An Explorative Study on Calcium Electroporation for Low-risk Basal Cell Carcinoma.

In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.

Investigating the efficacy and safety of calcipotriol/betamethasone dipropionate foam and laser microporation for psoriatic nail disease-A hybrid trial using a smartphone application, optical coherence tomography, and patient-reported outcome measures.

There is a lack of efficacious topical treatments for patients suffering from psoriatic nail disease (PND). We investigated the efficacy of Calcipotriol-Betamethasone Dipropionate (Cal/BD) foam with and without ablative fractional laser (AFL) in patients with PND. A total of 144 nails from 11 patients were treated in a 24-week long, open-label, randomized, intra-patient controlled proof-of-concept hybrid trial. In addition to daily Cal/BD foam application, half of each patient's psoriatic nails were randomized to receive optical coherence tomography (OCT)-guided AFL treatment at baseline, 6-, and 12-week follow-ups. In-clinic assessment (N-NAIL), patient-reported outcomes (PROMs), and drug consumption were supplemented by remote evaluation of 15 subclinical OCT features, smartphone app-based safety monitoring, and photo-based assessment (NAPSI). After 24 weeks of Cal/BD foam treatment, patients achieved a significant improvement (p < 0.001) in both clinical (N-NAIL -76%, NAPSI -68%) and subclinical (OCT -43%) PND severity as well as a 71% reduction in PROMs. AFL-assisted Cal/BD treatment led to higher clinical (N-NAIL -85%, NAPSI -78%) and OCT-assessed (-46%) reduction of PND signs than Cal/BD alone (N-NAIL -66%, NAPSI -58%, OCT -37%), but did not reach statistical significance. Smartphone app images documented adverse events and mild local skin reactions, particularly erythema (75%), laser-induced swelling (28%), and crusting (27%). This hybrid trial demonstrated a reduction in clinical NAPSI and N-NAIL scores, subclinical OCT features, and PROMs, suggesting that Cal/BD foam is a safe and efficacious treatment for PND. Larger trials are warranted to prove the clinical benefit of AFL pretreatment as a Cal/BD delivery enhancer.

Dermatologic Scar Assessment With Stereoscopic Imaging and Digital Three-Dimensional Models: A Validation Study.

BACKGROUND AND OBJECTIVE: We evaluated a new handheld stereoscopic imaging system capable of visualizing scars with digital three-dimensional (3D) models and providing automated morphometric estimates. The objective was to validate the repeatability and accuracy of intra- and inter-investigator scan results. STUDY DESIGN/MATERIALS AND METHODS: Engineered metal plates with depressed and elevated model scars (n = 72) were scanned six times by one investigator. In vivo hypertrophic and atrophic scars (n = 15) were scanned once by three investigators. The repeatability of morphometric estimates was assessed using coefficients of variation (CVs) to compare the variation among multiple scan results for both models and in vivo scars, with 0% reflecting a perfect match. Scar estimates from digital 3D reconstructions were compared with the known dimensions of physical model scars and with ruler measurements of in vivo scars. RESULTS: A total of 48 model scars and 12 in vivo scars were eligible for automated analyses with the imaging system's proprietary software. Intra-investigator scan results for the model scars were repeatable, with low variance for all parameters: volume, area, length, and depth/height (CV: 1.8-3.1%). By comparison, inter-investigator scans of real in vivo scars resulted in slightly higher median CVs (4.4-7.3%; P < 0.05). 3D model scar estimates correlated well with the known physical dimensions of model scars for all parameters (P < 0.001) and accurately reflected the measurements of in vivo scars (P < 0.001). The six in vivo scars situated on the chest and abdomen showed the highest inter-investigator variation, due to respiratory movement artifacts. CONCLUSION: Stereoscopic imaging of scars generates accurate and repeatable measurement estimates that show little intra- and inter-investigator-based assessment variation. The best results are achieved by minimizing subject movement. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

In Vivo Reflectance Confocal Microscopy of Gold Microparticles Deposited in the Skin. A Case Report on Cutaneous Chrysiasis.

Cutaneous chrysiasis is gold deposition in the dermis, described after parenteral administration of gold salts or after topical exposure to gold-containing materials. Gold microparticles (GMPs) have versatile therapeutic effects and are increasingly used in medicine. This case report describes the development of a blue-gray macule following the facial application of GMPs and laser treatment of acne vulgaris. Dermoscopy showed a nonspecific homogenous blue-gray pattern, gradually fading over an 8-month-period. Reflectance confocal microscopy (RCM) detected hyperreflective, subcellular particles in the papillary dermis, localized around hair follicles, eccrine glands, and inside macrophages. Histopathological evaluation, darkfield illumination with hyperspectral imaging, and neutron activation analysis confirmed the presence of GMPs in the dermis. RCM allowed non-invasive fast visualization of aggregates of hyperreflective particles in the dermis and can potentially be used for monitoring localized cutaneous chrysiasis and other metal deposition conditions over time. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Advancement through epidermis using tape stripping technique and Reflectance Confocal Microscopy.

The tape stripping technique is increasingly used in research regarding skin barrier function. However, number of tape strips varies between studies, and literature considering advancement into stratum corneum/epidermis in relation to number of tape strips is scarce. The aim of this pilot study was to assess the advancement through epidermis using tape stripping technique in healthy volunteers. A total of ten healthy volunteers were included. From all volunteers 0, 5, 15 and 35 consecutive tape strips (D-squame) were taken from four adjacent skin areas on the middle volar forearm, followed by Reflectance Confocal Microscopy (RCM) of the four areas to assess epidermal thickness. Squame Scan was used to determine amount of protein removed. Stratum corneum was completely removed in all volunteers after 35 tape strips. Advancement into epidermis was predominantly achieved by the first 15 tape strips, removing 25% of the total epidermis, whereas 35 tape strips removed 33% of epidermis. Protein removal per tape decreased with increasing depth. Information on advancement into the epidermis according to number of tape strips taken, is a significant step forward. The possibility to obtain samples from different layers of epidermis may lead to an improved understanding of skin barrier properties.

Acne Treatment With Light Absorbing Gold Microparticles and Optical Pulses: An Open-Label European Multi-Centered Study in Moderate to Moderately Severe Acne Vulgaris Patients.

BACKGROUND AND OBJECTIVE: Recently, a novel acne treatment based on selective photothermolysis of pilosebaceous units with follicular delivery of inert gold microparticles as an exogenous chromophore and diode laser pulses has been developed. To evaluate the efficacy and safety of a single monotherapy treatment regimen with gold microparticles and diode laser exposure in patients with moderate and moderately severe acne. Further, to evaluate the added benefit of a second treatment regimen combined with pharmaceutical acne treatment in patients with inadequate initial response. MATERIALS AND METHODS: Patients with moderate and moderately severe facial acne were recruited in this open-label, pilot study. A single treatment regimen consisted of three weekly facial treatments with topically applied gold microparticles and diode laser pulses. Outcome measures were the proportion of patients with ≥40% improvement in number of acne lesions (weighted lesion count [WLC]) at 12 weeks (single treatment regimen, primary outcome measure), 24, and 36 weeks from baseline (two treatment regimens), safety, and patient satisfaction. RESULTS: A total of 28 patients were enrolled in the study (18 males, 10 females, 19 patients with moderate acne severity, 9 with moderately severe, mean age: 19.8 years). Twenty-five patients underwent analysis for outcome measures. After a single monotherapy treatment regimen, 76% patients (19/25) achieved ≥40% reduction in WLC (mean WLC reduction: 63%; SD: 13%). Of the patients undergoing two treatment regimens (n = 9 patients), 56% experienced a reduction in acne lesion burden (WLC) ≥40% at 24 weeks and 89% 36 weeks post-baseline. Mean pain score was 4.0 (SD: 1.3), and transient erythema and perifollicular edema were commonly noted after treatment. Most patients (81%) were either "satisfied" or "very satisfied" with the treatment. CONCLUSION: Acne therapy based on selective photothermolysis with gold microparticles shows promise and may be used in treatment of moderate to moderately severe acne. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Oxytocin Effects on Pain Perception and Pain Anticipation.

There is an ongoing debate whether the neuropeptide oxytocin (OT) modulates pain processing in humans. This study differentiates behavioral and neuronal OT effects on pain perception and pain anticipation by using a Pavlovian conditioning paradigm. Forty-six males received intranasally administered OT in a randomized, double-blind, placebo-controlled group design. Although OT exerted no direct effect on perceived pain, OT was found to modulate the blood oxygen level-dependent response in the ventral striatum for painful versus warm unconditioned stimuli and to decrease activity in the anterior insula (IS) with repeated thermal pain stimuli. Regarding pain anticipation, OT increased responses to CSpain versus CSminus in the nucleus accumbens. Furthermore, in the OT condition increased correct expectations, particularly for the most certain conditioned stimuli (CS)-unconditioned stimuli associations (CSminus and CSpain) were found, as well as greatest deactivations in the right posterior IS in response to the least certain condition (CSwarm) with posterior IS activity and correct expectancies being positively correlated. In conclusion, OT seems to have both a direct effect on pain processing via the ventral striatum and by inducing habituation in the anterior IS as well as on pain anticipation by boostering associative learning in general and the neuronal conditioned fear of pain response in particular. PERSPECTIVE: The neuropeptide OT has recently raised the hope to offer a novel avenue for modulating pain experience. This study found OT to modulate pain processing and to facilitate the anticipation of pain, inspiring further research on OT effects on the affective dimension of the pain experience.

Transfollicular delivery of gold microparticles in healthy skin and acne vulgaris, assessed by in vivo reflectance confocal microscopy and optical coherence tomography.

INTRODUCTION: Topical application of gold microparticles (GMPs) for selective photothermolysis is a recently FDA-cleared therapy for acne vulgaris. Current evidence indicates the potential of optical imaging to non-invasively visualize GMPs and describe photothermal tissue effects. OBJECTIVES: To qualitatively and quantitatively describe GMP delivery in vivo and visualize laser-mediated thermal effects of GMPs in facial skin of acne patients and healthy participants, using reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). METHODS: Patients with facial acne (n = 14), and healthy participants (n = 7) were included. RCM and OCT images were acquired at baseline, after GMP application, and after diode laser exposure. All images were evaluated qualitatively and quantitatively with regards to GMP delivery in skin layers and morphological thermal effects. Lastly, skin biopsies were obtained to compare RCM and OCT findings to histology. RESULTS: GMPs were delivered equally in healthy participants and acne patients, and in lesional and non-lesional acne skin. In RCM images, GMPs appeared as hyperreflective aggregates inside hair follicles and eccrine ducts, corresponding to natural skin openings (NSOs). The fraction of NSOs with hyperreflective content increased significantly after GMP application compared to baseline (50-75% increase, P = 8.88 × 10-16 ). Similarly, in OCT images, GMPs appeared as hyperreflective columns inside hair follicles and were not detected in surrounding skin. GMPs reached a maximum depth of 920 µm (median 300 µm). After laser exposure, RCM and histology revealed selective perifollicular tissue changes around NSOs. CONCLUSION: Optical imaging visualizes GMP delivery and thermal tissue response following laser exposure and enables bedside monitoring of transfollicular microparticle delivery. Lasers Surg. Med. 51:430-438, 2019. © 2019 Wiley Periodicals, Inc.

Microneedle fractional radiofrequency-induced micropores evaluated by in vivo reflectance confocal microscopy, optical coherence tomography, and histology.

BACKGROUND: Microneedle fractional radiofrequency (MNRF) is a minimally invasive technique that delivers radiofrequency (RF) energy into the skin via microneedles. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) enable the characterization of device-tissue interactions in in vivo skin. The aim of this study is to describe MNRF-induced micropores using RCM and OCT imaging. MATERIALS AND METHODS: Five healthy participants were treated with a 7 × 7 array of 1500 µm microneedles on two adjacent areas of the right hip. One area received MNRF using high RF energy while the other underwent MNRF at low RF energy. Micropore morphology was evaluated qualitatively and quantitatively with RCM and OCT. To relate imaging with histology, one participant underwent punch biopsy in both areas. RESULTS: Reflectance confocal microscopy visualized shape, content, and thermal-induced coagulation zone (CZ) of MNRF micropores. At high RF energy, micropores showed concentric shape, contained hyperreflective granules, and coagulated tissue from epidermis to dermo-epidermal junction (diameter 63-85 µm). Micropores at low RF energy, presented with a stellate shape, no content and CZs that were visible only in epidermis (CZ thickness 9 µm, IQR 8-21 µm). Evaluating OCT, high RF energy showed deeper (150 µm), more easily identifiable micropores compared to low RF energy micropores (70 µm). Histology showed tissue coagulation to a depth of 1500 µm at high RF energy, while at low RF energy, disruption was only visible in epidermis. CONCLUSION: Microneedle fractional radiofrequency micropores show distinct characteristics in both RCM and OCT, depending on RF energy. These in vivo imaging modalities are complementary and allow combined, qualitative, and quantitative evaluation.

Acne vulgaris severity graded by in vivo reflectance confocal microscopy and optical coherence tomography.

INTRODUCTION: Acne is an inflammatory disease of the pilosebaceous unit, which can be investigated in vivo using reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). OBJECTIVES: By means of RCM and OCT to identify morphological characteristics of acne that may be associated with clinical acne severity. METHODS: Patients with mild to moderate facial acne (n = 14, Investigators Global Assessment scale, IGA 1-3), and healthy participants (n = 7, IGA 0) were included in this explorative study. A total of 108 RCM image blocks and 54 OCT scans (each RCM and OCT image measuring 6 × 6 mm) were captured from lesional-, perilesional, and lesion-free skin areas. Acne lesions, infundibular regions of follicles and inflammation degree were compared in acne patients and healthy participants. RESULTS: Combined use of RCM and OCT demonstrated infundibular morphology, acne lesions, and blood flow. RCM images of perilesional- and lesion-free skin in acne patients revealed follicle infundibula with hyperkeratinized borders and abundant keratin plugs, contrasting skin of healthy participants. Higher acne severity related to increased number of follicles with hyperkeratotic borders (P = 0.04) and keratin plugs (P = 0.006), increased infundibulum diameter (P < 0.001), increased density of inflammatory cells (P < 0.001), and blood flow (P = 0.03). Acne lesion morphology was not associated with acne severity. CONCLUSION: Combined use of RCM and OCT elucidated distinctive follicle infundibulum characteristics and inflammation degree that were associated with acne severity. Future trials may apply imaging techniques to support clinical acne grading, and monitor treatment efficacy. Lasers Surg. Med. 51:104-113, 2019. © 2018 Wiley Periodicals, Inc.

Trends in incidence, mortality, and causes of death associated with systemic sclerosis in Denmark between 1995 and 2015: a nationwide cohort study.

BACKGROUND: To investigate the incidence and the mortality-rates of systemic sclerosis (SSc), its primary causes of death, and the temporal trends in events in Denmark during the last decades. METHODS: Using the Danish National Patient Registry, we identified all persons aged ≥18 years with a first-time diagnosis of SSc (ICD-10 code M34, excluding M34.2) between 1995 and 2015. RESULTS: A total of 2778 incident SSc cases were identified. The mean age at time of SSc diagnosis was 56 (standard deviation 15) years and 76% were women. The overall incidence rate (per 1,000,000 person-years) of diagnosed SSc was 24.4 (95% confidence interval 23.6-25.4), with a slight increase over the study period, age- and sex-adjusted incidence rate ratio 1.02 (95% confidence interval 1.01-1.02) per 1-year increase. The 1-year all-cause mortality rate per 100 person-years decreased from 6.1 (3.1-12.2) in 1995 to 5.3 (2.5-11.1) in 2015, sex- and age-adjusted hazard ratio 0.96 (95% CI 0.94-0.98) per 1-year increase. Over the period, the average age at SSc diagnosis increased and the proportion of women decreased, whereas the burden of comorbidities increased. One fifth of all deaths were attributable to cardiovascular causes, a fourth to pulmonary diseases, and 15% were due to cancer. CONCLUSIONS: Within the last few decades, the incidence of SSc has increased and the 1-year mortality rate has decreased slightly in Denmark. Almost half of all deaths were attributable to cardiopulmonary causes.

Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media.

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in or in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).

Asymmetric Preparation of prim-, sec-, and tert-Amines Employing Selected Biocatalysts.

This account focuses on the application of ω-transaminases, lyases, and oxidases for the preparation of amines considering mainly work from our own lab. Examples are given to access α-chiral primary amines from the corresponding ketones as well as terminal amines from primary alcohols via a two-step biocascade. 2,6-Disubstituted piperidines, as examples for secondary amines, are prepared by biocatalytical regioselective asymmetric monoamination of designated diketones followed by spontaneous ring closure and a subsequent diastereoselective reduction step. Optically pure tert-amines such as berbines and N-methyl benzylisoquinolines are obtained by kinetic resolution via an enantioselective aerobic oxidative C-C bond formation.

Concise Chemoenzymatic Three Step Total Synthesis of Isosolenopsin Through Medium Engineering.

A short and efficient total synthesis of the alkaloid isosolenopsin and its enantiomer has been achieved. In the key step, a ω-transaminase catalyzed the regioselective mono-amination of the diketone pentadecane-2,6-dione which was obtained in a single step via Grignard reaction. Initial low conversions in the biotransformation could be overcome by optimisation of the reaction conditions employing suitable cosolvents. In the presence of 20 vol% DMF or n-heptane best results were obtained employing two enantio-complementary ω-transaminases originating from Arthrobacter between 30-40 °C; under these conditions conversions of >99% and perfect stereocontrol (ee > 99%) were achieved. Diastereostelective chemical reduction (H2/Pd/C) of the biocatalytic product gave the target compound. The linear three step synthesis provided the natural product isosolenopsin in diastereomerically pure form (ee > 99%, d.r. = 99:1) with an overall yield of 64%.

Inflammation-induced intussusceptive angiogenesis in murine colitis.

Intussusceptive angiogenesis is a morphogenetic process that forms new blood vessels by the division of a single blood vessel into two lumens. Here, we show that this process of intraluminal division participates in the inflammation-induced neovascularization associated with chemically induced murine colitis. In studies of both acute (4-7 days) and chronic (28-31 days) colitis, intravital microscopy of intravascular tracers demonstrated a twofold reduction in blood flow velocity. In the acute colitis model, the decreased velocity was associated with marked dilatation of the mucosal plexus. In contrast, chronic inflammation was associated with normal caliber vessels and duplication (and triplication) of the quasi-polygonal mucosal plexus. Scanning electron microscopy (SEM) of intravascular corrosion casts suggested that pillar formation and septation, previously linked to the morphogenetic process of intussusceptive angiogenesis, were present within days of the onset of inflammation. Four weeks after the onset of inflammation, SEM of vascular corrosion casts demonstrated replication of the mucosal plexus without significant evidence of sprouting angiogenesis. These data suggest that mucosal capillaries have comparable aggregate cross-sectional area in acute and chronic colitis; however, there is a significant increase in functional capillary density in chronic colitis. We conclude that intussusceptive angiogenesis is a fundamental mechanism of microvascular adaptation to prolonged inflammation.

Fibroblasts mediate induction of high mobility group box protein 1 in lung epithelial cancer cells by diffusible factors.

Cancer development is associated with the high mobility group box protein 1 (HMGB1), which modulates the transcriptional activity in the nucleus, but it is also present in the cytoplasm and outside the cell in certain conditions. As the progression of lung cancer is supported by mitogenic stimuli of stromal fibroblasts, we studied the impact of lung fibroblasts (WI-38) on the expression and localization of HMGB1 in lung epithelial cancer cells (H358). HMGB1 was mainly localized in the nucleus of non-mitotic H358 cells but highly distributed in the cytoplasm of mitotic cells. Conditioned medium (CM) from WI-38 fibroblasts enhanced the expression of HMGB1 at the mRNA and protein level compared to the control CM from H358 cells. In particular, the amount of cytoplasmic HMGB1 was elevated in response to fibroblast CM, which was reduced by inhibiting the basic fibroblast growth factor with blocking antibodies. Although cytoplasmic HMGB1 can be released from the cell, we did not determine a significant amount of extracellular HMGB1 in these conditions. This might partially be based on the sensitivity of HMGB1 to extracellular proteases as CM caused fast proteolysis of the cytoplasmic HMGB1 preparations. Our data suggest that diffusible factors of fibroblasts support the biological function of cancer cells via HMGB1 activation.

Lung level of HMBG1 is elevated in response to advanced glycation end product-enriched food in vivo.

High mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein that can be actively released from the cell in certain conditions thereby mediating cytokine-like function. While nuclear HMGB1 modulates the transcriptional activity of cells, extracellular HMGB1 partially acts via binding to the receptor for advanced glycation end products (RAGE), which is highly expressed in lung tissue. Therefore, we studied the impact of food-derived advanced glycation end products (AGEs), the Maillard reaction products, on the lung expression of HMGB1. Feeding rats with AGE-rich diet, containing either bread crust or coffee beverage, resulted in an upregulation of HMGB1 mRNA and protein especially in those animals receiving bread crust diet. The expression of RAGE was not influenced. Moreover, we revealed a positive correlation between an increased lung AGE level and HMGB1 protein expression in both animal groups receiving either bread crust or coffee extract but not in the control group. In contrast, the ageing-related AGE accumulation was not associated with an increased level of HMGB1 protein in lung tissue from senescent (100 wk) compared to young-adult (24 wk) rats. Our data suggest a physiological role of food- but not ageing-associated AGEs in the regulation of the HMGB1 expression in lung.

Duplicated cytoglobin genes in teleost fishes.

Cytoglobin is a recently discovered myoglobin-related O2-binding protein of vertebrates with uncertain function. It occurs as single-copy gene in mammals. Here, we demonstrate the presence of two paralogous cytoglobin genes (Cygb-1 and Cygb-2) in the teleost fishes Danio rerio, Oryzias latipes, Tetraodon nigroviridis, and Takifugu rubripes. The globin-typical introns at positions B12.2 and G7.0 are conserved in both genes, whereas the C-terminal exon found in mammalian cytoglobin is absent in the fish genes. Phylogenetic analyses show that the two cytoglobin genes diverged early in teleost evolution. This is confirmed by gene synteny analyses, which suggest a large-scale duplication event. Although both cytoglobin genes are highly conserved and have evolved under purifying selection, substitution rates are significantly higher in Cygb-1 than in Cygb-2. Similar to their mammalian ortholog, both fish cytoglobins are expressed in a broad range of tissues. However, Cygb-2 is more than 250-fold stronger expressed in neuronal tissues, suggesting a subfunctionalization of the two cytoglobin paralogs after gene duplication.

A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin families in animals.

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.