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Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Our goal was to test the feasibility of a new theranostic strategy in chronic epilepsy by targeting cathepsin function using novel cathepsin activity-based probes (ABPs). We assessed the biodistribution of fluorescent cathepsin ABPs in vivo, in vitro, and ex vivo, in rodents with pilocarpine-induced chronic epilepsy and naïve controls, in human epileptic tissue, and in the myeloid cell lines RAW 264.7 (monocytes) and BV2 (microglia). Distribution and localization of ABPs were studied by fluorescence scanning, immunoblotting, microscopy, and cross-section staining in anesthetized animals, in their harvested organs, in brain tissue slices, and in vitro. Blood-brain-barrier (BBB) efflux transport was evaluated in transporter-overexpressing MDCK cells and using an ATPase activation assay. Although the in vivo biodistribution of ABPs to both naïve and epileptic hippocampi was negligible, ex vivo ABPs bound cathepsins preferentially within epileptogenic brain tissue and colocalized with neuronal but not myeloid cell markers. Thus, our cathepsin ABPs are less likely to be of major clinical value in the diagnosis of chronic epilepsy, but they may prove to be of value in intraoperative settings and in CNS conditions with leakier BBB or higher cathepsin activity, such as status epilepticus., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response., Methods: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity., Results: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression., Conclusions: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Despite the common use of lipid-lowering medications, cardiovascular diseases continue to be a significant health concern. Atherosclerosis, one of the most frequent causes of cardiovascular morbidity, involves extensive inflammatory activity and remodeling of the vascular endothelium. This relentless inflammatory condition can ultimately give rise to clinical manifestations, such as ischemic heart disease or stroke. Accumulating evidence over the past decades implicates cysteine protease cathepsins in cardiovascular disorders. In particular, Cathepsins B, L, and S are over-expressed during vascular inflammation, and their activity is associated with impaired clinical outcomes. Here we took advantage of these molecular events to introduce a non-invasive detection and treatment approach to modulate vascular inflammation using a Photosensitizing quenched Activity-Based Probed (PS-qABP) that targets these proteases. Methods: We tested the application of this approach in LDL receptor-deficient mice and used non-invasive imaging and heart cross-section staining to assess the theranostic efficacy of this probe. Moreover, we used fresh human endarterectomy tissues to analyze cathepsin signals on gel, and verified cathepsin identity by mass spectrometry. Results: We showed that our PS-qABP can rapidly accumulate in areas of inflammatory atheromas in vivo , and application of light therapy profoundly reduced lesional immune cell content without affecting smooth muscle cell and collagen contents. Lastly, using human tissue samples we provided proof-of-concept for future clinical applications of this technology. Conclusions: Photodynamic therapy guided by cysteine cathepsin activity is an effective approach to reduce vascular inflammation and attenuate atherosclerosis progression. This approach could potentially be applied in clinical settings., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer., (© 2019 Weiss-Sadan et al.)

Background/aims: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood., Methods: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy., Results: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines., Conclusion: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1 BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1 BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTβ) and lymphotoxin receptor (LTβR) expression indicated that LTβR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTβR is sensitive to proteolysis, we analysed cathepsin activity in TSC1 BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTβR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics., (© 2018 John Wiley & Sons Ltd.)

X-ray CT instruments are among the most available, efficient, and cost-effective imaging modalities in hospitals. The field of CT molecular imaging is emerging which relies mainly on the detection of gold nanoparticles and iodine-containing compounds directed to tagging a variety of abundant biomolecules. Here for the first time we attempted to detect enzymatic activity, while the low sensitivity of CT scanners to contrast reagents made this a challenging task. Therefore, we developed a new class of nanosized cathepsin-targeted activity-based probes (ABPs) for functional CT imaging of cancer. ABPs are small molecules designed to covalently modify enzyme targets in an activity-dependent manner. Using a CT instrument, these novel probes enable detection of the elevated cathepsin activity within cancerous tissue, thus creating a direct link between biological processes and imaging signals. We present the generation and biochemical evaluation of a library of ABPs tagged with different sized gold nanoparticles (GNPs), with various ratios of cathepsin-targeting moiety and a combination of different polyethylene glycol (PEG) protective layers. The most potent and stable GNP-ABPs were applied for noninvasive cancer imaging in mice. Surprisingly, detection of CT contrast from the tumor had reverse correlation to GNP size and the amount of targeting moiety. Interestingly, TEM images of tumor sections show intercellular lysosomal subcellular localization of the GNP-ABPs. In conclusion, we demonstrate that the covalent linkage is key for detection using low sensitive imaging modalities and the utility of GNP-ABPs as a promising tool for enzymatic-based CT imaging.

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.

The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment might rely on simultaneous targeting of multiple genes, which can be achieved by RNA interference-based therapeutic strategies. Here we show a potent combination of microRNA and siRNA delivered by an efficient nanocarrier to PDAC tumors. Using proteomic-microRNA profiles and survival data of PDAC patients from TCGA, we found a novel signature for prolonged survival. Accordingly, we used a microRNA-mimic to increase miR-34a together with siRNA to silence PLK1 oncogene. For in vivo dual-targeting of this combination, we developed a biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA). APA-miRNA-siRNA polyplexes systemically administered to orthotopically inoculated PDAC-bearing mice showed no toxicity and accumulated at the tumor, resulting in an enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1. Taken together, our findings warrant this unique combined polyplex's potential as a novel nanotherapeutic for PDAC.

Background: The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins-4 and -13 are anti-inflammatory type-2 cytokines, signaling via the common interleukin-13 receptor α1 chain and the type-2 interleukin-4 receptor. The role of interleukin-13 receptor α1 in the heart is unknown., Methods and Results: We analyzed myocardial samples from human donors (n=136) and patients with end-stage heart failure (n=177). We found that the interleukin-13 receptor α1 is present in the myocardium and, together with the complementary type-2 interleukin-4 receptor chain Il4ra , is significantly downregulated in the hearts of patients with heart failure. Next, we showed that Il13ra1 -deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild-type mice (left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P =0.004, left ventricular end-diastolic diameter 4.2±0.2 versus 3.92±0.3; P =0.03). A bioinformatic analysis of mouse hearts indicated that interleukin-13 receptor α1 regulates critical pathways in the heart other than the immune system, such as extracellular matrix (normalized enrichment score=1.90; false discovery rate q=0.005) and glucose metabolism (normalized enrichment score=-2.36; false discovery rate q=0). Deficiency of Il13ra1 was associated with reduced collagen deposition under normal and pressure-overload conditions., Conclusions: The results of our studies in humans and mice indicate, for the first time, a role of interleukin-13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The caspases are a family of cysteine proteases that are key regulators of apoptosis and their activity may thus serve as a good marker to monitor cell death. We have developed a quenched fluorescent activity-based probe (qABP) that is selective for caspase-3 activity and emits a fluorescent signal after covalently modifying its target. The probe has a wide range of potential applications, e.g. in real-time imaging, FACS analysis or biochemical quantification of caspase activity in intact cells. Application of the probe allowed us to monitor caspase-3 activation after chemotherapy-treatment and to distinguish between apoptosis sensitive and resistant cells. Moreover, it enabled real-time high-resolution visualization of active caspase-3 during apoptosis. This led to the surprising finding that in cancerous cells active caspase-3 is not only found at the familiar cellular locations but also in mitochondria and the endoplasmic reticulum. Thus, our novel covalent probe allows high spatial and temporal resolution imaging of caspase-3 activation and may thus be used as an effective tool to study molecular mechanisms of programmed cell death in healthy and disease states.

Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

Fibrinogen has the potential of being used as a material to harvest and grow normal mesenchymal cells (fibroblasts, endothelial cells) or to trap cancer cells from a suspension with blood as a potential circulatory trap.Insoluble fibrinogen particles (iFP) were prepared from commercial Cohn fraction I paste (source: Kedrion). The sized iFP (~60-180 µm) were not soluble in physiologic buffers, exhibited a density of 1.2 ± 0.02, and did not aggregate or clump when mixed with whole blood or thrombin, but were degraded in lytic solutions.Cell culture studies indicated that the iFP could be used to harvest, expand and transfer normal, mammalian, attachment-dependent cells, notably fibroblasts and stem cells from bone marrow, as well as numerous cancer lines. Cells attached to iFP underwent logarithmic growth kinetics and could be transferred without trypsinization. Transplanted cancer cells-on-iFP generated characteristic tumors and retained their surface marker (by Western immuno-blot). An iFP 'cell-affinity' batch column was shown to trap MCF-7 cancer cells in the presence of red blood cells (RBCs) or serum.The scalable process for fabricating iFP retained the cell attachment properties of native fibrinogen. The results indicate that iFP has the potential to be used as a 3D cell culture matrix, and possibly to trap cancer cells from blood.

Introduction: Lysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo., Methods: Protein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S., Results: Cathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA., Conclusions: Based on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Background: The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance., Methods and Findings: The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2-5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells., Conclusions: Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDalpha1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using (32)P(i)-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldalpha1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldalpha1/delta double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants.

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plant's response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plant's response to high salinity and dehydration. A tomato (Lycopersicon esculentum) alpha-class PLD, LePLDalpha1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDalpha1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDalpha1 and AtPLDdelta were found to be activated in response to salt stress. Moreover, pldalpha1 and plddelta single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.

Desert plants are exposed to a combination of environmental stress conditions, including low water availability, extreme temperature fluctuations, high irradiance and nutrient deprivation. Studying desert plants within their natural habitat may therefore reveal novel mechanisms and strategies that enable plants to resist stressful conditions. We studied the acclimation of Retama raetam, an evergreen stem-assimilating desert plant, to growth within an arid dune ecosystem. Retama raetam contained two different populations of stems: those of the upper canopy, exposed to direct sunlight, and those of the lower canopy, protected from direct sunlight. During the dry season, stems of the upper canopy contained a very low level of a number of essential proteins, including the large and small subunits of rubisco, ascorbate peroxidase and the D1 subunit of the reaction centre of photosystem II. However, RNA encoding these proteins was present; cytosolic transcripts were associated with polysomes, while chloroplastic transcripts were not. Upon water application, as well as following the first rainfall of the season, these "photosynthetically suppressed" stems recovered and accumulated essential proteins within 6-24 h. In contrast, stems of the lower canopy contained the essential proteins throughout the dry season. We suggest that R. raetam uses an acclimation strategy of "partial plant dormancy" in order to survive the dry season. "Dormancy", as evident by the post-transcriptional suppression of gene expression, as well as the suppression of photosynthesis, was induced specifically in stems of the upper canopy which protect the lower canopy by shading.