Multifunctional DNA-Based Hydrogel Promotes Diabetic Alveolar Bone Defect Reconstruction.

Diabetic alveolar bone defect (DABD) causes persistent bacterial infection, prolonged inflammation, and delayed bone healing, making it a considerable clinical challenge. In this study, by integrating silver nanoclusters (AgNCs) and M2 macrophage-derived extracellular vesicles (M2EVs), a multifunctional DNA-based hydrogel, called Agevgel, is developed with antibacterial, anti-inflammatory, immunomodulatory, and osteogenic properties to promote DABD rebuilding. AgNCs are tightly embedded into the DNA scaffolds and exhibit effective anti-bacterial activity, while immunomodulatory M2EVs are encapsulated within the shape-variable DNA scaffolds and exhibit potent anti-inflammatory and osteogenic properties. The results reveal that Agevgel effectively prolongs the local retention time and bioactivity of M2EVs in vivo. In particular, the sustained release of M2EVs can last for at least 7 days when applying Agevgel to DABD. Compared to free M2EVs or Aggel (AgNCs encapsulated within the DNA hydrogel) treatments, the Agevgel treatment accelerates the defect healing rate of alveolar bone and dramatically improves the trabecular architecture. Mechanistically, Agevgel plays a key role in regulating macrophage polarization and promoting the expression of proliferative and osteogenic factors. In summary, Agevgel provides a comprehensive treatment strategy for DABD with a great clinical translational value, highlighting the application of DNA hydrogels as an ideal bioscaffolds for periodontal diseases.

Antibacterial and DNA-Based Hydrogels In Situ Block TNF-α to Promote Diabetic Alveolar Bone Rebuilding.

Alveolar bone injury under diabetic conditions can severely impede many oral disease treatments. Rebuilding diabetic alveolar bone in clinics is currently challenging due to persistent infection and inflammatory response. Here, an antibacterial DNA-based hydrogel named Agantigel is developed by integrating silver nanoclusters (AgNCs) and tumor necrosis factor-alpha (TNF-α) antibody into DNA hydrogel to promote diabetic alveolar bone regeneration. Agantigel can effectively inhibit bacterial growth through AgNCs while exhibiting negligible cytotoxicity in vitro. The sustained release of TNF-α antibody from Agantigel effectively blocks TNF-α and promotes M2 polarization of macrophages, ultimately accelerating diabetic alveolar bone regeneration in vivo. After 21 days of treatment, Agantigel significantly accelerates the defect healing rate of diabetic alveolar bone up to 82.58 ± 8.58% and improves trabecular architectures compared to free TNF-α (42.52 ± 15.85%). The results imply that DNA hydrogels are potential bio-scaffolds helping the sustained release of multidrug for treating DABI or other oral diseases.

The effects of first-line pharmacological treatments for reproductive outcomes in infertile women with PCOS: a systematic review and network meta-analysis.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in reproductive-age women. However, the efficacy and optimal therapeutic strategy for reproductive outcomes are still under debate. We conducted a systematic review and network meta-analysis to compare the efficacy of different first-line pharmacological therapies in terms of reproductive outcomes for women with PCOS and infertility. METHODS: A systematic retrieval of databases was conducted, and randomized clinical trials (RCTs) of pharmacological interventions for infertile PCOS women were included. The primary outcomes were clinical pregnancy and live birth, and the secondary outcomes were miscarriage, ectopic pregnancy and multiple pregnancy. A network meta-analysis based on a Bayesian model was performed to compare the effects of the pharmacological strategies. RESULTS: A total of 27 RCTs with 12 interventions were included, and all therapies tended to increase clinical pregnancy, especially pioglitazone (PIO) (log OR 3.14, 95% CI 1.56 ~ 4.70, moderate confidence), clomiphene citrate (CC) + exenatide (EXE) (2.96, 1.07 ~ 4.82, moderate confidence) and CC + metformin (MET) + PIO (2.82, 0.99 ~ 4.60, moderate confidence). Moreover, CC + MET + PIO (2.8, -0.25 ~ 6.06, very low confidence) could increase live birth most when compared to placebo, even without a significant difference. For secondary outcomes, PIO showed a tendency to increase miscarriage (1.44, -1.69 ~ 5.28, very low confidence). MET (-11.25, -33.7 ~ 0.57, low confidence) and LZ + MET (-10.44, -59.56 ~ 42.11, very low confidence) were beneficial for decreasing ectopic pregnancy. MET (0.07, -4.26 ~ 4.34, low confidence) showed a neutral effect in multiple pregnancy. Subgroup analysis demonstrated no significant difference between these medications and placebo in obese participants. CONCLUSIONS: Most first-line pharmacological treatments were effective in improving clinical pregnancy. CC + MET + PIO should be recommended as the optimal therapeutic strategy to improve pregnancy outcomes. However, none of the above treatments had a beneficial effect on clinical pregnancy in obese PCOS. TRIAL REGISTRATION: CRD42020183541; 05 July 2020.

Calcitriol, an Active Form of Vitamin D3, Mitigates Skin Barrier Dysfunction in Atopic Dermatitis NC/Nga Mice.

Atopic dermatitis and psoriasis are prevalent chronic inflammatory skin diseases that are characterized by dysfunctional skin barriers and substantially impact patients' quality of life. Vitamin D3 regulates immune responses and keratinocyte differentiation and improves psoriasis symptoms; however, its effects on atopic dermatitis remain unclear. Here, we investigated the effects of calcitriol, an active form of vitamin D3, on an NC/Nga mouse model of atopic dermatitis. We observed that the topical application of calcitriol decreased the dermatitis scores and epidermal thickness of NC/Nga mice with atopic dermatitis compared to untreated mice. In addition, both stratum corneum barrier function as assessed by the measurement of transepidermal water loss and tight junction barrier function as evaluated by biotin tracer permeability assay were improved following calcitriol treatment. Moreover, calcitriol treatment reversed the decrease in the expression of skin barrier-related proteins and decreased the expression of inflammatory cytokines such as interleukin (IL)-13 and IL-33 in mice with atopic dermatitis. These findings suggest that the topical application of calcitriol might improve the symptoms of atopic dermatitis by repairing the dysfunctional epidermal and tight junction barriers. Our results suggest that calcitriol might be a viable therapeutic agent for the treatment of atopic dermatitis in addition to psoriasis.

The Antimicrobial Peptide AMP-IBP5 Suppresses Dermatitis-like Lesions in a Mouse Model of Atopic Dermatitis through the Low-Density Lipoprotein Receptor-Related Protein-1 Receptor.

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) exhibits antimicrobial activities and immunomodulatory functions in keratinocytes and fibroblasts. However, its role in regulating skin barrier function remains unclear. Here, we investigated the effects of AMP-IBP5 on the skin barrier and its role in the pathogenesis of atopic dermatitis (AD). 2,4-Dinitrochlorobenzene was used to induce AD-like skin inflammation. Transepithelial electrical resistance and permeability assays were used to investigate tight junction (TJ) barrier function in normal human epidermal keratinocytes and mice. AMP-IBP5 increased the expression of TJ-related proteins and their distribution along the intercellular borders. AMP-IBP5 also improved TJ barrier function through activation of the atypical protein kinase C and Rac1 pathways. In AD mice, AMP-IBP5 ameliorated dermatitis-like symptoms restored the expression of TJ-related proteins, suppressed the expression of inflammatory and pruritic cytokines, and improved skin barrier function. Interestingly, the ability of AMP-IBP5 to alleviate inflammation and improve skin barrier function in AD mice was abolished in mice treated with an antagonist of the low-density lipoprotein receptor-related protein-1 (LRP1) receptor. Collectively, these findings indicate that AMP-IBP5 may ameliorate AD-like inflammation and enhance skin barrier function through LRP1, suggesting a possible role for AMP-IBP5 in the treatment of AD.

Carrot and carotene and multiple health outcomes: an umbrella review of the evidence.

In recent years, the benefits of carrots and carotene in different areas of health have been examined. The purpose of this umbrella review was to identify the associations between carrots and carotene and multiple health outcomes. The review considered evidence from meta-analyses of interventional and observational studies of carrots and carotene and any health outcome. We comprehensively searched Web of Science, PubMed, and Embase. For each association, we estimated the summary effect size using random and fixed effects models and the 95% confidence interval. A total of 1329 studies were searched, and 30 meta-analyses with 26 health outcomes were identified that met the eligibility criteria. Carrot intake was associated with a lower risk of multiple cancer outcomes including breast cancer, lung cancer, pancreatic cancer, gastric cancer, urothelial cancer, and prostate cancer. Carotene intake was associated with a lower risk of fracture, age-related cataract, sunburn, Alzheimer's disease, breast cancer, lung cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, and head and neck cancer (HNC). Serum carotene was inversely associated with all-cause mortality, breast cancer, and lung cancer. Our study revealed that carrot or carotene intake could reduce the risk of various negative health outcomes. © 2023 Society of Chemical Industry.

Candidalysin, a Virulence Factor of Candida albicans, Stimulates Mast Cells by Mediating Cross-Talk Between Signaling Pathways Activated by the Dectin-1 Receptor and MAPKs.

PURPOSE: Although mast cells (MCs) modulate the activity of effector cells during Candida albicans infection, their role in the pathogenesis of candidiasis remains unclear. Candidalysin, a C. albicans-derived peptide toxin, is a crucial factor in fungal infections. We aimed to investigate the effect of candidalysin on MC activation and the underlying molecular mechanism. METHODS: Serum from candidalysin-immunized mice was used to measure candidalysin expression in patients infected with C. albicans. MC degranulation and migration were evaluated by ß-hexosaminidase release assay and chemotaxis assay, respectively. EIA and ELISA were used to evaluate the production of eicosanoids and cytokines/chemokines, respectively. The production of nitric oxide (NO) was measured with a DAF-FM diacetate kit, while reactive oxygen species (ROS) production was analyzed by flow cytometry. MAPK activation was evaluated by Western blotting. RESULTS: We detected high candidalysin expression in the lesions of patients infected with C. albicans, and the MC number was increased in these lesions. LL-37 colocalized with MCs in the lesions of candidiasis patients. Candidalysin-enhanced MC accumulation in mice and treating LAD2 and HMC-1 cells with candidalysin induced their degranulation, migration, and production of pro- and anti-inflammatory cytokines/chemokines, eicosanoids, ROS, NO, and LL-37. Interestingly, C. albicans strains lacking candidalysin failed to induce MC activation. Moreover, candidalysin increased dectin-1 expression, and the inhibition of dectin-1 decreased MC activation. Downstream dectin-1 signaling involved the MAPK pathways. CONCLUSION: The finding that candidalysin causes cutaneous MC activation may improve our understanding of the role of MCs in the pathology of cutaneous C. albicans infection.

Antimicrobial peptide derived from insulin-like growth factor-binding protein 5 improves diabetic wound healing.

Impaired keratinocyte functions are major factors that are responsible for delayed diabetic wound healing. In addition to its antimicrobial activity, the antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) activates mast cells and promotes keratinocyte and fibroblast proliferation and migration. However, its effects on diabetic wound healing remain unclear. Human keratinocytes were cultured in normal or high glucose milieus. The production of angiogenic growth factor and cell proliferation and migration were evaluated. Wounds in normal and streptozotocin-induced diabetic mice were monitored and histologically examined. We found that AMP-IBP5 rescued the high glucose-induced attenuation of proliferation and migration as well as the production of angiogenin and vascular endothelial growth factors in keratinocytes. The AMP-IBP5-induced activity was mediated by the epidermal growth factor receptor, signal transducer and activator of transcription 1 and 3, and mitogen-activated protein kinase pathways, as indicated by the inhibitory effects of pathway-specific inhibitors. In vivo, AMP-IBP5 markedly accelerated wound healing, increased the expression of angiogenic factors and promoted vessel formation in both normal and diabetic mice. Overall, the finding that AMP-IBP5 accelerated diabetic wound healing by protecting against glucotoxicity and promoting angiogenesis suggests that AMP-IBP5 might be a potential therapeutic target for treating chronic diabetic wounds.

Preparation and characterization of a polyclonal antibody against PTEN-Long.

Phosphatase and tensin homolog-long (PTEN-L) is a translational isoform of PTEN, which exists in both intracellular and extracellular locations. Previous studies demonstrated that PTEN-L could inhibit oncogenesis due to its lipid phosphatase activity. However, recent studies found that PTEN-L could promote the proliferation of some types of cancer cells. Moreover, as a protein phosphatase, PTEN-L can suppress mitophagy by counteracting PTEN-induced putative kinase protein 1 (PINK1)-Parkin-mediated ubiquitin phosphorylation, namely, PTEN-L is critical for exploring the mitophagy progression and the treatment of mitochondrial diseases. Accounting for the critical functions of PTEN-L, its antibody can be used for the treatment or prognosis of tumors and mitochondrial diseases. Currently, the commercial antibody of PTEN-L is not available. In our study, the recombinant PTEN-L protein was expressed in Escherichia coli BL21 and used as an antigen to immunize Japan's big-eared white rabbit for the preparation of polyclonal antibody. The PTEN-L protein can be captured by PTEN-L antibody specifically and effectively. Taken together, a PTEN_L antibody is a valuable tool for further exploring the function of PTEN-L in oncogenesis and mitochondrial diseases, and it would be a new choice for the prognosis or treatment of cancer and mitochondrial diseases.

The Antimicrobial Peptides Human ß-Defensins Induce the Secretion of Angiogenin in Human Dermal Fibroblasts.

The skin produces a plethora of antimicrobial peptides that not only show antimicrobial activities against pathogens but also exhibit various immunomodulatory functions. Human ß-defensins (hBDs) are the most well-characterized skin-derived antimicrobial peptides and contribute to diverse biological processes, including cytokine production and the migration, proliferation, and differentiation of host cells. Additionally, hBD-3 was recently reported to promote wound healing and angiogenesis, by inducing the expression of various angiogenic factors and the migration and proliferation of fibroblasts. Angiogenin is one of the most potent angiogenic factors; however, the effects of hBDs on angiogenin production in fibroblasts remain unclear. Here, we investigated the effects of hBDs on the secretion of angiogenin by human dermal fibroblasts. Both in vitro and ex vivo studies demonstrated that hBD-1, hBD-2, hBD-3, and hBD-4 dose-dependently increased angiogenin production by fibroblasts. hBD-mediated angiogenin secretion involved the epidermal growth factor receptor (EGFR), Src family kinase, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-kappa B (NF-κB) pathways, as evidenced by the inhibitory effects of specific inhibitors for these pathways. Indeed, we confirmed that hBDs induced the activation of the EGFR, Src, JNK, p38, and NF-κB pathways. This study identified a novel role of hBDs in angiogenesis, through the production of angiogenin, in addition to their antimicrobial activities and other immunomodulatory properties.

Experimental and Clinical Evidence Suggests That Treatment with Betacellulin Can Alleviate Th2-Type Cytokine-Mediated Impairment of Skin Barrier Function.

Betacellulin (BTC) is a peptide ligand that belongs to the epidermal growth factor family, the members of which have been implicated in skin morphogenesis, homeostasis, repair, and angiogenesis; however, the role of BTC in the regulation of the skin barrier remains unknown. To examine the role of BTC in skin barrier function, we analyzed atopic dermatitis (AD) transcriptomic data from Gene Expression Omnibus (GEO) datasets, performed BTC immunohistochemistry using human skin tissues, and evaluated the effects of BTC on primary human keratinocytes by real-time PCR, Western blotting, and assay of the transepidermal electrical resistance (TER), a functional parameter to monitor the tight junction barrier. We found that the gene expression of BTC was downregulated in skin lesions from patients with AD, and this downregulated expression recovered following biological treatments. Consistently, the BTC protein levels were downregulated in the lesional skin of AD patients compared with the normal skin of healthy participants, suggesting that the BTC levels in skin might be a biomarker for the diagnosis and therapy of AD. Furthermore, in human keratinocytes, BTC knockdown reduced the levels of skin-derived antimicrobial peptides and skin barrier-related genes, whereas BTC addition enhanced their levels. Importantly, in human skin equivalents, BTC restored the increased tight junction permeability induced by Th2 cytokine IL-4/IL-13 treatment. In addition, specific inhibitors of epidermal growth factor receptor (EGFR) and protein kinase C (PKC) abolished the BTC-mediated improvement in skin barrier-related proteins in keratinocyte monolayers. Collectively, our findings suggest that treatment with BTC might improve the Th2-type cytokine-mediated impairment of skin barrier function through the EGFR/PKC axis and that BTC might be a novel potential biomarker and therapeutic target for the treatment of skin conditions characterized by the overproduction of Th2 cytokines and dysfunctional skin barriers, such as AD.

Identification of MDM2, YTHDF2 and DDX21 as potential biomarkers and targets for treatment of type 2 diabetes.

Type 2 diabetes (T2D) is a multifactorial and polygenetic disease, although its exact etiology remains poorly understood. The objective of this study was to identify key biomarkers and potential molecular mechanisms in the development of T2D. Human RNA-Seq datasets across different tissues (GSE18732, GSE41762, and GSE78721) were collected from the Gene Expression Omnibus (GEO) database and differentially expressed genes (DEGs) between T2D and controls were identified using differential analysis. A total of 90 overlapping DEGs were identified, among which YTHDF2, DDX21, and MDM2 were considered as key genes due to their central positions in the PPI network and the same regulatory pattern in T2D. Logistic regression analysis showed that low expression of the key genes increased the risk of T2D. Enrichment analysis revealed that the key genes are involved in various important biological functions and signaling pathways including Notch, Fork head box O (FOXO), and phosphoinositide 3-kinase (PI3K)-Akt. RT-qPCR and Western blot analysis showed that all three key genes were down-regulated in pancreatic islets of both prediabetic and diabetic mouse models. Finally, the insulin-sensitizer, pioglitazone was used to treat db/db mice and immunofluorescence analysis showed that the expression of all three key genes was significantly down-regulated in db/db islets, an effect that was overcome by pioglitazone treatment. Together, these results suggest that the identified key genes could be involved in the development of T2D and serve as potential biomarkers and therapeutic targets for this disease.

Exogenous factors in the pathogenesis of atopic dermatitis: Irritants and cutaneous infections.

Atopic dermatitis (AD) is a chronic relapsing inflammatory cutaneous disease that is often associated with other atopic symptoms, such as food allergy, allergic rhinitis and asthma, leading to significant morbidity and healthcare costs. The pathogenesis of AD is complicated and multifactorial. Although the aetiology of AD remains incompletely understood, recent studies have provided further insight into AD pathophysiology, demonstrating that the interaction among genetic predisposition, immune dysfunction and environmental provocation factors contributes to its development. However, the increasing prevalence of AD suggests that environmental factors such as irritation and cutaneous infection play a crucial role in triggering and/or aggravating the disease. Of note, AD skin is susceptible to bacterial, fungal and viral infections, and microorganisms may colonize the skin and aggravate AD symptoms. Overall, understanding the mechanisms by which these risk factors affect the cutaneous immunity of patients with AD is of great importance for developing a precision medicine approach for treatment. This review summarizes recent developments in exogenous factors involved in the pathogenesis of AD, with special emphasis on irritants and microbial infections.

Role of Antimicrobial Peptides in Skin Barrier Repair in Individuals with Atopic Dermatitis.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that exhibits a complex interplay of skin barrier disruption and immune dysregulation. Patients with AD are susceptible to cutaneous infections that may progress to complications, including staphylococcal septicemia. Although most studies have focused on filaggrin mutations, the physical barrier and antimicrobial barrier also play critical roles in the pathogenesis of AD. Within the physical barrier, the stratum corneum and tight junctions play the most important roles. The tight junction barrier is involved in the pathogenesis of AD, as structural and functional defects in tight junctions not only disrupt the physical barrier but also contribute to immunological impairments. Furthermore, antimicrobial peptides, such as LL-37, human b-defensins, and S100A7, improve tight junction barrier function. Recent studies elucidating the pathogenesis of AD have led to the development of barrier repair therapy for skin barrier defects in patients with this disease. This review analyzes the association between skin barrier disruption in patients with AD and antimicrobial peptides to determine the effect of these peptides on skin barrier repair and to consider employing antimicrobial peptides in barrier repair strategies as an additional approach for AD management.

Silencing of astrocyte elevated gene-1 inhibits proliferation and migration of melanoma cells and induces apoptosis.

Melanoma is an aggressive skin malignancy with a high mortality. Astrocyte elevated gene-1 (AEG-1), a downstream target of Ras and c-Myc, has been implicated in the development of multiple tumours, but its role in melanoma remains unclear. In the present study, the role of AEG-1 in melanoma was explored through AEG-1 silencing. Our results showed that silencing AEG-1 inhibited the proliferation of melanoma cells, induced cell cycle arrest, and reduced levels of cyclin A, cyclin B, cyclin D1, cyclin E, and cyclin-dependent kinase 2. AEG-1silencing also induced apoptosis in melanoma cells and altered the levels of cleaved caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein. Moreover, silencing AEG-1 suppressed the migration and invasion of melanoma cells, reduced the expressions and activities of matrix metallopeptidase (MMP)-2 and MMP-9, and inhibited the activation of the Wnt/ß-catenin signalling pathway in melanoma cells. Furthermore, in vivo experiments revealed that AEG-1 silencing inhibited the growth of melanoma xenografts in nude mice. In summary, our study demonstrates an oncogenic role of AEG-1 in melanoma and suggests that AEG-1 may serve as a potential therapeutic target in the treatment of melanoma.

Effect of an accelerating metro cabin on the diffusion of cough droplets.

Coronaviruses being capable of spreading through droplet contamination have raised significant concerns regarding high-capacity public rail transport, such as the metro. Within a rapidly moving railcar cabin, the internal airflow lags behind the bulkhead, generating internally induced airflow that accelerates droplet dispersion within a non-inertial reference system. This study investigates the impact of acceleration on the diffusion of cough droplets of varying sizes using computational fluid dynamics. The modified k-ε equation in ANSYS® Fluent was utilized to simulate droplet diffusion under different body orientations by adjusting the inertial force correction source term. Results indicate that droplets in the middle size range (50-175 µm) are primarily influenced by inertial forces, whereas smaller droplets (3.5-20 µm) are predominantly controlled by air drag forces. Regardless of facial orientation, the outlet of high-capacity public rail transport poses the highest risk of infection.

A DNA-based and bifunctional nanomedicine for alleviating multi-organ injury in sepsis under diabetic conditions.

Sepsis, defined as a life-threatening organ dysfunction, is associated with increased mortality in individuals with diabetes mellitus. In sepsis under diabetic conditions (SUDC), the superimposed inflammatory response and excessive production of reactive oxygen species (ROS) can cause severe damage to the kidney and liver, making it challenging to effectively repair multi-organ injury. In this study, we report the development of a DNA-based bifunctional nanomedicine, termed IL10-rDON, generated by assembling interleukin 10 (IL10) with rectangular DNA origami nanostructures (rDON) to address multi-organ dysfunction in SUDC. IL10-rDON was shown to predominantly accumulate in the kidney and liver of diabetic mice in vivo and effectively alleviate inflammatory responses through its anti-inflammatory IL10 component. In addition, the consumption of rDON itself significantly reduced excessive ROS in the liver and kidney. Serum and histological examinations further confirmed that IL10-rDON treatment could effectively improve liver and kidney function, as well as the survival of mice with SUDC. This study demonstrates an attractive antioxidant and anti-inflammatory nanomedicine for addressing acute liver and renal failure. The integration of rDON with therapeutic agents using DNA nanotechnology is a promising strategy for generating multifunctional nanomedicine to treat multi-organ dysfunction and other complicated diseases. STATEMENT OF SIGNIFICANCE: Sepsis under diabetic conditions (SUDC) leads to high mortality due to multiple organ failure such as acute liver and kidney injury. The anti-inflammatory cytokine interleukin 10 (IL10) holds great potential to treat SUDC, while disadvantages of IL-10 such as short half-life, non-specific distribution and lack of antioxidant activities limit its wide clinical applications. In this study, we developed a DNA-based, bifunctional nanomedicine (IL10-rDON) by assembling IL10 with rectangular DNA origami nanostructures (rDON). We found that IL10-rDON preferentially accumulated and sufficiently attenuated the increased levels of ROS and inflammation in the kidney and liver injury sites, and eventually improved the survival rate of mice with SUDC. Our finding provides new insights into the application of DNA-based nanomedicine in treating multi-organ failure.