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Competing Interests: Declaration of competing interest The authors declare no conflict of interest.

Introduction: This paper employs finite element analysis to assess the biomechanical behavior of surgically assisted rapid palatal expansion (SARPE) with a bone-borne transpalatal distractor (TPD) by varying surgical parameters., Material and Methods: Nine models were constructed to scrutinize the effects of pterygomaxillary disjunction (PMD), lateral osteotomy positioning, and TPD placement on displacement profiles and Von Mises stresses. These models encompassed variations such as no, unilateral or bilateral PMD, asymmetrical lateral osteotomy, and five TPD locations., Results: Performing a PMD reduces posterior resistance to transverse expansion, resulting in 10-20 % stress reduction around the maxillofacial complex. No significant changes in horizontal tipping were observed post-PMD. The asymmetric lateral osteotomy model exhibited larger displacements on the side with a more superiorly positioned osteotomy. Reduced stresses were observed at the maxillary body and medial pterygoid plate (superiorly), while increased stresses were observed at the medial (inferiorly) and lateral pterygoid plates. More posterior TPD placement facilitated more parallel expansion thus less horizontal tipping, albeit with increased vertical tipping., Discussion: SARPE procedures (distractor and osteotomy positions) can be tailored based on desired outcomes. PMD reduces stress within the maxillofacial complex but doesn't significantly affect tipping. Higher lateral osteotomies lead to increased displacements, more posterior distractors to more parallel expansion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Purpose: To compare the fit and fracture load of temporary fixed partial prostheses fabricated by means of a conventional direct technique, milling, or 3D printing., Materials and Methods: A maxillary right first premolar and molar were prepared on a Frasaco cast, which was then duplicated 40 times. In total, 10 provisional three-unit fixed prostheses (Protemp 4, 3M) were made using the conventional technique with a putty mold. The 30 remaining casts were scanned to design a provisional restoration using CAD software. A total of 10 designs were milled (CEREC MC X5/shaded PMMA Disk, Dentsply Sirona), while the other 20 were 3D printed with one of the two 3D printers (Asiga UV MAX or Nextdent 5100, C&B, Nextdent). Internal and marginal fit were examined using the replica technique. Next, the restorations were cemented on their respective casts and loaded until fracture using a universal testing machine. The location and propagation of the fracture were also evaluated., Results: 3D printing demonstrated the best internal fit. Nextdent (median internal fit: 132 μm) was significantly better compared to the milled (median internal fit: 185 μm; P = .006) and conventional restorations (median internal fit: 215 μm; P < .001), while the fit of Asiga (median internal fit: 152 μm) was only significantly better than the conventional restorations (P < .012). The lowest marginal discrepancy was found for the milled restorations (median marginal fit: 96 μm), but this was only significant when compared to the conventional group (median internal fit: 163 μm; P < .001). The conventional restorations demonstrated the lowest fracture load (median fracture load: 536 N), which was only significant when compared to Asiga (median fracture load: 892 N; P = .003)., Conclusions: Within the present in vitro study's limitations, CAD/CAM demonstrated superior fit and strength compared to the conventional technique.

Objectives: To provide a critical update identifying the knowledge gaps and controversies in medication-related osteonecrosis of the jaw (MRONJ) within the Belgian healthcare context and outline opportunities for improvement and research in these areas., Methods: A literature review was performed to identify guidelines from international clinical societies in oncology or oral and maxillofacial surgery on diagnosing, preventing, and treating MRONJ. The recommendations were critically assessed in light of recent developments in the field and confronted with the clinical experience of experts., Results: Despite progress in the diagnostic criteria of MRONJ, the continued need for an 8-week timeout period should be reconsidered. Furthermore, 3D imaging techniques should be introduced to improve diagnosis and staging. The staging system remains ambiguous regarding Stage 0 MRONJ, and ongoing confusion exists regarding the term non-exposed MRONJ. The prevention of MRONJ should be tailored, considering the individual patient's risk of MRONJ, frailty, and life expectancy. More research seems needed into the efficacy and safety of drug holidays, considering the risks of rebound remodeling on fractures. With renewed interest in surgical and adjunct management techniques, adequately designed clinical studies are needed to help translate trial outcomes into universally applicable treatment guidelines taking into account individual patient characteristics., Conclusions: Important knowledge gaps remain and hamper the development of clinical guidelines. Several controversies were identified where consensus is lacking, and further harmonization between stakeholders is necessary. Finally, the need for randomized controlled comparative clinical trials in MRONJ resonates harder than ever to identify the best treatment for individual patients.

Background: Profound variations in facial nerve branching, combined with the severe impact of facial palsy on the patient's quality of life, make surgery in this region challenging. Recent advancements in ultrasound (US) technology, including the improved visualization of small structures, have led to a sharp increase in its medical indications in various medical disciplines. We aimed to prove the feasibility of using ultra-high-frequency (UHF) US to visualize the facial nerve and to guide surgeons during surgery on and around the facial nerve., Methods: A cadaveric study was performed on one hemi-face with a UHF US imaging system and state-of-the-art transducers. Firstly, a transcutaneous US was performed, and the facial nerve branches of interest (zygomatic, buccal, and marginal mandibular branches) were marked using US-guided color-injections of filler mixed with methylene blue. Skin and subcutaneous fat were then removed to simulate the intraoperative field. Secondly, an "intraoperative" US examination was performed, and the same branches were marked by US-guided color-injections of filler mixed with indocyanine green. Anterograde facial nerve dissection was performed, and the distance between the nerve branches and the injected filler was measured., Results: All color-injections (mixed with both methylene blue and indocyanine green) were positioned right next to the nerve branches (<1 mm). The image quality of the US below the skin was observed to be far superior to that of the transcutaneous US., Conclusion: UHF US can be used to visualize the facial nerve with high precision both transcutaneously and intraoperatively (after elevation of the skin flap)., Competing Interests: The authors have no financial interest to declare in relation to the content of this article. Funding for this study was provided internally by the Department of Plastic, Reconstructive and Aesthetic Surgery, UZ Gent., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Hematological diseases, due to their complex nature and diverse manifestations, pose significant diagnostic challenges in healthcare. The pressing need for early and accurate diagnosis has driven the exploration of novel diagnostic techniques. Infrared (IR) spectroscopy, renowned for its noninvasive, rapid, and cost-effective characteristics, has emerged as a promising adjunct in hematological diagnostics. This review delves into the transformative role of IR spectroscopy and highlights its applications in detecting and diagnosing various blood-related ailments. We discuss groundbreaking research findings and real-world applications while providing a balanced view of the potential and limitations of the technique. By integrating advanced technology with clinical needs, we offer insights into how IR spectroscopy may herald a new era of hematological disease diagnosis.

This study investigated the incidence, risk factors, and outcome of medication-related osteonecrosis of the jaw after dental extractions in patients receiving antiresorptive agents for osteoporosis or bone metastases. 240 patients with a median drug exposure of 43 months were retrospectively studied. The incidence of MRONJ after dental extraction in the osteoporosis cohort was 2.7 % per person-year (95 % CI 1.6-4.6 %) (n = 13/126), and for the bone metastases cohort 26.4 % per person-year (95 % CI 20.4-34.2 %) (n = 58/114). 92 % of MRONJ cases were stage 1. Dental infection as the reason for extraction increased the osteonecrosis risk in the osteoporosis (OR 22.77; 95 % CI 2.85-181.62; p = 0.003) and bone metastases cohorts (OR 2.72; 95 % CI 1.28-5.81; p = 0.010). Using leukocyte and platelet-rich fibrin reduced this risk by 84 % (p = 0.003), as did antibiotics use by 86-93 % (p = 0.013). Within the bone metastases cohort, an interval since last administration of at least 3 months reduced risk of MRONJ (OR 0.83; 95 % CI 0.72-0.97; p = 0.018). Mucosal healing occurred in 11/13 patients (84.6 %; 95 % CI 54.5-98.1 %) with osteoporosis and 31/58 patients (53.4 %; 95 % CI 40.0-66.7 %) with bone metastases. In conclusion, though the MRONJ risk in this selected population taking antiresorptive agents and presenting to the Oral Maxillofacial Surgery clinic for a dental extraction is considerable and higher in those with dental infections, preventive measures such as antibiotics and use of LRPF membranes may significantly reduce that risk., (Copyright © 2023. Published by Elsevier Inc.)

Improvements in preoperative diagnostics and intraoperative techniques have made the surgical excision of benign parotid gland tumours less invasive. Extracapsular dissection (ECD) has become more popular in comparison to superficial parotidectomy (SP), the gold standard. Although clinical outcomes have been reported, reports on cost-effectiveness are limited. The aim of this retrospective study was to analyse the surgical outcomes and cost-effectiveness of ECD versus SP in benign parotid tumour surgery. A retrospective cohort of 161 patients treated between 2012 and 2020 was collected. Data concerning demographics, clinical outcomes, and cost-efficiency were recorded. Analysis of the 161 unilateral parotidectomy cases (59 SP, 102 ECD) showed a significantly longer operation time, anaesthesia time, and length of stay for SP patients (all P < 0.001). Regarding postoperative complications, transient facial nerve weakness (P < 0.001) and haematoma formation (P = 0.016) were more prevalent in the SP patients. The frequency of positive margins was lower for SP (P = 0.037). No case of recurrence was identified with either technique. ECD showed excellent clinical outcomes as well as a reduction in complications when compared to SP. ECD is a viable alternative for superficial benign parotid gland tumours after thorough preoperative clinical, pathological, and radiological examination. The reduction in operation, anaesthesia, and hospitalization times with ECD is likely to result in a gain in cost-effectiveness., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Salivary gland malignancies (SGMs) account for less than 5% of new diagnoses in head and neck tumors. If feasible, surgery is the preferred treatment modality. Nevertheless, some malignancies have a tendency of recurrence, with possible distant metastasis. Alternative treatment strategies, such as primary radiation or chemotherapeutics, often present low response rates. As a result, there is an unmet need for novel therapeutic approaches. Nowadays, target-based therapies (e.g., small inhibitors and immunotherapy) are used by the medical oncologist for possible treatment of advanced SGMs. Based on recent published trials, some novel treatments may provide additional disease control for some patients. However, sample sizes are small, the general findings are unsatisfactory, and a lot of uncertainties remain to be elucidated. Nevertheless, research shows that patients do not benefit from blind administration of systemic treatments and therefore a more personalized approach is highly needed. The aim of this review paper is to summarize the most recent advances in the biological understanding and molecular pathways of salivary gland cancers, the association of these pathways with the current treatments used and their implications for more personalized targeted-based therapies.

Background: Autologous facial fat grafting has gained popularity in recent years and is considered to be safe. This paper presents the case of a patient who died due to massive cerebral microfat embolism after facial fat grafting., Objectives: The aim of this study was to raise awareness and provide more evidence on the prevention and treatment of this potentially lethal complication of facial fat grafting., Methods: A detailed report was made of the case. Two online databases were searched for similar cases of facial fat embolism resulting in neurologic and/or visual symptoms. Thereafter a literature search was conducted to verify the etiology, current treatment options, and preventive measures., Results: Forty-nine cases with similar events were found in the literature. The most common injected area was the glabella (36.1%), and an average of 16.7 mL fat was injected. The main complications were visual impairment, with 88.5% of cases resulting in permanent monocular blindness, and neurologic symptoms, some of which never fully recovered. Including the present patient, 7 cases were fatal. Fat embolism can occur in the veins and arteries of the face. Two possible pathways for fat embolism exist: the macroscopic, mechanical pathway with immediate signs, and the microscopic, biochemical pathway with delayed symptoms. Mechanical embolectomy and corticosteroids are suggested treatment options but evidence for their efficacy is lacking. Several different preventive measures are described., Conclusions: Although facial fat grafting is considered a safe procedure, one should be aware of the risk of fat embolism. Underreporting of this adverse event is likely. With no effective treatment and often detrimental outcomes, preventive measures are of utmost importance to improve patient safety., (© 2021 The Aesthetic Society. Reprints and permission: journals.permissions@oup.com.)

Background: Only a few biomarkers have been evaluated for their prognostic value with regard to biochemical recurrence (BCR) following primary radical prostatectomy. We explored the possibilities of using near-infrared (NIR) spectroscopy as a prognostic biomarker for BCR-free survival (BCR-FS)., Methods: Tissue specimens from 82 prostate cancer patients were obtained. Formalin-fixed paraffin-embedded slides (hematoxylin-eosin-stained) were analyzed using NIR spectroscopy. Prognostic features for BCR-FS were determined following normalization of the spectra., Results: Several differences were found throughout the NIR spectrum for the patients with or without BCR, for both the first derivative and second derivative of the NIR spectrum. Following categorization and Cox regression analysis, spectral regions at 5236 cm -1 (first derivative; median BCR-FS not reached versus 3.2 years; HR high = 0.18 [0.08-0.39]; and p < 0.0001) and at 5956 cm -1 (second derivative; median BCR-FS not reached versus 3.8 years; HR low = 0.22 [0.10-0.48]; and p = 0.0002) showed prognostic properties for BCR-FS. The combination of both parameters further increased the prognostic value of NIR ( p < 0.0001)., Conclusions: We demonstrated NIR spectral variations between patients with or without BCR, which have been shown to have prognostic value. This easy-to-use technique could possibly further improve post-primary radical prostatectomy monitoring and swift referral to adjuvant local therapies. Further elaboration is highly recommended to fully elucidate these variations and to gain a deeper insight into the changing chemical and physical compositions of the prostate tumor architecture.

SGTs vary in histological behavior. Mucins, a major component in salivary glands, consist of a glycosylated and sialylated protein core. Rapid evolutions in glycobiology have demonstrated the important role of glycoproteins in cancer development. NIR spectroscopy is a method for the biochemical analysis of substrates. NIR spectra can be analyzed using specific chemometrics. Our aim was to explore the diagnostic possibilities of NIR spectroscopy in SGTs. 238 Hematoxylin and Eosine stained (H&E) SGT tissue sections were examined using NIR spectroscopy. 45 deparaffinized tissue sections were treated with neuraminidase to identify wavelengths in the NIR spectrum related to sialylation. NIR spectra were analyzed with chemometrics. NIR spectra could distinguish malignant SGTs from controls and benign SGTs. Prediction models based on the entire spectral range resulted in a 73.1% accurate classification of malignant SGTs and controls, while, based on neuraminidase experimental spectral peak differences (1436 nm; 1713 nm; 1783 nm; 1924 nm; 2032 nm; 2064 nm; 2178 nm; 2216 nm), an improved overall correct classification rate of 91.9% was obtained between healthy subjects and malignant tumors. H&E tissue section-based NIR spectroscopy can identify malignant SGTs from controls, promising an alternative method in the diagnosis of SGTs.

The aim of this systematic review was to evaluate the volumetric changes associated with different bone grafting techniques in the completely edentulous atrophic maxilla before dental implant placement. A search was performed according to the PRISMA guidelines. A PICO question was formed, and the PubMed, Scopus, Embase, and Cochrane Library databases were searched, covering the period 2000-2018. Relevant data were extracted from the results regarding study population, surgical details, technical information on volumetric data acquirement, and volumetric outcome after bone augmentation procedures before implant placement. Six articles with a combined population of 84 patients were included. All patients had a completely edentulous maxilla, with a crestal horizontal width of <3-4mm or a crestal vertical height of <6-7mm. The iliac bone and ascending ramus were most frequently used as grafts. Five of the six studies reported volumes of sinus inlay graft (SIG) and four reported volumes of lateral bone augmentation (LBA). Radiographic analyses of the augmented areas differed among the studies. Volume loss after bone augmentation procedures ranged from 5% to 50% for SIG and from 5% to 47% for LBA. All surgical augmentation techniques for the edentulous maxilla are prone to resorption; no procedure seemed to be superior, but some interesting observations were made., (Copyright © 2020 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Photosynthesis in bryophytes and lycophytes has received less attention than terrestrial plant groups. In particular, few studies have addressed the nonstomatal diffusion conductance to CO 2 g nsd of these plant groups. Their lower photosynthetic rate per leaf mass area at any given nitrogen concentration compared with vascular plants suggested a stronger limitation by CO 2 diffusion. We hypothesized that bryophyte and lycophyte photosynthesis is largely limited by low g nsd . Here, we studied CO 2 diffusion inside the photosynthetic tissues and its relationships with photosynthesis and anatomical parameters in bryophyte and lycophyte species in Antarctica, Australia, Estonia, Hawaii and Spain. On average, lycophytes and, specially, bryophytes had the lowest photosynthetic rates and nonstomatal diffusion conductance reported for terrestrial plants. These low values are related to their very thick cell walls and their low exposure of chloroplasts to cell perimeter. We conclude that the reason why bryophytes lie at the lower end of the leaf economics spectrum is their strong nonstomatal diffusion conductance limitation to photosynthesis, which is driven by their specific anatomical characteristics., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

This retrospective study was performed to verify the accuracy of horizontal and vertical repositioning of the maxilla in bimaxillary osteotomy with a focus on posterior vertical displacement. Data from 39 orthognathic patients undergoing bimaxillary surgery including a one-piece Le Fort I osteotomy with pitch rotation and advancement at the University Hospitals of Leuven (Belgium), between January 2015 and April 2016, were included in the study. Preoperative and 1-week postoperative lateral cephalograms were digitized and imported into cephalometric software. Horizontal and vertical measurements of dental landmarks were used to assess the accuracy of maxillary repositioning, and errors were reported in terms of the mean and absolute mean. The horizontal advancements were randomly under- and over-corrected an average of 1.4mm±1.2mm. Vertical repositioning of the anterior maxilla followed the planning. A tendency for under-correction was found for posterior vertical intrusion of the maxilla. The same tendency towards under-correction of posterior maxillary inferior repositioning was detected when planned movements were greater than 3mm. For all studied groups, no significant difference was found between the planning and the results achieved, validating the use of intermediate splints., (Copyright © 2018 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Orthognathic surgery is indicated for the treatment of significant skeletal malocclusions and facial dysmorphosis. Recent technological developments allow surgeons to virtually plan the orthognathic jaw movements. When producing intermediate splints, overlap between the upper and lower dentitions can occur. Autorotation of the mandible is often used as a solution for this problem. The purpose of this study was to present an optimized approach to fabricate digital intermediate splints based on the use of a scanned thick wax bite to overcome dental overlap during planning. Twenty patients (9 males and 11 females) undergoing bimaxillary orthognathic surgery participated in this prospective study. Planning of the maxillary movements was conducted by the same surgeon. The thick wax bite was scanned using an intraoral scanner and a special CBCT protocol. The fabrication of the intermediate splints was done by means of conventional model surgery and virtual 3D planning-printing. The splints were clinically assessed by comparing the digitally printed intermediate splints to the corresponding conventional set-up which served as the clinical standard. Moreover, reproducibility and comparison tests between the scanned wax bites were implemented. The root mean square (RMS) for the reproducibility and comparison tests was respectively 0,15 mm (SD 0,24 mm) and 0,19 mm (SD 0,08 mm). 19 out of the 20 intermediate digitally 3D printed splints were clinically accepted. This proposed protocol can be considered as an optimization to the current 3D planning protocols of bimaxillary orthognathic surgery., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Glycated keratin allows the monitoring of average tissue glucose exposure over previous weeks. In the present study, we wanted to explore if near-infrared (NIR) spectroscopy could be used as a non-invasive diagnostic tool for assessing glycation in diabetes mellitus., Methods: A total of 52 patients with diabetes mellitus and 107 healthy subjects were enrolled in this study. A limited number (n=21) of nails of healthy subjects were glycated in vitro with 0.278 mol/L, 0.556 mol/L and 0.833 mol/L glucose solution to study the effect of glucose on the nail spectrum. Consequently, the nail clippings of the patients were analyzed using a Thermo Fisher Antaris II Near-IR Analyzer Spectrometer and near infrared (NIR) chemical imaging. Spectral classification (patients with diabetes mellitus vs. healthy subjects) was performed using partial least square discriminant analysis (PLS-DA)., Results: In vitro glycation resulted in peak sharpening between 4300 and 4400 cm-1 and spectral variations at 5270 cm-1 and between 6600 and 7500 cm-1. Similar regions encountered spectral deviations during analysis of the patients' nails. Optimization of the spectral collection parameters was necessary in order to distinguish a large dataset. Spectra had to be collected at 16 cm-1, 128 scans, region 4000-7500 cm-1. Using standard normal variate, Savitsky-Golay smoothing (7 points) and first derivative preprocessing allowed for the prediction of the test set with 100% correct assignments utilizing a PLS-DA model., Conclusions: Analysis of protein glycation in human fingernail clippings with NIR spectroscopy could be an alternative affordable technique for the diagnosis of diabetes mellitus.

Objective: To present the hearing results of a 5-year longitudinal study in a pediatric population undergoing surgery for extensive cholesteatoma using a canal wall up (CWU) approach with bony obliteration of the mastoid and epitympanic space, with a standard residual rate of 5.8%, a recurrence rate of 2.9%, and all ears waterproof, free of otorrhea and all external ear canals patent and self-cleaning., Study Design: Retrospective consecutive study., Patients: Thirty-three children (≤18 yr) undergoing surgery for cholesteatoma (34 ears) between 1997 and 2009., Interventions: Therapeutic., Setting: Tertiary referral center., Main Outcome Measures: Hearing and gain in hearing at 1- and 5-year postsurgery: (1) pure-tone average (PTA), (2) pure-tone average high frequency, (3) pure-tone average including 3 kHz, (4) bone conduction at corresponding frequency averages, (5) gain at corresponding air conduction and bone conduction (gain at corresponding air conduction) frequency averages. (6) The Amsterdam Hearing Evaluation Plots were used to study the individual cases., Results: The Amsterdam Hearing Evaluation Plots at 5-year showed in 58.8% of patients a positive gain air conduction. In 23.5% a successful functional result was achieved, defined as an air-bone gap closure to 20 dBHL or less. In 6 patients (17.6%) a limited bone conduction deterioration was shown all limited to maximum 20 dBHL., Conclusion: The CWU bony obliteration tympanoplasty in a consecutive series of pediatric extensive cholesteatoma shows a similar to slightly improved hearing outcome as compared with CWU surgery without BOT. Although the series shows a clearly reduced reoperation rate and a significantly improved safety and hygienic outcome as compared with CWU without BOT, better hearing outcomes remain desirable in this group of children.

Objectives: Although HbA1c is a good diagnostic tool for diabetes, the precarity of the health system and the costs limit the use of this biomarker in developing countries. Fingernail clippings contain ±85% of keratins, which are prone to glycation. Nail keratin glycation may reflect the average glycemia over the last months. We explored if attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) can be used as a non-invasive tool for assessing glycation in diabetes., Design and Methods: Using ATR-FTIR spectroscopy, glycation and deglycation experiments with fructosamine 3-kinase allowed to identify the spectrum that corresponds with keratin glycation in fingernail clippings. Clippings of 105 healthy subjects and 127 diabetics were subjected to the standardized ATR-FTIR spectroscopy method., Results: In vitro glycation resulted in an increased absorption at 1047cm -1 . Following enzymatic deglycation, this peak diminished significantly, proving that the AUC between 970 and 1140cm -1 corresponded with glycated proteins. Within-run CV of the assay was 3%. Storage of nail clippings at 37°C for 2weeks did not significantly change results. In diabetics, glycated nail protein concentrations (median: 1.51μmol/g protein, IQR: 1.37-1.85μmol/g protein) were significantly higher than in the controls (median: 1.19μmol/g protein, IQR: 1.09-1.26μmol/g protein) (p<0.0001). ROC analysis yielded an AUC of 0.92 at a cut-off point of 1.28μmol/g nail (specificity: 82%; sensitivity: 90%). No correlation was observed between the glycated nail protein concentrations and HbA1c., Conclusions: Protein glycation analysis in fingernails with ATR-FTIR spectroscopy could be an alternative affordable technique for diagnosing and monitoring diabetes. As the test does not consume reagents, and the preanalytical phase is extremely robust, the test could be particularly useful in developing countries., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Although assessment of glycated nail proteins may be a useful marker for monitoring of diabetes, their nature and formation are still poorly understood. Besides a detailed anatomical analysis of keratin glycation, the usefulness of glycated nail protein assessment for monitoring diabetic complications was investigated., Methods: 216 patients (94 males, 122 females; mean age ± standard deviation: 75.0 ± 8.7 years) were enrolled. Glycation of nail and eye lens proteins was assessed using a photometric nitroblue tetrazolium-based assay. Following chromatographic separation of extracted nail proteins, binding and nonbinding fractions were analyzed using one-dimensional gel electrophoresis. Using a hand piece containing a latch-type-bur, a meticulous cutting of the nail plate into superficial and deep layers was performed, followed by a differential analysis of fructosamine., Results: Using SDS PAGE, four and two bands were identified among the nonglycated and glycated nail fraction respectively. Significantly lower fructosamine concentrations were found in the superficial nail layer (mean: 2.16 ± 1.37 μmol/g nails) in comparison with the deep layer (mean: 4.36 ± 2.55 μmol/g nails) (P<0.05). A significant higher amount of glycated eye lens proteins was found in diabetes mellitus patients (mean: 3.80 ± 1.57 μmol/g eye lens) in comparison with nondiabetics (mean: 3.35 ± 1.34 μmol/g eye lens) (P<0.05). A marked correlation was found between glycated nail and glycated eye lens proteins [y (glycated nail proteins) = 0.39 + 0.99 x (eye lens glycated proteins); r2 = 0.58, P<0.001]. The concentration of glycated eye lens proteins and the HbA1c level were found to be predictors of the concentration of glycated nail proteins., Conclusions: Glycation of nail proteins takes place in the deep layer of finger nails, which is in close contact with blood vessels and interstitial fluid. Glycation of nail proteins can be regarded as a representative marker for diabetic glycation-associated target organ damage.

Background: Gingivitis is a common inflammatory condition. We explored the value of flow cytometry of saliva in patients with periodontal inflammation., Methods: A cohort of 249 healthy adults (age range: 18-81 y; 2.5th to 97.5th percentile: 19-66 y) was investigated for caries, dental plaque and gingivitis. Saliva was analyzed using flow cytometry on a Sysmex UF-1000i®., Results: Sysmex UF-1000i® is capable to reproduce reliable measurements of cellular components in saliva. A statistically significant lower number of salivary bacteria was found in patients with gingivitis in comparison with healthy adults (p<0.0001). A significant difference in salivary leukocyte count was found between patients with different gingival index scores (p<0.0005). The gingivitis score was strongly dependent on the number of salivary leukocytes, the age of the patient and the degree of caries and dental plaque (r(2)=0.60, p<0.001). At a cut-off level of 10(3) leukocytes/μl saliva, an area under the curve of 0.82 was obtained with a sensitivity of 76% and a specificity of 78% in patients (>35 y) with a gingivitis score of 3., Conclusion: Flow cytometry is an alternative method to evaluate local inflammatory processes in the mouth with a sensitivity of 76% and a specificity of 78%., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research on the nematode Caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. This hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. It is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild C. elegans populations., Results: We used multiple wild C. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. The genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. The separation between the isolation sites was prevalent on all chromosomes, but chromosome V was the largest contributor to this variation. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Comparing polymorphic genes between the populations with a set of genes extracted from 19 different studies on gene expression in C. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation., Conclusions: We found that wild C. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. Studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in C. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild.

Nine isolates from Argyrolobium uniflorum, Lotus creticus , Medicago sativa (Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the 16S rRNA gene and the nodulation gene nodA. Analysis of the individual and concatenated gene sequences demonstrated that the nine new strains constituted three stable, well-supported (bootstrap and gene sequence similarity values) monophyletic clusters, A, B and C, all belonging to the branch of the genus Ensifer, regardless of the phylogenetic reconstruction method used (maximum likelihood, maximum-parsimony, neighbour-joining). The three groups were further characterized by API 100 auxanographic tests, host specificity and nodA gene sequence analysis. On the basis of these data, clusters A and C are suggested as representing two novel species within the genus Ensifer, for which the names Ensifer numidicus sp. nov. (type strain ORS 1407(T)=LMG 24690(T)=CIP 109850(T)) and Ensifer garamanticus sp. nov. (type strain ORS 1400(T)=LMG 24692(T)=CIP 109916(T)) are proposed. The cluster B strains were assigned to Ensifer adhaerens genomovar A.

There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA-DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (atpD, dnaK, gap, glnA, gltA, gyrB, pnp, recA, rpoB and thrC) was performed on 34 representatives of the genus Ensifer. Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA-DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA-DNA hybridization for the assessment of genetic relatedness between Ensifer species. Our MLSA data, confirmed by DNA-DNA hybridizations, support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

Three strains of a previously undescribed, Gram-positive, coryneform bacterium, which were isolated from cattle, were subjected to polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing revealed that the unknown isolates were members of the genus Arthrobacter and were phylogenetically closely related to Arthrobacter luteolus. However, DNA-DNA hybridization indicated that the strains belonged to a new sub-lineage within the genus Arthrobacter. The unknown isolates can be distinguished from related species by biochemical tests. It is proposed that the Arthrobacter-like bacteria of veterinary origin should be classified in the genus Arthrobacter as Arthrobacter gandavensis sp. nov., with the type strain LMG 21285(T) (=DSM 15046(T)).

The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.

Bacterial strains isolated from a large variety of necropsy samples of pigs and previously described as a phenotypical homogeneous group were shown to belong to the species Actinomyces hyovaginalis. This was unexpected because their colonial characteristics, as well as their origins, were very different from those originally reported for the vaginal strains on which the species description of A. hyovaginalis was based. Colonial morphology, as well as fermentation of cellobiose, reactions in hippurate and nitrate and production of beta-glucuronidase, allowed separation of the strains studied here from the vaginal strains. Analysis of tRNA intergenic length polymorphisms (tDNA-PCR), 16S rRNA-gene sequencing and DNA-DNA hybridizations were carried out and led to the proposal of a separate biotype within the species A. hyovaginalis. Since, the strains were isolated from different body sites, this biotype has been designated as the 'general' biotype of A. hyovaginalis, while the strains on which the original species description was based are designated as the 'vaginal' biotype.

DNA-DNA hybridizations were performed between Bradyrhizobium strains, isolated mainly from Faidherbia albida and Aeschynomene species, as well as Bradyrhizobium reference strains. Results indicated that the genus Bradyrhizobium consists of at least 11 genospecies, I to XI. The genospecies formed four subgeneric groups that were more closely related to each other (>40% DNA hybridization) than to other genospecies (<40% DNA hybridization): (i) genospecies I (Bradyrhizobium japonicum), III (Bradyrhizobium liaoningense), IV and V; (ii) genospecies VI and VIII; (iii) genospecies VII and IX; and (iv) genospecies II (Bradyrhizobium elkanii), X and XI. Photosynthetic Aeschynomene isolates were found to belong to at least two distinct genospecies in one subgeneric group. DNA-DNA hybridization data are compared with data from amplified fragment length polymorphism analysis and 165-23S rDNA spacer sequence analysis.

The sequences of the 16S-23S rDNA intergenic spacer region of 62 strains of Bradyrhizobium, including representatives of the three valid species, were determined. The majority of strains had a single rRNA operon type and all contained a tRNA(Ala) and a tRNA(Ile) gene. Analysis of the sequence data produced groupings in line with previously obtained AFLP data. DNA-DNA hybridizations were performed to determine to what extent spacer sequences and AFLP profiles reflected the overall genomic similarities. Strains belonging to the same AFLP group, and strains with spacer sequences diverging less than 4%, were found to belong to the same genospecies. More remote relationships (DNA homology levels of 40-60%) between species were reflected in the spacer sequence analysis, but not in the AFLP analysis. For the genus Bradyrhizobium, 16S-23S rDNA spacer sequence analysis provides taxonomic information similar, but not always identical to that obtained by DNA-DNA hybridizations. Our results indicate that this genus consists of a group of four highly related genospecies (Bradyrhizobium japonicum, Bradyrhizobium liaoningense and two other genospecies) and at least three other genospecies, one of which is Bradyrhizobium elkanii.

The 16S rDNA sequence of Bradyrhizobium liaoningense was determined and analysed together with sequences of other Bradyrhizobium species and related taxa. In addition, DNA-DNA hybridizations were performed between representative strains of the three Bradyrhizobium species. Bradyrhizobium liaoningense is genotypically highly related to Bradyrhizobium japonicum, whereas Bradyrhizobium elkanii is more distantly related to these two species. The fact that Afipia, Agromonas, Blastobacter, Nitrobacter and Rhodopseudomonas are phylogenetically more closely related to Bradyrhizobium japonicum than to Bradyrhizobium elkanii is discussed.

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization. It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96.2% sequence homology). The name Allorhizobium undicola gen. nov., sp. nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).

Twenty-four strains isolated mainly from infected respiratory tracts of pigeons were characterized by an integrated genotypic and phenotypic approach. An extensive biochemical examination using conventional tests and several API microtest systems indicated that all isolates formed a phenotypically homogeneous taxon with a DNA G + C content between 42 and 43 mol%. Whole-cell protein and fatty acid analysis revealed an unexpected heterogeneity which was confirmed by DNA-DNA hybridizations. Four main genotypic sub-groups (genomovars) were delineated. 16S rDNA sequence analysis of a representative strain indicated that this taxon belongs to the beta-subclass of the Proteobacteria with Taylorella equigenitalis as its closest neighbour (about 94.8% similarity). A comparison of phenotypic and genotypic characteristics of both taxa suggested that the pigeon isolates represented a novel genus for which the name Pelistega is proposed. In the absence of differential phenotypic characteristics between the genomovars, it was preferred to include all of the isolates into a single species, Pelistega europaea, and strain LMG 10982 was selected as the type strain. The latter strain belongs to fatty acid cluster I and protein electrophoretic sub-group 1, which comprise 13 and 5 isolates, respectively. It is not unlikely that the name P. europaea will be restricted in the future to organisms belonging to fatty acid cluster I, or even to protein electrophoretic sub-group 1, upon discovery of differential diagnostic features.