Intradermal Indocyanine Green for In Vivo Fluorescence Laser Scanning Microscopy of Human Skin: A Pilot Study

BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders

Evidence-based nanoscopic and molecular framework for excipient functionality in compressed orally disintegrating tablets

The work investigates the adhesive/cohesive molecular and physical interactions together with nanoscopic features of commonly used orally disintegrating tablet (ODT) excipients microcrystalline cellulose (MCC) and D-mannitol. This helps to elucidate the underlying physico-chemical and mechanical mechanisms responsible for powder densification and optimum product functionality. Atomic force microscopy (AFM) contact mode analysis was performed to measure nano-adhesion forces and surface energies between excipient-drug particles (6-10 different particles per each pair). Moreover, surface topography images (100 nm2-10 μm2) and roughness data were acquired from AFM tapping mode. AFM data were related to ODT macro/microscopic properties obtained from SEM, FTIR, XRD, thermal analysis using DSC and TGA, disintegration testing, Heckel and tabletability profiles. The study results showed a good association between the adhesive molecular and physical forces of paired particles and the resultant densification mechanisms responsible for mechanical strength of tablets. MCC micro roughness was 3 times that of D-mannitol which explains the high hardness of MCC ODTs due to mechanical interlocking. Hydrogen bonding between MCC particles could not be established from both AFM and FTIR solid state investigation. On the contrary, D-mannitol produced fragile ODTs due to fragmentation of surface crystallites during compression attained from its weak crystal structure. Furthermore, AFM analysis has shown the presence of extensive micro fibril structures inhabiting nano pores which further supports the use of MCC as a disintegrant. Overall, excipients (and model drugs) showed mechanistic behaviour on the nano/micro scale that could be related to the functionality of materials on the macro scale. © 2014 Al-khattawi et al

Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada

Background: Biomineralization processes have recently been applied in situ to protect and consolidate decayed ornamental stone of the Royal Chapel in Granada (Spain). While this promising method has demonstrated its efficacy regarding strengthening of the stone, little is known about its ecological sustainability.Methodology/Principal Findings: Here, we report molecular monitoring of the stone-autochthonous microbiota before and at 5, 12 and 30 months after the bio-consolidation treatment (medium/long-term monitoring), employing the well-known molecular strategy of DGGE analyses. Before the bio-consolidation treatment, the bacterial diversity showed the exclusive dominance of Actinobacteria (100%), which decreased in the community (44.2%) after 5 months, and Gamma-proteobacteria (30.24%) and Chloroflexi (25.56%) appeared. After 12 months, Gamma-proteobacteria vanished from the community and Cyanobacteria (22.1%) appeared and remained dominant after thirty months, when the microbiota consisted of Actinobacteria (42.2%) and Cyanobacteria (57.8%) only. Fungal diversity showed that the Ascomycota phylum was dominant before treatment (100%), while, after five months, Basidiomycota (6.38%) appeared on the stone, and vanished again after twelve months. Thirty months after the treatment, the fungal population started to stabilize and Ascomycota dominated on the stone (83.33%) once again. Members of green algae (Chlorophyta, Viridiplantae) appeared on the stone at 5, 12 and 30 months after the treatment and accounted for 4.25%, 84.77% and 16.77%, respectively.Conclusions: The results clearly show that, although a temporary shift in the bacterial and fungal diversity was observed during the first five months, most probably promoted by the application of the bio-consolidation treatment, the microbiota tends to regain its initial stability in a few months. Thus, the treatment does not seem to have any negative side effects on the stone-autochthonous microbiota over that time. The molecular strategy employed here is suggested as an efficient monitoring tool to assess the impact on the stone-autochthonous microbiota of the application of biomineralization processes as a restoration/conservation procedure.This work was supported by the European Regional Development Fund (ERDF), Junta de Andalucía (Spain) and the “Fortalecimiento de la I+D+i” program from the University of Granada, co-financed by grant RNM-3493 and Research Group BIO-103 from Junta de Andalucía, as well as by the Spanish Government through “José Castillejo” program from the “Ministerio de Educación, Cultura y Deporte” (I+D+i 2008-2011), and by the Austrian Science Fund (FWF) under Grant “Elise-Richter V194-B20”

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed

Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis

ABSTRACTAutomated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997–1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype l/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE

Impact of atrial fibrillation on the symptoms and echocardiographic evaluation of patients with aortic valve stenosis

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Aarne Koskelo foundation and Helsinki University Hospital EVO funding Introduction Atrial fibrillation (AF) is common among patients with aortic valve stenosis (AS) and can cause similar symptoms. AF may also contribute to diastolic dysfunction. Since AF may alter the echocardiographic profile and provoke symptoms independent of AS, it may complicate the evaluation of AS severity and decision of the time for valve intervention. The purpose of this study was to assess the effect of AF on symptoms and the echocardiographic evaluation of AS. Methods We followed 192 patients (46% female) with suspected severe symptomatic AS who were referred to our institution for intervention. Echocardiography and symptom questionnaires were performed at baseline and 3 months after the intervention. Aortic valve calcium score was measured using computed tomography in the 121 patients considered for transcatheter intervention (TAVR). Results 52 of the 192 patients (27%) had AF. Patients with AF were older (median 81.5 [IQR 74.5-86.3] vs. 74 [64.0-81.3] years) and more often underwent TAVR (81% vs. 54%). In patients with AF the aortic valve mean pressure gradient was lower (39.5 [31.8-48.3] mmHg) than in patients without AF (46 [39.8-52.3] mmHg, p = 0.006, Fig.1A) and a low-gradient phenotype was more common (50% vs. 25% had mean gradient &amp;lt; 40 mmHg). There were no significant differences between groups in AVA, median EF or the proportion of patients with EF &amp;lt;50% (27% vs 17%). The aortic valve calcium score was similar in patients with or without AF (Fig.1B) and, based on the calcium score, 86% (AF) vs. 88% (no AF) of the patients were classified likely or very likely to have severe AS. AF patients had more advanced diastolic dysfunction as demonstrated by higher average E/e’ ratio (15.5 [12.9-20.2] vs. 12.4 [9.90-14.0], p &amp;lt;0.001), left atrial volume index (55.3 [42.0-62.7] vs. 36.7 [32.4-48.7] ml/m2, p &amp;lt;0.001) and tricuspid regurgitation gradient (35.0 [29.0-43.0 vs. 25.0 [23.0-29.0] mmHg, p &amp;lt;0.001). AF patients had worse functional class at baseline (49% vs. 27% with NYHA III or higher, p = 0.01, Fig.2A). There was significant improvement in both groups on follow-up (71% vs 72% improved at least one class), with no difference between groups. Dyspnoea, chest pain and exercise intolerance were equally frequent in patients with and without AF (Fig.2B). After intervention symptoms decreased significantly in both groups. Only exercise intolerance remained more common among AF patients (35% vs. 17%, p = 0.01). Conclusions AS patients with AF present more often with low-gradient phenotype and diastolic dysfunction, which may hamper the evaluation of AS severity. However, calcium score can serve as an alternative tool in the assessment of AS severity particularly in patients with AF and low gradient. Patients with AF are more symptomatic at baseline, but their symptoms improve significantly after intervention and nearly as well as in those without AF. Multiple imaging tools may be needed in decision-making in these patients. Abstract Figure 1  Abstract Figure 2 </jats:sec