About 655 million persons worldwide are affected by osteoarthritis (OA), associated with excruciating chronic pain and disability in the elderly. Inflammation and cell death are two key events in OA and also key canonical functions of caspases. Since caspases play an important role in modulating inflammation, pharmacological caspase inhibitors have been widely utilized to study diseases involving inflammation in animal models and clinical trials. Among these, key caspases, which are responsible for activation of the corresponding pathways, include caspase-8 for the receptor mediated apoptotic pathway and caspase-1 as the key inflammatory caspase being involved in OA pathogenesis. OA- and non-OA chondrocytes were treated with caspase-1 and -8 inhibitors and TNF-α. RNA was isolated after 3 days from three biological replicates (n = 3) of each group and processed for single-end RNA sequencing (1 × 114 bp). Data were pre-processed and analyzed with R programming language v4.2.2. Downstream analysis included identification of differentially expressed genes providing a comprehensive overview of unique and shared transcriptional changes in both OA and non-OA cohorts.

Elevated atmospheric CO2 (EC) can enhance photosynthesis, biomass accumulation and water-use efficiency (WUE), but responses depend on water and nutrient availability and differ among tree species. Multifactorial studies addressing these interactions remain scarce. We examined interactive effects of EC (~700 μmol mol-1), nitrogen supply (nitrogen addition, N+; no nitrogen addition, N-) and water availability (well-watered, WW; drought-stressed, DS) on physiological and metabolic responses of more anisohydric European beech (Fagus sylvatica), which maintains stomatal opening longer during drought, and more isohydric Norway spruce (Picea abies), which closes stomata earlier to stabilise leaf water status. Across both species, EC produced the largest shifts in gas exchange and metabolite profiles, while water and nitrogen availability strongly modified these responses. EC generally increased net CO2 assimilation and WUE and reduced stomatal conductance, although responses differed between species and treatments; WUE was highest under EC×DS×N+. Species divergence was most evident in biochemical acclimation. In beech, EC markedly reduced photosynthetic capacity, reflected in lower Rubisco carboxylation capacity (VCmax) and electron transport rate (Jmax) across water and nitrogen treatments. This response was accompanied by lower succinate and foliar N and by resource-dependent shifts in amino acids, phenolic and stress-related metabolites, consistent with greater metabolic reorganization and photosynthetic downregulation. In contrast, spruce maintained comparatively stable VCmax and Jmax under EC, including under DS and N- conditions, together with comparatively stable primary metabolism and sustained phenolic-based defence. Overall, beech showed greater physiological and metabolic flexibility but stronger downregulation of photosynthetic capacity under EC, whereas spruce maintained a more conservative physiological strategy and greater biochemical stability. These contrasting responses highlight species-specific acclimation to CO2 enrichment and its modulation by water and nitrogen availability.

Caspase-12 is a molecule whose functions are still not well understood. Although its expression has been found in various tissues, specific roles have been described in only a few cases. These include the effect of caspase-12 on murine bone cell differentiation during craniofacial development. This work focused on the development of the limbs taking place through endochondral ossification, which precedes the formation of the cartilaginous growth plate. Caspase-12 was described here for the first time in growth plate chondrocytes during physiological development. Using pharmacological inhibition, caspase-12 was found to affect chondrogenesis. Limb-derived micromass cultures showed a significantly increased area of chondrogenic nodules after caspase-12 inhibition and there were changes in gene expression, the most significant of which was the reduction of Mmp9. These data point to potential new functions of caspase-12 in chondrogenesis.

PURPOSE/AIM:Caspase-1 inhibition is a promising option for degenerative joint diseases such as osteoarthritis; however, there is still a long way to go toward clinical use. One of the open challenges is associated with the non-inflammatory role of this caspase in the inflammatory environment as well as under physiological conditions. This study therefore focuses on two already pre-clinically tested caspase-1 inhibitors, VX-765 and VX-740, to specify their effects on chondrogenic cells. MATERIALS AND METHODS:The analysis was performed on mouse micromass cultures where chondrocyte differentiation, inflammatory cytokine release, and gene expression were examined. RESULTS:Our data indicate that the inhibitor VX-740 increases chondrogenesis, suggesting osteocalcin as a target molecule. In the inflammatory environment induced by IL-1β, there was an increase in chondrogenic nodules and partial compensation of differentiation for both investigated inhibitors. Morphological changes were not primarily due to changes in chondrogenic/osteogenic gene expression, but different levels of inflammatory molecules were found in the culture supernatant. While an increase in anti-inflammatory cytokine levels was observed with VX-765, a decrease in pro-inflammatory cytokines was recorded in the case of VX-740 treatment. CONCLUSIONS:The results demonstrate the differential effects of the caspase-1 inhibitors VX-765 and VX-740 on chondrogenic cell cultures and point to molecules that may be potential targets for use in the local treatment of osteoarthritis.

Plants face fluctuations in environmental conditions throughout their life cycles. Some of these conditions, such as CO2 concentration and increasing temperature, are closely linked to ongoing climate change. These conditions not only affect plant growth and development but also modify the response to sudden exposure to stressors through morphological, physiological, and biochemical acclimation. Understanding these responses is therefore important for defining adaptation strategies for future crop production. In this study, we tested the acclimation effect of light intensity (low, high) and CO2 concentration (low, ambient, elevated) on barley plants and its implications for subsequent responses to drought, heat, and their combination. The acclimation to the growth conditions induced numerous changes both in plant morphology and physiology. The whole-plant leaf area was stimulated by increasing light intensity and CO2 concentration. That led to increased whole-plant transpiration despite the trend of stomatal conductance was the opposite in comparison to leaf area. The increased whole-plant transpiration then increased the sensitivity of barley plants to the stress treatments. Similarly, the stimulatory effect of high light intensity on antioxidative capacity was not sufficient to improve barley performance under the stress treatments. The presented results show that for physiological or biochemical indicators of stress tolerance to be realistically used to evaluate the expected response to stress conditions, they must be related to the morphology of the whole plant, which influences both the severity of stress and the quantitative role of resistance mechanisms.

Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.

Hypoxia is relevant to several physiological and pathological processes and this also applies for the tooth. The adaptive response to lowering oxygen concentration is mediated by hypoxia-inducible factors (HIFs). Since HIFs were shown to participate in the promotion of angiogenesis, stem cell survival, odontoblast differentiation and dentin formation, they may play a beneficial role in the tooth reparative processes. Although some data were generated in vitro, little is known about the in vivo context of HIFs in tooth development. In order to contribute to this field, the mouse mandibular first molar was used as a model. The expression and in situ localisation of HIFs were examined at postnatal (P) days P0, P7, P14, using RT-PCR and immunostaining. The expression pattern of a broad spectrum of hypoxia-related genes was monitored by customised PCR Arrays. Metabolic aspects were evaluated by determination of the lactate level and mRNA expression of the mitochondrial marker Nd1. The results show constant high mRNA expression of Hif1a, increasing expression of Hif2a, and very low expression of Hif3a during early postnatal molar development. In the examined period the localisation of HIFs in the nuclei of odontoblasts and the subodontoblastic layer identified their presence during odontoblastic differentiation. Additionally, the lower lactate level and higher expression of mitochondrial Nd1 in advanced development points to decreasing glycolysis during differentiation. Postnatal nuclear localisation of HIFs indicates a hypoxic state in specific areas of dental pulp as oxygen demands depend on physiological events such as crown and root dentin mineralization.

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.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

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.

Drought resistance represents a complex of traits that are differently employed depending on drought type, severity and timing. Hence, a relatively comprehensive assessment of morphological and physiological pheno-typic response to drought is required. We evaluated the dynamic responses of six barley genotypes, representing a wide range of drought tolerance, to continuous drying and re-watering using non-invasively measured pa-rameters based on red-green-blue (RGB), thermal infrared and chlorophyll fluorescence imaging within an automated phenotyping platform. We identified three critical points in drought progress: i) 50% level of avail-able soil water, ii) wilting point, iii) full plant recovery after re-watering. However, the individual monitored parameters showed the potential to evaluate drought sensitivity at different points since the onset of drying. The correlation with relative yield response gradually increased for the side projected leaf area (SPA) and reached the maximum at the point of full recovery. The actual quantum yield of photosystem II (phi PSII) showed the highest correlation with a relative grain yield around wilting point. In contrast, the relative leaf temperature difference demonstrated a high correlation with yield response earlier, at 50% of available water. The highest correlations with the relative yield response were obtained for the colour RGB analysis at the wilting point and after recovery, particularly for khaki, beige, dark-green and olive-green colours. Multiple regression with parameters providing Pearson's correlation coefficient R > 0.5 slightly improved the estimation of relative yield response to drought but ensured significant improvement of absolute grain yield estimation under drought stress. This study shows that combining the phenotyping methods representing different morphological and physiological traits allows not only the assessment of drought tolerance (based on relative yield response to drought) which is crucial for selecting the genetic resources for the subsequent breeding process but also allows to test the yield performance of new genotypes under drought stress.

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.

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.

Cold acclimation, initiated by non-freezing low temperatures and light, is a natural strategy for increasing plant survival even at sub-zero temperatures. However, it remains unclear how the non-photochemical quenching processes, which are crucial for excessive light energy dissipation, are modulated during cold acclimation. We compared the effects of two weeks of acclimation to sub-optimal temperatures, at 10 degrees C (AC10) and 4 degrees C (AC4), with non-acclimated (NAC) Arabidopsis thaliana natural accessions grown at 21 degrees C, on their growth (rosette area), biochemistry (chlorophylls and epidermal flavonols), and physiology (CO2 assimilation rate, and quantum yields of photochemical and non-photochemical quenching processes). AC10 reduced rosette area in all (six) accessions, while chlorophylls and CO2 assimilation rate (Asat) decreased only in three accessions and it had no effect on maximum quantum yield (Fv/Fm). However, AC4 significantly decreased rosette area, chlorophylls, and Fv/Fm, in all accessions. Both AC10 and AC4 treatments increased the accumulation of epidermal flavonols in all acces-sions. In AC4 accessions, we found an increase in additional non-regulatory NPQ, phi f,d, and a decrease in the fraction of excitation energy used by PSII photochemistry, phi PSII. A similar irradiance resulted in a marginal difference in regulatory NPQ, phi npq, among NAC and AC10 or AC4 plants; however, AC10 plants have more energy-dependent fastest NPQ, phi qE, whereas AC4 predominates state transition quenching, phi qT. These variations in dissipation of absorbed light energy, when combined with reduced chlorophylls and accumulated flavonols, help to reduce the risk of photoinhibition in plants during cold periods. These findings provide new insights into how suboptimal temperature acclimation affects the regulation of NPQ molecular mechanisms in Arabidopsis thaliana natural accessions.

Summary 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 CO 2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO 2 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 CO 2 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 CO 2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO 2 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 CO 2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

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.

Probing insights into understanding photosynthetic processes via non-invasive means has an added advantage when used in phenotyping or precision agriculture. We employed Raman spectroscopy and fluorescence-based methods to investigate both the changes in the photosynthetic processes and the underlying protective mechanisms on Arabidopsis thaliana wild-type (WT), and ros1, which is a mutant of a repressor of transcriptional gene silencing, both grown under low light (LL: 100 mu mol m(-2) s(-1)) and high light (HL: 400 mu mol m(-2) s(-1)) regimes. Raman imaging detected a lower carotenoid intensity after two weeks in those plants grown under HL, compared to those grown under the LL regime; we interpret this as the result of oxidative damage of beta-carotene molecules. Further, the data revealed a significant depletion in carotenoids with enhanced phenolics around the midrib and tip of the WT leaves, but not in the ros1. On the contrary, small necrotic zones appeared after two weeks of HL in the ros1 mutant, pointing to the starting oxidative damage. The lower maximum quantum yield of the photochemistry (F-v/F-m) in the WT as well as in the ros1 mutant grown in HL (compared to those in the LL two weeks' post-exposure), indicates the HL partially inactivated photosystems. Chlorophyll a fluorescence imaging further showed high non-photochemical quenching (NPQ) in the plants grown under the HL regime for both the WT and the ros1 mutant, but the spatial heterogeneity of NPQ images was much higher in the HL-grown ros1 mutant. Fluorescence screening methods revealed significantly high values of chlorophyll proxies in the WT as well as in the ros1 mutant two weeks after in the HL compared to those under LL. The data generally revealed an increased accumulation of phenolics under HL in both the WT and ros1 mutant plants, but the proxies of anthocyanin and flavonols were significantly lower in the ros1 mutant than in the WT. The comparatively low accumulation of anthocyanin in the ros1 mutant compared to the WT supports the Raman data. We conclude that integrated use of these techniques can be efficiently applied for a better understanding of insights into photosynthetic mechanisms.

Among abiotic stressors, drought and enhanced UV 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 water deficit (WD) in selected grass ( Holcus mollis and Agrostis capillaris ) and herb species ( Hypericum maculatum and Rumex obtusifolius ). During 2011–2014, an outdoor manipulation experiment was conducted on the grassland ecosystem under mountainous conditions (Beskydy Mts; Czech Republic). Lamellar roofs were used to pass or exclude incident precipitation. In addition, the lamellas were made from acrylics either transmitting or blocking incident UV. Generally, both UV exposure and WD enhanced the accumulation of flavonols, while exposure to both factors resulted in additive or less than additive interactions. While UV stimulated an accumulation of flavonols in A. capillaris, H. mollis and H. maculatum , WD increased flavonols in R. obtusifolius . However, the UV-induced acclimation did not mitigate the negative effects of WD on CO 2 assimilation rate and subsequently morphological parameters. The study contributes to better understanding of plant responses to complex environmental conditions and will help for successful modelling forecasts of future climate change impacts.