Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

BACKGROUND: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging. RESULTS: Here, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. CONCLUSIONS: By reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Bone collagen tensile properties of the aging human proximal femur.

Despite the dominant role of bone mass in osteoporotic fractures, aging bone tissue properties must be thoroughly understood to improve osteoporosis management. In this context, collagen content and integrity are considered important factors, although limited research has been conducted on the tensile behavior of demineralized compact bone in relation to its porosity and elastic properties in the native mineralized state. Therefore, this study aims (i) at examining the age-dependency of mineralized bone and collagen micromechanical properties; (ii) to test whether, and if so to which extent, collagen properties contribute to mineralized bone mechanical properties. Two cylindrical cortical bone samples from fresh frozen human anatomic donor material were extracted from 80 proximal diaphyseal sections from a cohort of 24 female and 19 male donors (57 to 96 years at death). One sample per section was tested in uniaxial tension under hydrated conditions. First, the native sample was tested elastically (0.25 % strain), and after demineralization, up to failure. Morphology and composition of the second specimen was assessed using micro-computed tomography, Raman spectroscopy, and gravimetric methods. Simple and multiple linear regression were employed to relate morphological, compositional, and mechanical variables with age and sex. Macro-tensile properties revealed that only elastic modulus of native samples was age dependent whereas apparent elastic modulus was sex dependent (p < 0.01). Compositional and morphological analysis detected a weak but significant age and sex dependency of relative mineral weight (r = -0.24, p < 0.05) and collagen disorder ratio (I∼1670/I∼1640, r = 0.25, p < 0.05) and a strong sex dependency of bone volume fraction while generally showing consistent results in mineral content assessment. Young's modulus of demineralized bone was significantly related to tissue mineral density and Young's modulus of native bone. The results indicate that mechanical properties of the organic phase, that include collagen and non-collagenous proteins, are independent of donor age. The observed reduction in relative mineral weight and corresponding overall stiffer response of the collagen network may be caused by a reduced number of mineral-collagen connections and a lack of extrafibrillar and intrafibrillar mineralization that induces a loss of waviness and a collagen fiber pre-stretch.

Does tissue fixation change the mechanical properties of dry ovine bone extracellular matrix?

Tissue fixation is a prevalent method for bone conservation. Bone biopsies are typically fixed in formalin, dehydrated in ethanol, and infiltrated with polymethyl methacrylate (PMMA) Since some experiments can only be performed on fixed bone samples, it is essential to understand how fixation affects the measured material properties. The aim of this study was to quantify the influence of tissue fixation on the mechanical properties of cortical ovine bone at the extracellular matrix (ECM) level with state-of-the-art micromechanical techniques. A small section from the middle of the diaphysis of two ovine tibias (3.5 and 5.5 years old) was cut in the middle and polished on each side, resulting in a pair of mirrored surfaces. For each pair, one specimen underwent a fixation protocol involving immersion in formalin, dehydration with ethanol, and infiltration with PMMA. The other specimen (mirrored) was air-dried. Six osteons were selected in both pairs, which could be identified in both specimens. The influence of fixation on the mechanical properties was first analyzed using micropillar compression tests and nanoindentation in dry condition. Additionally, changes in the degree of mineralization were evaluated with Raman spectroscopy in both fixed and native bone ECM. Finally, micro tensile experiments were conducted in the 3.5-year fixed ovine bone ECM and compared to reported properties of unfixed dry ovine bone ECM. Interestingly, we found that tissue fixation does not alter the mechanical properties of ovine cortical bone ECM compared to experiments in dry state. However, animal age increases the degree of mineralization (p = 0.0159) and compressive yield stress (p = 0.041). Tissue fixation appears therefore as a valid conservation technique for investigating the mechanical properties of dehydrated bone ECM.

Tensile Mechanical Properties of Dry Cortical Bone Extracellular Matrix: A Comparison Among Two Osteogenesis Imperfecta and One Healthy Control Iliac Crest Biopsies.

Osteogenesis imperfecta (OI) is a genetic, collagen-related bone disease that increases the incidence of bone fractures. Still, the origin of this brittle mechanical behavior remains unclear. The extracellular matrix (ECM) of OI bone exhibits a higher degree of bone mineralization (DBM), whereas compressive mechanical properties at the ECM level do not appear to be inferior to healthy bone. However, it is unknown if collagen defects alter ECM tensile properties. This study aims to quantify the tensile properties of healthy and OI bone ECM. In three transiliac biopsies (healthy n = 1, OI type I n = 1, OI type III n = 1), 23 microtensile specimens (gauge dimensions 10 × 5 × 2 µm3) were manufactured and loaded quasi-statically under tension in vacuum condition. The resulting loading modulus and ultimate strength were extracted. Interestingly, tensile properties in OI bone ECM were not inferior compared to controls. All specimens revealed a brittle failure behavior. Fracture surfaces were graded according to their mineralized collagen fibers (MCF) orientation into axial, mixed, and transversal fracture surface types (FST). Furthermore, tissue mineral density (TMD) of the biopsy cortices was extracted from micro-computed tomogra[hy (µCT) images. Both FST and TMD are significant factors to predict loading modulus and ultimate strength with an adjusted R 2 of 0.556 (p = 2.65e-05) and 0.46 (p = 2.2e-04), respectively. The influence of MCF orientation and DBM on the mechanical properties of the neighboring ECM was further verified with quantitative polarized Raman spectroscopy (qPRS) and site-matched nanoindentation. MCF orientation and DBM were extracted from the qPRS spectrum, and a second mechanical model was developed to predict the indentation modulus with MCF orientation and DBM (R 2 = 67.4%, p = 7.73e-07). The tensile mechanical properties of the cortical bone ECM of two OI iliac crest biopsies are not lower than the one from a healthy and are primarily dependent on MCF orientation and DBM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Composition and micromechanical properties of the femoral neck compact bone in relation to patient age, sex and hip fracture occurrence.

Current clinical methods of bone health assessment depend to a great extent on bone mineral density (BMD) measurements. However, these methods only act as a proxy for bone strength and are often only carried out after the fracture occurs. Besides BMD, composition and tissue-level mechanical properties are expected to affect the whole bone's strength and toughness. While the elastic properties of the bone extracellular matrix (ECM) have been extensively investigated over the past two decades, there is still limited knowledge of the yield properties and their relationship to composition and architecture. In the present study, morphological, compositional and micropillar compression bone data was collected from patients who underwent hip arthroplasty. Femoral neck samples from 42 patients were collected together with anonymous clinical information about age, sex and primary diagnosis (coxarthrosis or hip fracture). The femoral neck cortex from the inferomedial region was analyzed in a site-matched manner using a combination of micromechanical testing (nanoindentation, micropillar compression) together with micro-CT and quantitative polarized Raman spectroscopy for both morphological and compositional characterization. Mechanical properties, as well as the sample-level mineral density, were constant over age. Only compositional properties demonstrate weak dependence on patient age: decreasing mineral to matrix ratio (p = 0.02, R2 = 0.13, 2.6 % per decade) and increasing amide I sub-peak ratio I∼1660/I∼1683 (p = 0.04, R2 = 0.11, 1.5 % per decade). The patient's sex and diagnosis did not seem to influence investigated bone properties. A clear zonal dependence between interstitial and osteonal cortical zones was observed for compositional and elastic bone properties (p < 0.0001). Site-matched microscale analysis confirmed that all investigated mechanical properties except yield strain demonstrate a positive correlation with the mineral fraction of bone. The output database is the first to integrate the experimentally assessed microscale yield properties, local tissue composition and morphology with the available patient clinical information. The final dataset was used for bone fracture risk prediction in-silico through the principal component analysis and the Naïve Bayes classification algorithm. The analysis showed that the mineral to matrix ratio, indentation hardness and micropillar yield stress are the most relevant parameters for bone fracture risk prediction at 70 % model accuracy (0.71 AUC). Due to the low number of samples, further studies to build a universal fracture prediction algorithm are anticipated with the higher number of patients (N > 200). The proposed classification algorithm together with the output dataset of bone tissue properties can be used for the future comparison of existing methods to evaluate bone quality as well as to form a better understanding of the mechanisms through which bone tissue is affected by aging or disease.

RNA-DNA interactomes of three prokaryotes uncovered by proximity ligation.

Proximity ligation approaches, which are widely used to study the spatial organization of the genome, also make it possible to reveal patterns of RNA-DNA interactions. Here, we use RedC, an RNA-DNA proximity ligation approach, to assess the distribution of major RNA types along the genomes of E. coli, B. subtilis, and thermophilic archaeon T. adornatum. We find that (i) messenger RNAs preferentially interact with their cognate genes and the genes located downstream in the same operon, which is consistent with polycistronic transcription; (ii) ribosomal RNAs preferentially interact with active protein-coding genes in both bacteria and archaea, indicating co-transcriptional translation; and (iii) 6S noncoding RNA, a negative regulator of bacterial transcription, is depleted from active genes in E. coli and B. subtilis. We conclude that the RedC data provide a rich resource for studying both transcription dynamics and the function of noncoding RNAs in microbial organisms.

Microscale 3D Printing and Tuning of Cellulose Nanocrystals Reinforced Polymer Nanocomposites.

The increasing demand for functional materials and an efficient use of sustainable resources makes the search for new material systems an ever growing endeavor. With this respect, architected (meta-)materials attract considerable interest. Their fabrication at the micro- and nanoscale, however, remains a challenge, especially for composites with highly different phases and unmodified reinforcement fillers. This study demonstrates that it is possible to create a non-cytotoxic nanocomposite ink reinforced by a sustainable phase, cellulose nanocrystals (CNCs), to print and tune complex 3D architectures using two-photon polymerization, thus, advancing the state of knowledge toward the microscale. Micro-compression, high-res scanning electron microscopy, (polarised) Raman spectroscopy, and composite modeling are used to study the structure-property relationships. A 100% stiffness increase is observed already at 4.5 wt% CNC while reaching a high photo-polymerization degree of ≈80% for both neat polymers and CNC-composites. Polarized Raman and the Halpin-Tsai composite-model suggest a random CNC orientation within the polymer matrix. The microscale approach can be used to tune arbitrary small scale CNC-reinforced polymer-composites with comparable feature sizes. The new insights pave the way for future applications where the 3D printing of small structures is essential to improve performances of tissue-scaffolds, extend bio-electronics applications or tailor microscale energy-absorption devices.

Metagenomic Insights into the Taxonomic and Functional Features of Traditional Fermented Milk Products from Russia.

Fermented milk products (FMPs) contain probiotics that are live bacteria considered to be beneficial to human health due to the production of various bioactive molecules. In this study, nine artisanal FMPs (kefir, ayran, khurunga, shubat, two cottage cheeses, bryndza, khuruud and suluguni-like cheese) from different regions of Russia were characterized using metagenomics. A metagenomic sequencing of ayran, khurunga, shubat, khuruud and suluguni-like cheese was performed for the first time. The taxonomic profiling of metagenomic reads revealed that Lactococcus species, such as Lc. lactis and Lc. cremoris prevailed in khuruud, bryndza, one sample of cottage cheese and khurunga. The latter one together with suluguni-like cheese microbiome was dominated by bacteria, affiliated to Lactobacillus helveticus (32-35%). In addition, a high proportion of sequences belonging to the genera Lactobacillus, Lactococcus and Streptococcus but not classified at the species level were found in the suluguni-like cheese. Lactobacillus delbrueckii, as well as Streptococcus thermophilus constituted the majority in another cottage cheese, kefir and ayran metagenomes. The microbiome of shubat, produced from camel's milk, was significantly distinctive, and Lentilactobacillus kefiri, Lactobacillus kefiranofaciens and Bifidobacterium mongoliense represented the dominant components (42, 7.4 and 5.6%, respectively). In total, 78 metagenome-assembled genomes with a completeness ≥ 50.2% and a contamination ≤ 8.5% were recovered: 61 genomes were assigned to the Enterococcaceae, Lactobacillaceae and Streptococcaceae families (the Lactobacillales order within Firmicutes), 4 to Bifidobacteriaceae (the Actinobacteriota phylum) and 2 to Acetobacteraceae (the Proteobacteria phylum). A metagenomic analysis revealed numerous genes, from 161 to 1301 in different products, encoding glycoside hydrolases and glycosyltransferases predicted to participate in lactose, alpha-glucans and peptidoglycan hydrolysis as well as exopolysaccharides synthesis. A large number of secondary metabolite biosynthetic gene clusters, such as lanthipeptides, unclassified bacteriocins, nonribosomal peptides and polyketide synthases were also detected. Finally, the genes involved in the synthesis of bioactive compounds like ß-lactones, terpenes and furans, nontypical for fermented milk products, were also found. The metagenomes of kefir, ayran and shubat was shown to contain either no or a very low count of antibiotic resistance genes. Altogether, our results show that traditional indigenous fermented products are a promising source of novel probiotic bacteria with beneficial properties for medical and food industries.

Microbial Communities of Artisanal Fermented Milk Products from Russia.

Fermented milk products (FMPs) have numerous health properties, making them an important part of our nutrient budget. Based on traditions, history and geography, there are different preferences and recipes for FMP preparation in distinct regions of the world and Russia in particular. A number of dairy products, both widely occurring and region-specific, were sampled in the households and local markets of the Caucasus republics, Buryatia, Altai, and the Far East and European regions of Russia. The examined FMPs were produced from cow, camel, mare's or mixed milk, in the traditional way, without adding commercial starter cultures. Lactate and acetate were the major volatile fatty acids (VFA) of the studied FMPs, while succinate, formate, propionate and n-butyrate were present in lower concentrations. Bacterial communities analyzed by 16S rRNA gene V4 fragment amplicon sequencing showed that Firmicutes (Lactococcus, Lactobacillus, Streptococcus, Lentilactobacillus and Leuconostoc) was the predominant phylum in all analyzed FMPs, followed by Proteobacteria (Acetobacter, Klebsiella, Pseudomonas and Citrobacter). Lactobacillus (mainly in beverages) or Lactococcus (mainly in creamy and solid products) were the most abundant community-forming genera in FMPs where raw milk was used and fermentation took place at (or below) room temperature. In turn, representatives of Streptococcus genus dominated the FMPs made from melted or pasteurized milk and fermented at elevated temperatures (such as ryazhenka, cottage cheese and matsoni-like products). It was revealed that the microbial diversity of koumiss, shubat, ryazhenka, matsoni-like products, chegen, sour cream and bryndza varied slightly within each type and correlated well with the same products from other regions and countries. On the other hand, the microbiomes of kefir, prostokvasha, ayran, cottage cheese and suluguni-like cheese were more variable and were shaped by the influence of particular factors linked with regional differences and traditions expressed in specificities in the production process. The microbial diversity of aarts, khurunga, khuruud, tan, ayran and suluguni-like cheese was studied here, to our knowledge, for the first time. The results of this study emphasize the overall similarity of the microbial communities of various FMPs on the one hand, and specificities of regional products on the other. The latter are of particular value in the age of globalization when people have begun searching for new and unusual products and properties. Speaking more specifically, these novel products, with their characteristic communities, might be used for the development of novel microbial associations (i.e., starters) to produce novel products with improved or unique properties.

Assessing minipig compact jawbone quality at the microscale.

Preclinical studies often require animal models for in vivo experiments. Particularly in dental research, pig species are extensively used due to their anatomical similarity to humans. However, there is a considerable knowledge gap on the multiscale morphological and mechanical properties of the miniature pigs' jawbones, which is crucial for implant studies and a direct comparison to human tissue. In the present work, we demonstrate a multimodal framework to assess the jawbone quantity and quality for a minipig animal model that could be further extended to humans. Three minipig genotypes, commonly used in dental research, were examined: Yucatan, Göttingen, and Sinclair. Three animals per genotype were tested. Cortical bone samples were extracted from the premolar region of the mandible, opposite to the teeth growth. Global morphological, compositional, and mechanical properties were assessed using micro-computed tomography (micro-CT) together with Raman spectroscopy and nanoindentation measurements, averaged over the sample area. Local mineral-mechanical relationships were investigated with the site-matched Raman spectroscopy and micropillar compression tests. For this, a novel femtosecond laser ablation protocol was developed, allowing high-throughput micropillar fabrication and testing without exposure to high vacuum. At the global averaged sample level, bone relative mineralization demonstrated a significant difference between the genotypes, which was not observed from the complementary micro-CT measurements. Moreover, bone hardness measured by nanoindentation showed a positive trend with the relative mineralization. For all genotypes, significant differences between the relative mineralization and elastic properties were more pronounced within the osteonal regions of cortical bone. Site-matched micropillar compression and Raman spectroscopy highlighted the differences between the genotypes' yield stress and mineral to matrix ratios. The methods used at the global level (averaged over sample area) could be potentially correlated to the medical tools used to assess jawbone toughness and morphology in clinics. On the other hand, the local analysis methods can be applied to quantify compressive bone mechanical properties and their relationship to bone mineralization.

Novel hyperthermophilic crenarchaeon Infirmifilum lucidum gen. nov. sp. nov., reclassification of Thermofilum uzonense as Infirmifilum uzonense comb. nov. and assignment of the family Thermofilaceae to the order Thermofilales ord. nov.

A novel hyperthermophilic crenarchaeon, strain 3507LTT, was isolated from a terrestrial hot spring near Tinguiririca volcano, Chile. Cells were non-motile thin, slightly curved filamentous rods. It grew at 73-93 °C and pH range of 5 to 7.5 with an optimum at 85 °C and pH 6.0-6.7. The presence of culture broth filtrate of another hyperthemophilic archaeon as well as yeast extract was obligatory for growth of the novel isolate. Strain 3507LTT is an anaerobic chemoorganoheterotroph, fermenting monosaccharides, disaccharides and polysaccharides (lichenan, starch, xanthan gum, xyloglucan, alpha-cellulose and amorphous cellulose). No growth stimulation was detected when nitrate, thiosulfate, selenate or elemental sulfur were added as the electron acceptors. The complete genome of strain 3507LTT consisted of a single circular chromosome with size of 1.63 Mbp. The DNA G+C content was 53.9%. According to the 16S rRNA gene sequence as well as conserved protein sequences phylogenetic analyses, strain 3507LTT together with Thermofilum uzonense formed a separate cluster within a Thermofilaceae family (Thermoproteales/Thermoprotei/Crenarchaeota). Based on phenotypic characteristics, phylogeny as well as AAI comparisons, a novel genus and species Infirmifilum lucidum strain 3507LTT (=VKM B-3376T = KCTC 15797T) gen. nov. sp. nov. was proposed. Its closest relative, Thermofilum uzonense strain 1807-2T should be reclassified as Infirmifilum uzonense strain 1807-2T comb. nov. Finally, based on phylogenomic and comparative genome analyses of representatives of Thermofilaceae family and other representatives of Thermoproteales order, a proposal of transfer of the family Thermofilaceae into a separate order Thermofilales ord. nov. was made.

Microtensile failure mechanisms in lamellar bone: Influence of fibrillar orientation, specimen size and hydration.

A mechanistic understanding of bone fracture is indispensable for developing improved fracture risk assessment in clinics. Since bone is a hierarchically structured material, gaining such knowledge requires analysis at multiple length scales. Here, the tensile response of cortical bone is characterized at the lamellar length scale under dry and hydrated conditions with the aim of investigating the influence of bone's microstructure and hydration on its microscale strength and toughness. For individual lamellae, bone strength strongly correlates with the underlying mineralized collagen fibrils orientation and shows a 2.3-fold increase compared to the macroscale. When specimen size is increased to a few lamellae, the influence of fibril orientation and the size effect on strength are significantly reduced. These findings highlight the critical influence of defects, such as canaliculi and interlamellar interfaces, when assessing larger volumes. Hydration leads up to a 3-fold strength decrease but activates several toughening mechanisms enabling inelastic deformation. In axial specimens, toughening is seen through fibril bridging and crack kinking. In transverse specimens, water presence leads to a progressive but stable crack growth parallel to the fibril orientation, suggesting crack-tip plasticity at the fibrillar interfaces. This work offers a better understanding of the role of interfaces, porosity, and hydration in crack initiation under tensile loading, which is a crucial step towards improved clinical management of disease-related bone fractures through multiscale modeling approaches. STATEMENT OF SIGNIFICANCE: Bone features a complex hierarchical structure which gives rise to several toughening mechanisms across several length scales. To better understand bone fracture, particularly the changes associated with age and disease, it is essential to investigate bone mechanical response at different levels of its hierarchical structure. For the first time, we were able to observe the nucleation of a single crack in hydrated bone lamellae under well-controlled uniaxial tensile loading conditions. These experiments highlight the role of water, interfaces, defects, and the ratio of defect to specimen size on bone's apparent strength and toughness. Such knowledge can be used in the future to develop multiscale models enabling improved clinical management of disease-related bone fractures.

Multiscale and multimodal X-ray analysis: Quantifying phase orientation and morphology of mineralized turkey leg tendons.

Fibrous biocomposites like bone and tendons exhibit a hierarchical arrangement of their components ranging from the macroscale down to the molecular level. The multiscale complex morphology, together with the correlated orientation of their constituents, contributes significantly to the outstanding mechanical properties of these biomaterials. In this study, a systematic road map is provided to quantify the hierarchical structure of a mineralized turkey leg tendon (MTLT) in a holistic multiscale evaluation by combining micro-Computed Tomography (micro-CT), small-angle X-ray scattering (SAXS), and wide-angle X-ray diffraction (WAXD). We quantify the interplay of the main MTLT components with respect to highly ordered organic parts such as fibrous collagen integrating inorganic components like hydroxyapatite (HA). The microscale fibrous morphology revealing different types of porous features and their orientation was quantified based on micro-CT investigations. The quantitative analysis of the alignment of collagen fibrils and HA crystallites was established from the streak-like signal in SAXS using the Ruland approach and the broadening of azimuthal profiles of the small and wide-angle diffraction peaks. It has been in general agreement that HA crystallites are co-aligned with the nanostructure of mineralized tissue. However, we observe relatively lower degree of orientation of HA crystallites compared to the collagen fibrils, which supports the recent findings of the structural interrelations within mineralized tissues. The generic multiscale characterization approach of this study is relevant to any hierarchically structured biomaterials or bioinspired materials from the µm-nm-Å scale. Hence, it gives the basis for future structure-property relationship investigations and simulations for a wide range of hierarchically structured materials. STATEMENT OF SIGNIFICANCE: Many fibrous biocomposites such as tendon, bone, and wood possess multiscale hierarchical structures, responsible for their exceptional mechanical properties. In this study, the 3-dimensional hierarchical structure, the degree of orientation and composition of mineralized tendon extracted from a turkey leg were quantified using a multimodal X-ray based approach combining small-angle X-ray scattering and wide-angle X-ray diffraction with micro-Computed Tomography. We demonstrate that hydroxyapatite (HA) domains are co-aligned with the nanostructure of mineralized tissue. However, the lower degree of orientation of HA crystallites was observed when compared to the collagen fibrils. The generic multiscale characterization approach of this study is relevant to any hierarchically structured biomaterials or bioinspired materials from the micrometer over the nanometer to the Angström scale level.

Compressive Strength of Iliac Bone ECM Is Not Reduced in Osteogenesis Imperfecta and Increases With Mineralization.

Osteogenesis imperfecta (OI) is an inheritable, genetic, and collagen-related disorder leading to an increase in bone fragility, but the origin of its "brittle behavior" is unclear. Because of its complex hierarchical structure, bone behaves differently at various length scales. This study aims to compare mechanical properties of human OI bone with healthy control bone at the extracellular matrix (ECM) level and to quantify the influence of the degree of mineralization. Degree of mineralization and mechanical properties were analyzed under dry conditions in 12 fixed and embedded transiliac crest biopsies (control n = 6, OI type I n = 3, OI type IV n = 2, and OI type III n = 1). Mean degree of mineralization was measured by microcomputed tomography at the biopsy level and the mineral-to-matrix ratio was assessed by Raman spectroscopy at the ECM level. Both methods revealed that the degree of mineralization is higher for OI bone compared with healthy control. Micropillar compression is a novel technique for quantifying post-yield properties of bone at the ECM level. Micropillars (d = 5 µm, h = 10 µm) were fabricated using focused ion beam milling and quasi-statically compressed to capture key post-yield properties such as ultimate strength. The qualitative inspection of the stress-strain curves showed that both OI and healthy control bone have a ductile response at the ECM level. The quantitative results showed that compressive strength is not reduced in OI bone and is increasing with OI severity. Nanoindentation measurements revealed that OI bone tends to have a higher Young's modulus, hardness, and dissipated energy compared with healthy bone. Micropillar strength and indentation modulus increased linearly and significantly (p < .0001) with mineral-to-matrix ratio. In conclusion, this study indicates that compressive mechanical properties of dry OI bone at the iliac crest are not inferior to healthy control at the ECM level and increase with mineralization. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Combining polarized Raman spectroscopy and micropillar compression to study microscale structure-property relationships in mineralized tissues.

Bone is a natural composite possessing outstanding mechanical properties combined with a lightweight design. The key feature contributing to this unusual combination of properties is the bone hierarchical organization ranging from the nano- to the macro-scale. Bone anisotropic mechanical properties from two orthogonal planes (along and perpendicular to the main bone axis) have already been widely studied. In this work, we demonstrate the dependence of the microscale compressive mechanical properties on the angle between loading direction and the mineralized collagen fibril orientation in the range between 0° and 82°. For this, we calibrated polarized Raman spectroscopy for quantitative collagen fibril orientation determination and validated the method using widely used techniques (small angle X-ray scattering, micro-computed tomography). We then performed compression tests on bovine cortical bone micropillars with known mineralized collagen fibril angles. A strong dependence of the compressive micromechanical properties of bone on the fibril orientation was found with a high degree of anisotropy for both the elastic modulus (Ea/Et=3.80) and the yield stress (σay/σty=2.54). Moreover, the post-yield behavior was found to depend on the MCF orientation with a transition between softening to hardening behavior at approximately 50°. The combination of methods described in this work allows to reliably determine structure-property relationships of bone at the microscale, which may be used as a measure of bone quality.

Siderite-based anaerobic iron cycle driven by autotrophic thermophilic microbial consortium.

Using a sample from a terrestrial hot spring (pH 6.8, 60 °C), we enriched a thermophilic microbial consortium performing anaerobic autotrophic oxidation of hydrothermal siderite (FeCO3), with CO2/bicarbonate as the electron acceptor and the only carbon source, producing green rust and acetate. In order to reproduce Proterozoic environmental conditions during the deposition of banded iron formation (BIF), we incubated the microbial consortium in a bioreactor that contained an unmixed anoxic layer of siderite, perfectly mixed N2/CO2-saturated liquid medium and microoxic (2% O2) headspace. Long-term incubation (56 days) led to the formation of magnetite (Fe3O4) instead of green rust as the main product of Fe(II) oxidation, the precipitation of newly formed metabolically induced siderite in the anoxic zone, and the deposition of hematite (Fe2O3) on bioreactor walls over the oxycline boundary. Acetate was the only metabolic product of CO2/bicarbonate reduction. Thus, we have demonstrated the ability of autotrophic thermophilic microbial consortium to perform a short cycle of iron minerals transformation: siderite-magnetite-siderite, accompanied by magnetite and hematite accumulation. This cycle is believed to have driven the evolution of the early biosphere, leading to primary biomass production and deposition of the main iron mineral association of BIF.

Hot in Cold: Microbial Life in the Hottest Springs in Permafrost.

Chukotka is an arctic region located in the continuous permafrost zone, but thermal springs are abundant there. In this study, for the first time, the microbial communities of the Chukotka hot springs (CHS) biofilms and sediments with temperatures 54-94 °C were investigated and analyzed by NGS sequencing of 16S rRNA gene amplicons. In microbial mats (54-75 °C), phototrophic bacteria of genus Chloroflexus dominated (up to 89% of all prokaryotes), while Aquificae were the most numerous at higher temperatures in Fe-rich sediments and filamentous "streamers" (up to 92%). The electron donors typical for Aquificae, such as H2S and H2, are absent or present only in trace amounts, and the prevalence of Aquificae might be connected with their ability to oxidize the ferrous iron present in CHS sediments. Armatimonadetes, Proteobacteria, Deinococcus-Thermus, Dictyoglomi, and Thermotogae, as well as uncultured bacteria (candidate divisions Oct-Spa1-106, GAL15, and OPB56), were numerous, and Cyanobacteria were present in low numbers. Archaea (less than 8% of the total community of each tested spring) belonged to Bathyarchaeota, Aigarchaeota, and Thaumarchaeota. The geographical location and the predominantly autotrophic microbial community, built on mechanisms other than the sulfur cycle-based ones, make CHS a special and unique terrestrial geothermal ecosystem.

Tenuifilum thalassicum gen. nov., sp. nov., a novel moderate thermophilic anaerobic bacterium from a Kunashir Island shallow hot spring representing a new family Tenuifilaceae fam. nov. in the class Bacteroidia.

A novel anaerobic moderately thermophilic bacterium, designated strain 38H-strT, was isolated from a 12m deep hot spring of the Kunashir Island shore. Gram-negative cells were non-spore-forming, motile, straight or curved filamentous rods, occasionally forming loops and knots. The strain grew at 20-65°C and pH range of 4.0-9.0 with an optimum at 50°C and pH 6.5-7.0. Strain 38H-strT required 0.5-2.5% NaCl (1.5% is an optimum) for growth. It was a chemoorganoheterotroph, growing on carbohydrates (starch, pullulan, alginate, laminarin, beta-glucan) or peptide mixtures and proteins (peptone, tryptone, gelatin, and α- or ß- keratins). Major products of glucose fermentation were acetate, hydrogen, and carbon dioxide. Major cellular fatty acids were iso- and anteiso-C15:0. Phosphatidylethanolamine, an unidentified phospholipid, and three unidentified polar lipids were detected in cellular lipids fractions. The quinone was MK-7. The size of complete genome of strain 38H-strT was 3.2 Mb; DNA G+C content was 38.3mol%. According to 16S rRNA gene sequence and conserved protein sequences phylogenies, strain 38H-strT represented a deeply branched lineage near the root of the class Bacteroidia. Based on phylogenetic analysis and phenotypic features the novel isolate was assigned to a novel family within the order Bacteroidales for which the name Tenuifilaceae fam. nov. is proposed. Strain 38H-strT (=DSM 100343T =VKM B-2964T) represents the first genus and species Tenuifilum thalassicum gen. nov., sp. nov.

Arenimonas fontis sp. nov., a bacterium isolated from Chukotka hot spring, Arctic region, Russia.

A moderately thermophilic, neutrophilic, aerobic, Gram-negative bacterium, strain 3729kT, was isolated from a thermal spring of the Chukotka Peninsula, Arctic region, Russia. It grew chemoorganoheterotrophically, utilizing proteinaceous substrates, including highly rigid keratins as well as various polysaccharides (glucomannan, locust bean gum, gum guar and xanthan gum). The major fatty acids of strain 3729kT were iso-C15 : 0 (60.9%), iso-C17 : 0 (12.0%), C16 : 0 (9.9%) and iso-C16 : 0 (7.4%). Isoprenoid quinones were Q-8 (95%) and Q-9 (5%). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and three unidentified polar lipids. Strain 3729kT was inhibited by chloramphenicol, neomycin, novobiocin, kanamycin, tetracycline, ampicillin and polymyxin B, but resistant to rifampicin, vancomycin and streptomycin. At the same time, strain 3729kT inhibited growth of Micrococcus luteus and its genome possessed genes for antimicrobial activity against Gram-positive bacteria (a single putative bacteriocin and several secreted lysozymes and peptidoglycan lytic transglycosylases). The DNA G+C content was 69.8 mol%. 16S rRNA gene sequence-based phylogenetic analysis placed strain 3729kT into a distinct species/genus-level branch within the family Xanthomonadaceae (Proteobacteria). Phylogenetic analysis of 120 conservative protein sequences of all Xanthomonadaceae with validly published names and publicly available genomic sequences supported a species-level position of strain 3729kT within the genus Arenimonas. Pairwise ANI values between strain 3729kT and other Arenimonas species were of 75-80 %, supporting the proposal of a novel species. Accordingly, Arenimonas fontis sp. nov., with the type strain 3729kT (=VMK В-3232Т=DSM 105847T), was proposed.

The first crenarchaeon capable of growth by anaerobic carbon monoxide oxidation coupled with H2 production.

The ability to grow by anaerobic CO oxidation with production of H2 from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505=VKM B-3180=KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO+H2O→CO2+H2; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH-ECH gene cluster). Compared to the [Ni,Fe]-CODH-ECH gene clusters occurring in the sequenced genomes of other H2-producing carboxydotrophs, the [Ni,Fe]-CODH-ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H2 production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.