An in vitro coculture approach to study the interplay between dental pulp cells and Streptococcus mutans.

AIM: To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. METHODOLOGY: Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-ß1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = .05). RESULTS: While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-ß1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. CONCLUSIONS: The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

Treatment of pulpal and apical disease: The European Society of Endodontology (ESE) S3-level clinical practice guideline.

BACKGROUND: The ESE previously published quality guidelines for endodontic treatment in 2006; however, there have been significant changes since not only in clinical endodontics but also in consensus and guideline development processes. In the development of the inaugural S3-level clinical practice guidelines (CPG), a comprehensive systematic and methodologically robust guideline consultation process was followed in order to produce evidence-based recommendations for the management of patients presenting with pulpal and apical disease. AIM: To develop an S3-level CPG for the treatment of pulpal and apical disease, focusing on diagnosis and the implementation of the treatment approaches required to manage patients presenting with pulpitis and apical periodontitis (AP) with the ultimate goal of preventing tooth loss. METHODS: This S3-level CPG was developed by the ESE, with the assistance of independent methodological guidance provided by the Association of Scientific Medical Societies in Germany and utilizing the GRADE process. A robust, rigorous and transparent process included the analysis of relevant comparative research in 14 specifically commissioned systematic reviews, prior to evaluation of the quality and strength of evidence, the formulation of specific evidence and expert-based recommendations in a structured consensus process with leading endodontic experts and a broad base of external stakeholders. RESULTS: The S3-level CPG for the treatment of pulpal and apical disease describes in a series of clinical recommendations the effectiveness of diagnosing pulpitis and AP, prior to investigating the effectiveness of endodontic treatments in managing those diseases. Therapeutic strategies include the effectiveness of deep caries management in cases with, and without, spontaneous pain and pulp exposure, vital versus nonvital teeth, the effectiveness of root canal instrumentation, irrigation, dressing, root canal filling materials and adjunct intracanal procedures in the management of AP. Prior to treatment planning, the critical importance of history and case evaluation, aseptic techniques, appropriate training and re-evaluations during and after treatment is stressed. CONCLUSION: The first S3-level CPG in endodontics informs clinical practice, health systems, policymakers, other stakeholders and patients on the available and most effective treatments to manage patients with pulpitis and AP in order to preserve teeth over a patient's lifetime, according to the best comparative evidence currently available.

Implications of root, pulp chamber, and canal anatomy on pulpotomy and revitalization procedures.

OBJECTIVES: This review aims to discuss the implications of anatomy of the root, pulp chamber, and canals on pulpotomy and revitalization procedures (RPs) as treatment alternatives to root canal treatment procedures. METHODS: This narrative review was undertaken to address two main questions - why remove vital pulp tissue in teeth with complex canal anatomy when it can be preserved? And why replace the necrotic pulp in teeth with mature roots with a synthetic material when we can revitalize? This review also aims to discuss anatomical challenges with pulpotomy and revitalization procedures. RESULTS: Maintaining the vitality of the pulp via partial or full pulpotomy procedures avoids the multiple potential challenges faced by clinicians during root canal treatment. However, carrying out pulpotomy procedures requires a meticulous understanding of the pulp chamber anatomy, which varies from tooth to tooth. Literature shows an increased interest in the application of RPs in teeth with mature roots; however, to date, the relation between the complexity of the root canal system and outcomes of RPs in necrotic multi-rooted teeth with mature roots is unclear and requires further robust comparative research and long-term follow-up. CONCLUSIONS: Whenever indicated, pulpotomy procedures are viable treatment options for vital teeth with mature roots; however, comparative, adequately powered studies with long-term follow-up are needed as a priority in this area. RPs show promising outcomes for necrotic teeth with mature roots that warrant more evidence in different tooth types with long-term follow-ups.  CLINICAL RELEVANCE: Clinicians should be aware of the pulp chamber anatomy, which is subject to morphological changes by age or as a defensive mechanism against microbial irritation, before practicing partial and full pulpotomy procedures. RP is a promising treatment option for teeth with immature roots, but more evidence is needed for its applications in teeth with mature roots. A universal consensus and considerably more robust evidence are needed for the standardization of RPs in teeth with mature roots.

Engineering the Future of Dental Health: Exploring Molecular Advancements in Dental Pulp Regeneration.

Protected by the surrounding mineralized barriers of enamel, dentin, and cementum, dental pulp is a functionally versatile tissue that fulfills multiple roles [...].

Present status and future directions: Endodontic management of traumatic injuries to permanent teeth.

The prognosis of traumatized teeth depends largely on the fate of the pulp and its treatment. This review aims to update the present status on the endodontic management of traumatic injuries to permanent teeth and to identify relevant research areas that could contribute to an improvement in diagnosis and treatment of traumatized permanent teeth. Future research should pay greater attention to (1) diagnostic methods to assess the perfusion of the pulp and enhance detection of tooth cracks and initial signs of root resorption; (2) improved materials for vital pulp treatment; (3) studies focusing on type and duration of splinting after root fractures; (4) antiresorptive intracanal medication in case of posttraumatic pulp necrosis and infection-related resorption and (5) long-term data on the apical barrier technique compared to revitalization.

A critical analysis of clinical research methods to study regenerative endodontics.

Regenerative endodontic treatment such as revitalization provides a treatment option for immature teeth with pulp necrosis. The main difference to the alternative procedure, the apical plug, is the induction of a blood clot inside the canal as a scaffold for healing and new tissue formation. Due to the biology-based and minimally-invasive nature of the treatment, revitalization has raised considerable interest in recent years. Whereas the procedure is fairly new and recommendations from endodontic societies have been in place only for a few years, the treatment protocol has evolved over the past two decades. Evidence has been created, not only from laboratory and animal work, but also from clinical studies including case reports, cohort studies and eventually prospective randomized controlled clinical trials, systematic reviews and meta-analyses. However, the research methods and clinical studies with subsequent reports oftentimes present with methodical limitations, which makes it difficult to objectively assess the value of this treatment modality. Several open questions remain, including the need for a more differentiated indication of revitalization after different traumatic injuries, the long-term prognosis of treated teeth and the true benefits for the patient. Therefore, this review aims to identify and reflect on such limitations, scrutinizing study design, diagnostic tools, procedural details and outcome parameters. A core outcome set is also proposed in this context, which can be considered in future clinical investigations. These considerations may lead to a more detailed and stringent planning and execution of future studies in order to create high-quality evidence for the treatment modality of revitalization and thus provide more robust data, create a larger body of knowledge for clinicians and further specify current recommendations.

Impact of access cavity cleaning on the seal of postendodontic composite restorations in vitro.

AIM: The aim of the study was to investigate the influence of cavity cleaning and conditioning on marginal integrity of directly placed post-endodontic composite class-I-restorations in vitro. METHODOLOGY: A total of 168 fully intact teeth without caries or fillings received pre-endodontic composite restorations (class-II) after their extraction. Occlusal endodontic access-cavities were prepared, and root canals were instrumented and filled with gutta-percha and an epoxy resin-based sealer. Prior to post-endodontic class-I-restoration, access cavities were completely contaminated with sealer, cleaned with alcohol and pre-treated as follows: cleaner only (alcohol), glycine-polishing, Al2 O3 sandblasting, carbide bur (immediate as well as delayed restoration). A positive control (not contaminated with sealer and adhesive used) and negative control (cleaner used but no adhesive) were established. Half of the teeth from each group were subjected to thermocycling and mechanical loading (TCML). Marginal integrity of post-endodontic restoration was evaluated in oro-vestibular or mesio-distal sections after AgNO3 dye penetration (DP) by standardized photomacroscopic imaging and expressed in per cent of margin length along all segments and separately for enamel, dentine and composite, respectively. Results were analysed non-parametrically (α = .05). RESULTS: No restorations or teeth fractured or debonded completely. Without TCML, the median DP of all segments was significantly higher for the negative control compared with all other groups in oro-vestibular cutting direction (53%; p = .002) and in mesio-distal cutting direction (51%; p ≤ .041). The other groups without TCML revealed 16%-24% DP (oro-vestibular) and 12%-24% DP (mesio-distal). With TCML, the median DP in oro-vestibular cutting direction for all segments ranged between 48% and 62% for all groups, a significant difference was only observed between glycine-polishing and carbide bur (p = .041). In mesio-distal cutting direction, the median DP in negative control was 69% with TCML and significantly higher compared with all other groups (p = .002). For all other groups, the median DP of all segments ranged between 28% and 40% with TCML without significant differences. Error rates method (k = 7) revealed a significant influence of TCML in general on penetration of all segments in both oro-vestibular and mesio-distal cutting directions. CONCLUSION: Additional access cavity pre-treatment after alcohol cleaning did not improve the marginal integrity of post-endodontic composite restorations. Thorough cleaning of the access cavity with alcohol seems to assure an acceptable marginal integrity to the tooth and restorative composite.

Advances in scaffolds used for pulp-dentine complex tissue engineering: A narrative review.

BACKGROUND: Pulp-dentine complex regeneration via tissue engineering is a developing treatment modality that aims to replace necrotic pulps with newly formed healthy tissue inside the root canal. Designing and fabricating an appropriate scaffold is a crucial step in such a treatment. OBJECTIVES: The present study aimed to review recent advances in the design and fabrication of scaffolds for de novo regeneration of pulp-dentine complexes via tissue engineering approaches. METHODS: A literature search was conducted using PubMed, Europe PMC, Scopus and Google Scholar databases. To highlight bioengineering techniques for de novo regeneration of pulp-dentine complexes, both in vitro and in vivo studies were included, and clinical studies were excluded. RESULTS: In the present review, four main classes of scaffolds used to engineer pulp-dentine complexes, including bioceramic-based scaffolds, synthetic polymer-based scaffolds, natural polymer-based scaffolds and composite scaffolds, are covered. Additionally, recent advances in the design, fabrication and application of such scaffolds are analysed along with their advantages and limitations. Finally, the importance of vascular network establishment in the success of pulp-dentine complex regeneration and strategies used to create scaffolds to address this challenge are discussed. DISCUSSION: In the tissue engineering platform, scaffolds provide structural support for cells to adhere and proliferate and also regulate cell differentiation and metabolism. Up to now, considerable progress has been achieved in the field of pulp-dentine complex tissue engineering, and a spectrum of scaffolds ranging from bioceramic-based to naturally derived scaffolds has been fabricated. However, in designing a suitable scaffold for engineering pulp-dentine complexes, a variety of characteristic parameters related to biological, structural, physical and chemical features should be considered. CONCLUSION: The variety of biomaterials and fabrication techniques provides a great opportunity to address some of the requirements for scaffolds in regenerative endodontics. However, more studies are required to develop an ideal scaffold for use in a clinical setting.

A Compilation of Study Models for Dental Pulp Regeneration.

Efforts to heal damaged pulp tissue through tissue engineering have produced positive results in pilot trials. However, the differentiation between real regeneration and mere repair is not possible through clinical measures. Therefore, preclinical study models are still of great importance, both to gain insights into treatment outcomes on tissue and cell levels and to develop further concepts for dental pulp regeneration. This review aims at compiling information about different in vitro and in vivo ectopic, semiorthotopic, and orthotopic models. In this context, the differences between monolayer and three-dimensional cell cultures are discussed, a semiorthotopic transplantation model is introduced as an in vivo model for dental pulp regeneration, and finally, different animal models used for in vivo orthotopic investigations are presented.

Molecular Biological Comparison of Dental Pulp- and Apical Papilla-Derived Stem Cells.

Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering.

Human Amnion Epithelial Cells: A Potential Cell Source for Pulp Regeneration?

The aim of this study was to analyze the suitability of pluripotent stem cells derived from the amnion (hAECs) as a potential cell source for revitalization in vitro. hAECs were isolated from human placentas, and dental pulp stem cells (hDPSCs) and dentin matrix proteins (eDMPs) were obtained from human teeth. Both hAECs and hDPSCs were cultured with 10% FBS, eDMPs and an osteogenic differentiation medium (StemPro). Viability was assessed by MTT and cell adherence to dentin was evaluated by scanning electron microscopy. Furthermore, the expression of mineralization-, odontogenic differentiation- and epithelial-mesenchymal transition-associated genes was analyzed by quantitative real-time PCR, and mineralization was evaluated through Alizarin Red staining. The viability of hAECs was significantly lower compared with hDPSCs in all groups and at all time points. Both hAECs and hDPSCs adhered to dentin and were homogeneously distributed. The regulation of odontoblast differentiation- and mineralization-associated genes showed the lack of transition of hAECs into an odontoblastic phenotype; however, genes associated with epithelial-mesenchymal transition were significantly upregulated in hAECs. hAECs showed small amounts of calcium deposition after osteogenic differentiation with StemPro. Pluripotent hAECs adhere on dentin and possess the capacity to mineralize. However, they presented an unfavorable proliferation behavior and failed to undergo odontoblastic transition.

Inflammatory Response Mechanisms of the Dentine-Pulp Complex and the Periapical Tissues.

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.

Inflammation in the Human Periodontium Induces Downregulation of the α1- and ß1-Subunits of the sGC in Cementoclasts.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α1- and ß1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α1- and ß1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α1- and ß1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

Pathophysiological mechanisms of root resorption after dental trauma: a systematic scoping review.

BACKGROUND: The objective of this scoping review was to systematically explore the current knowledge of cellular and molecular processes that drive and control trauma-associated root resorption, to identify research gaps and to provide a basis for improved prevention and therapy. METHODS: Four major bibliographic databases were searched according to the research question up to February 2021 and supplemented manually. Reports on physiologic, histologic, anatomic and clinical aspects of root resorption following dental trauma were included. Duplicates were removed, the collected material was screened by title/abstract and assessed for eligibility based on the full text. Relevant aspects were extracted, organized and summarized. RESULTS: 846 papers were identified as relevant for a qualitative summary. Consideration of pathophysiological mechanisms concerning trauma-related root resorption in the literature is sparse. Whereas some forms of resorption have been explored thoroughly, the etiology of others, particularly invasive cervical resorption, is still under debate, resulting in inadequate diagnostics and heterogeneous clinical recommendations. Effective therapies for progressive replacement resorptions have not been established. Whereas the discovery of the RANKL/RANK/OPG system is essential to our understanding of resorptive processes, many questions regarding the functional regulation of osteo-/odontoclasts remain unanswered. CONCLUSIONS: This scoping review provides an overview of existing evidence, but also identifies knowledge gaps that need to be addressed by continued laboratory and clinical research.

Labile heme impairs hepatic microcirculation and promotes hepatic injury.

Sepsis is a life-threatening clinical syndrome defined as a deregulated host response to infection associated with organ dysfunction. Mechanisms underlying the pathophysiology of septic liver dysfunction are incompletely understood. Among others, the iron containing tetrapyrrole heme inflicts hepatic damage when released into the circulation during systemic inflammation and sepsis. Accordingly, hemolysis and decreased concentrations of heme-scavenging proteins coincide with an unfavorable outcome of critically ill patients. As the liver is a key organ in heme metabolism and host response to infection, we investigated the impact of labile heme on sinusoidal microcirculation and hepatocellular integrity. We here provide experimental evidence that heme increases portal pressure via a mechanism that involves hepatic stellate cell-mediated sinusoidal constriction, a hallmark of microcirculatory failure under stress conditions. Moreover, heme exerts direct cytotoxicity in vitro and aggravates tissue damage in a model of polymicrobial sepsis. Heme binding by albumin, a low-affinity but high-capacity heme scavenger, attenuates heme-mediated vasoconstriction in vivo and prevents heme-mediated cytotoxicity in vitro. We demonstrate that fractions of serum albumin-bound labile heme are increased in septic patients. We propose that heme scavenging might be used therapeutically to maintain hepatic microcirculation and organ function in sepsis.

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli.

BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

Hepatic Vitamin A Content Investigation Using Coherent Anti-Stokes Raman Scattering Microscopy.

Standard techniques for examining the distribution of vitamin A in liver either require staining or lead to rapid photobleaching of the molecule. A potentially better alternative approach is to use coherent anti-Stokes Raman scattering (CARS) microscopy; a fast, label-free, non-disruptive imaging method that provides contrast based on molecular vibrations. This contribution evaluates the viability of CARS microscopy for imaging vitamin A within thick hepatic tissue under physiological conditions by tuning into its characteristic vibrational band in the fingerprint region. Additional information about the morphology and architecture of the tissue was acquired using second harmonic generation (SHG) and multi-photon excited fluorescence (MPEF) to help mapping the intra-lobular positions of the vitamin A droplets. We demonstrate the capability of our multimodal imaging framework to selectively image lipid-soluble vitamin A droplets deep in bulk liver tissue with a high contrast while co-registering a complementary morphological background that clearly visualizes hepatic lobules. The results obtained envisage the good prospect of the technique for in vivo studies assessing vitamin A distribution heterogeneity and how it is affected by the progression of hepatic diseases.

Hepatic cirrhosis and recovery as reflected by Raman spectroscopy: information revealed by statistical analysis might lead to a prognostic biomarker.

Hepatic cirrhosis is a severe chronic disease of the liver accompanied by massive changes in the physiology of the cells constituting the hepatic tissue. Success or failing of a therapeutic effort is difficult to recognize because of its late manifestation in the tissue morphology. In this study, the complex course of hepatic cirrhosis and its regression is followed in a rodent carbon tetrachloride model. Raman spectroscopy, which senses molecular vibrations and reflects the molecular composition of a sample, was applied to gain label- and destruction-free insights into the process of cirrhosis and to conclude on the hepatic disease state on the cellular level. Hematoxylin and eosin staining and immunofluorescence labeling were used to obtain complementary information. False color images derived from maps of Raman spectra by spectral unmixing revealed individual nucleus positions giving structural information. Spectral data unraveled chemical changes associated with liver damage on the cellular level. Upon carbon tetrachloride treatment, a higher lipid content and the presence of catabolic products indicated cirrhosis in tissue samples. Furthermore, 77 % of the Raman spectra recorded from treated rats were classified as diseased, whereas 96 % of the Raman spectra recorded from untreated rats were classified as healthy. Importantly, samples from rats that experienced a recovery period revealed a chemical composition highly similar to the ones from healthy rats while morphologically clear signs of tissue damage were still obvious. Eighty-three percent of their Raman spectra were classified as healthy. The vibrational fingerprint of tissue provides characteristic information that might serve as prognostic biomarker. Graphical Abstract Images of hematoxylin and eosin stained tissue slices together with an average Raman spectrum belonging to healthy and cirrhotic rat liver, respectively, visualize morphological and spectral differences between the two states of the liver.

Role of the Chemical Environment beyond the Coordination Site: Structural Insight into Fe(III) Protoporphyrin Binding to Cysteine-Based Heme-Regulatory Protein Motifs.

The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme-protein interactions is still missing. We report an in-depth structural analysis of Fe(III) heme-peptide complexes by a combination of UV/Vis, resonance Raman, and 2D-NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine-based peptides display different heme-binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme-unbound state. Thus, the heme-binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme-regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.

Making a big thing of a small cell--recent advances in single cell analysis.

Single cell analysis is an emerging field requiring a high level interdisciplinary collaboration to provide detailed insights into the complex organisation, function and heterogeneity of life. This review is addressed to life science researchers as well as researchers developing novel technologies. It covers all aspects of the characterisation of single cells (with a special focus on mammalian cells) from morphology to genetics and different omics-techniques to physiological, mechanical and electrical methods. In recent years, tremendous advances have been achieved in all fields of single cell analysis: (1) improved spatial and temporal resolution of imaging techniques to enable the tracking of single molecule dynamics within single cells; (2) increased throughput to reveal unexpected heterogeneity between different individual cells raising the question what characterizes a cell type and what is just natural biological variation; and (3) emerging multimodal approaches trying to bring together information from complementary techniques paving the way for a deeper understanding of the complexity of biological processes. This review also covers the first successful translations of single cell analysis methods to diagnostic applications in the field of tumour research (especially circulating tumour cells), regenerative medicine, drug discovery and immunology.