Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Growth, physiology, and stomatal parameters of plant polyploids grown under ice age, present-day, and future CO2 concentrations.

Polyploidy plays an important role in plant evolution, but knowledge of its eco-physiological consequences, such as of the putatively enlarged stomata of polyploid plants, remains limited. Enlarged stomata should disadvantage polyploids at low CO2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO2 uptake. We observed the growth, physiology, and epidermal cell features of 15 diploids and their polyploid relatives cultivated under glacial, present-day, and potential future atmospheric CO2 concentrations (200, 400, and 800 ppm respectively). We demonstrated some well-known polyploidy effects, such as faster growth and larger leaves, seeds, stomata, and other epidermal cells. The stomata of polyploids, however, tended to be more elongated than those of diploids, and contrary to common belief, they had no negative effect on the CO2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO2 concentrations. Higher polyploids with large genomes also showed increased operational stomatal conductance and consequently, a lower water-use efficiency. Our results point to a possible decrease in growth superiority of polyploids over diploids in a current and future high CO2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

The effect of elevated CO2 on photosynthesis is modulated by nitrogen supply and reduced water availability in Picea abies.

It is assumed that the stimulatory effects of elevated CO2 concentration ([CO2]) on photosynthesis and growth may be substantially reduced by co-occurring environmental factors and the length of CO2 treatment. Here, we present the study exploring the interactive effects of three manipulated factors ([CO2], nitrogen supply and water availability) on physiological (gas-exchange and chlorophyll fluorescence), morphological and stoichiometric traits of Norway spruce (Picea abies) saplings after 2 and 3 years of the treatment under natural field conditions. Such multifactorial studies, going beyond two-way interactions, have received only limited attention until now. Our findings imply a significant reduction of [CO2]-enhanced rate of CO2 assimilation under reduced water availability which deepens with the severity of water depletion. Similarly, insufficient nitrogen availability leads to a down-regulation of photosynthesis under elevated [CO2] being particularly associated with reduced carboxylation efficiency of the Rubisco enzyme. Such adjustments in the photosynthesis machinery result in the stimulation of water-use efficiency under elevated [CO2] only when it is combined with a high nitrogen supply and reduced water availability. These findings indicate limited effects of elevated [CO2] on carbon uptake in temperate coniferous forests when combined with naturally low nitrogen availability and intensifying droughts during the summer periods. Such interactions have to be incorporated into the mechanistic models predicting changes in terrestrial carbon sequestration and forest growth in the future.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

Interactive effects of UV radiation and water deficit on production characteristics in upland grassland and their estimation by proximity sensing.

An increase in extreme weather and changes in other conditions associated with ongoing climate change are exposing ecosystems to a very wide range of environmental drivers that interact in ways which are not sufficiently understood. Such uncertainties in how ecosystems respond to multifactorial change make it difficult to predict the impacts of environmental change on ecosystems and their functions. Since water deficit (WD) and ultraviolet radiation (UV) trigger similar protective mechanisms in plants, we tested the hypothesis that UV modulates grassland acclimation to WD, mainly through changes in the root/shoot (R/S) ratio, and thus enhances the ability of grassland to acquire water from the soil and hence maintain its productivity. We also tested the potential of spectral reflectance and thermal imaging for monitoring the impacts of WD and UV on grassland production parameters. The experimental plots were manipulated by lamellar shelters allowing precipitation to pass through or to be excluded. The lamellas were either transmitting or blocking the UV. The results show that WD resulted in a significant decrease in aboveground biomass (AB). In contrast, belowground biomass (BB), R/S ratio, and total biomass (TB) increased significantly in response to WD, especially in UV exclusion treatment. UV exposure had a significant effect on AB and BB, but only in the last year of the experiment. The differences in the effect of WD between years show that the effect of precipitation removal is largely influenced by the potential evapotranspiration (PET) in a given year and hence mainly by air temperatures, while the resulting effect on production parameters is best correlated with the water balance given by the difference between precipitation and PET. Canopy temperature and selected spectral reflectance indices showed a significant response to WD and also significant relationships with morphological (AB, R/S) and biochemical (C/N ratio) parameters. In particular, the vegetation indices NDVI and RDVI provided the best correlations of biomass changes caused by WD and thus the highest potential to remotely sense drought effects on terrestrial vegetation.

UV radiation and drought interact differently in grass and forb species of a mountain grassland.

Among abiotic stressors, drought and enhanced ultraviolet radiation (UV) received a lot of attention, because of their potential to impair plant growth. Since drought and UV induce partially similar protective mechanisms, we tested the hypothesis that UV ameliorates the effect of reduced water availability (WA) in selected grass (Holcus mollis and Agrostis capillaris) and forb species (Hypericum maculatum and Rumex acetosa). During 2011-2014, an outdoor manipulation experiment was conducted on a mountain grassland ecosystem (Beskydy Mts; Czech Republic). Lamellar shelters were used to pass (WAamb) or exclude (WA-) incident precipitation in order to simulate reduced water availability (WA). In addition, the lamellas were made from acrylics either transmitting (UVamb) or blocking (UV-) incident UV. Generally, both UV exposure and reduced WA enhanced epidermal UV-screening, while exposure to both factors resulted in less than additive interactions. Although UV radiation increased epidermal UV-screening rather in the grass (up to 29 % in A. capillaris) than forb (up to 12 % in H. maculatum) species and rather in well-watered than reduced WA plants, such acclimation response did not result in significant alleviation of reduced WA effects on gas exchange and morphological parameters. The study contributes to a better understanding of plant responses to complex environmental conditions and will help for successful modelling forecasts of future climate change impacts.

Single and interactive effects of variables associated with climate change on wheat metabolome.

One of the key challenges linked with future food and nutritional security is to evaluate the interactive effect of climate variables on plants' growth, fitness, and yield parameters. These interactions may lead to unique shifts in the morphological, physiological, gene expression, or metabolite accumulation patterns, leading to an adaptation response that is specific to future climate scenarios. To understand such changes, we exposed spring wheat to 7 regimes (3 single and 4 combined climate treatments) composed of elevated temperature, the enhanced concentration of CO2, and progressive drought stress corresponding to the predicted climate of the year 2100. The physiological and metabolic responses were then compared with the current climate represented by the year 2020. We found that the elevated CO2 (eC) mitigated some of the effects of elevated temperature (eT) on physiological performance and metabolism. The metabolite profiling of leaves revealed 44 key metabolites, including saccharides, amino acids, and phenolics, accumulating contrastingly under individual regimes. These metabolites belong to the central metabolic pathways that are essential for cellular energy, production of biosynthetic pathways precursors, and oxidative balance. The interaction of eC alleviated the negative effect of eT possibly by maintaining the rate of carbon fixation and accumulation of key metabolites and intermediates linked with the Krebs cycle and synthesis of phenolics. Our study for the first time revealed the influence of a specific climate factor on the accumulation of metabolic compounds in wheat. The current work could assist in the understanding and development of climate resilient wheat by utilizing the identified metabolites as breeding targets for food and nutritional security.

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways.

Caspase-8 is the key component of the receptor-mediated (extrinsic) apoptotic pathway. Immunological localization of active caspase-8 showed its presence in osteoblasts, including non-apoptotic ones. Further in vivo exploration of caspase-8 functions in the bone is hindered by the fact that the caspase-8 knock-out is lethal prenatally. Examinations were thus performed using individual cell populations in vitro. In this study, caspase-8 was eliminated by the CRISPR/cas9 technology in MC3T3-E1 cells, the most common in vitro model of osteoblastic populations. The aim of the work was to specify the consequences of caspase-8 deficiency on non-apoptotic pathways. The impact on the osteogenic gene expression of the osteoblastic cells along with alterations in proliferation, caspase cascades and rapamycin induced autophagy response were evaluated. Osteogenic differentiation of caspase-8 deficient cells was inhibited as these cells displayed a decreased level of mineralization and lower activity of alkaline phosphatase. Among affected osteogenic genes, based on the PCR Array, major changes were observed for Ctsk, as down-regulated, and Gdf10, as up-regulated. Other significantly down-regulated genes included those coding osteocalcin, bone morphogenetic proteins (-3, -4 and -7), collagens (-1a1, -14a1) or Phex. The formation of autophagosomes was not altered in rapamycin-treated caspase-8 deficient cells, but expression of some autophagy-related genes, including Tnfsf10, Cxcr4, Dapk1 and Igf1, was significantly downregulated. These data provide new insight into the effects of caspase-8 on non-apoptotic osteogenic pathways.

Autophagy-related proteases accompany the transition of pre-chondrogenic cells into chondroblasts.

BACKGROUND: Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium. METHODS: PCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture. RESULTS: Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression. CONCLUSIONS: The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

General Caspase Inhibition in Primary Chondrogenic Cultures Impacts Their Transcription Profile Including Osteoarthritis-Related Factors.

OBJECTIVE: The knowledge about functions of caspases, usually associated with cell death and inflammation, keeps expanding also regarding cartilage. Active caspases are present in the growth plate, and caspase inhibition in limb-derived chondroblasts altered the expression of osteogenesis-related genes. Caspase inhibitors were reported to reduce the severity of cartilage lesions in osteoarthritis (OA), and caspase-3 might represent a promising biomarker for OA prognosis. The objective of this investigation was to decipher the transcriptomic regulation of caspase inhibition in chondrogenic cells. DESIGN: Limb-derived chondroblasts were cultured in the presence of 2 different inhibitors: Z-VAD-FMK (FMK) and Q-VD-OPH (OPH). A whole transcriptome RNA sequencing was performed as the key analysis. RESULTS: The analysis revealed a statistically significant increase in the expression of 252 genes in the FMK samples and 163 genes in the OPH samples compared with controls. Conversely, there was a significant decrease in the expression of 290 genes in the FMK group and 188 in the OPH group. Among the top up- and downregulated genes (more than 10 times changed), almost half of them were associated with OA. Both inhibitors displayed the highest upregulation of the inflammatory chemokine Ccl5, the most downregulated gene was the one for mannose receptors Mrc1. CONCLUSIONS: The obtained datasets pointed to a significant impact of caspase inhibition on the expression of several chondro-/osteogenesis-related markers in an in vitro model of endochondral ossification. Notably, the list of these genes included some encoding for factors associated with cartilage/bone pathologies such as OA.

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules, and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism.

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.

Impact of FasL Stimulation on Sclerostin Expression and Osteogenic Profile in IDG-SW3 Osteocytes.

The Fas ligand (FasL) is known from programmed cell death, the immune system, and recently also from bone homeostasis. As such, Fas signalling is a potential target of anti-osteoporotic treatment based on the induction of osteoclastic cell death. Less attention has been paid to osteocytes, although they represent the majority of cells within the mature bone and are the key regulators. To determine the impact of FasL stimulation on osteocytes, differentiated IDG-SW3 cells were challenged by FasL, and their osteogenic expression profiles were evaluated by a pre-designed PCR array. Notably, the most downregulated gene was the one for sclerostin, which is the major marker of osteocytes and a negative regulator of bone formation. FasL stimulation also led to significant changes (over 10-fold) in the expression of other osteogenic markers: Gdf10, Gli1, Ihh, Mmp10, and Phex. To determine whether these alterations involved caspase-dependent or caspase-independent mechanisms, the IDG-SW3 cells were stimulated by FasL with and without a caspase inhibitor: Q-VD-OPh. The alterations were also detected in the samples treated by FasL along with Q-VD-OPh, pointing to the caspase-independent impact of FasL stimulation. These results contribute to an understanding of the recently emerging pleiotropic effects of Fas/FasL signalling and specify its functions in bone cells.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

Caspase Inhibition Affects the Expression of Autophagy-Related Molecules in Chondrocytes.

Objective. Caspases, cysteine proteases traditionally associated with apoptosis and inflammation, have recently been identified as important regulators of autophagy and reported within the growth plate, a cartilaginous part of the developing bone. The aim of this research was to identify novel autophagy-related molecules affected by inhibition of pro-apoptotic caspases in chondrocytes. Design. Chondrocyte micromasses derived from mouse limb buds were treated with pharmacological inhibitors of caspases. Autophagy-related gene expression was examined and possible novel molecules were confirmed by real-time polymerase chain reaction and immunocytofluorescence. Individual caspases inhibitors were used to identify the effect of specific caspases. Results. Chondrogenesis accompanied by caspase activation and autophagy progression was confirmed in micromass cultures. Expression of several autophagy-associated genes was significantly altered in the caspases inhibitors treated groups with the most prominent decrease for Pik3cg and increase of Tnfsf10. The results showed the specific pro-apoptotic caspases that play a role in these effects. Importantly, use of caspase inhibitors mimicked changes triggered by an autophagy stimulator, rapamycin, linking loss of caspase activity to an increase in autophagy. Conclusion. Caspase inhibition significantly affects regulation of autophagy-related genes in chondrocytes cultures. Detected markers are of importance in diagnostics and thus the data presented here open new perspectives in the field of cartilage development and degradation.

Caspase-12 Is Present During Craniofacial Development and Participates in Regulation of Osteogenic Markers.

Caspases are evolutionary conserved proteases traditionally known as participating in apoptosis and inflammation but recently discovered also in association with other processes such as proliferation or differentiation. This investigation focuses on caspase-12, ranked among inflammatory caspases but displaying other, not yet defined functions. A screening analysis pointed to statistically significant (P < 0.001) increase in expression of caspase-12 in a decisive period of mandibular bone formation when the original mesenchymal condensation turns into vascularized bone tissue. Immunofluorescence analysis confirmed the presence of caspase-12 protein in osteoblasts. Therefore, the osteoblastic cell line MC3T3-E1 was challenged to investigate any impact of caspase-12 on the osteogenic pathways. Pharmacological inhibition of caspase-12 in MC3T3-E1 cells caused a statistically significant decrease in expression of some major osteogenic genes, including those for alkaline phosphatase, osteocalcin and Phex. This downregulation was further confirmed by an alkaline phosphatase activity assay and by a siRNA inhibition approach. Altogether, this study demonstrates caspase-12 expression and points to its unknown physiological engagement in bone cells during the course of craniofacial development.

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR.

In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott-Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells.

Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2, -3, -6, -7, -8 and -9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells.

Spatial and Temporal Variability of Plant Leaf Responses Cascade after PSII Inhibition: Raman, Chlorophyll Fluorescence and Infrared Thermal Imaging.

The use of photosystem II (PSII) inhibitors allows simulating cascade of defense and damage responses, including the oxidative stress. In our study, PSII inhibiting herbicide metribuzin was applied to the leaf of the model plant species Chenopodium album. The temporally and spatially resolved cascade of defense responses was studied noninvasively at the leaf level by combining three imaging approaches: Raman spectroscopy as a principal method, corroborated by chlorophyll a fluorescence (ChlF) and infrared thermal imaging. ChlF imaging show time-dependent transport in acropetal direction through veins and increase of area affected by metribuzin and demonstrated the ability to distinguish between fast processes at the level of electron transport (1 - Vj) from slow processes at the level of non-photochemical energy dissipation (NPQ) or maximum efficiency of PSII photochemistry (Fv/Fm). The high-resolution resonance Raman images show zones of local increase of carotenoid signal 72 h after the herbicide application, surrounding the damaged tissue, which points to the activation of defense mechanisms. The shift in the carotenoid band indicates structural changes in carotenoids. Finally, the increase of leaf temperature in the region surrounding the spot of herbicide application and expanding in the direction to the leaf tip proves the metribuzin effect on slow stomata closure.

Application of organic carbon affects mineral nitrogen uptake by winter wheat and leaching in subsoil: Proximal sensing as a tool for agronomic practice.

We tested the hypothesis that application of stable forms of organic carbon (C) into the soil reduces leaching of nitrogen (N). We also examined the potential to estimate N leaching employing N-sensitive spectral reflectance indices. During three growing seasons 2013-2015, field experiment at two experimental sites combining application of distinct N doses (0 (N0), 35 (N35), 70 (N70), and 140 (N140) kg N ha-1) and two stable forms of organic C (lignohumate and compost) was established to measure N uptake by winter wheat and its leaching to subsoil layers. The spectral reflectance at canopy level was measured simultaneously with N content in leaf dry matter at the beginning of the grain filling phase. At full maturity, the above-ground biomass, grain yield, and grain protein content were evaluated. That data was used to calculate N uptake in grain. The N140 dose led to increased N uptake by grain of 64% and 73% in the wetter years 2013 and 2014, respectively, and even by 118% in the drier year 2015 in comparison with the N0 treatment. N leaching to subsoil increased substantially with higher N dose, but only in wetter years 2013 (by 74%) and 2014 (by 87%). By contrast, no effect of N dose on leached N was found in the dry year 2015. The application of organic C along with the N140 dose substantially reduced N leaching by 26% and 29% in 2014 and 2015, respectively. Moreover, we demonstrated that normalized red-edge spectral reflectance index (NRERI) is able to predict N uptake by wheat and it can serve as an indicator of N leaching in heavy-rainfall years. Our results thus point towards possible agronomic practices and use of remote-sensing techniques to reduce groundwater contamination by N-based fertilizers.