Bacterial Lipoproteins Shift Cellular Metabolism to Glycolysis in Macrophages Causing Bone Erosion.

Belonging to a group of membrane proteins, bacterial lipoproteins (LPPs) are defined by a unique lipid structure at their N-terminus providing the anchor in the bacterial cell membrane. In Gram-positive bacteria, LPPs play a key role in host immune activation triggered through a Toll-like receptor 2 (TLR2)-mediated action resulting in macrophage stimulation and subsequent tissue damage demonstrated in in vivo experimental models. Yet the physiologic links between LPP activation, cytokine release, and any underlying switches in cellular metabolism remain unclear. In this study, we demonstrate that Staphylococcus aureus Lpl1 not only triggers cytokine production but also confers a shift toward fermentative metabolism in bone marrow-derived macrophages (BMDMs). Lpl1 consists of di- and tri-acylated LPP variants; hence, the synthetic P2C and P3C, mimicking di-and tri-acylated LPPs, were employed to reveal their effect on BMDMs. Compared to P3C, P2C was found to shift the metabolism of BMDMs and the human mature monocytic MonoMac 6 (MM6) cells more profoundly toward the fermentative pathway, as indicated by lactate accumulation, glucose consumption, pH reduction, and oxygen consumption. In vivo, P2C caused more severe joint inflammation, bone erosion, and lactate and malate accumulation than P3C. These observed P2C effects were completely abrogated in monocyte/macrophage-depleted mice. Taken together, these findings now solidly confirm the hypothesized link between LPP exposure, a macrophage metabolic shift toward fermentation, and ensuing bone destruction. IMPORTANCE Osteomyelitis caused by S. aureus is a severe infection of the bone, typically associated with severe bone function impairment, therapeutic failure, high morbidity, invalidity, and occasionally even death. The hallmark of staphylococcal osteomyelitis is the destruction of the cortical bone structures, yet the mechanisms contributing to this pathology are hitherto poorly understood. One bacterial membrane constituent found in all bacteria is bacterial lipoproteins (LPPs). Previously, we have shown that injection of purified S. aureus LPPs into wild-type mouse knee joints caused a TLR2-dependent chronic destructive arthritis but failed to elicit such effect in monocyte/macrophage-depleted mice. This observation stirred our interest in investigating the interaction of LPPs and macrophages and analyzing the underlying physiological mechanisms. This ascertainment of LPP-induced changes in the physiology of macrophages provides an important clue in the understanding of the mechanisms of bone disintegration, opening novel avenues to manage the course of S. aureus disease.

Saving Money and Reducing Waste With a Tailored Hand Surgery Kit.

BACKGROUND AND OBJECTIVES: There is growing interest in containing cost and decreasing waste in the operating room. As part of a quality improvement initiative, we redesigned the supply kit used for 2 common surgical procedures (carpal tunnel release and trigger finger release) performed under local anesthesia. METHODS: A hand surgeon, a medical student, and an operating room nurse examined each item that would be necessary for performing carpal tunnel release and trigger finger release. A new disposable supply kit was formulated on the basis of their recommendations and was implemented over a 7-month period. Cost savings and waste avoidance were calculated. RESULTS: The streamlined kit ($43.40) produced a 53% cost savings relative to the standard hand pack ($92.83) per case. The local pack (2.896 kg) was 41% lighter than the standard pack (4.938 kg), translating to significant waste avoidance. The local hand pack was used for 46 cases from September 2020 to April 2021, saving a total of $2246.78 and avoiding 94 kg of waste. There have been no noted interruptions in delivery of surgical care. CONCLUSION: Our redesign of the local hand pack led to substantial cost savings and waste avoidance. We believe there are many opportunities for surgical teams to use similar strategies to decrease cost and environmental waste.

Phenol-soluble modulin α and ß display divergent roles in mice with staphylococcal septic arthritis.

Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMß which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmß). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMß has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMß strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMß play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMß protects arthritis development.

Ex vivo Vitamin D supplementation improves viscoelastic profiles in prostate cancer patients.

BACKGROUND: Increased risk of thromboembolic events is associated with prostate cancer, specifically linked to activation of tissue factor. Vitamin D has potential anticoagulant effects by the downregulation of tissue factor expression. OBJECTIVES: To evaluate the effects on clot formation, the morphological and viscoelastic profiles of prostate cancer patients, before and after ex vivo supplementation of Vitamin D was studied. METHODS: Participants were recruited into a metastatic, non-metastatic and reference group. Whole blood samples were treated ex vivo with a dose of 0.5µg/kg Calcitriol. Clot kinetics were assessed using Thromboelastography®. Morphology of the blood components were studied using scanning electron microscopy (SEM). RESULTS: Results from the Thromboelastography® and SEM indicated no major differences between the non-metastatic group before and after treatment compared to the reference group. The Thromboelastography® showed that the metastatic group had an increased viscoelastic profile relating to a hypercoagulable state. Visible changes with regards to platelet activation and fibrin morphology were demonstrated with SEM analysis of the metastatic group. The viscoelastic and morphological properties for the non-metastatic group after treatment improved to be comparable to the reference group. CONCLUSION: Vitamin D supplementation may lead to a more favorable viscoelastic profile, with less dangerous clots forming.

Lipoproteins Cause Bone Resorption in a Mouse Model of Staphylococcus aureus Septic Arthritis.

Septic arthritis, most often caused by Staphylococcus aureus, is a rapidly progressive and destructive joint disease with substantial mortality and morbidity. Staphylococcus aureus lipoproteins (Lpps) are known to induce arthritis and bone destruction. Here, we aimed to investigate the bone resorptive effect of S. aureus Lpps in a murine arthritis model by intra-articular injection of purified S. aureus Lpps, synthetic lipopeptides, and live S. aureus strains. Analyses of the bone mineral density (BMD) of the distal femur bone were performed. Intra-articular injection of both live S. aureus and purified S. aureus Lpps were shown to significantly decrease total- and trabecular BMD. Liquid chromatography-mass spectrometry analyses revealed that the Lpps expressed by S. aureus SA113 strain contain both diacyl and triacyl lipid moieties. Interestingly, synthetic diacylated lipopeptide, Pam2CSK4, was more potent in inducing bone resorption than synthetic triacylated lipopeptide, Pam3CSK4. Modified lipoproteins lacking the lipid moiety were deprived of their bone resorptive abilities. Monocyte depletion by clodronate liposomes fully abrogated the bone resorptive capacity of S. aureus lipoproteins. Our data suggest that S. aureus Lpps induce bone resorption in locally-induced murine arthritis, an effect mediated by their lipid-moiety through monocytes/macrophages.

Fetal Alcohol Spectrum Disorder-Issues of Misdiagnosis and Missed Diagnosis in Black Youth: A Case Report.

Fetal alcohol spectrum disorder (FASD) is an umbrella term used to describe the set of conditions that result from prenatal alcohol exposure (PAE) that lead to cognitive impairment, neurodevelopmental delays, socioemotional and behavioral problems, medical complications, and/or secondary disabilities. In addition, various internalizing and externalizing disorders share similar symptoms with FASD, resulting in misdiagnoses and/or missed diagnosis of FASD. This is amplified for Black youths due to the later onset of referral for assessment and lower frequency of referral to specialty clinics. This clinical case report depicts a misdiagnosis and a missed diagnosis of FASD in a 10-year-old African American patient, who was referred for neuropsychological evaluation. Diagnoses at the time of referral included attention-deficit/hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and disruptive mood dysregulation disorder (DMDD). Upon completion of a comprehensive evaluation, the patient's diagnoses changed to neurodevelopmental disorder associated with prenatal alcohol exposure, intellectual disability (ID), ADHD, and unspecified depressive disorder, leading to referral to appropriate interventions. The goal of this clinical case report is to increase clinician understanding of FASD and its clinical presentation, inform clinicians about the diagnostic and systemic factors that contribute to misdiagnosis and missed diagnosis of FASD, and to demonstrate the importance of an accurate diagnosis of FASD. By depicting the diagnostic challenges in an African American youth, the authors hope to bring awareness to the racial and ethnic disparities in the diagnosis of neurodevelopmental disabilities, specifically FASD in minority youth.

Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway.

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Thioredoxin system-mediated regulation of mutant Kras associated pancreatic neoplasia and cancer.

Peroxiredoxin-1 (Prdx1), a member of the thioredoxin (Txn) system, is overexpressed and correlates with poor prognosis in pancreatic cancer patients and can suppress Kras signaling through redox-mediated inhibition of ERK and AKT in lung and breast cancer. Its redox function is maintained by Txn and sulfiredoxin (Srxn), and its tumor promoting functions are activated by post-translational modification. We studied the role of the Txn system in pancreatic neoplasia and cancer by determining how it regulates the phosphorylation of Kras effectors and by determining its association with patient survival. We found that elevated Prdx1 nuclear localization significantly correlated with better patient survival. Our data also demonstrate that the expression of the Txn system is dysregulated, with elevated Prdx1 expression and significantly decreased Txn and Srxn expression in pancreatic lesions of targeted mutant Kras mouse models. This correlated with distinct differences in the interconversion of Prdx1 oligomers that affect its ability to regulate ERK and AKT phosphorylation. Our data also suggest that Prdx1 post-translational modification and oligomerization suppress Prdx1 mediated redox regulation of ERK phosphorylation. We observed distinct differences in Txn expression and in the ability of pTyr-Prdx1 to bind to pERK in a PanIN model of pancreatic neoplasia as compared to an IPMN model, indicating a distinct difference in the function of post-translationally modified Prdx1 in cells with less Txn expression. Modified Txn system function and post-translational regulation may therefore play a significant role in pancreatic tumorigenesis by altering Kras effector phosphorylation and inhibiting the tumor suppressive redox functions of Prdx1.

Ablation of 5-lipoxygenase mitigates pancreatic lesion development.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), which continues to have a dismal prognosis, is associated with a pronounced fibroinflammatory response. Inflammation in vivo can be mediated by 5-lipoxygenase (5LO), an enzyme that converts omega-6 fatty acids (FA) to eicosanoids, including leukotriene B4 (LTB4). We have previously shown that diets rich in omega-6 FA increase pancreatic lesions and mast cell infiltration in EL-Kras mice. In this study, we evaluated the role of 5LO in generating higher levels of LTB4 from human cells and in mediating lesion development and mast cell infiltration in EL-Kras mice. MATERIALS AND METHODS: Human pancreatic ductal epithelial and cancer cells were treated with omega-6 FA in vitro. EL-Kras mice lacking 5LO (EL-Kras/5LO(-/-)) mice were generated and fed standard chow or omega-6 FA diets. Pancreatic lesion frequency and mast cell infiltration were compared with EL-Kras/5LO(+/+) mice. Human PDAC tumors were evaluated for 5LO expression and mast cells. RESULTS: Human pancreatic ductal epithelial and pancreatic cancer cells treated with omega-6 FA generated increased LTB4 levels in vitro. EL-Kras/5LO(-/-) mice developed fewer pancreatic lesions and had decreased mast cell infiltration when compared with EL-Kras/5LO(+/+) mice. Human PDAC tumors with increased 5LO expression demonstrate increased mast cell infiltration. Additionally, diets rich in omega-6 FA failed to increase pancreatic lesion development and mast cell infiltration in EL-Kras/5LO(-/-) mice. CONCLUSIONS: The expansion of mutant Kras-induced lesions via omega-6 FA is dependent on 5LO, and 5LO functions downstream of mutant Kras to mediate inflammation, suggesting that 5LO may be a potential chemopreventive and therapeutic target in pancreatic cancer.

Nrf1 and Nrf2 transcription factors regulate androgen receptor transactivation in prostate cancer cells.

Despite androgen deprivation therapy (ADT), persistent androgen receptor (AR) signaling enables outgrowth of castration resistant prostate cancer (CRPC). In prostate cancer (PCa) cells, ADT may enhance AR activity through induction of oxidative stress. Herein, we investigated the roles of Nrf1 and Nrf2, transcription factors that regulate antioxidant gene expression, on hormone-mediated AR transactivation using a syngeneic in vitro model of androgen dependent (LNCaP) and castration resistant (C4-2B) PCa cells. Dihydrotestosterone (DHT) stimulated transactivation of the androgen response element (ARE) was significantly greater in C4-2B cells than in LNCaP cells. DHT-induced AR transactivation was coupled with higher nuclear translocation of p65-Nrf1 in C4-2B cells, as compared to LNCaP cells. Conversely, DHT stimulation suppressed total Nrf2 levels in C4-2B cells but elevated total Nrf2 levels in LNCaP cells. Interestingly, siRNA mediated silencing of Nrf1 attenuated AR transactivation while p65-Nrf1 overexpression enhanced AR transactivation. Subsequent studies showed that Nrf1 physically interacts with AR and enhances AR's DNA-binding activity, suggesting that the p65-Nrf1 isoform is a potential AR coactivator. In contrast, Nrf2 suppressed AR-mediated transactivation by stimulating the nuclear accumulation of the p120-Nrf1 which suppressed AR transactivation. Quantitative RT-PCR studies further validated the inductive effects of p65-Nrf1 isoform on the androgen regulated genes, PSA and TMPRSS2. Therefore, our findings implicate differential roles of Nrf1 and Nrf2 in regulating AR transactivation in PCa cells. Our findings also indicate that the DHT-stimulated increase in p65-Nrf1 and the simultaneous suppression of both Nrf2 and p120-Nrf1 ultimately facilitates AR transactivation in CRPC cells.

Loss of Nrf2 accelerates ionizing radiation-induced bone loss by upregulating RANKL.

Radiation therapy is an integral part of treatment for cancer patients; however, major side effects of this modality include aberrant bone remodeling and bone loss. Ionizing radiation (IR) is a major external factor that contributes to a significant increase in oxidative stress such as reactive oxygen species (ROS), has been implicated in osteoporotic phenotypes, and has been implicated in osteoporotic phenotypes, bone loss, and fracture risk. One of the major cellular defenses against heightened oxidative stress is mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master transcription factor that regulates induction of antioxidant gene expression and phase II antioxidant enzymes. Our objective was to test the hypothesis that loss of functional Nrf2 increases radiation-induced bone loss. We irradiated (single dose, 20Gy) the hindlegs of age- and sex-matched Nrf2(+/+) and Nrf2(-/-) mice. After 1 month, microCT analysis and histology revealed a drastic overall decrease in the bone volume after irradiation of mice lacking Nrf2. Although radiation exposure led to bone loss in mice with intact Nrf2, it was dramatically enhanced by loss of Nrf2. Furthermore, in the absence of Nrf2, a decrease in osteoblast mineralization was noted in calvarial osteoblasts compared with wild-type controls, and treatment with a common antioxidant, N-acetyl-l-cysteine (NAC), was able to rescue the mineralization. As expected, we observed a higher number of osteoclasts in Nrf2(-/-) mice compared to Nrf2(+/+) mice, and after irradiation, the trend remained the same. RT-PCR analysis of calvarial osteoblasts revealed that in the absence of Nrf2, the expression of RANKL was increased after irradiation. Interestingly, RANKL expression was suppressed when the calvarial osteoblasts were treated with NAC before IR exposure. Taken together, our data suggest that loss of Nrf2 leads to heightened oxidative stress and increased susceptibility to radiation-induced bone loss.

Studies on molecular mechanisms of growth inhibitory effects of thymoquinone against prostate cancer cells: role of reactive oxygen species.

Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.

The nrf1 and nrf2 balance in oxidative stress regulation and androgen signaling in prostate cancer cells.

Reactive oxygen species (ROS) signaling has recently sparked a surge of interest as being the molecular underpinning for cancer cell survival, but the precise mechanisms involved have not been completely elucidated. This review covers the possible roles of two ROS-induced transcription factors, Nrf1 and Nrf2, and the antioxidant proteins peroxiredoxin-1 (Prx-1) and Thioredoxin-1 (Txn-1) in modulating AR expression and signaling in aggressive prostate cancer (PCa) cells. In androgen independent (AI) C4-2B cells, in comparison to the parental androgen dependent (AD) LNCaP cells, we present evidence of high Nrf1 and Prx-1 expression and low Nrf2 expression in these aggressive PCa cells. Furthermore, in DHT treated C4-2B cells, increased expression of the p65 (active) isoform of Nrf1 correlated with enhanced AR transactivation. Our findings implicate a crucial balance of Nrf1 and Nrf2 signaling in regulating AR activity in AI-PCa cells. Here we will discuss how understanding the mechanisms by which oxidative stress may affect AR signaling may aid in developing novel therapies for AI-PCa.