the right lung, and histopathological analysis of the remaining lung were evaluated in all animals., Results: Videothermometry in real time allowed identification of temperature variance of right lung after pneumectomy, indicating a significant decrease in temperature during evaluation. There was a statistical difference between M0 and M1, M1 and M2 and M0 and M2 (p<0.004) in GS, and significant difference between M0 and M1, M1 and M2, and M2 and M0 with p<0.0001 in GP., Conclusions: Left pneumonectomy in rats shows initial histopathological changes after 60 minutes of its completion, indicating a possible compensation beginning. The open-field videothermometry in real time proved to be efficient identifying the temperature changes of the remaining lung.

Purpose: Functional endoscopic sinus surgery (FESS) is considered standard surgical therapy for fungus ball of the maxillary sinus. However, recent findings have indicated an odontogenic etiology, which requires simultaneous treatment of the dental origin. This study presents the authors' treatment results of fungus ball of the maxillary sinus using a combination of FESS and an endoscopically assisted osteoplastic approach through the anterior wall of the maxillary sinus, enabling simultaneous treatment of the dental origin., Materials and Methods: A cohort of 22 patients with histopathologically confirmed fungus ball of the maxillary sinus was retrospectively analyzed. Clinical records and medical imaging data were reviewed to evaluate the etiology, clinical and radiologic findings, and postoperative outcome., Results: Only 15 patients presented nonspecific clinical symptoms compatible with chronic unilateral maxillary sinusitis. Computed tomography visualized complete opacity of the maxillary sinus in 11 patients and intralesional hyperdensity in 12 patients. An odontogenic association was verified in 18 patients. Twenty-one patients underwent endoscopically assisted osteoplastic surgery through the anterior maxillary sinus wall. In 12 cases, the assumed persistent odontogenic source was treated simultaneously. Depending on the patency of the ostiomeatal complex, the accompanying chronic sinusitis was treated by FESS., Conclusions: The present data support the assumption of an odontogenic etiology of fungus ball of the maxillary sinus. Hence, surgical management requires simultaneous treatment of the fungal mass, the odontogenic origin of the disease, and the accompanying chronic sinusitis. To properly treat fungus ball, the authors present a modern treatment concept, using a minimally invasive endoscopically assisted osteoplastic approach through the anterior maxillary wall, for sufficient and necessary surgical treatment., (Copyright © 2018 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from 'electrostatic' or 'base stacking' influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I⋅C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I⋅C and two A⋅T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1'-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2'-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I⋅C pairs has a geometry similar to that of the reference duplex with eight G⋅C and two A⋅T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.

Purpose: To investigate the safety and clinical, hemodynamic and tissue improvement ability in mini pigs undergoing cell and gene therapy for the treatment of acute myocardial infarction., Methods: Thirty-two mini pigs Br1 lineage, 12 months old, undergoing induction of acute myocardial infarction by occlusion of the diagonal branch of the paraconal coronary. They were divided into 4 groups: one control group and 3 treatment groups (cell therapy and gene cell therapy). Echocardiography reviews were performed on three occasions and histopathological analysis was performed after 4 weeks. Analysis of variance (ANOVA), Tukey and Wilcoxon tests, were performed., Results: Association of vascular endothelial growth factor (VEGF) with angiopoietin1 (Ang1) presented satisfactory results in the improvement of ventricular function following ischemic cardiomyopathy in mini pigs when compared to the results of the other treated groups., Conclusion: The therapy with VEGF and the combination of VEGF with Ang1, promoted recovered function of the myocardium, characterized by reduced akinetic area and induction of neovascularization.

Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler's murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo , as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination.

Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here, we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Purpose: To perform technically the laparoscopic sleeve gastrectomy (LSG) using a unique Intragastric Single Port (IGSG) in animal swine model, evidencing an effective and safe procedure, optimizing the conventional technique., Methods: IGSG was performed in 4 minipigs, using a percutaneous intragastric single port located in the pre-pyloric region. The gastric stapling of the greater curvature started from the pre-pyloric region towards the angle of His by Endo GIA™ system and the specimen was removed through the single port. In the postoperative day 30, the animals were sacrificed and submitted to autopsy., Results: All procedures were performed without conversion, and all survived 30 days. The mean operative time was 42 min. During the perioperative period no complications were observed during invagination and stapling. No postoperative complications occurred. Post-mortem examination showed no leaks or infectious complications., Conclusion: Intragastric Single Port is a feasible procedure that may be a suitable alternative technique of sleeve gastrectomy for the treatment of morbid obesity.

Purpose: To investigate the efficacy of allogeneic mesenchymal stem-cells and autologous mononuclear cells to promote sensorimotor recovery and tissue rescue., Methods: Female rabbits were submitted to the epidural balloon inflation method and the intravenous cells administrations were made after 8 hours or seven days after injury induction. Sensorimotor evaluation of the hindlimbs was performed, and the euthanasia was made thirty days after the treatment. Spinal cords were stained with hematoxylin and eosin., Results: Both therapies given 8 hours after the injury promoted the sensorimotor recovery after a week. Only the group treated after a week with mononuclear cells showed no significant recovery at post-injury day 14. In the days 21 and 28, all treatments promoted significant recovery. Histopathological analysis showed no difference among the experimental groups. Our results showed that both bone marrow-derived cell types promoted significant sensorimotor recovery after injury, and the treatment made at least a week after injury is efficient., Conclusion: The possibilities of therapy with bone marrow-derived cells are large, increasing the therapeutic arsenal for the treatment of spinal cord injury.

Background: Observational studies are the most frequently published studies in literature. When randomized controlled trials cannot be conducted because of ethical or practical considerations, an observational study design is the first choice. The STROBE Statement (STrengthening the Reporting of OBservational studies in Epidemiology) was developed to provide guidance on how to adequately report observational studies., Objectives: The objectives were 1) to evaluate the quality of reporting of observational studies of otorhinolaryngologic literature using the STROBE Statement checklist, 2) to compare the quality of reporting of observational studies in the top 5 Ear, Nose, Throat (ENT) journals versus the top 5 general medical journals and 3) to formulate recommendations to improve adequate reporting of observational research in otorhinolaryngologic literature., Methods: The top 5 general medical journals and top 5 otorhinolaryngologic journals were selected based on their ISI Web of Knowledge impact factors. On August 3rd, 2015, we performed a PubMed search using different filters to retrieve observational articles from these journals. Studies were selected from 2010 to 2014 for the general medical journals and from 2015 for the ENT journals. We assessed all STROBE items to examine how many items were reported adequately for each journal type., Results: The articles in the top 5 general medical journals (n = 11) reported a mean of 69.2% (95% confidence interval (CI): 65.8%-72.7%; median 70.6%), whereas the top 5 ENT journals (n = 29) reported a mean of 51.4% (95% CI: 47.7%-55.0%; median 50.0%). The two journal types reported STROBE items significantly different (p < .001)., Conclusion: Quality of reporting of observational studies in otorhinolaryngologic articles can considerably enhance. The quality of reporting was better in general medical journals compared to ENT journals. To improve the quality of reporting of observational studies, we recommend authors and editors to endorse and actively implement the STROBE Statement., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Asymmetries of the jaw and orthodontic abnormalities are suspected as long-term consequences of positional cranial deformity. But only few data exist on this issue. As plagiocephaly is a common problem in infancy, potential functional impairments should be investigated to initiate appropriate measures if necessary. The aim of our study was to compare the orthodontic situation in primary dentition of children with positional plagiocephaly and children without cranial deformities., Material and Methods: Fifty children treated by helmet therapy for plagiocephaly and 50 non-affected children (age 1.98-5.69 years) were examined in a cross-sectional study. Orthodontic parameters of all dimensions were assessed and analyzed., Results: Children of the plagiocephalic group showed more often orthodontic alterations compared to the others. Especially the frequencies of a class II malocclusion (36 vs. 14 %), an edge-to edge bite (28 vs. 12 %), and deviations of the midline (38 vs. 16 %) were conspicuous. However, none of the differences was significant (p > 0.003). Of all observed mandibular asymmetries, 69 % appeared as a shift to the contralateral side of the former flattened occipital region., Conclusion: Positional head deformity might be associated in some cases with a higher prevalence of occlusal abnormalities in primary dentition., Clinical Relevance: Positional plagiocephaly interfaces medicine and dentistry. As it is a common disorder, this etiology has to be considered in the prevention and therapy of malocclusion.

Objective: To evaluate the effectiveness of type 1 tympanoplasty with one-piece composite cartilage-perichondrium (CCP) grafts compared to temporalis fascia (TF) grafts for tympanic membrane (TM) closure and hearing improvement in adult patients with a subtotal TM perforation and chronic otitis media (COM)., Data Sources: PubMed, Embase, Cochrane Library., Review Methods: A systematic search was conducted. Relevance and validity of selected articles were assessed. Studies that scored moderate or high on relevance were included, and relevant data for both outcomes were extracted. For the outcome of TM closure, absolute risk differences (RD), relative risks, and number needed to treat with their respective 95% confidence intervals were calculated when possible., Results: We retrieved 3,783 unique studies. Ten studies satisfied the eligibility criteria. Four studies of moderate validity showed RD ranging from 0.08 to 0.13 in favor of the CCP graft compared to the TF graft for TM closure 1 year or more postoperatively, but this was not statistically significant. Five studies of moderate to high validity showed no clinically relevant difference in hearing improvement between both intervention groups at a minimum follow-up of 3 months. The relative air-bone gap closure ranged from 5.7 to 11.5 dB in the TF group and from 8.9 to 12.7 dB in the CCP group., Conclusions: There is no evidence of superiority of one-piece CCP grafting over TF grafting in type 1 tympanoplasty regarding complete closure of a subtotal perforated TM 1 year or more postoperatively or hearing improvement at a minimum of 3 months follow-up. Laryngoscope, 126:1662-1670, 2016., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: To systematically review the literature on cochlear implantation (CI) for children with unilateral hearing loss (UHL)., Data Sources: PubMed, Cochrane, CINAHL, and Embase databases were searched for articles up to June 29, 2015 for UHL, children and CI, and all of their synonyms., Methods: After screening of titles, abstracts, and full texts for eligible articles, directness of evidence (DoE) and risk of bias (RoB) were assessed for the included articles. Study characteristics and data on our outcomes of interest (speech perception in noise, sound localization, quality of life, and speech and language development) were extracted., Results: In total, 296 unique articles were retrieved, of which five articles satisfied the eligibility criteria. All of these articles were case series or case reports and had a low to moderate DoE and a high RoB. In these studies, heterogeneous findings were reported in small patient samples. Speech perception in noise and localization ability improved in most patients. Although only measured in one study each, quality of life and speech and language development improved. Most of these results were not statistically significant., Conclusions: No firm conclusions can be drawn on the effectiveness of CI in children with UHL, due to heterogeneous findings, small sample sizes, and the lack of high Level of Evidence studies. Based on the findings of this systematic review, cochlear implantation may be an effective treatment option in children with UHL. Laryngoscope, 126:713-721, 2016., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Hypoxia inducible factors (HIFs) play vital roles in cellular maintenance of oxygen homeostasis. These transcription factors are responsible for the expression of genes involved in angiogenesis, metabolism, and cell proliferation. Here, we generate a detailed mathematical model for the enzyme kinetics of α-ketoglutarate-dependent HIF prolyl 4-hydroxylase domain (PHD) dioxygenases to simulate our in vitro data showing synergistic PHD inhibition by succinate and hypoxia in experimental models of succinate dehydrogenase loss, which phenocopy familial paraganglioma. Our mathematical model confirms the inhibitory synergy of succinate and hypoxia under physiologically-relevant conditions. In agreement with our experimental data, the model predicts that HIF1α is not stabilized under atmospheric oxygen concentrations, as observed. Further, the model confirms that addition of α-ketoglutarate can reverse PHD inhibition by succinate and hypoxia in SDH-deficient cells., (© 2015. Published by The Company of Biologists Ltd.)

Background: Systematic reviews (SRs) and meta-analyses (MAs) provide the highest possible level of evidence. However, poor conduct or reporting of SRs and MAs may reduce their utility. The PRISMA Statement (Preferred Reporting Items for Systematic reviews and Meta-Analyses) was developed to help authors report their SRs and MAs adequately., Objectives: Our objectives were to (1) evaluate the quality of reporting of SRs and MAs and their abstracts in otorhinolaryngologic literature using the PRISMA and PRISMA for Abstracts checklists, respectively, (2) compare the quality of reporting of SRs and MAs published in Ear Nose Throat (ENT) journals to the quality of SRs and MAs published in the 'gold standard' Cochrane Database of Systematic Reviews (CDSR), and (3) formulate recommendations to improve reporting of SRs and MAs in ENT journals., Methods: On September 3, 2014, we searched the Pubmed database using a combination of filters to retrieve SRs and MAs on otorhinolaryngologic topics published in 2012 and 2013 in the top 5 ENT journals (ISI Web of Knowledge 2013) or CDSR and relevant articles were selected. We assessed how many, and which, PRISMA (for Abstracts) items were reported adequately per journal type., Results: We identified large differences in the reporting of individual items between the two journal types with room for improvement. In general, SRs and MAs published in ENT journals (n = 31) reported a median of 54.4% of the PRISMA items adequately, whereas the 49 articles published in the CDSR reported a median of 100.0 adequately (difference statistically significant, p < 0.001). For abstracts, medians of 41.7% for ENT journals and 75.0% for the CDSR were found (p < 0.001)., Conclusion: The reporting of SRs and MAs in ENT journals leaves room for improvement and would benefit if the PRISMA Statement were endorsed by these journals.

Background: Individuals with single-sided deafness (SSD) have problems with speech perception in noise, localisation of sounds and with communication and social interaction in their daily life. Current treatment modalities (Contralateral Routing of Sound systems [CROS] and Bone Conduction Devices [BCD]) do not restore binaural hearing. Based on low level of evidence studies, CROS and BCD do not improve speech perception in noise or sound localisation. In contrast, cochlear implantation (CI) may overcome the limitations of CROS and BCD, as binaural input can be restored. Promising results have previously been achieved on speech perception in noise, sound localisation, tinnitus and quality of life., Methods and Design: A single-center Randomised Controlled Trial (RCT) was designed to compare all treatment strategies for SSD. One hundred and twenty adult single-sided deaf patients (duration of deafness >3 months and maximum 10 years; pure tone average at 0.5, 1, 2, 4 kHz, deaf ear: threshold equal to or more than 70 dB, better ear: threshold of maximum 30 dB) will be included in this trial and randomised to CI, 'first BCD, then CROS' or 'first CROS, then BCD'-groups. After the trial period, patients in the two latter groups may choose with which treatment option they continue. Outcomes of interest are speech perception in noise, sound localization, tinnitus and quality of life. These outcomes will be measured during a baseline visit and at follow up visits, which will take place at 6, 12, 18, 24, 36, 48 and 60 months after onset of treatment. Furthermore, an economic evaluation will be performed and adverse events will be monitored., Discussion: This RCT allows for a comparison between the two current treatment modalities for single-sided deafness and a new promising treatment strategy, CI, on a range of health outcomes: speech perception in noise, sound localization, tinnitus and quality of life. Additionally, we will be able to answer the question if the additional costs of CI are justified by increased benefits, when compared to current treatment strategies. This study will inform health policy makers with regard to reimbursement of CI., Trial Registration: Netherlands Trial Register (www.trialregister.nl): NTR4580.

Background: Randomised Controlled Trials (RCTs) are the preferred study design when comparing therapeutical interventions in medicine. To improve clarity, consistency and transparency of reporting RCTs, the Consolidated Standards of Reporting Trials (CONSORT) statement was developed., Objectives: (1) To assess the quality of reports and abstracts of RCTs in otorhinolaryngologic literature by using CONSORT checklists, (2) to compare the quality of reports and abstracts of otorhinolaryngologic RCTs between the top 5 general medical journals and top 5 otorhinolaryngologic journals, and (3) to formulate recommendations for authors and editors of otorhinolaryngologic ('ENT') journals., Methods: Based on 2012 ISI Web of Knowledge impact factors, the top 5 general medical and ENT journals were selected. On 25 June 2014, using a highly sensitive Cochrane RCT filter and ENT filter, possibly relevant articles since January 1st, 2010 were retrieved and relevant RCTs were selected. We assessed how many CONSORT items were reported adequately in reports and abstracts and compared the two journal types., Results: Otorhinolaryngologic RCTs (n = 15) published in general medical journals reported a mean of 92.1% (95% confidence interval: 89.5%-94.7%) of CONSORT items adequately, whereas RCTs (n = 18) published in ENT journals reported a mean of 71.8% (66.7%-76.8%) adequately (p < 0.001). For abstracts, means of 70.0% (63.7%-76.3%) and 32.3% (26.6-38.0%) were found respectively (p < 0.001). Large differences for specific items exist between the two journal types., Conclusion: The quality of reporting of RCTs in otorhinolaryngologic journals is suboptimal. RCTs published in general medical journals have a higher quality of reporting than RCTs published in ENT journals. We recommend authors to report their trial according to the CONSORT Statement and advise editors to endorse the CONSORT Statement and implement the CONSORT Statement in the editorial process to ensure more adequate reporting of RCTs and their abstracts.

Objective: A systematic review of the literature to evaluate the clinical outcome of cochlear implantation for patients with single-sided deafness (SSD) or asymmetrical hearing loss (AHL)., Data Sources: We searched the PubMed, Embase, Cochrane Library, and CINAHL databases from their inception up to December 10, 2013 for SSD or AHL and cochlear implantation or their synonyms., Study Selection: In total, 781 articles were retrieved, of which 15 satisfied the eligibility criteria. Our outcomes of interest were speech perception in noise, sound localization, quality of life (QoL), and tinnitus., Data Extraction: Critical appraisal showed that six studies reported on less than five patients or that they carried a low directness of evidence or a high risk of bias. Therefore, we extracted the data of nine studies (n = 112). Patient numbers, age, duration of deafness, classification of deafness, pure tone audiometry, follow-up duration, and outcome measurements were extracted from all nine articles., Data Synthesis: Because of large heterogeneity between studies, we were not able to pool data in a meta-analysis. We therefore summarized the results of the studies specified per outcome., Conclusion: There are no high-level-of-evidence studies concerning cochlear implantation in patients with SSD or AHL. Current literature suggests important benefits of cochlear implantation regarding sound localization, QoL, and tinnitus. Varying results were reported for speech perception in noise, possibly caused by the large clinical heterogeneity between studies. Larger and high-quality studies are certainly warranted.

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3' modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

The parents' point of view regarding positional cranial deformities and helmet therapy has not been the subject of scientific interest yet. However, carer acceptance is a key factor for therapeutic success. We therefore investigated parental perception. The parents of 218 infants were included in a telephone survey; 122 children had undergone helmet therapy and 96 had not. Satisfaction with the outcome, treatment-associated problems, and parental stress were investigated using a structured questionnaire. The great majority (90.8%) of caregivers were satisfied with the outcome, regardless of whether or not helmet therapy was used. Retrospectively, 76% of the parents of infants who had not undergone helmet therapy would decide against helmet therapy again. Therapy was either temporarily stopped (27.0%) or terminated (4.9%) in 31.9% of infants treated with a helmet. Major problems were sweating (51.1%) and skin lacerations (30.9%). The parents indicated minor (54.9%) or even great (25.4%) personal strain. Conflict with others (38.5%), stress for the child (30.3%), and a financial burden (36.9%) were mentioned most frequently. There appear to be more parental problems than expected associated with helmet therapy. Medical experts should take this into consideration. The indication for a helmet should be evaluated critically and the potential parental burdens should be addressed during counselling., (Copyright © 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Objectives/hypothesis: Systematically review the literature on the clinical outcome of bone conduction devices (BCD) and contralateral routing of sound systems (CROSS) for patients with single-sided deafness (SSD)., Data Sources: PubMed, Embase, Cochrane Library, and CINAHL databases were searched up to April 7, 2014., Review Methods: All studies investigating BCD and CROSS for patients with SSD on speech perception in noise, sound localization, or quality of life were selected and critically appraised., Results: In total, 46 articles were retrieved, of which six satisfied the eligibility criteria. Critical appraisal showed that five studies (n = 91) carried a moderate to high directness of evidence and a low to moderate risk of bias. Subsequently, study characteristics and outcome measurements were extracted. Due to large heterogeneity between studies, pooling of data was not feasible. Studies did not show a clear advantage of BCD or CROSS on speech perception in noise. BCD and CROSS lead to the same sound localization ability as the unaided condition. Quality of life did not differ significantly between conditions; however, subjective speech communication did improve., Conclusion: No high level of evidence studies compare BCD and CROSS in patients with SSD. Literature showed no beneficial effect of BCD or CROSS regarding speech perception in noise and sound localization. Subjective speech communication demonstrated a moderate improvement with BCD and CROSS. High evidence studies comparing all treatment options for single-sided deafness should be conducted., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

The clinical impact of epidermal growth factor receptor (EGFR) (E746-A750del) mutation and human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) is unclear. EGFR (E746-A750del) expression was analyzed in OSCC specimens (n=161) by immunohistochemistry. The expression results were correlated with clinical characteristics and impact on survival. Using INNO-LiPA Extra, high-risk HPV types were genotyped and analyzed in 211 OSCC specimens. Positive EGFR (E746-A750del) expression (n=40/161, 25%) was not associated with any clinicopathological characteristics, prognostic factors, social habits (smoking, alcohol consumption), or tumour-specific survival. HPV16 DNA was detected in three out of 211 samples (HPV16-positive: n=3/211, 1.4%). This study shows that mutation-specific EGFR (E746-A750del) expression and HPV do not appear to be relevant to the survival of patients with OSCC., (Copyright © 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Hypothesis: Making a cochleostomy with a laser can affect the inner ear function., Background: Different types of lasers can be used to create a fenestration in the footplate of the stapes during stapedotomy. Because of variations in absorption spectra of the laser light in various tissues or fluids, each laser has its own characteristics and possible side effects., Materials and Methods: The basal turns of the cochleae of 20 guinea pigs were fenestrated using 4 types of lasers (thulium, KTP, CO2, diode; all groups n = 4). A control group (n = 4) was included to correct for the effects of the surgery alone. At 3 different time points, acoustically evoked compound action potentials (CAPs) were recorded at 5 frequencies and at different sound pressure levels. N1-P2 amplitudes were measured, and subsequently, thresholds were calculated. A repeated measures analysis of variance was used to investigate differences between groups., Results: There was a decrease in CAP amplitudes and an increase in CAP thresholds after cochleostomy with each laser. The increase in thresholds was significantly larger for higher frequencies. The thulium laser evoked the largest threshold shifts, the KTP laser the smallest with the CO2, and diode lasers in intermediate positions. Overall, there was an increase in latencies after treatment., Conclusion: Laser treatment on or near the cochlea can cause damage to the sensitivity of the cochlea for sound. The thulium laser seems to be the worst choice in this respect.

This work probes the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. Here we explore the relationship between base stacking, functional group occupancy of the DNA minor and major grooves, and DNA mechanical properties. We study double-helical DNA molecules substituting either inosine for guanosine or 2,6-diaminopurine for adenine. These DNA variants, respectively, remove or add an amino group from the DNA minor groove, with corresponding changes in hydrogen-bonding and base stacking energy. Using the techniques of ligase-catalyzed cyclization kinetics, atomic force microscopy, and force spectroscopy with optical tweezers, we show that these DNA variants have bending persistence lengths within the range of values reported for sequence-dependent variation of the natural DNA bases. Comparison with seven additional DNA variants that modify the DNA major groove reveals that DNA bending stiffness is not correlated with base stacking energy or groove occupancy. Data from circular dichroism spectroscopy indicate that base analog substitution can alter DNA helical geometry, suggesting a complex relationship among base stacking, groove occupancy, helical structure, and DNA bend stiffness., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have been measured by CE. DNA charge density was modified either by appending positively or negatively charged groups to the thymine residues in a 98 bp DNA molecule, or by replacing some of the negatively charged phosphate internucleoside linkers in small ssDNA or dsDNA oligomers with positively charged phosphoramidate linkers. Mobility ratios were calculated for each dataset by dividing the mobility of a charge variant by the mobility of its unmodified parent DNA. Mobility ratios essentially eliminate the effect of the BGE on the observed mobility, making it possible to compare analytes measured under different experimental conditions. Neutral moieties attached to the thymine residues in the 98-bp DNA molecule had little or no effect on the mobility ratios, indicating that bulky substituents in the DNA major groove do not affect the mobility significantly. The mobility ratios observed for the thymine-modified and linker-modified DNA charge variants increased approximately linearly with the logarithm of the fractional negative charge of the DNA. Mobility ratios calculated from previous studies of linker-modified DNA charge variants and small multicharged organic molecules also increased approximately linearly with the logarithm of the fractional negative charge of the analyte. The results do not agree with the Debye-Hückel-Onsager theory of electrophoresis, which predicts that the mobility of an analyte should depend linearly on analyte charge, not the logarithm of the charge, when the frictional coefficient is held constant., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed ∼100-fold, with up to an additional ∼10-fold repression (∼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding., (© The Author(s) 2014. Published by Oxford University Press.)

Background: The main advantage of using the KTP (potassium-titanyl-phosphate) laser for stapedotomy instead of the conventional micropick instrument is the smaller risk for mechanical damage. However, the KTP laser could theoretically inflict damage to inner ear structures. We hypothesize that KTP laser light [wavelength (λ) = 532 nm] is hardly absorbed in perilymph but well absorbed in solid structures. The aim of this pilot study was to assess if damage occurred after KTP laser cochleostomy in an animal model and, if so, to what extent and at which settings., Materials and Methods: In six guinea pigs, a KTP laser cochleostomy at the basal turn was created. Laser settings of 1, 3 and 5 W and 100 ms pulse time (n = 2 each) were used. Histological preparations were studied for damage to neuroendothelial cells and intrascalar blood., Results: No damage to inner ear neuroendothelial cells was observed, even at the highest power. Blood clots in the scala tympani from vessels in the cochlear wall were seen. The effects were minimal in the lowest, currently clinically used settings., Conclusion: KTP laser cochleostomy gives no damage to inner ear neuroendothelial cells but may cause intrascalar hemorrhages., (© 2014 S. Karger AG, Basel.)

The molecular structure of the DNA double helix has been known for 60 years, but we remain surprisingly ignorant of the balance of forces that determine its mechanical properties. The DNA double helix is among the stiffest of all biopolymers, but neither theory nor experiment has provided a coherent understanding of the relative roles of attractive base stacking forces and repulsive electrostatic forces creating this stiffness. To gain insight, we have created a family of double-helical DNA-like polymers where one of the four normal bases is replaced with various cationic, anionic or neutral analogs. We apply DNA ligase-catalyzed cyclization kinetics experiments to measure the bending and twisting flexibilities of these polymers under low salt conditions. Interestingly, we show that these modifications alter DNA bending stiffness by only 20%, but have much stronger (5-fold) effects on twist flexibility. We suggest that rather than modifying DNA stiffness through a mechanism easily interpretable as electrostatic, the more dominant effect of neutral and charged base modifications is their ability to drive transitions to helical conformations different from canonical B-form DNA.

Background: The treatment of complex orbital floor fractures with extensive orbital prolapse remains a surgical challenge in craniomaxillofacial traumatology and is still controversially discussed. Reduction of orbital tissue via a transcutaneous or transconjunctival approach alone can be very difficult and lead to unsatisfying results., Methods: Over a 3-year-period, we enrolled 13 patients who underwent endoscopy-assisted reconstruction of isolated orbital floor fractures via a combined subciliary and transantral approach. Patient data, imaging and ophthalmologic examination were reviewed prospectively., Results: Ten patients underwent primary surgical treatment, 3 patients had secondary surgical treatment because of unsatisfactory results of primary surgical intervention. All patients had an uneventful postoperative course without ophthalmologic deterioration, no further surgical procedures were necessary., Conclusions: The additional use of an endoscopy-assisted transantral approach provides a reliable treatment modality in selected cases. To our knowledge, this is the only study of patients treated with a combined subciliary and transantral approach. Special emphasis was given to postoperative functional results, a short algorithm for use of an additional transantral endoscopy-assisted approach is presented., (Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.

The intrinsic curvature of seven 98 bp DNA molecules containing up to four centrally located A6-tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ∼200 mM, regardless of whether the cation is a hydrophilic ion like Na+, NH4+, or Tris+ or a hydrophobic ion like tetrabutylammonium. The decrease in A-tract curvature with increasing ionic strength, along with the known decrease in A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.

Structuring protein foods to control the textural properties receives growing attention nowadays. It requires decoupling of the product properties such as water holding capacity and the mechanical properties from the actual protein concentration in the product. From an application point of view, both increasing and lowering the protein content in the food are interesting. Foods enriched with proteins are important due to their reported health benefits, but increasing the protein content in food products generally leads to products that are firmer and have a more rubbery mouth-feel than the regular products, making them less attractive. A reduced protein content, for example in meat- or cheese-analogues, is relevant because it leads to a lower caloric intake per serving and it enhances its economic potential. Decoupling of the protein concentration and product properties can be obtained by changing the internal structure of those food products. This paper outlines the use of protein aggregates and particles in a protein matrix as a tool to obtain different textural properties of a model protein product. Whey protein isolate (WPI) was taken as a model protein. However, further investigation of WPI microparticles should focus on a better understanding of their swelling behaviour in the protein matrix to fully use the potential of those protein particles as a tool to decouple product properties and actual protein concentration.