A shedding analysis after AAV8 CNS injection revealed fragmented viral DNA without evidence of functional AAV particles in mice.

Adeno-associated viruses (AAV) are commonly used in the scientific field due to their diverse application range. However, AAV shedding, the release of virions from the host organism, can impact the safety of AAV-based approaches. An increasing number of authorities require the characterization of vector shedding in clinical trials. Recently, shedding of transduced laboratory animals has also gained attention regarding the necessary disposal measures of their waste products. However, no explicit international regulations for AAV-shedding waste exist. Generating insights into shedding dynamics becomes increasingly relevant to help authorities develop adequate regulations. To date, knowledge of AAV vector shedding in mice is very limited. Moreover, confirmation of functional shed AAV particles in mice is missing. Therefore, we examined feces, urine, and saliva of mice after CNS injection with AAV2/8. It revealed the presence of viral DNA fragments via qPCR for up to 4 days after injection. To examine AAV functionality we performed nested PCR and could not detect full-length viral genomes in any but two collected feces samples. Furthermore, a functional infection assay did not reveal evidence of intact AAV particles. Our findings are supposed to contribute murine shedding data as a foundation to help establish still lacking adequate biosafety regulations in the context of AAV shedding.

Cerebellar contribution to threat probability in a SCA6 mouse model.

Fear and anxiety have proven to be essential during the evolutionary process. However, the mechanisms involved in recognizing and categorizing threat probability (i.e. low to high) to elicit the appropriate defensive behavior are yet to be determined. In this study, we investigated the cerebellar contribution in evoking appropriate defensive escape behavior using a purely cerebellar, neurodegenerative mouse model for spinocerebellar ataxia type 6 which is caused by an expanded CAG repeat in exon 47 of the P/Q type calcium channel α1A subunit. These mice overexpress the carboxy terminus (CT) of the P/Q type calcium channel containing an expanded 27 CAG repeat specifically in cerebellar Purkinje cells (CT-longQ27PC). We found that our CT-longQ27PC mice exhibit anxiolytic behavior in the open field, elevated plus maze and light/dark place preference tests, which could be recovered with more threatening conditions such as brighter lighting, meowing sounds and an ultrasound repellent. Their innate fear to find safety in the Barnes maze and visual cliff tests was also diminished with subsequent trials, which could be partially recovered with an ultrasound repellent in the Barnes maze. However, under higher threat conditions such as in the light/dark place preference with ultrasound repellent and in the looming tests, CT-longQ27PC mice responded with higher defensive escape behaviors as controls. Moreover, CT-longQ27PC mice displayed increased levels of CT-labeled aggregates compared with controls. Together these data suggest that cerebellar degeneration by overexpression of CT-longQ27PC is sufficient to impair defensive escape responses in those mice.

Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells.

Increased lactate levels in the tissue microenvironment are a well-known feature of chronic inflammation. However, the role of lactate in regulating T cell function remains controversial. Here, we demonstrate that extracellular lactate predominantly induces deregulation of the Th17-specific gene expression program by modulating the metabolic and epigenetic status of Th17 cells. Following lactate treatment, Th17 cells significantly reduced their IL-17A production and upregulated Foxp3 expression through ROS-driven IL-2 secretion. Moreover, we observed increased levels of genome-wide histone H3K18 lactylation, a recently described marker for active chromatin in macrophages, in lactate-treated Th17 cells. In addition, we show that high lactate concentrations suppress Th17 pathogenicity during intestinal inflammation in mice. These results indicate that lactate is capable of reprogramming pro-inflammatory T cell phenotypes into regulatory T cells.

Evaluation of an individual anamnesis tool for teaching risk-oriented prevention - a pilot study in undergraduate dental students.

BACKGROUND: A comprehensive medical history is needed to establish and ensure a high standard in dental care; however, it is challenging to draw clinical consequences on the variety of potential diseases and medications, especially for dental students. Aim of this observational study was to investigate, whether undergraduate dental students using an analog anamnesis tool for risk classification would be more confident and have more knowledge in risk classification than other students in the same year of study. METHODS: A cohort of 48 fifth year dental students was included and allocated into two groups based on their curriculum-related division (group A: n = 25, group B: n = 23). Group A received a teaching event and provision of an analog anamnesis tool for risk classification; group B received neither a teaching event nor the anamnesis tool. At baseline and after two weeks (follow-up), questionnaires regarding self-perceived confidence with risk classification, questions on different disease, medications and lifestyle factors and a task with 15 medical histories of prepared patient cases were applied. The data was statistically analyzed using Mann-Whitney or Wilcoxon test. RESULTS: In group comparison of the differences between baseline and follow-up regarding self-perceived confidence, significantly higher improvement was noted in group A compared to group B for all questions (p < 0.05). With regard to knowledge, the group comparison revealed that the differences in all of the four tasks were significantly higher in group A compared to group B (pi ≤ 0.01). Thereby, the different tasks in group A differed between baseline and follow-up as follows: Risk of complications: 49.04 ± 13.59 vs. 67.96 ± 17.22, p < 0.01, Risk of oral diseases: 48.77 ± 13.57 vs. 63.44 ± 16.78, p = 0.01, Indication of antibiotic prophylaxis: 75.70 ± 13.45 vs. 87.97 ± 10.37, p < 0.01 and the Medical history task on 15 patient cases: 58.45 ± 4.74 vs. 71.47 ± 9.54, p < 0.01. CONCLUSION: The applied analog anamnesis tool supported an increase in students´ confidence with issues related to at-risk patients alongside with their knowledge in risk classification. The applied anamnesis tool can be recommended for improving teaching of risk management for undergraduate dental students.

STAndard Reporting of CAries Detection and Diagnostic Studies (STARCARDDS).

AIM: The aim of this paper is to present recommendations from an international workshop which evaluated the methodology and reporting of caries diagnostic studies. As a unique feature, this type of studies is focused on caries lesion detection and assessment, and many of them are carried out in vitro, because of the possibility of histological validation of the whole caries spectrum. This feature is not well covered in the existing reporting STARD guideline within the EQUATOR Network. PARTICIPANTS AND METHODS: An international working group of 13 cariology researchers was formed. The STARD checklist was reviewed and modified for caries detection and diagnosis purposes, in a three-step process of evaluation, consensual modification, and delivery during three 2-day workshops over 18 months. Special attention was paid to reporting requirements of caries studies that solely focus on reliability. RESULTS: The STARD checklist was modified in 14/30 items, with an emphasis on issues of sample selection (tooth selection in in vitro studies), blinding, and detailed reporting of results. CONCLUSION: Following STARCARDDS (STAndard Reporting of CAries Detection and Diagnostic Studies) is expected to result in complete reporting of study design and methodology in future caries diagnosis and detection experiments both in vivo and in vitro, thus allowing for better comparability of studies and higher quality of systematic reviews. CLINICAL RELEVANCE: Standardization of caries diagnostic studies leads to a better comparability among future studies, both in vivo and in vitro.

Arrest of root caries with an adjuvant chlorhexidine-fluoride varnish over a 12-months observation period: a QLF-analyzed, placebo-controlled, randomized, clinical trial (RCT).

This study aimed at evaluating the effectiveness of an adjuvant chlorhexidine-fluoride varnish (Cervitec F) for prevention and arrest of root caries on elderly participants using quantitative light-induced fluorescence (QLF). 23 participants with two or three non-cavitated root carious lesions were included and assigned to three groups of different varnishes (CF: Cervitec F, P: placebo, DP: Duraphate). Agents were applied once to root surface at baseline and in follow-up after 3, 6 and 9 months. The lesions were assessed clinically and with QLF. QLF-images were analyzed regarding fluorescence loss (ΔF), lesion volume (ΔQ) and bacterial activity (ΔR) before (t0), after 14 days (t1), 6- (t2) and 12-months (t3). CF showed a significant difference between t0 and t3: ∆F (- 12.51 [15.41] vs. - 7.80 [16.72], p = 0.012), ∆Q (- 2339.97 (20,898.30) vs. - 751.82 (5725.35), p < 0.001), ∆R (23.80 [41.70] vs. 7.07 [37.50], p = 0.006). Independently of the varnish application, preventive care seems positively influence the root caries progress. Although within CF group the strongest effect was observed, no superiority of a specific varnish application was confirmed over a 12-months QLF observation period. Extra topical fluoride can help remineralise dentin lesions and QLF can be used as a measurement method to determine changes in the dentin lesions.

A video- and feedback-based approach to teaching communication skills in undergraduate clinical dental education: The student perspective.

OBJECTIVE: Student evaluation of a communication-education programme that combined patient consultation videos with peer- or expert-based feedback. METHODS: A self-perception questionnaire was given to undergraduate dental students who had undertaken a curricular communication training and feedback programme, in which each participant was videoed three times during a patient interview or consultation, subsequently receiving either peer (PG) or expert feedback (EG). The questionnaire used feedback programme content to evaluate student perspectives, ascertaining whether the students experienced a gain in knowledge and professional conversational skills, whether videos or feedback helped them improve more and general questions about the structure and content of the curriculum. Differences between feedback groups were analysed (Mann-Whitney U test). RESULTS: 45 participants (peer group: n = 23, expert group: n = 22) took part in the study. The peer group experienced watching their video (38%) and receiving feedback (33%) as their major beneficial aspect to improve communication skills (watching fellow student´s video: 17%, providing feedback: 12%). The expert group experienced the major effect in receiving expert feedback (73%, video watching: 27%). The students said that communication skills should be a core competency in dental education learning outcomes (PG: 4.48 ± 0.67, EG: 4.41 ± 0.67; P > 0.05) and that communication curricula should include video-based feedback (PG: 3.91 ± 0.73, EG: 4.00 ± 0.93; P > 0.05). CONCLUSION: Watching videos and both receiving and providing peer group feedback were experienced as helpful in improving students' communication skills. These findings suggest that a longitudinal communication curriculum, which includes both video-based peer feedback and correlated self-reflection, is a promising learning approach for dental education.

Systematic review and meta-analysis of diagnostic methods for occlusal surface caries.

AIM: This systematic review and meta-analysis aimed to assess the diagnostic performance of commonly used methods for occlusal caries diagnostics, such as visual examination (VE), bitewing radiography (BW) and laser fluorescence (LF), in relation to their ability to detect (dentin) caries under clinical and laboratory conditions. MATERIALS AND METHODS: A systematic search of the literature was performed to identify studies meeting the inclusion criteria using the PIRDS concept (N = 1090). A risk of bias (RoB) assessment tool was used for quality evaluation. Reports with low/moderate RoB, well-matching thresholds for index and reference tests and appropriate reporting were included in the meta-analysis (N = 37; 29 in vivo/8 in vitro). The pooled sensitivity (SE), specificity (SP), diagnostic odds ratio (DOR) and areas under ROC curves (AUCs) were computed. RESULTS: SP ranged from 0.50 (fibre-optic transillumination/caries detection level) to 0.97 (conventional BW/dentine detection level) in vitro. AUCs were typically higher for BW or LF than for VE. The highest AUC of 0.89 was observed for VE at the 1/3 dentin caries detection level; SE (0.70) was registered to be higher than SP (0.47) for VE at the caries detection level in vivo. CONCLUSION: The number of included studies was found to be low. This underlines the need for high-quality caries diagnostic studies that further provide data in relation to multiple caries thresholds. CLINICAL RELEVANCE: VE, BW and LF provide acceptable measures for their diagnostic performance on occlusal surfaces, but the results should be interpreted with caution due to the limited data in many categories.

Systematic review and meta-analysis of diagnostic studies of proximal surface caries.

AIM: This systematic review and meta-analysis aimed to assess the diagnostic accuracy and reliability of commonly used caries detection methods for proximal caries diagnostics. Visual examination (VE), bitewing radiography (BWR), laser fluorescence (LF), and fibre-optic transillumination (FOTI) were considered in detail. MATERIAL AND METHODS: PRISMA guidelines for the reporting of systematic reviews and meta-analyses were applied. The mnemonic PIRDS (problem, index test, reference test, diagnostic and study type) concept was used to guide the literature search. Next, studies that met the inclusion criteria were stepwise selected and evaluated for their quality with a risk of bias (RoB) assessment tool. Studies with low/moderate bias and sufficient reporting were considered for meta-analysis. The pooled sensitivity (SE), specificity (SP), diagnostic odds ratio (DOR), and area under the ROC curve (AUC) were calculated. RESULTS: From 129 studies meeting the selection criteria, 31 in vitro studies and five clinical studies were finally included in the meta-analysis. The AUC values for in vitro VE amounted to 0.84 (caries detection) and 0.85 (dentin caries detection). BWR ranged in vitro from 0.55 to 0.82 (caries detection) and 0.81-0.92 (dentin caries detection). LF showed higher AUC values for overall caries detection (0.91) and dentin caries detection (0.83) than did other methods. Clinical data are limited. CONCLUSION: The number of diagnostic studies with low/moderate RoB was found to be low and indicates a need for high-quality, well-designed caries diagnostic studies. CLINICAL RELEVANCE: BWR and LF showed good diagnostic performance on proximal surfaces. However, because of the low number of includable clinical studies, these data should be interpreted with caution.

Evaluation of an educational concept for risk-oriented prevention in undergraduate dental education.

BACKGROUND: Aim of this observational study with a three-month follow-up was to evaluate an educational concept for risk-oriented prevention applied by fifth-year undergraduate dental students. METHODS: Dental students from two clinical treatment courses of the last undergraduate year were included. The subjects were divided into two groups according to their assignment to the two clinical classes. Group A received a sequence of seminars, including the basics of a risk classification system (RCS) with the theoretical background and case studies in the context of preventive dentistry. Thereby, 1) a theoretical seminar (background, RCS, cases) and 2) the transfer of the RCS on a clinical patient case chosen by the student, and its presentation within a discussion round was applied. Group B served as a comparison group with students who did not receive any of teaching events in terms of RCS. The self-perceived knowledge and importance of RCS, as well as objective knowledge (qualitative questions), were assessed with a standardized questionnaire at baseline and after 3 months. RESULTS: Out of 90 students at baseline, 79 (group A: 39, group B: 40) were re-evaluated after 3 months. At this follow-up, Group A estimated their confidence in handling the medication (p = 0.02), the RCS (p < 0.01), and in identifying the risk of oral diseases (p = 0.02) higher than group B. Furthermore, group A felt it was more important to identify patients at risk (p = 0.02), the risk of complications (p = 0.02) and to apply an RCS (p = 0.03). At follow-up, group A exhibited more correct answers of qualitative questions than group B regarding risk of complications (p < 0.01) and bacteremia (p < 0.01). Group A felt more confident with at-risk patients and more competent concerning RCS than group B (p < 0.01). CONCLUSION: The concept for educating risk-oriented prevention increased the self-perceived skills and the knowledge of undergraduate dental students after 3 months within a clinical treatment course.

Visualization of the pulp chamber roof and residual dentin thickness by spectral-domain optical coherence tomography in vitro.

The aim of this study was to evaluate the ability of spectral-domain optical coherence tomography (SD-OCT) to display the roof of the pulp chamber and to estimate the residual dentin thickness (RDT) of the pulp complex. The roots of 20 extracted human molars were embedded in epoxy resin, and crowns were longitudinally sectioned in the mesial-distal direction, exposing the pulp chamber. The coronal part of the crown was removed up to an RDT to the pulp chamber roof of 2 mm. Samples were imaged by SD-OCT from coronal view and by light microscopy (LM) in the sagittal plane. Using a microtome, dentin was subsequently removed in four levels from the occlusal aspect in steps of 250 µm. At each level, RDT was documented and measured by both methods. The data were compared (Spearman's rho correlation coefficient, Wilcoxon signed-rank test). Using OCT, the roof of the pulp chamber was first displayed at a maximum RDT of 1.94 mm. The minimal RDT that could be imaged by OCT was 0.06 mm. Values from both methods were strongly correlated (r, 0.83-0.95; pi ≤ 0.05) and differed significantly for large RDTs (dentin levels 1, 2; pi < 0.05) but not for small RDTs (levels 3, 4; pi ≥ 0.226). The roof of the dental pulp chamber could be already visualized by SD-OCT with a RDT of 1.94 mm. Therefore, the method could be a useful diagnostic tool during the preparation of deep dentin cavities and might help to preserve the integrity of the pulp chamber.

Correction to: Internal and marginal adaptation of high-viscosity bulk-fill composites in class II cavities placed with different adhesive strategies.

In the original publication of the article the paragraph "The four resin-based composites…" appearing below the "Materials and methods" section is incomplete. The correct paragraph is provided in this correction below.

Internal and marginal adaptation of high-viscosity bulk-fill composites in class II cavities placed with different adhesive strategies.

The aim of this study was to evaluate internal and marginal adaptation of high-viscosity bulk-fill composites to enamel and dentin with a self-etch (SE) and an etch-and-rinse adhesive (ER) without and with artificial aging. 128 MOD cavities in extracted human molars were prepared, randomly assigned to 8 groups (n = 16), bonded with either OptiBond FL (OFL, ER) or Xeno V+ (X, SE), and restored in 4 mm horizontal bulk layers with SonicFill (SF), Tetric EvoCeram Bulk fill (TEC), and x-tra fil (XF). Incremental layering technique with Premise (P) served as control. Half of the specimens each (n = 8) were subjected either to water storage (1 day, 37 °C) or prolonged storage (180 days) and thereafter thermocycling. Replicas were analyzed for marginal gap formation. After sectioning, interfacial adhesive defects were assessed. Results were statistical analyzed. (1) Internal adaptation: Except for TEC/OFL at enamel without artificial aging, no significant differences between bulk-fill composites and the control were observed. All groups at dentin with OFL showed less internal adhesive defects than that with X (p < 0.05). (2) Marginal adaptation: No significant differences were observed between bulk-fill composites and control except for P after artificial aging (p > 0.05). All other composites, regardless of artificial aging, formed significantly more marginal gaps at enamel with X compared to with OFL (p ≤ 0.05). Simplified restorations with high-viscosity bulk-fill composite showed comparable internal and marginal adaptation to incrementally placed fillings. A superiority of the 3-step ER approach was seen compared to the 1-step SE adhesive irrespective of the bulk-fill composite used.

Optical coherence tomography to evaluate variance in the extent of carious lesions in depth.

Evaluation of variance in the extent of carious lesions in depth at smooth surfaces within the same ICDAS code group using optical coherence tomography (OCT) in vitro and in vivo. (1) Verification/validation of OCT to assess non-cavitated caries: 13 human molars with ICDAS code 2 at smooth surfaces were imaged using OCT and light microscopy. Regions of interest (ROI) were categorized according to the depth of carious lesions. Agreement between histology and OCT was determined by unweighted Cohen's Kappa and Wilcoxon test. (2) Assessment of 133 smooth surfaces using ICDAS and OCT in vitro, 49 surfaces in vivo. ROI were categorized according to the caries extent (ICDAS: codes 0-4, OCT: scoring based on lesion depth). A frequency distribution of the OCT scores for each ICDAS code was determined. (1) Histology and OCT agreed moderately (κ = 0.54, p ≤ 0.001) with no significant difference between both methods (p = 0.25). The lesions (76.9% (10 of 13)) _were equally scored. (2) In vitro, OCT revealed caries in 42% of ROI clinically assessed as sound. OCT detected dentin-caries in 40% of ROIs visually assessed as enamel-caries. In vivo, large differences between ICDAS and OCT were observed. Carious lesions of ICDAS codes 1 and 2 vary largely in their extent in depth.

No difference between manual and different power toothbrushes with and without specific instructions in young, oral healthy adults-results of a randomized clinical trial.

BACKGROUND: The aim of this randomized clinical study was to detect the effect of an instruction within a group using oscillating-rotating (OR), sonic-active (SA), or manual toothbrushes (MTB) in young, oral healthy adults. METHODS: One hundred fifty participants were randomly assigned into six groups (n = 25): with (OR-I, SA-I, MTB-I) and without instruction (OR-NI, SA-NI, MTB-NI). Participants in I subgroups received one standardized instruction of the toothbrush system. At baseline (t0), after 2 (t1), 4 (t2), and 12 weeks (t3), plaque indices including modified Quigley-Hein Index (QHI) and Marginal Plaque Index (MPI) as well as inflammation indices including Papilla Bleeding Index (PBI) and Gingival Index (GI) were assessed. Kruskal-Wallis test, Friedman test, and chi-square or Fisher's exact test (p < 0.05) were used for statistical analysis. RESULTS: One hundred thirty-one participants completed the follow-up and were analyzed: OR-I = 21, OR-NI = 22, SA-I = 22, SA-NI = 22, MTB-I = 22, and MTB-NI = 22. Within groups between t0 and t3, OR and SA systems showed a significant plaque reduction, irrespective of instruction (p i < 0.05). In MTB-I and in SA-NI subgroups, a reduction of GI was detected, while an improvement in PBI within SA-I was found (p i < 0.05). Thereby, after 12 weeks, gingival inflammation and plaque indices were comparable between all subgroups (p i > 0.05). Irrespective of the toothbrush system used, only QHI was positively influenced by instruction (p < 0.01). CONCLUSION: The used toothbrush as well as the presence or absence of a single brush-specific instruction has no influence on plaque removal and reduction of gingival inflammation in young, oral healthy adults in an observation period of 12 weeks. CLINICAL RELEVANCE: A single instruction might bring no benefit in this patient group, independently of the used toothbrush system.

Correction to: No difference between manual and different power toothbrushes with and without specific instructions in young, oral healthy adults-results of a randomized clinical trial.

The type of used rotating-oscillating toothbrush was incorrectly assigned; correct used rotating-oscillating toothbrush is Pro1000 Precision Clean, Procter&Gamble GmbH, Schwalbach, Germany.

Evaluation of Plasmatic Kisspetin-10 as a Biomarker for Malignancy and Subtype Differentiation in Small Renal Tumours.

OBJECTIVE: To evaluate Kisspeptin-10 (Kiss-10) in patients with small renal tumours (SRTs) and controls. MATERIAL AND METHODS: Kiss-10 was measured in preoperative plasma samples in a cohort of 143 patients with unilateral renal tumours smaller than or equal to 4 cm and 40 age-matched controls by a competitive ELISA test kit. The cohort of patients included 56 patients with clear cell renal cell carcinoma (ccRCC), 43 with papillary RCC (pRCC), 12 with chromophobe RCC (chRCC) and 32 with oncocytomas. RESULTS: Kiss-10 was detected in all patients and controls. SRT patients revealed significantly higher Kiss-10 levels than controls (mean value 10.04 vs. 6.37 pmol/l, p < 0.001). In SRT patients, Kiss-10 was detected at significantly different concentrations between the subgroups (p = 0.021). The highest concentration was observed in those with oncocytomas (11.50 pmol/) followed by chRCC, pRCC and ccRCC patients (9.89, 10.01 and 9.25 pmol/l, respectively). Receiver operating characteristic curve analyses revealed an area under the curve (AUC) of 0.82 for the comparison of all tumours vs. controls (p < 0.001) and an AUC of 0.671 for all malignant tumours vs. oncoytomas (p = 0.003). CONCLUSION: This study shows that Kiss-10 levels are significantly altered by malignancy and tumour subtypes even in patients with SRTs. Kiss-10 therefore deserves further attention as a plasmatic biomarker for renal tumours.

Use of AC impedance spectroscopy for monitoring sound teeth and incipient carious lesions.

OBJECTIVE: The objective of this study was to assess the ability of alternating current impedance spectroscopy technique (ACIST) to monitor sound tooth surfaces and incipient carious lesions. SUBJECTS AND METHODS: Two hundred ninety-two teeth were examined in 30 patients. Occlusal surfaces were classified according to International Caries Detection and Assessment System (ICDAS). Two consecutive ACIST measurements at the investigation sites already visually examined were taken (t1). Examinations were repeated after 6 (t2) and 12 months (t3). Reproducibility of ACIST findings was calculated with the intra-class correlation coefficient. Values of the ACIST measurements were categorized, and kappa values were calculated. Spearman correlation coefficients (r s) were calculated for correlations between ICDAS findings and ACIST measurements. To test whether ACIST detected changes similarly to ICDAS, Wilcoxon's test was used (α = 0.05). RESULTS: Intra-class correlation coefficient values of ACIST measurements ranged between 0.88 and 0.98. Kappa values for ACIST categories were 0.66-0.80. Rank correlation coefficient of ICDAS and ACIST readings was 0.38-0.65 at different time intervals (p < 0.01). Significant differences could be shown for ICDAS findings between t1/t2 (p = 0.001), t2/t3 (p = 0.021), and over the total duration of the study (t1/t3, p < 0.001). No significant differences between the various examination periods were found for the impedance measurements (p > 0.05). CONCLUSIONS: ACIST exhibited in vivo high reproducibility but moderate correlation to visual findings at each time of examination. CLINICAL RELEVANCE: ACIST can be used for monitoring sound teeth and early carious lesions although its suitability as a single detection method is limited since not all changes could be detected with respect to visual findings.

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells.

OBJECTIVES: To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. RESULTS: Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. CONCLUSION: Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

Effect of simulated pulpal fluid circulation on intrapulpal temperature following irradiation with an Nd:YVO4 laser.

It is suggested that pulpal fluid circulation has an impact on pulp temperature increase during heat-generating dental treatment procedures. Thus, the aim of the study was to assess the effect of a simulated pulpal fluid circulation on temperature changes inside the pulp chamber following laser irradiation of the tooth surface. Twenty freshly extracted human multirooted teeth were included and cross-sectioned along the long axis exposing two root canals each. The pulp chamber and root canals were cleaned from remaining soft tissues to achieve access for a temperature sensor and two cannulas to allow fluid circulation. Cross sections were glued together, and the roots were encased with silicone impression material to ensure the position of the connected devices. Each tooth was irradiated by employing a neodymium-doped yttrium orthovanadate (Nd:YVO4) laser at 1,064 nm with a pulse duration of 9 ps and a repetition rate of 500 kHz. A commercially available scanning system (SCANcube 7, SCANLAB) deflected the beam by providing rectangular irradiated areas of 0.5 mm edge length. Measurements were performed with four different settings for fluid circulation: without any water and with water (23 °C) at a flow rate of 6, 3, and 0 ml/min. The primary outcome measure was the maximum temperature difference (ΔT) after laser irradiation. Highest temperature changes (median 3.6 K, range 0.5-7.1 K) could be observed without any fluid inside the pulp chamber. Water without circulation decreased ΔT values statistically significantly (median 1.4 K, range 0.2-4.9 K) (p < 0.05). Lowest temperature changes could be observed with a water flow rate of 6 ml/min (median 0.8 K, range 0.2-3.7 K) (p < 0.05). Pulpal fluid circulation has a cooling effect on temperature increase caused by laser irradiation of dental hard tissues. Studies on heat generation during dental treatment procedures should include this aspect to assess a potential thermal injury of pulp tissue.