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Tempeh, a soybean product from Indonesia, is created through fermentation by Rhizopus spp. and associated bacteria. Here, we aim to get an overview of the variability of the tempeh microbiota across Indonesia and disentangle influencing factors. We found high variability in bacterial abundance (10 3 - 10 9 copies g -1 ), richness (n ASV = 40 - 175 ASVs), and diversity (H' = 0.9 - 3.5) in tempeh. The primary factor affecting this variation was the region, where the tempeh was produced. Interestingly, tempeh samples obtained from geographically close areas tended to share similar bacterial profiles, suggesting a "terroir" of tempeh. Additionally, tempeh wrapped in banana leaves had a higher abundance of enterobacteria in comparison to tempeh wrapped in plastic but also tended to have a higher total bacterial and lactobacilli abundance. Despite all variability, the tempeh core microbiome consists Lactobacillales and Enterobacteriales. This study demonstrates a high variability of bacterial diversity in traditional tempeh from local producers highlighting a strong regional influence across Indonesia., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Despite that climate change is currently one of the most pervasive challenges, its effects on the plant-associated microbiome is still poorly studied. The aim of this study was to evaluate the impact of the independent and combinatory effect of climate warming and drought on the microbiome assembly of oak from seed to seedling. In a multifactorial experimental set up, acorns were subjected to different temperatures (15 °C, 20 °C, and 25 °C) and soil moisture levels (drought (15%) and control (60%)) from germination until the seedling stage, after which the bacterial and fungal communities associated to the rhizosphere and phyllosphere were characterized by amplicon sequencing and qPCR. The results showed a stronger effect of temperature on fungal than on bacterial diversity and the effect was more pronounced in the phyllosphere. Under drought condition, temperature had a significantly negative effect on phyllosphere fungal diversity. In the rhizosphere, temperature had a significant effect on the fungal community composition which was primarily caused by species turnover. Regardless of temperature, Actinobacteriota was significantly enriched in drought, a group of bacteria known to increase plant drought tolerance. This study provides new insights into the effect of climate change on the plant microbiome in natural ecosystems., (© 2024. The Author(s).)

Exposure to air pollution affects health of all organisms on earth but the impact on the plant microbiome is less understood. Here, we link the Air Quality Index with the dust and apple epiphytic and endophytic microbiome across the city of Graz (Austria). The microbiome of the apple episphere, peel endosphere and pulp endosphere, and surrounding dust was analyzed. Our results show that the fungal communities were more influenced by air quality than bacterial communities. Bacterial communities, instead, were more specific for the individual sample types, especially noticeable in the pulp endosphere. The microbiome of each sample type was comprised of distinct microbial communities. Overall, the bacterial communities were highly dominated by Proteobacteria followed by Bacteroidota and Actinobacteriota, and the fungal communities were dominated by Ascomycota followed by Basidiomycota. With lower air quality, the relative abundance of the fungal orders Hypocreales and Pleosporales decreased in the apple episphere and the peel endosphere, respectively. Interestingly, an unexpectedly high level of similarity was observed between the bacterial communities of dust and peel endosphere, while the epiphytic bacterial community was significantly different compared to the other samples. We suggested that dust served as a potential microbial colonization route for the fruit microbiome as most bacteria (55%) colonizing the peel endosphere originated from dust. In conclusion, air quality affects the microbiome of edible plants, which can cause health consequences in humans. Therefore, this knowledge should be considered in urban and horticultural farming strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the last two decades, new discoveries from microbiome research have changed our understanding of human health. It became evident that daily habits and lifestyle choices shape the human microbiome and ultimately determine health or disease. Therefore, we developed 'Tiny Biome Tales' (https://microbiome.gamelabgraz.at/), a science pedagogy video game designed like a scientific review based exclusively on peer-reviewed articles, to teach about the influence of lifestyle choices on the human microbiome during pregnancy, early and adult life, and related health consequences. Despite the scientific character, it can be played by a broad audience. Here, we also present a scientific assessment and showed that playing the game significantly contributed to knowledge gain. The innovative style of the 'gamified review' represents an ideal platform to disseminate future findings from microbiome research by updating existing and adding new scenes to the game., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The holobiont Holobiont theory is more than 80 years old, while the importance of microbial communities for plant holobionts was already identified by Lorenz Hiltner more than a century ago. Both concepts are strongly supported by results from the new field of microbiome research. Here, we present ecological and genetic features of the plant holobiont that underpin principles of a shared governance between hosts and microbes and summarize the relevance of plant holobionts in the context of global change. Moreover, we uncover knowledge gaps that arise when integrating plant holobionts in the broader perspective of the holobiome as well as one and planetary health concepts. Action is needed to consider interacting holobionts at the holobiome scale, for prediction and control of microbiome function to improve human and environmental health outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Diversity of the gut microbiota is crucial for human health. However, whether fruit and vegetable associated bacteria contribute to overall gut bacterial diversity is still unknown. We reconstructed metagenome-assembled genomes from 156 fruit and vegetable metagenomes to investigate the prevalence of associated bacteria in 2,426 publicly available gut metagenomes. The microbiomes of fresh fruits and vegetables and the human gut are represented by members in common such as Enterobacterales, Burkholderiales , and Lactobacillales . Exposure to bacteria via fruit and vegetable consumption potentially has a beneficial impact on the functional diversity of gut microbiota particularly due to the presence of putative health-promoting genes for the production of vitamin and short-chain fatty acids. In the human gut, they were consistently present, although at a low abundance, approx. 2.2%. Host age, vegetable consumption frequency, and the diversity of plants consumed were drivers favoring a higher proportion. Overall, these results provide one of the primary links between the human microbiome and the environmental microbiome. This study revealed evidence that fruit and vegetable-derived microbes could be found in the human gut and contribute to gut microbiome diversity.

An efficient algorithm to obtain the solutions for n -th order terms of perturbation expansions in absorption, scattering, and cross-coupling for light propagating in human tissue is presented. The proposed solution is free of any approximations and makes possible fast and efficient estimates of mammographic, optical tomographic, and fluorescent images, applying a perturbation order of 30 and more. The presented analysis sets the general limits for the applicability of the perturbation approach as a function of tumor size and optical properties of the human tissue. The convergence tests of the efficient calculations for large absorbing objects show excellent agreement with the reference data from finite element method calculations. The applicability of the theory is demonstrated in experiments on breast-like phantoms with high absorbing and low-scattering lesions.

Despite evidence that the microbiome extends host genetic and phenotypic traits, information on how the microbiome is transmitted and maintained across generations remains fragmented. For seed-bearing plants, seeds harbor a distinct microbiome and play a unique role by linking one generation to the next. Studies on microbial inheritance, a process we suggest including both vertical transmission and the subsequent migration of seed microorganisms to the new plant, thus become essential for our understanding of host evolutionary potential and host-microbiome coevolution. We propose dividing the inheritance process into three stages: (i) plant to seed, (ii) seed dormancy, and (iii) seed to seedling. We discuss the factors affecting the assembly of the microbiome during the three stages, highlight future research directions, and emphasize the implications of microbial inheritance for fundamental science and society., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microbiomes have an immense potential to enhance plant resilience to various biotic and abiotic stresses. However, intrinsic microbial communities respond to changes in their host's physiology and environment during plant's life cycle. The potential of the inherent plant microbiome has been neglected for a long time, especially for the postharvest period. Currently, close to 50% of all produced fruits and vegetables are lost either during production or storage. Biological control of spoilage and storage diseases is still lacking sufficiency. Today, novel multiomics technologies allow us to study the microbiome and its responses on a community level, which will help to advance current classic approaches and develop more effective and robust microbiome-based solutions for fruit and vegetable storability, quality, and safety., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Seed microbiota influence germination and plant health and have the potential to improve crop performance, but the factors that determine their structure and functions are still not fully understood. Here, we analysed the impact of plant-related and external factors on seed endophyte communities of 10 different oilseed rape (Brassica napus L.) cultivars from 26 field sites across Europe. All seed lots harboured a high abundance and diversity of endophytes, which were dominated by six genera: Ralstonia, Serratia, Enterobacter, Pseudomonas, Pantoea, and Sphingomonas. The cultivar was the main factor explaining the variations in bacterial diversity, abundance and composition. In addition, the latter was significantly influenced by diverse biotic and abiotic factors, for example host germination rates and disease resistance against Plasmodiophora brassicae. A set of bacterial biomarkers was identified to discriminate between characteristics of the seeds, for example Sphingomonas for improved germination and Brevundimonas for disease resistance. Application of a Bayesian community approach suggested vertical transmission of seed endophytes, where the paternal parent plays a major role and might even determine the germination performance of the offspring. This study contributes to the understanding of seed microbiome assembly and underlines the potential of the microbiome to be implemented in crop breeding and biocontrol programmes., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: A detailed understanding of antimicrobial resistance trends among all human-related environments is key to combat global health threats. In food science, however, the resistome is still little considered. Here, we studied the apple microbiome and resistome from different cultivars (Royal Gala and Braeburn) and sources (freshly harvested in South Africa and exported apples in Austrian supermarkets) by metagenomic approaches, genome reconstruction and isolate sequencing., Results: All fruits harbor an indigenous, versatile resistome composed of 132 antimicrobial resistance genes (ARGs) encoding for 19 different antibiotic classes. ARGs are partially of clinical relevance and plasmid-encoded; however, their abundance within the metagenomes is very low (≤ 0.03%). Post-harvest, after intercontinental transport, the apple microbiome and resistome was significantly changed independently of the cultivar. In comparison to fresh apples, the post-harvest microbiome is characterized by higher abundance of Enterobacteriales, and a more diversified pool of ARGs, especially associated with multidrug resistance, as well as quinolone, rifampicin, fosfomycin and aminoglycoside resistance. The association of ARGs with metagenome-assembled genomes (MAGs) suggests resistance interconnectivity within the microbiome. Bacterial isolates of the phyla Gammaproteobacteria, Alphaproteobacteria and Actinobacteria served as representatives actively possessing multidrug resistance and ARGs were confirmed by genome sequencing., Conclusion: Our results revealed intrinsic and potentially acquired antimicrobial resistance in apples and strengthen the argument that all plant microbiomes harbor diverse resistance features. Although the apple resistome appears comparatively inconspicuous, we identified storage and transport as potential risk parameters to distribute AMR globally and highlight the need for surveillance of resistance emergence along complex food chains., (© 2022. The Author(s).)

A synthetic route for oxidation-sensitive core-multishell (osCMS) nanocarriers was established, and their drug loading and release properties were analyzed based on their structural variations. The nanocarriers showed a drug loading of 0.3-3 wt % for the anti-inflammatory drugs rapamycin and dexamethasone and the photosensitizer meso -tetra-hydroxyphenyl-porphyrin ( m THPP). Oxidative processes of the nanocarriers were probed in vitro by hydrogen peroxide, and the degradation products were identified by infrared spectroscopy supported by ab initio calculations, yielding mechanistic details on the chemical changes occurring in redox-sensitive nanocarriers. Oxidation-triggered drug release of the model drug Nile Red measured and assessed by time-dependent fluorescence spectroscopy showed a release of up to 80% within 24 h. The drug delivery capacity of the new osCMS nanocarriers was tested in ex vivo human skin with and without pretreatments to induce local oxidative stress. It was found that the delivery of m THPP was selectively enhanced in skin under oxidative stress. The number and position of the thioether groups influenced the physicochemical as well as drug delivery properties of the carriers.

Understanding the plant microbiome is a key for plant health and controlling pathogens. Recent studies have shown that plants are responsive towards natural and synthetic sound vibration (SV) by perception and signal transduction, which resulted in resistance towards plant pathogens. However, whether or not native plant microbiomes respond to SV and the underlying mechanism thereof remains unknown. Within the present study we compared grapevine-associated microbiota that was perpetually exposed to classical music with a non-exposed control group from the same vineyard in Stellenbosch, South Africa. By analyzing the 16S rRNA gene and ITS fragment amplicon libraries we found differences between the core microbiome of SV-exposed leaves and the control group. For several of these different genera, e.g., Bacillus, Kocuria and Sphingomonas , a host-beneficial or pathogen-antagonistic effect has been well studied. Moreover, abundances of taxa identified as potential producers of volatile organic compounds that contribute to sensory characteristics of wines, e.g., Methylobacterium , Sphingomonas , Bacillus and Sporobolomyces roseus , were either increased or even unique within the core music-exposed phyllosphere population. Results show an as yet unexplored avenue for improved plant health and the terroir of wine, which are important for environmentally friendly horticulture and consumer appreciation. Although our findings explain one detail of the long-term positive experience to improve grapevine's resilience by this unusual but innovative technique, more mechanistic studies are necessary to understand the whole interplay.

Recent studies indicate that seed microbiomes affect germination and plant performance. However, the interplay between seed microbiota and plant health is still poorly understood. To get a complete picture of the system, a comprehensive analysis is required, comprising culture-dependent and culture-independent techniques. In this chapter, we provide a combination of methods that are established and optimized for the analysis of the seed microbiome. These include methods to: (1) activate and cultivate dormant seed microbiota, (2) analyze microbiota in germinated seeds (with and without substrate), (3) quantify microbial DNA via real-time PCR, (4) deplete host DNA for amplicon and metagenome analysis, and (5) visualize seed endophytes in microtomed sections using fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). A deep understanding of the seed microbiome and its functions can help in developing new seed treatments and breeding strategies for sustainable agriculture.

Postharvest food decay is one major issue for today's food loss along the supply chain. Hot water treatment (HWT), a sustainable method to reduce pathogen-induced postharvest fruit decay, has been proven to be effective on a variety of crops. However, the microbiome response to HWT is still unknown, and the role of postharvest microbiota for fruit quality is largely unexplored. To study both, we applied a combined approach of metabarcoding analysis and real time qPCR for microbiome tracking. Overall, HWT was highly effective in reducing rot symptoms on apples under commercial conditions, and induced only slight changes to the fungal microbiota, and insignificantly affected the bacterial community. Pathogen infection, however, significantly decreased the bacterial and fungal diversity, and especially rare taxa were almost eradicated in diseased apples. Here, about 90% of the total fungal community was composed by co-occurring storage pathogens Neofabraea alba and Penicillium expansum. Additionally, the prokaryote to eukaryote ratio, almost balanced in apples before storage, was shifted to 0.6% bacteria and 99.4% fungi in diseased apples, albeit the total bacterial abundance was stable across all samples. Healthy stored apples shared 18 bacterial and 4 fungal taxa that were not found in diseased apples; therefore, defining a health-related postharvest microbiome. In addition, applying a combined approach of HWT and a biological control consortium consisting of Pantoea vagans 14E4, Bacillus amyloliquefaciens 14C9 and Pseudomonas paralactis 6F3, were proven to be efficient in reducing both postharvest pathogens. Our results provide first insights into the microbiome response to HWT, and suggest a combined treatment with biological control agents., (Copyright © 2019 Wassermann, Kusstatscher and Berg.)

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1 -/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9β1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The plant microbiota is crucial for plant health and growth. Recently, vertical transmission of a beneficial core microbiota was identified for crop seeds, but for native plants, complementary mechanisms are almost completely unknown., Methods: We studied the seeds of eight native plant species growing together for centuries under the same environmental conditions in Alpine meadows (Austria) by qPCR, FISH-CLSM, and amplicon sequencing targeting bacteria, archaea, and fungi., Results: Bacteria and fungi were determined with approx. 10 10 gene copy numbers g -1 seed as abundant inhabitants. Archaea, which were newly discovered as seed endophytes, are less and represent only 1.1% of the signatures. The seed microbiome was highly diversified, and all seeds showed a species-specific, highly unique microbial signature, sharing an exceptionally small core microbiome. The plant genotype (species) was clearly identified as the main driver, while different life cycles (annual/perennial) had less impact on the microbiota composition, and fruit morphology (capsule/achene) had no significant impact. A network analysis revealed significant co-occurrence patterns for bacteria and archaea, contrasting with an independent fungal network that was dominated by mutual exclusions., Conclusions: These novel insights into the native seed microbiome contribute to a deeper understanding of seed microbial diversity and phytopathological processes for plant health, and beyond that for ecosystem plasticity and diversification within plant-specific microbiota.

Apples are among the most consumed fruits world-wide. They represent a source of direct human exposure to bacterial communities, which is less studied. We analyzed the apple microbiome to detect differences between tissues and the impact of organic and conventional management by a combined approach of 16S rRNA gene amplicon analysis and qPCR, and visualization using fluorescence in situ hybridization and confocal laser scanning microscopy (FISH-CLSM). Each apple fruit harbors different tissues (stem, peel, fruit pulp, seeds, and calyx), which were colonized by distinct bacterial communities. Interestingly, fruit pulp and seeds were bacterial hot spots, while the peel was less colonized. In all, approximately 10 8 16S rRNA bacterial gene copy numbers were determined in each g apple. Abundances were not influenced by the management practice but we found a strong reduction in bacterial diversity and evenness in conventionally managed apples. In addition, despite the similar structure in general dominated by Proteobacteria (80%), Bacteroidetes (9%), Actinobacteria (5%), and Firmicutes (3%), significant shifts of almost 40% of bacterial genera and orders were monitored. Among them, especially bacterial signatures known for health-affecting potential were found to be enhanced in conventionally managed apples. Our results suggest that we consume about 100 million bacterial cells with one apple. Although this amount was the same, the bacterial composition was significantly different in conventionally and organically produced apples.

The adsorption thermodynamics of 4-(dimethylamino)pyridine (DMAP) and its five divalent derivatives di-DMAP- n (2 ≤ n ≤ 6) with gradually increasing methylene-spacer lengths n binding to planar gold surfaces has been studied by surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT). SERS intensities of the totally symmetrical breathing mode of the pyridine ring at approximately 1007 cm -1 are used to monitor the surface coverage of the DMAP and di-DMAP- n ligands on gold surfaces at different concentrations. The equilibrium constant as a measure of the binding affinity is obtained from these measurements by using a modified Langmuir isotherm. Due to multivalent binding to the gold substrate, a characteristic enhancement of the binding affinity of di-DMAP- n compared to the monovalent DMAP is observed for all divalent species. First principles calculations of the di-DMAP- n ligands on an ideal Au(111) surface model as well as step terrace models have been performed to understand the adsorption structures and the multivalent binding enhancements. Furthermore, Raman spectra of the adsorbed molecules have been studied by first principles calculations to correlate the binding affinities to experimentally determined adsorption constants. The joint experimental and theoretical investigation of an oscillatory behavior of the binding affinity as a function of the methylene-spacer length in mono- and divalent 4-(dimethylamino)pyridines reveals that the molecular architecture plays an important role for the structure-function interplay of multivalently bound adsorbates.

Plant health is strongly connected with plants´ microbiome. In case of raw-eaten plants, the microbiome can also affect human health. To study potential impacts on health issues of both hosts, the microbiome composition of seven different Brassica vegetables, originating from different food processing pathways, was analyzed by a combined approach of amplicon sequencing, metagenomic mining and cultivation. All Brassica vegetables harbored a highly diverse microbiota as identified by 16S rRNA gene amplicon sequencing. The composition of the microbiota was found to be rather driven by the plant genotype than by the processing pathway. We characterized isolates with potential cancer-preventing properties by tracing myrosinase activity as well as isolates with biological control activity towards plant pathogens. We identified a novel strain with myrosinase activity and we found bacterial myrosinase genes to be enriched in rhizosphere and phyllosphere metagenomes of Brassica napus and Eruca sativa in comparison to the surrounding soil. Strains which were able to suppress plant pathogens were isolated from naturally processed vegetables and represent a substantial part (4.1%) of all vegetable microbiomes. Our results shed first light on the microbiome of edible plants and open the door to harnessing the Brassica microbiome for plant disease resistance and human health.

The influence of electrical excess charges on the crystallization from supersaturated aqueous sodium chloride solutions is reported. This is accomplished by efflorescence studies on single levitated microdroplets using optical and electrodynamic levitation. Specifically, a strong increase in efflorescence humidity is observed as a function of the droplet's negative excess charge, ranging up to -2.1 pC, with a distinct threshold behavior, increasing the relative efflorescence humidity, at which spontaneous nucleation occurs, from 44% for the neutral microparticle to 60%. These findings are interpreted by using molecular dynamics simulations for determining plausible structural patterns located near the particle surface that could serve as suitable precursors for the formation of critical clusters overcoming the nucleation barrier. These results, facilitating heterogeneous nucleation in the case of negatively charged microparticles, are compared to recent work on charge-induced nucleation of neat supercooled water, where a distinctly different nucleation behavior as a function of droplet charge has been observed.

The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments., (© 2016 The Author(s).)

Mononuclear osteoclast precursors circulate in the monocyte fraction of peripheral blood and form multinuclear cells with all osteoclastic phenotypic characteristics when cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL). The method to obtain osteoclast precursors from peripheral blood is simple but the number of recovered osteoclasts is often largely insufficient for functional analyses. The original aim of this study was to develop a rapid and efficient method that could overcome the donor variability and enrich the osteoclast precursors from a small volume of peripheral blood as a basis for future clinical studies to correlate the differentiation potential of circulating osteoclast precursors with bone lesions in cancer patients. We improved the efficiency of osteoclastogenesis by reducing isolation and purification times and overcame the use of flow cytometry and immunomagnetic purification procedures. In our culture system the osteoclast number was increased several-fold and the precursors were able to reach a full differentiation within seven days of culture. Both age as well as gender differences in osteoclastogenesis efficiency were no longer evident by processing limited volume blood samples with this simple and rapid method.

Proteolysis of the extracellular matrix (ECM) is a key event in tumor growth and progression. The breakdown of ECM can lead to the generation of bioactive fragments that promote cell growth and spread. EMILIN1, a multidomain glycoprotein expressed in several tissues, exerts a crucial regulatory function through the engagement of α4/α9 integrins. Unlike the majority of ECM molecules that elicit a proliferative program, the signals emitting from EMILIN1 engaged by α4/α9β1 integrins are antiproliferative. In this study, aimed to demonstrate if the suppressor role of EMILIN1 was related to its structural integrity, we tested the possibility that EMILIN1 could be specifically cleaved. Among the proteolytic enzymes released in the tumor microenvironment we showed that neutrophil elastase cleaved EMILIN1 in three/four major fragments. The consequence of this proteolytic process was the impairment of its anti-proliferative role. Accordingly, EMILIN1 was digested in sarcomas and ovarian cancers. Sarcoma specimens were infiltrated by neutrophils (PMNs) and stained positively for elastase. The present findings highlight the peculiar activity of PMN elastase in disabling EMILIN1 suppressor function., (Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a "guiding" molecule in migration of lymphatic endothelial cells.

The evidence that EMILIN1 (Elastic Microfibril Interface Located proteIN) deficiency in Emilin1(-/-) mice caused dermal and epidermal hyperproliferation and an abnormal lymphatic phenotype prompted us to hypothesize the involvement of this extracellular matrix component in tumor development and in lymphatic metastasis. Using the 12-dimethylbenz(α)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage model of skin carcinogenesis, we found that Emilin1(-/-) mice presented an accelerated formation, a higher incidence, and the development of a larger number of tumors compared with their wild-type littermates. EMILIN1-negative tumors showed more Ki67-positive proliferating cells and higher levels of pErk1/2. In these tumors, PTEN expression was lower. Emilin1(-/-) mice displayed enhanced lymphangiogenesis both in the tumor and in the sentinel lymph nodes. Accordingly, tumor growth and lymph node metastasis of transplanted syngenic tumors were also increased in Emilin1(-/-) mice. In vitro transmigration assays through lymphatic endothelial cells showed that EMILIN1 deficiency greatly facilitated tumor cell trafficking. Overall, these data established that EMILIN1 exerts a protective role in tumor growth, in tumor lymphatic vessel formation, as well as in metastatic spread to lymph nodes and reinforced the importance of its presence in the microenvironment to determine the tumor phenotype.

Introduction: The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions., Methods: MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs., Results: MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion., Conclusions: These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation.

EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.

The control of bone resorption is crucial in osteolytic diseases. Once attached to bone, osteoclasts (OCs) initiate the resorption process through the activation of a complex cascade of morphological and biochemical changes. Hyaluronan (HA), an extracellular glycosaminoglycan long non-branching polysaccharide, is expressed in bone matrices. Here we demonstrate that HA counter-balances the erosion activity of human mature OCs by significantly reducing their degradative potential. HA treatment of fully differentiated OCs derived from human peripheral blood monocytes inhibited migration on collagen as well as bone resorption. HA-mediated effects were primarily due to TRAcP, MMP-9, and cathepsin K down-regulation and to the increased levels of TIMP-1, a natural MMP-9 inhibitor. Binding of HA to mature OCs was entirely mediated by CD44: function-blocking anti-CD44 antibodies fully abrogated HA effects, and the engagement of HA receptor caused a rapid de-phosphorylation of Ser325 in the CD44 cytoplasmic tail. The inhibitory action by HA was associated with a transient up-phosphorylation of Pyk2, a novel persistent phosphorylation of p38 and the down-regulation of NFATc1 transcription factor. Our results provide a direct evidence for the involvement of CD44 in the HA-dependent regulation of OC activity and suggest a signaling pathway that could be unique in OC function inhibition., (Copyright © 2010 Wiley-Liss, Inc.)

Coherent excitation of a superposition of Rydberg states in neon by the 13th harmonic of an intense 804 nm pulse and the formation of a wave packet is reported. Pump-probe experiments are performed, where the 3d-manifold of the 2p6-->2p5 (2P3/2) 3d [1/2]1- and 2p6-->2p5 (2P3/2) 3d [3/2]1-transitions are excited by an extreme ultraviolet (XUV) radiation pulse, which is centered at 20.05 eV photon energy. The temporal evolution of the excited state population is probed by ionization with a time-delayed 804 nm pulse. Control of coherent transient excitation and wave packet dynamics in the XUV-regime is demonstrated, where the spectral phase of the 13th harmonic is used as a control parameter. Modulation of the phase is achieved by propagation of the XUV-pulse through neon of variable gas density. The experimental results indicate that phase-shaped high-order harmonics can be used to control fundamental coherent excitation processes in the XUV-regime.

Enhanced expression levels of NG2 proteoglycan in presurgical original lesions of soft-tissue sarcoma (STS) patients defines with 55% probability the immediate (i.e., within 12 months postsurgery) risk in these individuals to develop postsurgical secondary lesions, independently of any other clinical trait. It, therefore, provides a molecular factor that alone prospects a particularly unfavorable clinical outcome in such patients. Evaluation of the timing of metastasis formation in patients with high and low levels of NG2 in their primitive lesions further stratified the patients in subsets with diverse lag phases in the occurrence of metastatic disease. In our cohort of high-grade STS cases, transcription of NG2 also showed a 81-fold amplification in metastatic lesions, when compared to primitive ones, and this gene overexpression was accompanied by an abundant but nonuniform in situ expression of its product. In a similar manner as seen in primitive lesions, patients with higher levels of metastatic NG2 encountered a significantly more dismal clinical course. Multivariate analysis asserted that in these individuals upregulation of NG2 represented an absolute independent prognostic parameter. Therefore, minimally invasive assessment of the transcription levels of the NG2 gene represents a parameter capable of predicting the arising of metastatic disease within a definite postsurgery time interval, and affords in adjunct in the definition of life expectance in STS patients.

Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.

We analysed 21 samples of malignant fibrous histiocytoma (MFH) distinguished into the two principal morphological categories ('spindle cell' and the 'pleomorphic' subtypes). The aim of our study was to verify if a distinction between the two subclasses of MFH in terms of expression/activation of protein profiles could support and extend the morphological criteria. For this purpose, we carried out an immunohistochemical and immunoblotting analysis of proteins that could be relevant in sarcoma biology and potential diagnostic and therapeutical targets such as matrix metalloproteinases (MMPs) and molecules related to adhesive and proliferative properties. Our analysis revealed that MMP-1, MMP-9 expression and p27(kip1) cytoplasmic localisation can be considered valid parameters in the classification and potential explanation of the aggressive behaviour of this non-homogeneous group of MFH.

Optical mammography is one of several new techniques for breast cancer detection and characterization presently under development for clinical use that provide information other than morphologic, in particular on the biochemical and metabolic state of normal and diseased tissue. In breast tissue, scattering of red to near infrared (NIR) light dominates absorption and NIR light may penetrate several centimeters through the breast. Optical mammography avoids the use of ionizing radiation and offers the power of diffuse optical spectroscopy. However, because of strong light scattering, spatial resolution of optical mammography is generally low. The paper reviews the results of a clinical study on scanning time-domain optical mammography comprising 154 patients carrying a total of 102 carcinomas validated by histology. Ninety two of these tumors were detected in optical mammograms retrospectively and for 87 of the detected tumors optical properties and tissue parameters were derived. In addition developments on instrumentation and data analysis are covered and possible improvements of optical mammography are briefly discussed.

Using a triple wavelength (670 nm, 785 nm, 843/884 nm) scanning laser-pulse mammograph we recorded craniocaudal and mediolateral projection optical mammograms of 154 patients, suspected of having breast cancer. From distributions of times of flight of photons recorded at typically 1000-2000 scan positions, optical mammograms were derived displaying (inverse) photon counts in selected time windows, absorption and reduced scattering coefficients or total haemoglobin concentration and blood oxygen saturation. Optical mammograms were analysed by comparing them with x-ray and MR mammograms, including results of histopathology, attributing a subjective visibility score to each tumour assessed. Out of 102 histologically confirmed tumours, 72 tumours were detected retrospectively in both optical projection mammograms, in addition 20 cases in one projection only, whereas 10 tumours were not detectable in any projection. Tumour contrast and contrast-to-noise ratios of mammograms of the same breast, but derived from measured DTOFs by various methods were quantitatively compared. On average, inverse photon counts in selected time windows, including total photon counts, provide highest tumour contrast and contrast-to-noise ratios. Based on the results of the present study we developed a multi-wavelength, multi-projection scanning time-domain optical mammograph with improved spectral and spatial (angular) sampling, that allows us to record entire mammograms simultaneously at various offsets between the transmitting fibre and receiving fibre bundle and provides first results for illustration.

A novel scanning tunneling microscope manipulation scheme for a controlled molecular transport of weakly adsorbed molecules is demonstrated. Single sexiphenyl molecules adsorbed on a Ag(111) surface at 6 K are shot towards single silver atoms by excitation with the tip. To achieve atomically straight shooting paths, an electron resonator consisting of linear standing-wave fronts is constructed. The sexiphenyl manipulation signals reveal a pi ring flipping as the molecule moves from the hcp to fcc site. Ab initio calculations show an incorporation of the Ag atom below the center of a pi ring.

Using a dual-wavelength (670 nm, 785 nm) time-domain scanning instrument we have recorded optical mammograms of 93 patients suspected of having breast cancer which was subsequently assessed histologically. Among 65 histologically confirmed carcinomas, 54 were detectable in at least one of two optical mammograms recorded of each tumour-bearing breast in craniocaudal and mediolateral projection. Optical mammograms were based on photon counts in selected time windows of measured distributions of times of flight of photons. Optical properties of 50 carcinomas investigated at both wavelengths were derived by modelling the breast as partially homogeneous infinite slab with an embedded spherical inhomogeneity representing the tumour and by calculating the diffraction of photon density waves. In selected cases, additional information about the location of the tumour along the compression direction was used that was obtained from scans at selected offsets between source and detector optical fibres. A correlation plot of haemoglobin concentration and blood oxygen saturation of tumours and healthy tissue shows good separation between both kinds of tissue. The majority of carcinomas exhibited increased total haemoglobin concentration compared to healthy tissue.