EMPathways2: Estimation of Enzyme Expression and Metabolic Pathway Activity Using RNA-Seq Reads.

In this chapter, we outline an approach to analyzing metatranscriptomic data, focusing on the assessment of differential enzyme expression and metabolic pathway activities using a novel bioinformatics software tool, EMPathways2. The analysis pipeline commences with raw data originating from a sequencer and concludes with an output of enzyme expressions and an estimate of metabolic pathway activities. The initial step involves aligning specific transcriptomes assembled from RNA-Seq data using Bowtie2 and acquiring gene expression data with IsoEM2. Subsequently, the pipeline proceeds to quality assessment and preprocessing of the input data, ensuring accurate estimates of enzymes and their differential regulation. Upon completion of the preprocessing stage, EMPathways2 is employed to decipher the intricate relationships between genes, enzymes, and pathways. An online repository containing sample data has been made available, alongside custom Python scripts designed to modify the output of the programs within the pipeline for diverse downstream analyses. This chapter highlights the technical aspects and practical applications of using EMPathways2, which facilitates the advancement of transcriptome data analysis and contributes to a deeper understanding of the complex regulatory mechanisms underlying living systems.

Estimating Enzyme Expression and Metabolic Pathway Activity in Borreliella-Infected and Uninfected Mice.

Evaluating changes in metabolic pathway activity is essential for studying disease mechanisms and developing new treatments, with significant benefits extending to human health. Here, we propose EMPathways2, a maximum likelihood pipeline that is based on the expectation-maximization algorithm, which is capable of evaluating enzyme expression and metabolic pathway activity level. We first estimate enzyme expression from RNA-seq data that is used for simultaneous estimation of pathway activity levels using enzyme participation levels in each pathway. We implement the novel pipeline to RNA-seq data from several groups of mice, which provides a deeper look at the biochemical changes occurring as a result of bacterial infection, disease, and immune response. Our results show that estimated enzyme expression, pathway activity levels, and enzyme participation levels in each pathway are robust and stable across all samples. Estimated activity levels of a significant number of metabolic pathways strongly correlate with the infected and uninfected status of the respective rodent types.

A rigorous benchmarking of alignment-based HLA typing algorithms for RNA-seq data.

Accurate identification of human leukocyte antigen (HLA) alleles is essential for various clinical and research applications, such as transplant matching and drug sensitivities. Recent advances in RNA-seq technology have made it possible to impute HLA types from sequencing data, spurring the development of a large number of computational HLA typing tools. However, the relative performance of these tools is unknown, limiting the ability for clinical and biomedical research to make informed choices regarding which tools to use. Here we report the study design of a comprehensive benchmarking of the performance of 12 HLA callers across 682 RNA-seq samples from 8 datasets with molecularly defined gold standard at 5 loci, HLA-A, -B, -C, -DRB1, and -DQB1. For each HLA typing tool, we will comprehensively assess their accuracy, compare default with optimized parameters, and examine for discrepancies in accuracy at the allele and loci levels. We will also evaluate the computational expense of each HLA caller measured in terms of CPU time and RAM. We also plan to evaluate the influence of read length over the HLA region on accuracy for each tool. Most notably, we will examine the performance of HLA callers across European and African groups, to determine discrepancies in accuracy associated with ancestry. We hypothesize that RNA-Seq HLA callers are capable of returning high-quality results, but the tools that offer a good balance between accuracy and computational expensiveness for all ancestry groups are yet to be developed. We believe that our study will provide clinicians and researchers with clear guidance to inform their selection of an appropriate HLA caller.

Loss of function STK11 alterations and poor outcomes in non-small-cell lung cancer: Literature and case series of US Veterans.

Emerging evidence suggests that STK11 alterations, frequently found in non-small-cell lung cancers, may be prognostic and/or predictive of response to therapy, particularly immunotherapy. STK11 affects multiple important cellular pathways, and mutations lead to tumor growth by creating an immunosuppressive and altered metabolic environment through changes in AMPK, STING, and vascular endothelial growth factor pathways. We illustrate the questions surrounding STK11 genomic alteration in NSCLC with a case series comprising six United States Veterans from a single institution. We discuss the history of STK11, review studies on its clinical impact, and describe putative mechanisms of how loss of STK11 might engender resistance to immunotherapy or other therapies. While the exact impact of STK11 alteration in non-small-cell lung cancer remain to be fully elucidated, future research and ongoing clinical trials will help us better understand its role in cancer development and devise more effective treatment strategies.

Large Necrotic Mass in the Small Bowel.

Melanoma of unknown primary (MUP) is a melanoma found in sites other than the skin, mucosa, or eye. It is most commonly present in lymph nodes and least frequently in visceral organs. Diagnosis is difficult given its internal presentation and is often late stage with poor prognosis. We report an 89-year-old man who presented with weakness, a 27-kg weight loss, and gastrointestinal bleeding. He was found to have a large necrotic mass in the small bowel consistent with melanoma. Although he had extensive skin, anogenital, and ocular examinations, no cutaneous lesion was identified, therefore leading to a diagnosis of MUP.

Alcohol Withdrawal Mimicking Neuroleptic Malignant Syndrome.

Long-standing, heavy alcohol use can lead to alcohol dependence, which predisposes to alcohol withdrawal if alcohol consumption is suddenly decreased or stopped. Alcohol withdrawal syndrome is characterized by a hyperadrenergic response, with symptoms ranging from mild tremulousness to delirium tremens. We report a 55-year-old male presenting with hyperthermia, tachycardia, tachypnea, altered consciousness, tremors, rigidity, diaphoresis, elevated creatinine kinase, and myoglobinuria. The diagnosis of alcohol withdrawal was made due to a history of alcohol use disorder with the last drink two days ago and no history of any medication or drug intake prior to admission. He was treated with benzodiazepines with an improvement in his condition.

Streptococcus gordonii Empyema: A Rare Presentation of Streptococcus gordonii Infection.

Empyema is often caused by Streptococcus pneumoniae, Staphylococcus aureus, and a variety of gram-negative organisms as well as anaerobes. Streptococcus gordonii (S. gordonii) is among some of the initial colonizers of the periodontal environment that is recognized to cause bacterial endocarditis. However, there are only a few case reports of S. gordonii causing empyema in the literature. We report the case of a 75-year-old male who presented with coughing up blood-tinged sputum. Physical examination revealed decreased breath sounds in the right lung base. Chest X-ray demonstrated a lower, right-sided, loculated pleural effusion. He underwent ultrasound-guided chest tube placement. The pleural fluid culture grew S. gordonii. He was started on ampicillin/ sulbactam. The follow-up computed tomography (CT) scan showed no significant improvement. Given his inability to improve with antibiotics and chest tube drainage, he was referred to an advanced care center for decortication of lung tissue.

Fulminant Diabetes in a Patient with Advanced Melanoma on Nivolumab.

BACKGROUND: Anti-PD-1 agents were approved for advanced melanoma after the landmark trial Checkmate-037. Anti-PD-1 agents can breach immunologic tolerance. Fulminant diabetes is an immune endocrinopathy that results from a violent immune attack leading to complete destruction of pancreatic beta cells in genetically predisposed people. We present a rare case of fulminant diabetes precipitated by anti-PD-1 immunotherapy. CASE: A 61-year-old male with advanced melanoma presented with a three-day history of nausea, vomiting, and malaise. He was started on nivolumab and ipilimumab. After the third dose, he developed a generalized rash and was prescribed high-dose prednisone. Labs revealed potassium 9.5 mmol/L, sodium 127 mmol/L, bicarbonate <10 mmol/L, blood glucose 1211 mg/dL, anion gap >31 mmol, arterial blood pH 7.14, and beta-hydroxybutyrate 13.7 mmol/L. He was diagnosed with diabetic ketoacidosis. Hemoglobin A1C was 6.9%. C-peptide was undetectable (<0.1 ng/ml). Glutamic acid decarboxylase autoantibodies, zinc transporter 8 autoantibodies, insulin autoantibodies, islet antigen 2 autoantibodies, and islet cell antibodies were all negative. CONCLUSION: Anti-PD-1 immunotherapy is effective in cancers refractory to standard chemotherapy. These agents can precipitate autoimmune disorders. As the use of anti-PD-1 agents is expected to rise, physicians should be educated about the potential side effects. We recommend conducting routine blood glucose checks in patients on these agents.

Metastatic Sternal Osteosarcoma: A Rare Tumor.

Osteosarcoma is the most common primary malignant tumor of the long bones. However, primary osteosarcoma of the chest wall, particularly the sternum, is an extremely rare occurrence. We report a 36-year-old male presenting with a hard, immobile, palpable, anterior chest wall mass. A computed tomographic (CT) scan showed a large destructive anterior mediastinal mass involving the manubrium and sternum with multiple bilateral calcified lung masses, pleural effusions and partially calcified aortopulmonary, right hilar and subcarinal lymphadenopathy. Incisional biopsy of the mass revealed grade 2 chondroblastic osteosarcoma. The patient underwent one cycle of chemotherapy with ifosfamide and palliative radiation. Unfortunately, the patient was unable to tolerate ifosfamide and developed severe nausea and vomiting requiring the discontinuation of chemotherapy. Given his metastatic disease and inability to tolerate standard chemotherapy, he was referred to a comprehensive cancer center for advanced clinical trials.