Methods to analyze mitochondrial protein translocation in plant mitochondria.

Complex processes have evolved in plants to import proteins into mitochondria. Investigating these processes in plants provides insights into the specialised machinery and pathways that have evolved to cope with; (1) the immobile nature of plants that results in exposure to environmental stresses, and (2) the more complex cell environment due to the presence of plastids, the most prevalent being chloropalst in leaves. In this chapter, we present detailed protocols for the isolation of respiratory competent, coupled mitochondria from Arabidopsis thaliana, conducting protein import assays, and analyzing protein assembly into large multi-subunit complexes.

Cancer-associated fibroblasts are the main contributors to epithelial-to-mesenchymal signatures in the tumor microenvironment.

Epithelial-to-mesenchymal transition (EMT) is associated with tumor initiation, metastasis, and drug resistance. However, the mechanisms underlying these associations are largely unknown. We studied several tumor types to identify the source of EMT gene expression signals and a potential mechanism of resistance to immuno-oncology treatment.

The biogenesis and regulation of the plant oxidative phosphorylation system.

Mitochondria are central organelles for respiration in plants. At the heart of this process is oxidative phosphorylation (OXPHOS) system, which generates ATP required for cellular energetic needs. OXPHOS complexes comprise of multiple subunits that originated from both mitochondrial and nuclear genome, which requires careful orchestration of expression, translation, import, and assembly.

Cancer cell survival depends on collagen uptake into tumor-associated stroma.

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma.

The mitochondrial LYR protein SDHAF1 is required for succinate dehydrogenase activity in Arabidopsis.

Succinate dehydrogenase (SDH, complex II), which plays an essential role in mitochondrial respiration and tricarboxylic acid metabolism, requires the assembly of eight nuclear-encoded subunits and the insertion of various cofactors. Here, we report on the characterization of an Arabidopsis thaliana leucine-tyrosine-arginine (LYR) protein family member SDHAF1, (At2g39725) is a factor required for SDH activity. SDHAF1 is located in mitochondria and can fully complement the yeast SDHAF1 deletion strain.

Paired 18F-Fluorodeoxyglucose (18F-FDG), and 64Cu-Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (64Cu-ATSM) PET Scans in Dogs with Spontaneous Tumors and Evaluation for Hypoxia-Directed Therapy.

Hypoxia is associated with neoplastic tissue, protecting cancer cells from death by irradiation and chemotherapy. Identification of hypoxic volume of tumors could optimize patient selection for hypoxia-directed medical, immunological, and radiation therapies. Clostridium novyi-NT (CNV-NT) is an oncolytic bacterium derived from attenuated wild-type Clostridium novyi spores, which germinates exclusively in the anaerobic core of tumors with low-oxygen content.

Digital pathology and artificial intelligence in translational medicine and clinical practice.

Traditional pathology approaches have played an integral role in the delivery of diagnosis, semi-quantitative or qualitative assessment of protein expression, and classification of disease. Technological advances and the increased focus on precision medicine have recently paved the way for the development of digital pathology-based approaches for quantitative pathologic assessments, namely whole slide imaging and artificial intelligence (AI)-based solutions, allowing us to explore and extract information beyond human visual perception. Within the field of immuno-oncology, the application of such methodologies in drug development and translational research have created invaluable opportunities for deciphering complex pathophysiology and the discovery of novel biomarkers and drug targets.

Intratumoral Injection of -NT Spores in Patients with Treatment-refractory Advanced Solid Tumors.

Purpose: Intratumorally injected -NT (nontoxic; lacking the alpha toxin), an attenuated strain of , replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation.

Patients And Methods: This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of -NT across 6 dose cohorts (1 × 10 to 3 × 10 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose.

Results: Among 24 patients, a single intratumoral injection of -NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses.

Aluminum Phosphide Poisoning Presenting like Acute Myocardial Infarction in a Young Girl.

Aluminium phosphide poisoning is very common in India. It is one of the most fatal poisons. The clinical spectrum of poisoning varies depending upon the dosage and duration of consumption.

Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site.

Immune response to C. novyi-NT immunotherapy.

Clostridium novyi-NT (CVN-NT) spores germinate in hypoxic regions of tumors and have successfully cured induced neoplasia in mouse models and resulted in objective tumor responses in naturally developing neoplasia in the dog. The objective of this pilot, descriptive, prospective, clinical investigation, was to evaluate and describe the immune response to CNV-NT spores to better understand which immune pathways might play a role in the response to this bacteriolytic immunotherapy. Intratumoral injection of CNV-NT spores result in increased phagocytosis and NK cell-like function after treatment.

Identification of immunologic and clinical characteristics that predict inflammatory response to C. Novyi-NT bacteriolytic immunotherapy.

Background: Clostridium novyi-NT (CNV-NT), has shown promise as a bacterolytic therapy for solid tumors in mouse models and in dogs with naturally developing neoplasia. Factors that impact the immunologic response to therapy are largely unknown. The goal of this pilot study was to determine if plasma immune biomarkers, immune cell function, peripheral blood cytological composition and tumor characteristics including evaluation of a PET imaging surrogate of tumor tissue hypoxia could predict which dogs with naturally developing naïve neoplasia would develop an inflammatory response to CNV-NT.

Repeated Measures of Blood and Breath Ammonia in Response to Control, Moderate and High Protein Dose in Healthy Men.

Ammonia physiology is important to numerous disease states including urea cycle disorders and hepatic encephalopathy. However, many unknowns persist regarding the ammonia response to common and potentially significant physiologic influences, such as food. Our aim was to evaluate the dynamic range of ammonia in response to an oral protein challenge in healthy participants.

First-in-Class ERK1/2 Inhibitor Ulixertinib (BVD-523) in Patients with MAPK Mutant Advanced Solid Tumors: Results of a Phase I Dose-Escalation and Expansion Study.

Ulixertinib (BVD-523) is an ERK1/2 kinase inhibitor with potent preclinical activity in BRAF- and RAS-mutant cell lines. In this multicenter phase I trial (NCT01781429), 135 patients were enrolled to an accelerated 3 + 3 dose-escalation cohort and six distinct dose-expansion cohorts. Dose escalation included 27 patients, dosed from 10 to 900 mg twice daily and established the recommended phase II dose (RP2D) of 600 mg twice daily.

Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates.

Understanding the pharmacology of microbiome-based therapeutics is required to support the development of new medicines. Strains of E. coli Nissle (EcN) were genetically modified and administered to cynomolgus monkeys at doses of 1 × 10 and 1 × 10 colony-forming units (CFU)/day for 28 days.

Targeting the MAPK Signaling Pathway in Cancer: Promising Preclinical Activity with the Novel Selective ERK1/2 Inhibitor BVD-523 (Ulixertinib).

Aberrant activation of signaling through the RAS-RAF-MEK-ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting.

Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature.

A Prospective Clinical Trial Combining Radiation Therapy With Systemic Immunotherapy in Metastatic Melanoma.

Purpose: Local radiation therapy (RT) combined with systemic anti-cytotoxic T-lymphocyte-associated protein-4 immunotherapy may enhance induction of systemic antimelanoma immune responses. The primary objective of the present trial was to assess the safety and efficacy of combining ipilimumab with RT in patients with stage IV melanoma. The secondary objectives included laboratory assessment of induction of antimelanoma immune responses.

[(124)I]FIAU: Human dosimetry and infection imaging in patients with suspected prosthetic joint infection.

Introduction: Fialuridine (FIAU) is a nucleoside analog that is a substrate for bacterial thymidine kinase (TK). Once phosphorylated by TK, [(124)I]FIAU becomes trapped within bacteria and can be detected with positron emission tomography/computed tomography (PET/CT). [(124)I]FIAU PET/CT has been shown to detect bacteria in patients with musculoskeletal bacterial infections.

MRI-monitored intra-tumoral injection of iron-oxide labeled Clostridium novyi-NT anaerobes in pancreatic carcinoma mouse model.

Objectives: To validate the feasibility of labeling Clostridium novyi-NT (C.novyi-NT) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled C.novyi-NT during percutaneous injection procedures.

Effect of short-term physical exercise on serum total testosterone levels in young adults.

The effect of short term (12 weeks) physical exercise on serum total testosterone level was evaluated in 30 young male adults, aged 18-27 years (mean age 21.67±2.26 years).

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses.

Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection.

Systematic repurposing screening in xenograft models identifies approved drugs with novel anti-cancer activity.

Approved drugs target approximately 400 different mechanisms of action, of which as few as 60 are currently used as anti-cancer therapies. Given that on average it takes 10-15 years for a new cancer therapeutic to be approved, and the recent success of drug repurposing for agents such as thalidomide, we hypothesized that effective, safe cancer treatments may be found by testing approved drugs in new therapeutic settings. Here, we report in-vivo testing of a broad compound collection in cancer xenograft models.

Evaluation of 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) as an ex vivo bacterial detection agent.

The nucleoside analogue, 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) is a substrate for thymidine kinase (TK), which is commonly expressed in bacteria. It is currently being investigated in clinical studies as an in vivo bacterial infection detection agent. In developing countries where imaging facilities are not readily available, deploying such technology can be a big hurdle.