MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements.

The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of anti-ageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.

An unusual histologic variant of necrobiotic xanthogranuloma.

Necrobiotic xanthogranuloma (NXG) usually shows a stereotypical histopathologic presentation. However, few unusual cases have been published. We present a patient with NXG showing exceptional histopathologic features. NXG in our patient presents with exclusively dermal granulomatous inflammation mimicking interstitial granuloma annulare. Not only subcutaneous involvement, but also, evident zones of degenerated collagen, foam cells, and cholesterol clefts were missing. Moreover, the case shows overlaps with recently published granulomatous scleromyxedema. Some common clinical and histopathologic features of NXG and scleromyxedema might be based on shared underlying paraproteinemia.

Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases.

Multiphoton tomography (MPT) is an in vivo imaging technique with very high spatial resolution and efforts are made to combine MPT with other non-invasive imaging methods. The goals of the present study were the description of the features of different dermatological entities as seen in MPT and confocal laser scanning microscopy (CLSM) comparison of these two novel techniques and the 'classical' diagnostic measures visual inspection, dermoscopy and histology with respect to the strengths and weaknesses of the different methods and the potential benefit from their combined implementation. After study approval by the local Ethics Committee, 47 patients (31 male, 16 female, age range: 24-88 years) were recruited from the Department of Dermatology of the University Hospital Jena. In this work, we present an illustrative selection of eleven cases from a clinical study combining in vivo MPT with in vivo CLSM. The patients presented with a broad range of dermatological disorders including seborrheic keratoses, angioma, actinic keratoses, melanocytic nevi, malignant melanoma, psoriasis, pemphigus vulgaris and scarring. Both methods, CLSM and MPT, were found to be suitable for in vivo imaging of superficial skin layers and may therefore be useful in dermatological practice for the diagnosis of skin diseases. However, both methods differ in their technical and physical principles. Thus, despite of many similarities concerning the morphological presentation of cells and tissues, important differences are recognized. Synergies of the combination of CLSM and MPT may be obtained by combined implementation in order to benefit from the fast overview given by CLSM and the detailed imaging of skin structures by MPT.

Erythema elevatum diutinum - a chronic leukocytoclastic vasculitis microscopically indistinguishable from granuloma faciale?

BACKGROUND: As the sequential inflammatory changes are the same in erythema elevatum diutinum (EED) and granuloma faciale (GF), histopathologic distinction may be difficult. METHODS: All available cases from 1998 to 2009 with the diagnosis of EED and GF were collected and reviewed, both clinically and histopathologically. Nine cases of EED and 41 cases of GF were reviewed in a blinded fashion using a checklist of 26 histopathologic criteria. RESULTS: Only four of the evaluated criteria showed differences between GF and EED. High density of the infiltrate was noted in 97% of cases of GF but only in 56% of cases of EED. Eosinophils were the predominant cell type in 59% of cases of GF but in none of the cases of EED. Plasma cells were more frequent in GF (64%) than in EED (22%), and granulomas were never found in GF but in 22% of EED. A zone of perijunctional sparing (Grenz zone) was observed in about three quarters of the cases in both the groups. CONCLUSIONS: The histopathology of GF and EED is very similar and overlapping. The presence of a Grenz zone and patterned fibrosis does not distinguish the two diseases. However, granulomatous nodules are only seen in EED, and a predominance of eosinophils in the infiltrate favors a diagnosis of GF.

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography.

BACKGROUND: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography. OBJECTIVES: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. METHODS: Thirty healthy volunteers aged 21-82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth-resolved intensity curves after correlation with multiphoton images. RESULTS: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. CONCLUSION: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.

Depth-resolved measurement of the dermal matrix composition by multiphoton laser tomography.

BACKGROUND: In the last years, multiphoton laser tomography (MLT) has emerged as a promising tool for non-invasive diagnostics in dermatology and other medical specialties. The present work is dedicated to the question to what degree the measurement depth and the thickness of the epidermis influence the evaluation of dermal matrix composition and if recommendations for future measurement procedures can be given. METHODS: In a study group of 30 healthy volunteers aged 21-82 years multiphoton depth-resolved measurements of autofluorescence and second harmonics have been performed in order to evaluate the dermal matrix composition. RESULTS: Characteristic intensity curves depending on the penetration depth were derived and differences between age groups were found. CONCLUSION: With the present work we provide evidence for the accuracy of the measurement of dermal matrix composition by MLT and give detailed advice for the measurement procedure. Furthermore, we propose the use of depth-dependent emission intensity curves for monitoring of anti-aging treatment.

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.

Intrinsic, solar and sunbed-induced skin aging measured in vivo by multiphoton laser tomography and biophysical methods.

BACKGROUND: Skin aging is accelerated by extrinsic factors, particularly actinic damage. Over the last decades, both clinical and pathological differences between intrinsic and actinic aging have been characterized. In this work, we aimed at quantifying skin aging by non-invasive in vivo methods. METHODS: Young healthy volunteers using indoor tanning facilities and aged people were compared with appropriate controls by measurements of skin elasticity with the Cutometer and the Reviscometer and by semi-quantitative evaluation of the dermal matrix composition by the multiphoton laser tomograph DermaInspect. RESULTS: We found differences between the sun-protected volar forearm and the dorsal side as well as between young and old test persons with all three methods. No significant differences were found between the skin of indoor-tanned test persons and control. Also, gender had no influence on the severity of skin aging. CONCLUSION: The most consistent results were obtained with the DermaInspect. The considerable inter-individual variation due to the cross-sectional design of the study may have disguised the factual skin damage caused by tanning beds.

Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network.

BACKGROUND: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. OBJECTIVES: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. METHODS: In vivo MLT was used to image two-photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age-dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. RESULTS: The resulting MLT-based Dermis Morphology Score is correlated with age (R2 = -0.90) and with the previously published SHG to AF Ageing Index of Dermis (R2 = 0.66). The two groups of young (age 21-38) and old (age 66-84) volunteers showed a significant difference in MLT score values (P < 0.001). CONCLUSIONS: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi-quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.

Non-invasive imaging techniques in the diagnosis of skin diseases.

INTRODUCTION: In recent years, there has been an increasing interest in non-invasive imaging techniques in clinical and investigational dermatology. Besides the growing number of biophysical methods for the assessment of skin properties, novel imaging tools have emerged and classical imaging methods are substantially improved. AREAS COVERED: This review focuses on well established and some of the most promising imaging techniques for application in dermatology and cutaneous research. These comprise dermoscopy, sonography, confocal microscopy, multiphoton tomography and optical coherence tomography. Non-invasive imaging techniques are presented concerning their functional principles, the history of their development and their current clinical and research application. Advantages and limitations of each of the methods are discussed. EXPERT OPINION: Available non-invasive imaging techniques in dermatology show substantial differences concerning their limitations and opportunities, potential clinical applicability and practicability. Also, the current data available differ largely between these methods due to different levels of experience in research and clinical application. Future research will not only aim at improving current technical limitations, but also investigate the potential synergistic effects of combining two or more techniques in order to enhance their diagnostic impact.