Heterologous Production of the D-Cycloserine Intermediate O-acetyl-L-serine in Human Lung Cancer Cells

According to the World Health Organization, Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, we seek to develop a genetic prophylaxis for TB. We have determined D-cycloserine to be the optimal target for this approach due to its relatively short six-enzyme biosynthetic pathway. D-CS is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB in human cells, we endeavored to express DcsE in human cells and test its functionality. We overexpressed DcsE tagged with FLAG and GFP in A549 lung cancer cells as determined using fluorescence microscopy. We observed that purified DcsE catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, DcsE synthesized in human cells is a functional enzyme capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards DCS production in human cells.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced fatty acid oxidation through Metformin and Baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response and ultimate tissue scarring. Energy balance may be crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs Metformin (AMPk activator) and Baicalin (Cpt1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID19 patients that had been previously treated with Metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-beta-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two new indole derivatives IND6 and IND8 with AMPK activating capacity. Consistently, a reduced stay in the intensive care unit was observed in COVID-19 patients previously exposed to Metformin. Baicalin also reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19, while in vitro both drugs improved mitochondrial function and prevented TGF-beta-induced renal epithelial cell dedifferentiation. Our results support that strategies based on energy supply may prove useful in the prevention of COVID-19-induced lung and renal damage.Copyright © 2023

PATHOPHYSIOLOGY OF LACTIC ACIDOSIS IN PATIENTS WITH ASTHMA

SESSION TITLE: Challenges in Asthma SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 10:15 am - 11:10 am INTRODUCTION: Asthma is a chronic illness affecting 334 million people worldwide[1]. Asthma affects the respiratory gas exchange, which plays a significant role in acid-base balance. Acid-base disorders in asthma involve respiratory alkalosis, respiratory acidosis, and AG acidosis[2]. CASE PRESENTATION: A 37 years old Hispanic male with a PMH of intermittent asthma presents with progressive dyspnea for three days, worse with activity and decreases with rest. He reported no [cough, fever, rhinorrhea, chest pain]. No orthopnea. He is vaccinated for COVID ( 2 Pfizer doses), has no sickness exposure, and works as a driver. The patient is not a smoker. Physical Exam: Blood pressure 124/72 mmHg. Heart Rate 100 PPM. Temperature 97.1 F.Respiratory Rate 21BPM.SPO2 90% General appearance: acute distress with nasal flaring. Heart: Normal S1, S2. RRR. Lung: Poor air entry with diffuse wheeze bilaterally. He was placed on a 6 LPM NC. CBC and differential were unremarkable. He was started on methylprednisone, Ceftriaxone, and azithromycin. The patient was started on inhaled Salbutamol and Budesonide. Chest X-ray was unremarkable, Chemistry was unremarkable except for elevated Lactic acid 4.7, There was no concern for reduced tissue perfusion or hypoxia, with no evidence of an infectious process because both viral and bacterial causes for pneumonia were excluded, and antibiotics were stopped. A serial lactic acid level trend was 4.5/4.3/ 4.1/ 4 on the first day, while on the next day, it was 3.1/ 2.9/ 2.7/ 2.5/ 3.5, we stopped trending his lactic acid level. He improved and was discharged on an oral taper steroid and inhaled steroids with a B2 agonist. DISCUSSION: There are two types of Lactic acidosis in patients with asthma: 1- Type-A results from impaired oxygen delivery to tissues and reduced tissue perfusion in severe acute asthma may be accompanied by reduced cardiac output. 2- Type B where oxygen delivery is normal, but the cellular function is impaired due to increased norepinephrine in plasma, increasing metabolic rate and lactate production, drugs like beta-agonists increase glycogenolysis leading to an increased pyruvate concentration;pyruvate is converted to lactic acid. B2 agonist increases lipolysis and increases Acetyl CoA, this increase in Acetyl CoA inhibits the conversion of pyruvate to Acetyl CoA, increasing pyruvate which will be converted to lactic acid[2], Theophylline is a non-selective 5'-phosphodiesterase inhibitor and potentiates the activity of ß-adrenergic agents by increasing the intracellular concentration of cAMP, Glucocorticoids are also known to increase the ß-receptor's sensitivity to ß-adrenergic agonists. CONCLUSIONS: Providers are increasingly challenged by hyperlactatemia,it is not harmful but elevated Lactic acid levels and clearance rate is used for prognostication,hyperlactatemia might be misleading,and all possible causes of elevated lactic acid levels must be explored. Reference #1: 10.5334/aogh.2412 Reference #2: https://doi.org/10.3390/jcm8040563 Reference #3: Edwin B. Liem, Stephen C. Mnookin, Michael E. Mahla;Albuterol-induced Lactic Acidosis. Anesthesiology 2003;99:505–506 doi: https://doi.org/10.1097/00000542-200308000-00036 DISCLOSURES: No relevant relationships by Vasudev Malik Daliparty No relevant relationships by Abdallah Khashan No relevant relationships by Samer Talib No relevant relationships by MATTHEW YOTSUYA

Social isolation promotes mammary cancer recurrence, increases IL6/JAK/STAT3 signaling and causes mitochondrial dysfunction in tamoxifen treated rats, and a traditional herbal mixture reverses all these changes

Social isolation is associated with increased risk and mortality from many diseases, such as breast cancer. Socially isolated breast cancer survivors have a 43% higher risk of recurrence and a 64% higher risk of breast cancer-specific mortality than socially integrated survivors. Since Covid-19 has dramatically increased the incidence of social isolation, it is important to determine if social isolation affects the response to endocrine therapy and/or recurrence after the therapy is completed. Since previous studies indicate that social isolation increases circulating inflammatory cytokines, we investigated if an anti-inflammatory herbal mixture Jaeumkanghwa-tang (JGT) prevents the adverse effects of social isolation on breast cancer mortality. Estrogen receptor positive mammary tumors were initiated with 7,12-dimethylbenz[a]anthracene. When a rat developed a palpable mammary tumor, it was either socially isolated (SI) by housing it singly or a rat was allowed to remain group-housed (GH). Tamoxifen (340ppm via diet) or tamoxifen + JGT (500ppm via drinking water) started when the first mammary tumor reached a size of 11 mm in diameter. Tamoxifen administration ended when a complete response to this therapy had lasted for 9 weeks (corresponds to 5 years in women). During tamoxifen therapy, social isolation non-significantly reduced the rate of complete responses to 21%, from 31% in GH group (p>0.05). After the therapy was completed, SI significantly increased local mammary tumor recurrence (p<0.001;45% GH vs 75% SI). RNAseq analysis was performed in the mammary glands. Gene set enrichment analysis (GSEA) of transcriptome showed that the increased recurrence risk in socially isolated rats was associated with an enrichment of IL6/JAK/STAT3 signaling: this result was confirmed in the tumors. In addition, oxidative phosphorylation (OXPHOS) pathway was suppressed: the suppressed genes included those involved in mitochondrial pyruvate transport and conversion of pyruvate to acetyl CoA as well as genes in the TCA cycle and mediating electron transport in mitochondrial complexes I-IV. Social isolation also increased the expression of inflammatory receptor for advanced glycation end-products (RAGE) (p≤0.05). Consumption of an anti-inflammatory JGT inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling and prevented the increased risk of mammary cancer recurrence in socially isolated animals. The percentage of recurrences in the SI rats dropped from 75% without JGT to 22% with JGT (p<0.001). Breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following endocrine therapy using tools that inhibit IL6/JAK/STAT3 inflammatory cytokine signaling and correct disrupted OXPHOS and mitochondrial dysfunction.