KRCC1, a modulator of the DNA damage response.

The lysine-rich coiled-coil 1 (KRCC1) protein is overexpressed in multiple malignancies, including ovarian cancer, and overexpression correlates with poor overall survival. Despite a potential role in cancer progression, the biology of KRCC1 remains elusive. Here, we characterize the biology of KRCC1 and define its role in the DNA damage response and in cell cycle progression. We demonstrate that KRCC1 associates with the checkpoint kinase 1 (CHK1) upon DNA damage and regulates the CHK1-mediated checkpoint. KRCC1 facilitates RAD51 recombinase foci formation and augments homologous recombination repair. Furthermore, KRCC1 is required for proper S-phase progression and subsequent mitotic entry. Our findings uncover a novel component of the DNA damage response and a potential link between cell cycle, associated damage response and DNA repair.

Self-Therapeutic Nanomaterials: Applications in Biology and Medicine.

Over past decades, nanotechnology has contributed to the biomedical field in areas including detection, diagnosis, and drug delivery via opto-electronic properties or enhancement of biological effects. Though generally considered inert delivery vehicles, a plethora of past and present evidence demonstrates that nanomaterials also exude unique intrinsic biological activity based on composition, shape, and surface functionalization. These intrinsic biological activities, termed self-therapeutic properties, take several forms, including mediation of cell-cell interactions, modulation of interactions between biomolecules, catalytic amplification of biochemical reactions, and alteration of biological signal transduction events. Moreover, study of biomolecule-nanomaterial interactions offers a promising avenue for uncovering the molecular mechanisms of biology and the evolution of disease. In this review, we observe the historical development, synthesis, and characterization of self-therapeutic nanomaterials. Next, we discuss nanomaterial interactions with biological systems, starting with administration and concluding with elimination. Finally, we apply this materials perspective to advances in intrinsic nanotherapies across the biomedical field, from cancer therapy to treatment of microbial infections and tissue regeneration. We conclude with a description of self-therapeutic nanomaterials in clinical trials and share our perspective on the direction of the field in upcoming years.

Controlling Nanoparticle Uptake in Innate Immune Cells with Heparosan Polysaccharides.

We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles. Competition with other glycosaminoglycan types inhibited the endocytosis of HEP-coated nanoparticles only partially. We further found that nanoparticle uptake into innate immune cells can be controlled by more than 3 orders of magnitude via systematically varying the HEP surface density. Our results suggest a substantial potential for HEP-coated nanoparticles to target innate immune cells for efficient intracellular delivery, including into the cytoplasm. This HEP nanoparticle surface engineering technology may be broadly used to develop efficient nanoscale devices for drug and gene delivery as well as possibly for gene editing and immuno-engineering applications.

Sepsis in the era of data-driven medicine: personalizing risks, diagnoses, treatments and prognoses.

Sepsis is a series of clinical syndromes caused by the immunological response to infection. The clinical evidence for sepsis could typically attribute to bacterial infection or bacterial endotoxins, but infections due to viruses, fungi or parasites could also lead to sepsis. Regardless of the etiology, rapid clinical deterioration, prolonged stay in intensive care units and high risk for mortality correlate with the incidence of sepsis. Despite its prevalence and morbidity, improvement in sepsis outcomes has remained limited. In this comprehensive review, we summarize the current landscape of risk estimation, diagnosis, treatment and prognosis strategies in the setting of sepsis and discuss future challenges. We argue that the advent of modern technologies such as in-depth molecular profiling, biomedical big data and machine intelligence methods will augment the treatment and prevention of sepsis. The volume, variety, veracity and velocity of heterogeneous data generated as part of healthcare delivery and recent advances in biotechnology-driven therapeutics and companion diagnostics may provide a new wave of approaches to identify the most at-risk sepsis patients and reduce the symptom burden in patients within shorter turnaround times. Developing novel therapies by leveraging modern drug discovery strategies including computational drug repositioning, cell and gene-therapy, clustered regularly interspaced short palindromic repeats -based genetic editing systems, immunotherapy, microbiome restoration, nanomaterial-based therapy and phage therapy may help to develop treatments to target sepsis. We also provide empirical evidence for potential new sepsis targets including FER and STARD3NL. Implementing data-driven methods that use real-time collection and analysis of clinical variables to trace, track and treat sepsis-related adverse outcomes will be key. Understanding the root and route of sepsis and its comorbid conditions that complicate treatment outcomes and lead to organ dysfunction may help to facilitate identification of most at-risk patients and prevent further deterioration. To conclude, leveraging the advances in precision medicine, biomedical data science and translational bioinformatics approaches may help to develop better strategies to diagnose and treat sepsis in the next decade.

Disabling partners in crime: Gold nanoparticles disrupt multicellular communications within the tumor microenvironment to inhibit ovarian tumor aggressiveness.

The tumor microenvironment (TME) plays a key role in the poor prognosis of many cancers. However, there is a knowledge gap concerning how multicellular communication among the critical players within the TME contributes to such poor outcomes. Using epithelial ovarian cancer (EOC) as a model, we show how crosstalk among cancer cells (CC), cancer associated fibroblasts (CAF), and endothelial cells (EC) promotes EOC growth. We demonstrate here that co-culturing CC with CAF and EC promotes CC proliferation, migration, and invasion in vitro and that co-implantation of the three cell types facilitates tumor growth in vivo. We further demonstrate that disruption of this multicellular crosstalk using a gold nanoparticle (GNP) inhibits these pro-tumorigenic phenotypes in vitro as well as tumor growth in vivo. Mechanistically, GNP treatment reduces expression of several tumor-promoting cytokines and growth factors, resulting in inhibition of MAPK and PI3K-AKT activation and epithelial-mesenchymal transition - three key oncogenic signaling pathways responsible for the aggressiveness of EOC. The current work highlights the importance of multicellular crosstalk within the TME and its role for the aggressive nature of EOC, and demonstrates the disruption of these multicellular communications by self-therapeutic GNP, thus providing new avenues to interrogate the crosstalk and identify key perpetrators responsible for poor prognosis of this intractable malignancy.

MicroRNA-195 controls MICU1 expression and tumor growth in ovarian cancer.

MICU1 is a mitochondrial inner membrane protein that inhibits mitochondrial calcium entry; elevated MICU1 expression is characteristic of many cancers, including ovarian cancer. MICU1 induces both glycolysis and chemoresistance and is associated with poor clinical outcomes. However, there are currently no available interventions to normalize aberrant MICU1 expression. Here, we demonstrate that microRNA-195-5p (miR-195) directly targets the 3' UTR of the MICU1 mRNA and represses MICU1 expression. Additionally, miR-195 is under-expressed in ovarian cancer cell lines, and restoring miR-195 expression reestablishes native MICU1 levels and the associated phenotypes. Stable expression of miR-195 in a human xenograft model of ovarian cancer significantly reduces tumor growth, increases tumor doubling times, and enhances overall survival. In conclusion, miR-195 controls MICU1 levels in ovarian cancer and could be exploited to normalize aberrant MICU1 expression, thus reversing both glycolysis and chemoresistance and consequently improving patient outcomes.

Strategies for Delivering Nanoparticles across Tumor Blood Vessels.

Nanoparticle transport across tumor blood vessels is a key step in nanoparticle delivery to solid tumors. However, the specific pathways and mechanisms of this nanoparticle delivery process are not fully understood. Here, the biological and physical characteristics of the tumor vasculature and the tumor microenvironment are explored and how these features affect nanoparticle transport across tumor blood vessels is discussed. The biological and physical methods to deliver nanoparticles into tumors are reviewed and paracellular and transcellular nanoparticle transport pathways are explored. Understanding the underlying pathways and mechanisms of nanoparticle tumor delivery will inform the engineering of safer and more effective nanomedicines for clinical translation.

Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.

Mutations in the human cystathionine beta synthase (CBS) gene are known to cause endothelial dysfunction responsible for cardiovascular and neurovascular diseases. CBS is the predominant hydrogen sulfide (H2 S)-producing enzyme in endothelial cells (ECs). Recently, H2 S was shown to attenuate ROS and improve mitochondrial function. Mitochondria are metabolic organelles that actively transform their ultrastructure to mediate their function. Therefore, we questioned whether perturbation of CBS/H2 S activity could drive mitochondrial dysfunction via mitochondrial dynamics in ECs. Here we demonstrate that silencing CBS induces mitochondria fragmentation, attenuates efficient oxidative phosphorylation, and decreases EC function. Mechanistically, CBS silencing significantly elevates ROS production, thereby leading to reduced mitofusin 2 (MFN2) expression, decouple endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death. These defects were significantly rescued by the treatment of H2 S donors. Taken together our data highlights a novel signaling axis that mechanistically links CBS with mitochondrial function and ER-mitochondrial tethering and could be considered as a new therapeutic approach for the intervention of EC dysfunction-related pathologies.

Ubiquitin-binding associated protein 2 regulates KRAS activation and macropinocytosis in pancreatic cancer.

Macropinocytosis supports the metabolic requirement of RAS-transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS-transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin-binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2-deletion construct, we demonstrate that the UBA-domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP-bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS-driven cancers.

KRCC1: A potential therapeutic target in ovarian cancer.

Using a systems biology approach to prioritize potential points of intervention in ovarian cancer, we identified the lysine rich coiled-coil 1 (KRCC1), as a potential target. High-grade serous ovarian cancer patient tumors and cells express significantly higher levels of KRCC1 which correlates with poor overall survival and chemoresistance. We demonstrate that KRCC1 is predominantly present in the chromatin-bound nuclear fraction, interacts with HDAC1, HDAC2, and with the serine-threonine phosphatase PP1CC. Silencing KRCC1 inhibits cellular plasticity, invasive properties, and potentiates apoptosis resulting in reduced tumor growth. These phenotypes are associated with increased acetylation of histones and with increased phosphorylation of H2AX and CHK1, suggesting the modulation of transcription and DNA damage that may be mediated by the action of HDAC and PP1CC, respectively. Hence, we address an urgent need to develop new targets in cancer.

Hydrogen sulfide signaling in mitochondria and disease.

Hydrogen sulfide can signal through 3 distinct mechanisms: 1) reduction and/or direct binding of metalloprotein heme centers, 2) serving as a potent antioxidant through reactive oxygen species/reactive nitrogen species scavenging, or 3) post-translational modification of proteins by addition of a thiol (-SH) group onto reactive cysteine residues: a process known as persulfidation. Below toxic levels, hydrogen sulfide promotes mitochondrial biogenesis and function, thereby conferring protection against cellular stress. For these reasons, increases in hydrogen sulfide and hydrogen sulfide-producing enzymes have been implicated in several human disease states. This review will first summarize our current understanding of hydrogen sulfide production and metabolism, as well as its signaling mechanisms; second, this work will detail the known mechanisms of hydrogen sulfide in the mitochondria and the implications of its mitochondrial-specific impacts in several pathologic conditions.-Murphy, B., Bhattacharya, R., Mukherjee, P. Hydrogen sulfide signaling in mitochondria and disease.

Nanoparticle Interactions with the Tumor Microenvironment.

Compared to normal tissues, the tumor microenvironment (TME) has a number of aberrant characteristics including hypoxia, acidosis, and vascular abnormalities. Many researchers have sought to exploit these anomalous features of the TME to develop anticancer therapies, and several nanoparticle-based cancer therapeutics have resulted. In this Review, we discuss the composition and pathophysiology of the TME, introduce nanoparticles (NPs) used in cancer therapy, and address the interaction between the TME and NPs. Finally, we outline both the potential problems that affect TME-based nanotherapy and potential strategies to overcome these challenges.

Gold Nanoparticles Disrupt Tumor Microenvironment - Endothelial Cell Cross Talk To Inhibit Angiogenic Phenotypes in Vitro.

It is currently recognized that perpetual cross talk among key players in tumor microenvironment such as cancer cells (CCs), cancer associated fibroblasts (CAFs), and endothelial cells (ECs) plays a critical role in tumor progression, metastasis, and therapy resistance. Disruption of the cross talk may be useful to improve the outcome of therapeutics for which limited options are available. In the current study we investigate the use of gold nanoparticles (AuNPs) as a therapeutic tool to disrupt the multicellular cross talk within the TME cells with an emphasis on inhibiting angiogenesis. We demonstrate here that AuNPs disrupt signal transduction from TME cells (CCs, CAFs, and ECs) to ECs and inhibit angiogenic phenotypes in vitro. We show that conditioned media (CM) from ovarian CCs, CAFs, or ECs themselves induce tube formation and migration of ECs in vitro. Migration of ECs is also induced when ECs are cocultured with CCs, CAFs, or ECs. In contrast, CM from the cells treated with AuNPs or cocultured cells pretreated with AuNPs demonstrate diminished effects on ECs tube formation and migration. Mechanistically, AuNPs deplete ∼95% VEGF165 from VEGF single-protein solution and remove up to ∼45% of VEGF165 from CM, which is reflected on reduced activation of VEGF-Receptor 2 (VEGFR2) as compared to control CM. These results demonstrate that AuNPs inhibit angiogenesis via blockade of VEGF-VEGFR2 signaling from TME cells to endothelial cells.

Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Deregulation of mitochondrial morphogenesis, a dynamic equilibrium between mitochondrial fusion and fission processes, is now evolving as a key metabolic event that fuels tumor growth and therapy resistance. However, fundamental knowledge underpinning how cancer cells reprogram mitochondrial morphogenesis remains incomplete. Here, we report that cystathionine ß-synthase (CBS) reprograms mitochondrial morphogenesis in ovarian cancer (OvCa) cells by selectively regulating the stability of mitofusin 2 (MFN2). Clinically, high expression of both CBS and MFN2 implicates poor overall survival of OvCa patients, and a significant association between CBS and MFN2 expression exists in individual patients in the same data set. The silencing of CBS by small interfering RNA or inhibition of its catalytic activity by a small molecule inhibitor creates oxidative stress that activates JNK. Activated JNK phosphorylates MFN2 to recruit homologous to the E6-AP carboxyl terminus' domain-containing ubiquitin E3 ligase for its degradation via the ubiquitin-proteasome system. Supplementation with hydrogen sulfide or glutathione (the catalytic products of CBS enzymatic activity), anti-oxidants, or a JNK inhibitor restores MFN2 expression. In CBS-silenced orthotopic xenograft tumor tissues, MFN2 but not MFN1 is selectively downregulated. In summary, this report reveals a role for deregulated mitochondrial morphogenesis in OvCa, suggests one of the mechanisms for this deregulation, and provides a way to correct it through modulation of the metabolic enzyme CBS.-Chakraborty, P. K., Murphy, B., Mustafi, S. B., Dey, A., Xiong, X., Rao, G., Naz, S., Zhang, M., Yang, D., Dhanasekaran, D. N., Bhattacharya, R., Mukherjee, P. Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Probing Cellular Processes Using Engineered Nanoparticles.

Nanoparticles, the building blocks of nanotechnology, have been widely utilized in various biomedical applications, such as detection, diagnosis, imaging, and therapy. However, another emerging, albeit under-represented, area is the employment of nanoparticles as tools to understand cellular processes (e.g., oxidative stress-induced signaling cascades). Such investigations have enormous potential to characterize a disease from a different perspective and unravel some new features that otherwise would have remained a mystery. In this review, we summarize the intrinsic biological properties of unmodified as well surface modified nanoparticles and discuss how such properties could be utilized to interrogate biological processes and provide a perspective for future evolution of this field.

MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.

BMI1, a new target of CK2α.

BACKGROUND: The polycomb group protein, BMI1 plays important roles in chromatin modification, stem cell function, DNA damage repair and mitochondrial bioenergetics. Such diverse cellular functions of BMI1 could be, in part, due to post-translational modifications, especially phosphorylation. To date, AKT has been reported as a kinase that by site specific phosphorylation of BMI1 modulates its oncogenic functions. METHODS: Immunoprecipitation in conjunction with kinase assay and mass spectrometry was used to determine association with and site specific phosphorylation of BMI1 by CK2α. Functional implications of the BMI1/CK2α axis was examined in cancer cells utilizing siRNA and exogenous gene expression followed by biochemical and phenotypic studies. Correlations between expression of CK2α and BMI1 were determined from cell lines and formalin fixed paraffin embedded tissues representing the normal fallopian tube epithelium and high grade serous ovarian cancer samples. RESULTS: Here we report that CK2α, a nuclear serine threonine kinase, phosphorylates BMI1 at Serine 110 as determined by in-vitro/ex-vivo kinase assay and mass spectrometry. In ovarian cancer cell lines, expression of CK2α correlated with the phospho-species, as well as basal BMI1 levels. Preventing phosphorylation of BMI1 at Serine 110 significantly decreased half-life and stability of the protein. Additionally, re-expression of the phosphorylatable but not non-phosphorylatable BMI1 rescued clonal growth in endogenous BMI1 silenced cancer cells leading us to speculate that CK2α-mediated phosphorylation stabilizes BMI1 and promotes its oncogenic function. Clinically, compared to normal fallopian tube epithelial tissues, the expression of both BMI1 and CK2α were significantly higher in tumor tissues obtained from high-grade serous ovarian cancer patients. Among tumor samples, the expression of BMI1 and CK2α positively correlated (Spearman coefficient = 0.62, P = 0.0021) with each other. CONCLUSION: Taken together, our findings establish an important regulatory role of CK2α on BMI1 phosphorylation and stability and implicate the CK2α/BMI1 axis in ovarian cancer.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

The polycomb complex proto-oncogene BMI1 [B lymphoma Mo-MLV insertion region 1 homolog (mouse)] is essential for self-renewal of normal and cancer stem cells. BMI1-null mice show severe defects in growth, development, and survival. Although BMI1 is known to exert its effect in the nucleus via repression of 2 potent cell-cycle regulators that are encoded by the Ink4a/Arf locus, deletion of this locus only partially rescues BMI1-null phenotypes, which is indicative of alternate mechanisms of action of BMI1. Here, we show that an extranuclear pool of BMI1 localizes to inner mitochondrial membrane and directly regulates mitochondrial RNA (mtRNA) homeostasis and bioenergetics. These mitochondrial functions of BMI1 are independent of its previously described nuclear functions because a nuclear localization-defective mutant BMI1 rescued several bioenergetic defects that we observed in BMI1-depleted cells, for example, mitochondrial respiration, cytochrome c oxidase activity, and ATP production. Mechanistically, BMI1 coprecipitated with polynucleotide phosphorylase, a ribonuclease that is responsible for decay of mtRNA transcripts. Loss of BMI1 enhanced ribonuclease activity of polynucleotide phosphorylase and reduced mtRNA stability. These findings not only establish a novel extranuclear role of BMI1 in the regulation of mitochondrial bioenergetics, but also provide new mechanistic insights into the role of this proto-oncogene in stem cell differentiation, neuronal aging, and cancer.-Banerjee Mustafi, S., Aznar, N., Dwivedi, S. K. D., Chakraborty, P. K., Basak, R., Mukherjee, P., Ghosh, P., Bhattacharya, R. Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

Hepatoma derived growth factor (HDGF) dynamics in ovarian cancer cells.

As a leading cause of cancer death among women, identification of pathophysiologically-relevant biomarkers for ovarian cancer is important. The heparin binding, hepatoma-derived growth factor (HDGF) is overexpressed in ovarian cancer cell lines and may have prognostic value, but the mechanism by which this predominantly nuclear protein is secreted or functions is unknown. In this study, we focused on the circumstances under which HDGF is released by cells and the functional relevance of extracellular HDGF in the context of ovarian cancer. Immunofluorescence imaging showed nuclear localization of HDGF in ovarian cells, but unlike what is reported for other cell types, HDGF was minimally secreted into the media. However, HDGF was passively released by necrotic and late apoptotic cells. Extracellular HDGF was functionally relevant as it stimulated phosphorylation of ERK 1/2 and P38 in both non-cancer and ovarian cancer cells, and enhanced cellular migration. Overall, our study uncovers a novel function of HDGF as a messenger of cellular condition (alarmin) which in-turn modulates cellular function-aspects that could be used as a biomarker for ovarian cancer.