PXL01 alters macrophage response with no effect on axonal outgrowth or Schwann cell response after nerve repair in rats.

Background: Adjunctive pharmacological treatment may improve nerve regeneration. We investigated nerve regeneration processes of PXL01 - a lactoferrin-derived peptide - after repair of the sciatic nerve in healthy Wistar rats.Materials & methods: PXL01, sodium hyaluronate (carrier) or sodium chloride was administered around the repair. After 6 days axonal outgrowth, Schwann cell response, pan- (CD68) and pro-healing (CD206) macrophages in sciatic nerve, sensory neuronal response in dorsal root ganglia (DRG) and expression of heat shock protein 27 (HSP27) in sciatic nerves and DRGs were analyzed.Results: Despite a lower number of pan-macrophages, other investigated variables in sciatic nerves or DRGs did not differ between the treatment groups.Conclusion: PLX01 applied locally inhibits inflammation through pan-macrophages in repaired sciatic nerves without any impact on nerve regeneration or pro-healing macrophages.

[Box: see text].

Genetic Ablation of STE20-Type Kinase MST4 Does Not Alleviate Diet-Induced MASLD Susceptibility in Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its advanced subtype, metabolic dysfunction-associated steatohepatitis (MASH), have emerged as the most common chronic liver disease worldwide, yet there is no targeted pharmacotherapy presently available. This study aimed to investigate the possible in vivo function of STE20-type protein kinase MST4, which was earlier implicated in the regulation of hepatocellular lipotoxic milieu in vitro, in the control of the diet-induced impairment of systemic glucose and insulin homeostasis as well as MASLD susceptibility. Whole-body and liver-specific Mst4 knockout mice were generated by crossbreeding conditional Mst4fl/fl mice with mice expressing Cre recombinase under the Sox2 or Alb promoters, respectively. To replicate the environment in high-risk subjects, Mst4-/- mice and their wild-type littermates were fed a high-fat or a methionine-choline-deficient (MCD) diet. Different in vivo tests were conducted in obese mice to describe the whole-body metabolism. MASLD progression in the liver and lipotoxic damage to adipose tissue, kidney, and skeletal muscle were analyzed by histological and immunofluorescence analysis, biochemical assays, and protein and gene expression profiling. In parallel, intracellular fat storage and oxidative stress were assessed in primary mouse hepatocytes, where MST4 was silenced by small interfering RNA. We found that global MST4 depletion had no effect on body weight or composition, locomotor activity, whole-body glucose tolerance or insulin sensitivity in obese mice. Furthermore, we observed no alterations in lipotoxic injuries to the liver, adipose, kidney, or skeletal muscle tissue in high-fat diet-fed whole-body Mst4-/- vs. wild-type mice. Liver-specific Mst4-/- mice and wild-type littermates displayed a similar severity of MASLD when subjected to an MCD diet, as evidenced by equal levels of steatosis, inflammation, hepatic stellate cell activation, fibrosis, oxidative/ER stress, and apoptosis in the liver. In contrast, the in vitro silencing of MST4 effectively protected primary mouse hepatocytes against ectopic lipid accumulation and oxidative cell injury triggered by exposure to fatty acids. In summary, these results suggest that the genetic ablation of MST4 in mice does not mitigate the initiation or progression of MASLD and has no effect on systemic glucose or insulin homeostasis in the context of nutritional stress. The functional compensation for the genetic loss of MST4 by yet undefined mechanisms may contribute to the apparent discrepancy between in vivo and in vitro phenotypic consequences of MST4 silencing.

Knockout of STE20-type kinase TAOK3 does not attenuate diet-induced NAFLD development in mice.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD), the primary hepatic consequence of obesity, is affecting about 25% of the global adult population. The aim of this study was to examine the in vivo role of STE20-type protein kinase TAOK3, which has been previously reported to regulate hepatocellular lipotoxicity in vitro, in the development of NAFLD and systemic insulin resistance in the context of obesity. METHODS: Taok3 knockout mice and wild-type littermates were challenged with a high-fat diet. Various in vivo tests were performed to characterize the whole-body metabolism. NAFLD progression in the liver, and lipotoxic damage in adipose tissue, kidney, and skeletal muscle were compared between the genotypes by histological assessment, immunofluorescence microscopy, protein and gene expression profiling, and biochemical assays. Intracellular lipid accumulation and oxidative/ER stress were analyzed in cultured human and mouse hepatocytes where TAOK3 was knocked down by small interfering RNA. The expression of TAOK3-related STE20-type kinases was quantified in different organs from high-fat diet-fed Taok3-/- and wild-type mice. RESULTS: TAOK3 deficiency had no impact on body weight or composition, food consumption, locomotor activity, or systemic glucose or insulin homeostasis in obese mice. Consistently, Taok3-/- mice and wild-type littermates developed a similar degree of high-fat diet-induced liver steatosis, inflammation, and fibrosis, and we detected no difference in lipotoxic damage of adipose tissue, kidney, or skeletal muscle when comparing the two genotypes. In contrast, the silencing of TAOK3 in vitro markedly suppressed ectopic lipid accumulation and metabolic stress in mouse and human hepatocytes. Interestingly, the hepatic mRNA abundance of several TAOK3-related kinases, which have been previously implicated to increase the risk of NAFLD susceptibility, was significantly elevated in Taok3-/- vs. wild-type mice. CONCLUSIONS: In contrast to the in vitro observations, genetic deficiency of TAOK3 in mice failed to mitigate the detrimental metabolic consequences of chronic exposure to dietary lipids, which may be partly attributable to the activation of liver-specific compensation response for the genetic loss of TAOK3 by related STE20-type kinases.

STE20-type kinases MST3 and MST4 promote the progression of hepatocellular carcinoma: Evidence from human cell culture and expression profiling of liver biopsies.

Hepatocellular carcinoma (HCC) is one of the most fatal and fastest growing malignancies. Recently, nonalcoholic steatohepatitis (NASH), characterized by liver steatosis, inflammation, cell injury (hepatocyte ballooning), and different stages of fibrosis, has emerged as a major catalyst for HCC. Because the STE20-type kinases, MST3 and MST4, have been described as critical molecular regulators of NASH pathophysiology, we here focused on determining the relevance of these proteins in human HCC. By analyzing public datasets and in-house cohorts, we found that hepatic MST3 and MST4 expression was positively correlated with the incidence and severity of HCC. We also found that the silencing of both MST3 and MST4, but also either of them individually, markedly suppressed the tumorigenesis of human HCC cells including attenuated proliferation, migration, invasion, and epithelial-mesenchymal transition. Mechanistic investigations revealed lower activation of STAT3 signaling in MST3/MST4-deficient hepatocytes and identified GOLGA2 and STRIPAK complex as the binding partners of both MST3 and MST4. These findings reveal that MST3 and MST4 play a critical role in promoting the progression of HCC and suggest that targeting these kinases may provide a novel strategy for the treatment of liver cancer.

Silencing of STE20-type kinase TAOK1 confers protection against hepatocellular lipotoxicity through metabolic rewiring.

BACKGROUND: NAFLD has become the leading cause of chronic liver disease worldwide afflicting about one quarter of the adult population. NASH is a severe subtype of NAFLD, which in addition to hepatic steatosis connotes liver inflammation and hepatocyte ballooning. In light of the exponentially increasing prevalence of NAFLD, it is imperative to gain a better understanding of its molecular pathogenesis. The aim of this study was to examine the potential role of STE20-type kinase TAOK1 -a hepatocellular lipid droplet-associated protein-in the regulation of liver lipotoxicity and NAFLD etiology. METHODS: The correlation between TAOK1 mRNA expression in liver biopsies and the severity of NAFLD was evaluated in a cohort of 62 participants. Immunofluorescence microscopy was applied to describe the subcellular localization of TAOK1 in human and mouse hepatocytes. Metabolic reprogramming and oxidative/endoplasmic reticulum stress were investigated in immortalized human hepatocytes, where TAOK1 was overexpressed or silenced by small interfering RNA, using functional assays, immunofluorescence microscopy, and colorimetric analysis. Migration, invasion, and epithelial-mesenchymal transition were examined in TAOK1-deficient human hepatoma-derived cells. Alterations in hepatocellular metabolic and pro-oncogenic signaling pathways were assessed by immunoblotting. RESULTS: We observed a positive correlation between the TAOK1 mRNA abundance in human liver biopsies and key hallmarks of NAFLD (i.e., hepatic steatosis, inflammation, and ballooning). Furthermore, we found that TAOK1 protein fully colocalized with intracellular lipid droplets in human and mouse hepatocytes. The silencing of TAOK1 alleviated lipotoxicity in cultured human hepatocytes by accelerating lipid catabolism (mitochondrial ß-oxidation and triacylglycerol secretion), suppressing lipid anabolism (fatty acid influx and lipogenesis), and mitigating oxidative/endoplasmic reticulum stress, and the opposite changes were detected in TAOK1-overexpressing cells. We also found decreased proliferative, migratory, and invasive capacity, as well as lower epithelial-mesenchymal transition in TAOK1-deficient human hepatoma-derived cells. Mechanistic studies revealed that TAOK1 knockdown inhibited ERK and JNK activation and repressed acetyl-CoA carboxylase (ACC) protein abundance in human hepatocytes. CONCLUSIONS: Together, we provide the first experimental evidence supporting the role of hepatic lipid droplet-decorating kinase TAOK1 in NAFLD development through mediating fatty acid partitioning between anabolic and catabolic pathways, regulating oxidative/endoplasmic reticulum stress, and modulating metabolic and pro-oncogenic signaling.

Dendritic Nanogels Directed Dual-Encapsulation Topical Delivery System of Antimicrobial Peptides Targeting Skin Infections.

Antimicrobial peptides (AMPs) are promising antibacterial agents in the fight against multidrug resistant pathogens. However, their application to skin infections is limited by the absence of a realizable topical delivery strategy. Herein, a hybrid hierarchical delivery system for topical delivery of AMPs is accomplished through the incorporation of AMPs into dendritic nanogels (DNGs) and their subsequent embedding into poloxamer gel. The high level of control over the crosslink density and the number of chosen functionalities makes DNGs ideal capsules with tunable loading capacity for DPK-060, a human kininogen-derived AMP. Once embedded into the poloxamer gel, DPK-060 encapsulated in DNGs displays a slower release rate compared to those entrapped directly in the gels. In vitro EpiDerm Skin Irritation Tests show good biocompatibility, while MIC and time-kill curves reveal the potency of the peptide toward Staphylococcus aureus. Anti-infection tests on ex vivo pig skin and in vivo mouse infection models demonstrate that formulations with 0.5% and 1% AMPs significantly inhibit the growth of S. aureus. Similar outcomes are observed for an in vivo mouse surgical site infection model. Importantly, when normalizing the bacteria inhibition to released/free DPK-060 at the wound site, all formulations display superior efficacy compared to DPK-060 in solution.

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage.

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating ß-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.

GCKIII kinases in lipotoxicity: Roles in NAFLD and beyond.

Nonalcoholic fatty liver disease (NAFLD) is defined by excessive accumulation of lipid droplets within hepatocytes. The STE20-type kinases comprising the germinal center kinase III (GCKIII) subfamily - MST3, MST4, and STK25 - decorate intrahepatocellular lipid droplets and have recently emerged as critical regulators of the initiation and progression of NAFLD. While significant advancement has been made toward deciphering the role of GCKIII kinases in hepatic fat accumulation (i.e., steatosis) as well as the aggravation of NAFLD into its severe form nonalcoholic steatohepatitis (NASH), much remains to be resolved. This review provides a brief overview of the recent studies in patient cohorts, cultured human cells, and mouse models, which have characterized the function of MST3, MST4, and STK25 in the regulation of hepatic lipid accretion, meta-inflammation, and associated cell damage in the context of NAFLD/NASH. We also highlight the conflicting data and emphasize future research directions that are needed to advance our understanding of GCKIII kinases as potential targets in the therapy of NAFLD and its comorbidities. Conclusions: Several lines of evidence suggest that GCKIII proteins govern the susceptibility to hepatic lipotoxicity and that pharmacological inhibition of these kinases could mitigate NAFLD development and aggravation. Comprehensive characterization of the molecular mode-of-action of MST3, MST4, and STK25 in hepatocytes as well as extrahepatic tissues is important, especially in relation to their impact on carcinogenesis, to fully understand the efficacy as well as safety of GCKIII antagonism.

Silencing of STE20-type kinase STK25 in human aortic endothelial and smooth muscle cells is atheroprotective.

Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.

Antagonizing STK25 Signaling Suppresses the Development of Hepatocellular Carcinoma Through Targeting Metabolic, Inflammatory, and Pro-Oncogenic Pathways.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most fatal and fastest-growing cancers. Recently, nonalcoholic steatohepatitis (NASH) has been recognized as a major catalyst for HCC. Thus, additional research is critically needed to identify mechanisms involved in NASH-induced hepatocarcinogenesis, to advance the prevention and treatment of NASH-driven HCC. Because the sterile 20-type kinase serine/threonine kinase 25 (STK25) exacerbates NASH-related phenotypes, we investigated its role in HCC development and aggravation in this study. METHODS: Hepatocarcinogenesis was induced in the context of NASH in Stk25 knockout and wild-type mice by combining chemical procarcinogens and a dietary challenge. In the first cohort, a single injection of diethylnitrosamine was combined with a high-fat diet-feeding. In the second cohort, chronic administration of carbon tetrachloride was combined with a choline-deficient L-amino-acid-defined diet. To study the cell-autonomous mode of action of STK25, we silenced this target in the human hepatocarcinoma cell line HepG2 by small interfering RNA. RESULTS: In both mouse models of NASH-driven HCC, the livers from Stk25-/- mice showed a markedly lower tumor burden compared with wild-type controls. We also found that genetic depletion of STK25 in mice suppressed liver tumor growth through reduced hepatocellular apoptosis and decreased compensatory proliferation, by a mechanism that involves protection against hepatic lipotoxicity and inactivation of STAT3, ERK1/2, and p38 signaling. Consistently, silencing of STK25 suppressed proliferation, apoptosis, migration, and invasion in HepG2 cells, which was accompanied by lower expression of the markers of epithelial-mesenchymal transition and autophagic flux. CONCLUSIONS: This study provides evidence that antagonizing STK25 signaling hinders the development of NASH-related HCC and provides an impetus for further analysis of STK25 as a therapeutic target for NASH-induced HCC treatment in human beings.

Evaluation of LL-37 in healing of hard-to-heal venous leg ulcers: A multicentric prospective randomized placebo-controlled clinical trial.

Many patients with venous leg ulcers do not reach complete healing with compression treatment alone, which is current standard care. This clinical trial HEAL LL-37 was a phase IIb double-blind, randomized, placebo-controlled study, with the aim to evaluate the efficacy and safety of a new drug LL-37 for topical administration, in combination with compression therapy, in 148 patients suffering from hard-to-heal venous leg ulcers. The study had three arms, consisting of two groups treated with LL-37 at concentrations of 0.5 or 1.6 mg/mL, and a placebo cohort. Patients had a mean age of 67.6 years, a median ulcer duration of 20.3 months, and a mean wound size at the time of randomization of 11.6 cm2 . Efficacy analysis performed on the full study population did not identify any significant improvement in healing in patients treated with LL-37 as compared with the placebo. In contrast, a post hoc analysis revealed statistically significant improvement with LL-37 treatment in several interrelated healing parameters in the subgroup of patients with large target wounds (a wound area of at least 10 cm2 at randomization), which is a known negative prognostic factor for healing. The study drug was well tolerated and safe in both dose strengths. In summary, this clinical trial did not detect any significant differences in healing of venous lower leg ulcers in the entire study cohort comparing patients treated with LL-37 versus placebo. A subgroup analysis provided an interesting observation that LL-37 could offer a treatment benefit in patients with large ulcers, exigently warranting a further study adequately powered to statistically assess the treatment outcome in this patient group.

STE20-type kinase TAOK3 regulates hepatic lipid partitioning.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), defined by excessive lipid storage in hepatocytes, has recently emerged as a leading global cause of chronic liver disease. The aim of this study was to examine the role of STE20-type protein kinase TAOK3, which has previously been shown to associate with hepatic lipid droplets, in the initiation and aggravation of human NAFLD. METHODS: The correlation between TAOK3 mRNA expression and the severity of NAFLD was investigated in liver biopsies from 62 individuals. In immortalized human hepatocytes, intracellular fat deposition, lipid metabolism, and oxidative and endoplasmic reticulum stress were analyzed when TAOK3 was overexpressed or knocked down by small interfering RNA. Subcellular localization of TAOK3 was characterized in human and mouse hepatocytes by immunofluorescence microscopy. RESULTS: We found that the TAOK3 transcript levels in human liver biopsies were positively correlated with the key lesions of NAFLD (i.e., hepatic steatosis, inflammation, and ballooning). Overexpression of TAOK3 in cultured human hepatocytes exacerbated lipid storage by inhibiting ß-oxidation and triacylglycerol secretion while enhancing lipid synthesis. Conversely, silencing of TAOK3 attenuated lipid deposition in human hepatocytes by stimulating mitochondrial fatty acid oxidation and triacylglycerol efflux while suppressing lipogenesis. We also found aggravated or decreased oxidative/endoplasmic reticulum stress in human hepatocytes with increased or reduced TAOK3 levels, respectively. The subcellular localization of TAOK3 in human and mouse hepatocytes was confined to intracellular lipid droplets. CONCLUSIONS: This study provides the first evidence that hepatic lipid droplet-coating kinase TAOK3 is a critical regulatory node controlling liver lipotoxicity and susceptibility to NAFLD.

STE20-Type Protein Kinase MST4 Controls NAFLD Progression by Regulating Lipid Droplet Dynamics and Metabolic Stress in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) has emerged as a leading cause of chronic liver disease worldwide, primarily because of the massive global increase in obesity. Despite intense research efforts in this field, the factors that govern the initiation and subsequent progression of NAFLD are poorly understood, which hampers the development of diagnostic tools and effective therapies in this area of high unmet medical need. Here we describe a regulator in molecular pathogenesis of NAFLD: STE20-type protein kinase MST4. We found that MST4 expression in human liver biopsies was positively correlated with the key features of NAFLD (i.e., hepatic steatosis, lobular inflammation, and hepatocellular ballooning). Furthermore, the silencing of MST4 attenuated lipid accumulation in human hepatocytes by stimulating ß-oxidation and triacylglycerol secretion, while inhibiting fatty acid influx and lipid synthesis. Conversely, overexpression of MST4 in human hepatocytes exacerbated fat deposition by suppressing mitochondrial fatty acid oxidation and triacylglycerol efflux, while enhancing lipogenesis. In parallel to these reciprocal alterations in lipid storage, we detected substantially decreased or aggravated oxidative/endoplasmic reticulum stress in human hepatocytes with reduced or increased MST4 levels, respectively. Interestingly, MST4 protein was predominantly associated with intracellular lipid droplets in both human and rodent hepatocytes. Conclusion: Together, our results suggest that hepatic lipid droplet-decorating protein MST4 is a critical regulatory node governing susceptibility to NAFLD and warrant future investigations to address the therapeutic potential of MST4 antagonism as a strategy to prevent or mitigate the development and aggravation of this disease.

Silencing of STE20-type kinase MST3 in mice with antisense oligonucleotide treatment ameliorates diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is emerging as a leading cause of chronic liver disease worldwide. Despite intensive nonclinical and clinical research in this field, no specific pharmacological therapy is currently approved to treat NAFLD, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies have identified STE20-type kinase MST3, which localizes to intracellular lipid droplets, as a critical regulator of ectopic fat accumulation in human hepatocytes. Here, we explored whether treatment with Mst3-targeting antisense oligonucleotides (ASOs) can promote hepatic lipid clearance and mitigate NAFLD progression in mice in the context of obesity. We found that administration of Mst3-targeting ASOs in mice effectively ameliorated the full spectrum of high-fat diet-induced NAFLD including liver steatosis, inflammation, fibrosis, and hepatocellular damage. Mechanistically, Mst3 ASOs suppressed lipogenic gene expression, as well as acetyl-CoA carboxylase (ACC) protein abundance, and substantially reduced lipotoxicity-mediated oxidative and endoplasmic reticulum stress in the livers of obese mice. Furthermore, we found that MST3 protein levels correlated positively with the severity of NAFLD in human liver biopsies. In summary, this study provides the first in vivo evidence that antagonizing MST3 signaling is sufficient to mitigate NAFLD progression in conditions of excess dietary fuels and warrants future investigations to assess whether MST3 inhibitors may provide a new strategy for the treatment of patients with NAFLD.

Depletion of protein kinase STK25 ameliorates renal lipotoxicity and protects against diabetic kidney disease.

Diabetic kidney disease (DKD) is the most common cause of severe renal disease worldwide and the single strongest predictor of mortality in diabetes patients. Kidney steatosis has emerged as a critical trigger in the pathogenesis of DKD; however, the molecular mechanism of renal lipotoxicity remains largely unknown. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase 25 (STK25) as a critical regulator of ectopic lipid storage in several metabolic organs prone to diabetic damage. Here, we demonstrate that overexpression of STK25 aggravates renal lipid accumulation and exacerbates structural and functional kidney injury in a mouse model of DKD. Reciprocally, inhibiting STK25 signaling in mice ameliorates diet-induced renal steatosis and alleviates the development of DKD-associated pathologies. Furthermore, we find that STK25 silencing in human kidney cells protects against lipid deposition, as well as oxidative and endoplasmic reticulum stress. Together, our results suggest that STK25 regulates a critical node governing susceptibility to renal lipotoxicity and that STK25 antagonism could mitigate DKD progression.

Antimicrobial peptides as therapeutic agents: opportunities and challenges.

The rapid development of microbial resistance to conventional antibiotics has accelerated efforts to find anti-infectives with a novel mode-of-action, which are less prone to bacterial resistance. Intense nonclinical and clinical research is today ongoing to evaluate antimicrobial peptides (AMPs) as potential next-generation antibiotics. Currently, multiple AMPs are assessed in late-stage clinical trials, not only as novel anti-infective drugs, but also as innovative product candidates for immunomodulation, promotion of wound healing, and prevention of post-operative scars. The efforts to translate AMP-based research findings into pharmaceutical product candidates are expected to accelerate in coming years due to technological advancements in multiple areas, including an improved understanding of the mechanism-of-action of AMPs, smart formulation strategies, and advanced chemical synthesis protocols. At the same time, it is recognized that cytotoxicity, low metabolic stability due to sensitivity to proteolytic degradation, and limited oral bioavailability are some of the key weaknesses of AMPs. Furthermore, the pricing and reimbursement environment for new antimicrobial products remains as a major barrier to the commercialization of AMPs.

Lipid droplet-associated kinase STK25 regulates peroxisomal activity and metabolic stress response in steatotic liver.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25-/- versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.

Characterization of the in vitro, ex vivo, and in vivo Efficacy of the Antimicrobial Peptide DPK-060 Used for Topical Treatment.

Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical in vitro, ex vivo, and in vivo antimicrobial activity, as well as the potential to cause skin irritation, of human kininogen-derived antimicrobial peptide DPK-060 in different formulations designed for topical delivery. We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro (minimum microbicidal concentration <5 µg/ml). We also found that DPK-060 in poloxamer gel significantly suppressed microbial survival in an ex vivo wound infection model using pig skin and in an in vivo mouse model of surgical site infection (≥99 or ≥94% reduction in bacterial counts was achieved with 1% DPK-060 at 4 h post-treatment, respectively). Encapsulation of DPK-060 in different types of lipid nanocapsules or cubosomes did not improve the bactericidal potential of the peptide under the applied test conditions. No reduction in cell viability was observed in response to administration of DPK-060 in any of the formulations tested. In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, adsorption of the peptide to nanocarriers failed to show any additional benefits.

Protein kinase MST3 modulates lipid homeostasis in hepatocytes and correlates with nonalcoholic steatohepatitis in humans.

Ectopic lipid storage in the liver is considered the main risk factor for nonalcoholic steatohepatitis (NASH). Understanding the molecular networks controlling hepatocellular lipid deposition is therefore essential for developing new strategies to effectively prevent and treat this complex disease. Here, we describe a new regulator of lipid partitioning in human hepatocytes: mammalian sterile 20-like (MST) 3. We found that MST3 protein coats lipid droplets in mouse and human liver cells. Knockdown of MST3 attenuated lipid accumulation in human hepatocytes by stimulating ß-oxidation and triacylglycerol secretion while inhibiting fatty acid influx and lipid synthesis. We also observed that lipogenic gene expression and acetyl-coenzyme A carboxylase protein abundance were reduced in MST3-deficient hepatocytes, providing insight into the molecular mechanisms underlying the decreased lipid storage. Furthermore, MST3 expression was positively correlated with key features of NASH (i.e., hepatic lipid content, lobular inflammation, and hepatocellular ballooning) in human liver biopsies. In summary, our results reveal a role of MST3 in controlling the dynamic metabolic balance of liver lipid catabolism vs. lipid anabolism. Our findings highlight MST3 as a potential drug target for the prevention and treatment of NASH and related complex metabolic diseases.-Cansby, E., Kulkarni, N. M., Magnusson, E., Kurhe, Y., Amrutkar, M., Nerstedt, A., Ståhlman, M., Sihlbom, C., Marschall, H.-U., Borén, J., Blüher, M., Mahlapuu, M. Protein kinase MST3 modulates lipid homeostasis in hepatocytes and correlates with nonalcoholic steatohepatitis in humans.

Targeted Delivery of Stk25 Antisense Oligonucleotides to Hepatocytes Protects Mice Against Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide. Currently, no specific pharmacologic therapy is available for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase (STK)25 as a critical regulator of hepatic lipid partitioning and NAFLD/NASH. Here, we studied the metabolic benefit of liver-specific STK25 inhibitors on NAFLD development and progression in a mouse model of diet-induced obesity. METHODS: We developed a hepatocyte-specific triantennary N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide (ASO) targeting Stk25 and evaluated its effect on NAFLD features in mice after chronic exposure to dietary lipids. RESULTS: We found that systemic administration of hepatocyte-targeting GalNAc-Stk25 ASO in obese mice effectively ameliorated steatosis, inflammatory infiltration, hepatic stellate cell activation, nutritional fibrosis, and hepatocellular damage in the liver compared with mice treated with GalNAc-conjugated nontargeting ASO, without any systemic toxicity or local tolerability concerns. We also observed protection against high-fat-diet-induced hepatic oxidative stress and improved mitochondrial function with Stk25 ASO treatment in mice. Moreover, GalNAc-Stk25 ASO suppressed lipogenic gene expression and acetyl-CoA carboxylase protein abundance in the liver, providing insight into the molecular mechanisms underlying repression of hepatic steatosis. CONCLUSIONS: This study provides in vivo nonclinical proof-of-principle for the metabolic benefit of liver-specific inhibition of STK25 in the context of obesity and warrants future investigations to address the therapeutic potential of GalNAc-Stk25 ASO in the prevention and treatment of NAFLD.