Tourniquet Use and Local Tissue Concentrations of Cefazolin During Total Knee Arthroplasty: A Randomized Clinical Trial.

Importance: Prophylactic administration of antibiotics before skin incision is an important component in the prevention of periprosthetic joint infection in arthroplasty surgery. For antibiotics to be effective, the local tissue concentration (LTC) must exceed the minimum inhibitory concentration of typical infecting organisms; however, the LTC of cefazolin during arthroplasty is poorly understood. Objective: To compare the systemic concentration of cefazolin in serum with the LTC in fat, synovium, and bone during primary total knee arthroplasty (TKA) while assessing the effect of tourniquet inflation. Design, Setting, and Participants: This prospective randomized clinical trial was conducted from March 1, 2022, to June 30, 2023, in patients undergoing TKA at a single academic center. Intervention: Total knee arthroplasty with or without a limb tourniquet. Main Outcomes and Measures: Systemic blood and local tissues from the surgical site (fat, synovium, and bone) were harvested at regular intervals during the surgery. The primary outcome was the LTC of cefazolin, quantified using the liquid chromatography-tandem mass spectrometry technique. Results: A total of 59 patients were included in the study, with 29 in the tourniquet group (mean [SD] age, 69.3 [9.6] years; 23 [79.3%] female) and 30 in the no tourniquet group (mean [SD] age, 69.9 [9.7] years; 21 [70.0%] female). In patients undergoing TKA without a tourniquet, the mean concentration of cefazolin in serum was 71.9 µg/mL (95% CI, 66.4-77.5 µg/mL), whereas the mean LTCs were 13.9 µg/g (95% CI, 12.1-15.7 µg/g) in fat, 27.7 µg/g (95% CI, 24.3-31.0 µg/g) in synovium, and 17.7 µg/g (95% CI, 14.8-20.5 µg/g) in bone. For patients undergoing TKA with a tourniquet, the mean concentration of cefazolin in serum was 72.0 µg/mL (95% CI, 66.3-77.7 µg/mL), and the mean LTCs were 9.9 µg/g (95% CI, 8.7-11.1 µg/g) in fat, 21.8 µg/g (95% CI, 18.7-25.0 µg/g) in synovium, and 13.0 µg/g (95% CI, 10.8-15.2 µg/g) in bone. The use of a tourniquet resulted in significantly lower mean LTCs by 60 minutes after cefazolin infusion (10.8 µg/g [95% CI, 9.1-12.4 µg/g] vs 16.9 µg/g [95% CI, 14.1-19.6 µg/g], P = .001 in fat; 18.9 µg/g [95% CI, 14.1-23.6 µg/g] vs 25.8 µg/g [95% CI, 21.4-30.3 µg/g], P = .03 in synovium; and 11.8 µg/g [95% CI, 9.3-14.2 µg/g] vs 19.4 µg/g [95% CI, 14.5-24.4 µg/g], P = .007 in bone). Conclusions and Relevance: In this randomized clinical trial, the concentration of cefazolin was lower in local tissues (fat, synovium, and bone) than in systemic blood, and the use of a limb tourniquet further significantly reduced these concentrations. Although the current prophylactic dosing regimen for cefazolin provides sufficient serum concentrations, the levels in the periarticular tissue during TKA may be insufficient to prevent periprosthetic joint infection. Trial Registration: ClinicalTrials.gov Identifier: NCT05604157.

Targeting fatty acid oxidation enhances response to HER2-targeted therapy.

Metabolic reprogramming, a hallmark of tumorigenesis, involves alterations in glucose and fatty acid metabolism. Here, we investigate the role of Carnitine palmitoyl transferase 1a (Cpt1a), a key enzyme in long-chain fatty acid (LCFA) oxidation, in ErbB2-driven breast cancers. In ErbB2+ breast cancer models, ablation of Cpt1a delays tumor onset, growth, and metastasis. However, Cpt1a-deficient cells exhibit increased glucose dependency that enables survival and eventual tumor progression. Consequently, these cells exhibit heightened oxidative stress and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Inhibiting Nrf2 or silencing its expression reduces proliferation and glucose consumption in Cpt1a-deficient cells. Combining the ketogenic diet, composed of LCFAs, or an anti-ErbB2 monoclonal antibody (mAb) with Cpt1a deficiency significantly perturbs tumor growth, enhances apoptosis, and reduces lung metastasis. Using an immunocompetent model, we show that Cpt1a inhibition promotes an antitumor immune microenvironment, thereby enhancing the efficacy of anti-ErbB2 mAbs. Our findings underscore the importance of targeting fatty acid oxidation alongside HER2-targeted therapies to combat resistance in HER2+ breast cancer patients.

Metastatic breast cancer cells are metabolically reprogrammed to maintain redox homeostasis during metastasis.

Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors.

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.

The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.