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Supplementary table showing demographic information and protein docing.

Influence of SH3GL2 overexpression in BCa cells.

Legends for Supplementary Figures 1-6.

Purpose: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers.Experimental Design: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development.Results: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis–associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors.Conclusions: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348–60. ©2016 AACR.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the M. avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin (IL)-1β, IL-18, IL-1α and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1β production by macrophages infected with MAC. TLR2 and TWIK2 were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all 4 cytokines, whereas a TWIK2 inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. We suggest that an attenuated TWIK2-mediated IL-1 response may increase susceptibility to NTM pulmonary infection.

Background: Nontuberculous mycobacteria (NTM) causes disseminated disease in patients with immunodeficiency and pulmonary disease in individuals without obvious immunodeficiency. There is no clear information on how NTM pulmonary disease (NTMPD) develops in immunocompetent hosts. This profile study was initiated to gain insight into the immunological factors that predispose persons to pulmonary NTM infections.Methods: Blood samples were obtained from 15 pairs of NTMPD patients and age-matched healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the heat-killed M. avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in PBMCs culture supernatants and plasma using multiplex immunoassays. The mRNA expression of TLR2, P2X7R, TWIK2, THIK2 and TREK1 was measured by quantitative real-time PCR. In mechanistic studies, blood samples were obtained from healthy volunteers who had not been diagnosed or treated for NTM.Results: Interleukin (IL)-1β, IL-18, IL-1α and IL-10 production were significantly reduced in response to MAC in PBMCs of NTMPD patients compared with PBMCs of their healthy household contacts. RANTES was the only chemokine significantly elevated in the plasma of NTMPD patients compared with plasma of their healthy household contacts. TLR2 and TWIK2 expression were impaired in response to MAC in PBMCs of NTMPD patients compared with PBMCs of their healthy household contacts. RANTES had no effect on IL-1β production by macrophages infected with MAC. A TLR2 inhibitor decreased IL-1β, IL-18, IL-1α and IL-10 production by MAC-stimulated PBMCs and monocytes. A TWIK2 inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes.Conclusions: These findings suggest that impaired TWIK2 results in decreased production of IL-1 family cytokines (IL-1β, IL-18, and IL-1α) in response to MAC, consequentially may increase susceptibility to NTM pulmonary infection.

Nontuberculous mycobacteria (NTM) infection is common in patients with structural lung damage. To address how NTM infection is established and causes lung damage, we established an NTM mouse model by intranasal inoculation of clinical isolates ofM.intracellulare. During the 39-week course of infection, the bacteria persistently grew in the lung and caused progressive granulomatous and fibrotic lung damage with mortality exceeding 50%. Lung neutrophils were significantly increased at 1 week postinfection, reduced at 2 weeks postinfection and increased again at 39 weeks postinfection. IL-17A was increased in the lungs at 1–2 weeks of infection and reduced at 3 weeks postinfection. Depletion of neutrophils during early (0–2 weeks) and late (32–34 weeks) infection had no effect on mortality or lung damage in chronically infected mice. However, neutralization of IL-17A during early infection significantly reduced bacterial burden, fibrotic lung damage, and mortality in chronically infected mice. Since it is known that IL-17A regulates matrix metalloproteinases (MMPs) and that MMPs contribute to the pathogenesis of pulmonary fibrosis, we determined the levels of MMPs in the lungs ofM.intracellulare-infected mice. Interestingly, MMP-3 was significantly reduced by anti-IL-17A neutralizing antibody. Moreover,in vitrodata showed that exogenous IL-17A exaggerated the production of MMP-3 by lung epithelial cells uponM.intracellulareinfection. Collectively, our findings suggest that early IL-17A production precedes and promotes organized pulmonaryM.intracellulareinfection in mice, at least in part through MMP-3 production.

BACKGROUND Burnout and anxiety compromise physical and mental well-being of nurses and jeopardize patient safety. Personal, professional, and workplace characteristics have been associated with burnout and anxiety across diverse practice settings, yet none in rural, community trauma centers. We sought to identify the severity and predictors of burnout and anxiety in the trauma nursing staff of a rural Level I trauma center. METHODS A convenience sample of trauma nurses from the emergency department (ED), intensive care unit (ICU), and trauma ward was voluntarily surveyed using a demographic questionnaire, the Maslach Burnout Inventory (MBI) subscales: depersonalization (DP), emotional exhaustion (EE), and reduced personal accomplishment, as well as the Generalized Anxiety Disorder seven-item (GAD-7) scale. Multivariable linear regression identified the significant predictors of burnout and anxiety. RESULTS Ninety-six nurses completed surveys (response rate: 83.5%). Married or divorced status, and ICU or trauma ward job assignments were associated with significantly lower adjusted DP scores. Thus, the model-predicted score for a single ED nurse was 15 versus a predicted score of 7 for a divorced ICU or trauma ward nurse, p < .001 for each group. The GAD-7 model demonstrated that race/ethnicity (Asian compared with White, coefficient: -5.06, p = .03), number of children (2 compared with 0, coefficient: -2.54, p = .02), and job tenure (5-10 years vs.

Twelve patients were treated with omadacycline (OMC) as part of a multidrug regimen for Mycobacterium abscessus. The majority of infections were of pulmonary origin (7/12; 58.3%). The median (interquartile range) duration of OMC was 6.2 (4.2–11.0) months. Clinical success occurred in 9/12 (75.0%) patients. Three patients experienced a possible adverse effect while on therapy.

Background: Increasing numbers of patients are being diagnosed with bronchiectasis, yet much remains to be elucidated about this heterogeneous patient population. We sought to determine the relationship between nutrition and health outcomes in non-cystic fibrosis (non-CF) bronchiectasis, using data from the U.S. Bronchiectasis Nontuberculous Mycobacterial Research Registry (U.S. BRR). Methods: This was a retrospective, observational, longitudinal study using 5-year follow-up data from the BRR. Bronchiectasis was confirmed on computed tomography (CT). We stratified patients into nutrition categories using body mass index (BMI), and correlated BMI to markers of disease severity. Results: Overall, n = 496 patients (mean age 64.6­ ± 13 years; 83.3% female) were included. At baseline 12.3% (n = 61) were underweight (BMI < 18.5kg/m(2)), 63.9% (n = 317) had normal weight (BMI ≥ 18.5kg/m(2) and

// Julie V. Philley 1 , Anbarasu Kannan 2 , David E. Griffith 1 , Megan S. Devine 1 , Jeana L. Benwill 1 , Richard J. Wallace, Jr. 1, 3 , Barbara A. Brown-Elliott 3 , Foram Thakkar 1 , Varsha Taskar 1 , James G. Fox 1 , Ammar Alqaid 1 , Hernaina Bains 1 , Sudeep Gupta 4 , Santanu Dasgupta 2 1 Departments of Medicine, The University of Texas Health Science Center at Tyler, Texas, USA 2 Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Texas, USA 3 The Mycobacteria/Nocardia Research Laboratory Department of Microbiology, The University of Texas Health Science Center at Tyler, Texas, USA 4 Medical Oncology, Tata Memorial Center, Mumbai, India Correspondence to: Santanu Dasgupta, email: santanu.dasgupta@uthct.edu Keywords: Exosomes, breast cancer, nontuberculous Mycobacterium, ECM1, biomarker Received: July 10, 2016 Accepted: January 10, 2017 Published: February 01, 2017 ABSTRACT Bronchiectasis Nontuberculous mycobacterium (NTM nb ) infection is an emerging health problem in breast cancer (BCa) patients. We measured sera exosome proteome in BCa-NTM nb subjects and controls by Mass Spectroscopy. Extracellular matrix protein 1 (ECM1) was detected exclusively in the circulating exosomes of 82% of the BCa-NTM nb cases. Co-culture of ECM1 + exosomes with normal human mammary epithelial cells induced epithelial to mesenchymal transition accompanied by increased Vimentin/CDH1 expression ratio and Glutamate production. Co-culture of the ECM1 + exosomes with normal human T cells modulated their cytokine production. The ECM1 + exosomes were markedly higher in sera obtained from BCa-NTM nb subjects. Exclusive expression of APN, APOC4 and AZGP1 was evident in the circulating exosomes of these BCa-NTM nb cases, which predicts disease prevalence independent of the body max index in concert with ECM1. Monitoring ECM1, APN, APOC4 and AZGP1 in the circulating exosomes could be beneficial for risk assessment, monitoring and surveillance of BCa-NTM nb .

Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication.

Cervical cancer develops through persistent infection with high-risk human papilloma virus (hrHPV) and is a leading cause of death among women worldwide and in the United States. Periodic surveillance through hrHPV and Pap smear-based testing has remarkably reduced cervical cancer incidence worldwide and in the USA. However, considerable discordance in the occurrence and outcome of cervical cancer in various populations exists. Lack of adequate health insurance appears to act as a major socioeconomic burden for obtaining cervical cancer preventive screening in a timely manner, which results in disparate cervical cancer incidence. On the other hand, cervical cancer is aggressive and often detected in advanced stages, including African American and Hispanic/Latina women. In this context, our knowledge of the underlying molecular mechanism and genetic basis behind the disparate cervical cancer outcome is limited. In this review, we shed light on our current understanding and knowledge of racially disparate outcomes in cervical cancer.

Mycobacterial disease is commonly encountered during transplant evaluations and can be a major source of morbidity and mortality among transplant recipients. The impact of TB and NTM around the world has gained increased awareness, and the global impact of disease varies from country to country thus making mycobacterial epidemiology an important aspect of disease management. Risk factors for both tuberculosis (TB) and nontuberculous mycobacteria (NTM) must be assessed by transplant providers, and a low index of suspicion should warrant a prompt and accurate work-up to identify disease. Evaluation for active and latent TB is necessary prior to transplant. Preventing drug resistance for both TB and NTM plays a critical role in treatment options and outcomes. Adequate laboratory services are an essential component of drug regimens and often require reference laboratory assistance. Treatment regimens for mycobacterial disease require multiple antibiotics and frequently are associated with a host of drug-drug interactions. Knowledge of mycobacterial disease diagnosis, treatment regimens, and susceptibility patterns are essential to transplant evaluation and success.

The spectrum of nontuberculous mycobacterial (NTM) infections has become an increasingly recognized cause of clinical concern in transplant recipients. While episodic isolation of NTM is often common among certain solid organ transplants such as lung recipients, there is sufficient evidence to support that serious infections can result in all transplant groups. As NTM are ubiquitous in the environment, and exposure to such bacteria is universally unavoidable, clinicians providing care for transplant populations must be up to date regarding the diagnosis and management of infections caused by NTM and maintain a high index of suspicion as clinical signs and symptoms may be insidious.

Background Omadacycline (OMC) is an aminomethylcycline antibiotic in the tetracycline class that has been Food and Drug Administration-approved for acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. OMC has been shown to have potent in vitro activity against a broad-spectrum of Gram-positive and Gram-negative organisms, as well as Nontuberculous Mycobacteria (NTM). Due to it’s unique activity and availability as an oral agent, off-label use of OMC has been increasing. We evaluated the real-world effectiveness and safety of OMC for a variety of infections. Methods This was a multicenter, retrospective, observational study that was conducted from January 2020 to June 2020. We included all patients ≥ 18 years of age that received OMC for ≥ 72 hours for any indication and/or pathogen. The primary outcome was clinical success, defined as a lack of 30-day (non-NTM) or 90-day (NTM) mortality or microbiologic recurrence and absence of therapy escalation or alteration. Reasons for OMC utilization and incidence of potential adverse effects attributable to OMC were also analyzed. Results A total of 18 patients were included from six geographically distinct academic health systems (median age: 56 (IQR, 49-60.5) years; 61% male; 72% Caucasian). The majority of OMC use was in NTM (61%; 100% Mycobacterium abscessus) and in Acinetobacter baumannii (22%) for bone/joint (39%) and respiratory tract (33%) infections. OMC was used primarily in the outpatient setting alone (83%) and most isolates did not have OMC susceptibility conducted (89%). Clinical success was reported in 83% of the total population (71% non-NTM and 91% NTM). The majority of patients were prescribed OMC due to antimicrobial resistance to previous antibiotic(s) (61%) and/or due to OMC’s availability as an oral agent (44%). Three patients experienced side effects while on therapy (serum creatinine elevation, AST/ALT increase, and gastrointestinal distress). Conclusion OMC appears to be effective and well-tolerated for a variety of infections caused by various pathogens, including M. abscessus and A. baumannii. Disclosures Michael J. Rybak, PharmD, MPH, PhD, Paratek (Grant/Research Support)

Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare, is widely distributed in the environment, and exposure to these organisms is inevitable. Despite widespread exposure, the disease is unusual in immunocompetent hosts. During the past two decades, MAC represents a common cause of progressive pulmonary disease in the United States. To gain insight into the immunologic factors that predispose persons to MAC infections, we evaluated the immune responses of HIV negative (-) MAC patients with pulmonary diseases in comparison to their healthy HIV− household contacts (HHCs). Whole blood from 20 pairs of HIV− MAC patients and healthy HHCs was immunolabeled, and the percentages of various immune cell populations were determined by flow cytometry. Among various cell types, MAC patients have significantly lower ratio of monocytes compared to healthy HHCs. In 11 pairs of HIV− MAC patients and healthy HHCs, peripheral blood mononuclear cells (PBMC) of MAC patients produced lower levels of interleukin (IL)-10 and IL-1 family (IL-1β, IL-18, IL-1α) out of 34 cytokines and chemokines measured by multiplex ELISA upon MAC stimulation, compared with that of healthy HHCs. In two MAC patients, PBMC restricted MAC growth less efficiently compared with their healthy HHCs despite there was no deficiency in restricting MAC growth by monocyte derived macrophages of six MAC patients. Our findings suggest that the interactions among various immune cells are required for efficient clearance of MAC by macrophages in MAC patients. Studies are underway to pinpoint the nature of defective immune responses that prevents efficient clearance of MAC by macrophages of MAC patients.

Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication.

Rationale: Improved therapeutic options are needed for patients with treatment-refractory nontuberculous mycobacterial lung disease caused by Mycobacterium avium complex (MAC). Objectives: To evaluate the efficacy and safety of daily amikacin liposome inhalation suspension (ALIS) added to standard guideline-based therapy (GBT) in patients with refractory MAC lung disease. Methods: Adults with amikacin-susceptible MAC lung disease and MAC-positive sputum cultures despite at least 6 months of stable GBT were randomly assigned (2:1) to receive ALIS with GBT (ALIS + GBT) or GBT alone. Once-daily ALIS was supplied in single-use vials delivering 590 mg amikacin to the nebulizer. The primary endpoint was culture conversion, defined as three consecutive monthly MAC-negative sputum cultures by Month 6. Measurements and Main Results: Enrolled patients (ALIS + GBT, n = 224; GBT-alone, n = 112) were a mean 64.7 years old and 69.3% female. Most had underlying bronchiectasis (62.5%), chronic obstructive pulmonary disease (14.3%), or both (11.9%). Culture conversion was achieved by 65 of 224 patients (29.0%) with ALIS + GBT and 10 of 112 (8.9%) with GBT alone (odds ratio, 4.22; 95% confidence interval, 2.08-8.57; P < 0.001). Patients in the ALIS + GBT arm versus GBT alone were more likely to achieve conversion (hazard ratio, 3.90; 95% confidence interval, 2.00-7.60). Respiratory adverse events (primarily dysphonia, cough, and dyspnea) were reported in 87.4% of patients receiving ALIS + GBT and 50.0% receiving GBT alone; serious treatment-emergent adverse events occurred in 20.2% and 17.9% of patients, respectively. Conclusions: Addition of ALIS to GBT for treatment-refractory MAC lung disease achieved significantly greater culture conversion by Month 6 than GBT alone, with comparable rates of serious adverse events. Clinical trial registered with www.clinicaltrials.gov (NCT02344004).

Nontuberculous mycobacterial (NTM) lung infections are increasingly recognized as a cause of chronic pulmonary disease. This article focuses on the most common NTM species known to cause human lung disease and the treatment options currently available. The diagnosis of NTM lung disease is also discussed, emphasizing the necessity for treating clinicians to have sufficient familiarity of the mycobacteria laboratory to provide optimal patient management.

Rapidly growing mycobacteria (RGM) are divided into six major groups including the two clinically most important groups, M. chelonae/M. abscessus complex and Mycobacterium fortuitum group. This chapter discusses infections associated with these groups with an emphasis on pulmonary disease. Phenotypic and molecular laboratory identification methods are reviewed as accurate organism identification is necessary for optimal RGM lung disease patient management. Antimicrobial susceptibility patterns for the most common pathogenic RGM species are discussed with an emphasis on the impact of inducible macrolide resistance found in many RGM species and subspecies. Because of antibiotic resistance, the RGM are frequently difficult to treat successfully. Current therapeutic approaches are reviewed with an emphasis on antibiotic options in the context of both innate and acquired antibiotic resistance.

Nontuberculous mycobacterial lung disease (NTM), including Mycobacterium avium complex (MAC), is a growing health problem in North America and worldwide. Little is known about the molecular alterations occurring in the tissue microenvironment during NTM pathogenesis. Utilizing next generation sequencing, we sequenced sputum and matched lymphocyte DNA in 15 MAC patients for a panel of 19 genes known to harbor cancer susceptibility associated mutations. Thirteen of 15 NTM subjects had a diagnosis of breast cancer (BCa) before or after NTM infection. Thirty three percent (4/12) of these NTM-BCa cases exhibited at least 3 somatic mutations in sputa compared to matched lymphocytes. Twenty four somatic mutations were detected with at least one mutation in ATM, ERBB2, BARD1, BRCA1, BRCA2, AR, TP53, PALB2, CASP8, BRIP1, NBN and TGFB1 genes. All four NTM-BCa patients harboring somatic mutations also exhibited 15 germ line BRCA1 and BRCA2 mutations. The two NTM subjects without BCa exhibited twenty somatic mutations spanning BRCA1, BRCA1, BARD1, BRIP1, CHEK2, ERBB2, TP53, ATM, PALB2, TGFB1 and 3 germ line mutations in BRCA1 and BRCA2 genes. A single copy loss of STK11 and AR gene was noted in NTM-BCa subjects. Periodic screening of sputa may aid to develop risk assessment biomarkers for neoplastic diseases in NTM patients.

Background/aims The nontuberculous mycobacterial lung disease (NTM), caused by Mycobacterium avium complex (MAC) is an increasing health problem in the USA and worldwide. The NTM disease is prevalent in Caucasian women with a current diagnosis or history of breast cancer (BCa), posing a significant challenge towards treatment. We hypothesize that NTM affected women with considerable therapeutic resistance may harbor pathogenic microbes other than nontuberculous mycobacterium, aiding in disease progression and therapeutic resistance. Methods We assessed microbiome diversity in sputa from healthy women, women with nontuberculous mycobacterial lung disease (NTM) and women with both nontuberculous mycobacterial lung disease and breast cancer (NTM-BCa). First, we collected sputa and isolated DNA from sputa of these healthy women and women with NTM and NTM-BCa. We also isolated DNA from sera derived extracellular vesicles from women with NTM-BCa. To identify diverse pathogenic microbes in various groups of subjects, we then performed 16S rDNA sequencing. Data analysis was performed utilizing the analytical pipelines at the Center for Metagenomic and Microbiome Research (CMMR), Baylor College of Medicine. Results A large community of resident microbes, including bacteria, virus, Archeas and Fungi live in the human body are being increasingly recognized as the key components of human health and disease. We identified a diverse microbiome community in the sputa and the extracellular vesicles dominated by Streptococcus, Haemophillus, Veillonella, Neisseria, Prevotella, Fusobacterium, Bacteroides, Allistipes, Faecalibacterium and Staphylococcus in women with nontuberculous mycobacterial lung disease as well as women with both nontuberculous mycobacterial lung disease and breast cancer. Some of these genera, including Fusobacterium, Bacteroides, and Allistipes have estrobolome activity and associated with breast and other neoplasms. Conclusion This work confirms the presence of a distinct pathogenic microbiome other than nontuberculous mycobacteria in the sputa and the circulating extracellular vesicles of these patients. This information could be useful for better therapeutic design to treat the NTM patients.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

MDA-9/Syntenin is a small PDZ domain containing scaffolding protein with diverse array of functions regulating membrane trafficking, cell adhesion, neural, and synaptic development, ubiquitination, and exosome biogenesis. An appreciable number of studies also established a pivotal role of MDA-9/Syntenin in cancer development and progression. In this review, we will discuss the dynamic role of MDA-9/Syntenin in regulating normal and abnormal fate of various cellular processes.

BACKGROUND Bedaquiline is an oral antimycobacterial agent belonging to a new class of drugs called diarylquinolines. It has low equivalent minimal inhibitory concentrations for Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) lung disease, especially Mycobacterium avium complex (MAC) and Mycobacterium abscessus (Mab). Bedaquiline appears to be effective for the treatment of multidrug-resistant TB but has not been tested clinically for NTM disease. METHODS We describe a case series of off-label use of bedaquiline for treatment failure lung disease caused by MAC or Mab. Only patients whose insurance would pay for the drug were included. Fifteen adult patients were selected, but only 10 (six MAC, four Mab) could obtain bedaquiline. The 10 patients had been treated for 1 to 8 years, and all were on treatment at the start of bedaquiline therapy. Eighty percent had macrolide-resistant isolates (eight of 10). The patients were treated with the same bedaquiline dosage as that used in TB trials and received the best available companion drugs (mean, 5. 0 drugs). All patients completed 6 months of therapy and remain on bedaquiline. RESULTS Common side effects included nausea (60%), arthralgias (40%), and anorexia and subjective fever (30%). No abnormal ECG findings were observed with a mean corrected QT interval lengthening of 2. 4 milliseconds at 6 months. After 6 months of therapy, 60% of patients (six of 10) had a microbiologic response, with 50% (five of 10) having one or more negative cultures. CONCLUSIONS This small preliminary report demonstrates potential clinical and microbiologic activity of bedaquiline in patients with advanced MAC or Mab lung disease but the findings require confirmation with larger studies.

BACKGROUND Isolation of Mycobacterium abscessus subspecies abscessus (MAA) is common during Mycobacterium avium complex (MAC) lung disease therapy, but there is limited information about the clinical significance of the MAA isolates. METHODS We identified 53 of 180 patients (29%) treated for MAC lung disease who had isolation of MAA during MAC lung disease therapy. Patients were divided into those without (group 1) and those with (group 2) MAA lung disease. RESULTS There were no significant demographic differences between patients with and without MAA isolation or between groups 1 and 2. Group 1 and 2 patients had similar total sputum cultures obtained ( P = .7; 95% CI, −13.4 to 8.6) and length of follow-up ( P = .8; 95% CI, −21.5 to 16.1). Group 2 patients had significantly more total positive cultures for MAA (mean±SD, 15.0 ± 11.1 vs 1.2 ± 0.4; P P > .0001), and were significantly more likely to meet all three American Thoracic Society diagnostic criteria for nontuberculous mycobacterial disease (21 of 21 [100%] vs 0 of 32 [0%]; P P CONCLUSIONS Microbiologic and clinical follow-up after completion of MAC lung disease therapy is required to determine the significance of MAA isolated during MAC lung disease therapy. Single MAA isolates are not likely to be clinically significant.

The most common nontuberculous mycobacterial (NTM) lung pathogen, Mycobacterium avium complex (MAC), requires antibiotic treatment regimens that are long and often arduous. M kansasii is the slowly growing NTM pathogen with the most predictably successful treatment outcomes, whereas other slowly growing NTM pathogens such as M xeonpi, M szulgai, and M malmoense are less predictably responsive to antibiotic regimens. M simiae is the most difficult of the common slowly growing NTM pathogens to eradicate. Surgical intervention for slowly growing mycobacterial lung infection has proved beneficial for some patients, but the optimal candidates and timing for surgical intervention remain unknown.

Human papilloma virus-16 (HPV-16) associated oropharyngeal cancer (HPVOPC) is increasing alarmingly in the United States. We performed whole genome sequencing of a 44 year old, male HPVOPC subject diagnosed with moderately differentiated tonsillar carcinoma. We identified new somatic mutation in MUC16 (A.k.a. CA-125), MUC12, MUC4, MUC6, MUC2, SIRPA, HLA-DRB1, HLA-A and HLA-B molecules. Increased protein expression of MUC16, SIRPA and decreased expression of HLA-DRB1 was further demonstrated in this HPVOPC subject and an additional set of 15 HPVOPC cases. Copy number gain (3 copies) was also observed for MUC2, MUC4, MUC6 and SIRPA. Enhanced expression of MUC16, SIRPA and HPV-16-E7 protein was detectable in the circulating exosomes of numerous HPVOPC subjects. Treatment of non-tumorigenic mammary epithelial cells with exosomes derived from aggressive HPVOPC cells harboring MUC16, SIRPA and HPV-16-E7 proteins augmented invasion and induced epithelial to mesenchymal transition (EMT) accompanied by an increased expression ratio of the EMT markers Vimentin/E-cadherin. Exosome based screening of key HPVOPC associated molecules could be beneficial for early cancer diagnosis, monitoring and surveillance.

Rapidly growing mycobacteria (RGM) compose approximately one-half of the currently validated mycobacterial species and are divided into six major groups, including the Mycobacterium fortuitum group, M. chelonae/M. abscessus complex, M. smegmatis group, M. mucogenicum group, M. mageritense / M. wolinskyi , and the pigmented RGM. This review discusses each group and highlights the major types of infections associated with each group. Additionally, phenotypic and molecular laboratory identification methods, including gene sequencing, mass spectrometry, and the newly emerging whole-genome sequencing, are detailed, along with a discussion of the current antimicrobial susceptibility methods and patterns of the most common pathogenic species.

Treatment of pulmonary infections caused by Mycobacterium avium complex (MAC) and Mycobacterium kansasii involves multidrug oral therapy with a macrolide (azithromycin or clarithromycin), ethambutol, and a rifamycin (rifampin or rifabutin). Patients with M. kansasii rapidly respond to a regimen of intermittent (three times weekly) or daily administration of this three-drug regimen. Patients with MAC respond more slowly and often require adjustment of the multidrug regimen because of drug intolerance. The usual treatment for patients with MAC nodular disease takes 15–18 months, with a goal of 12 months of negative cultures. Recent studies support the use of a three-times weekly oral treatment regimen for patients with macrolide-susceptible nodular MAC disease. Patients with upper lobe fibro-cavitary MAC, macrolide-resistant MAC, or severe nodular bronchiectatic disease are usually treated with a daily multidrug regimen supplemented with an injectable antibiotic (amikacin or streptomycin) or, most recently, inhaled preparations of amikacin. Patients with cavitary changes and/or those with macrolide-resistant isolates are often associated with poor treatment response and may require surgical resection in addition to their drug therapy. In contrast to patients with lung disease due to MAC and M. kansasii, the presence of a functional erythromycin ribosomal methylase (erm) gene in the majority of isolates of Mycobacterium abscessus (M. abscessus subsp. abscessus) blocks the activity of macrolides and precludes an effective oral drug regimen for most of these patients. Treatment regimens for macrolide-resistant M. abscessus require long-term intravenous access and parenteral drug combinations of amikacin, cefoxitin, imipenem, and/or tigecycline. Because of the inconvenience of dosing cefoxitin, a regimen of imipenem and amikacin may be preferred. Cure with these agents is infrequent because of the long-term toxicity and expense of these agents. Other treatment options are currently dismal. The role of newer antimicrobials such as tedizolid and bedaquiline has not been evaluated. Approximately 15 % of isolates of subsp. abscessus and all isolates of subsp. massiliense (infrequent in the USA) have a nonfunctional erm gene and are macrolide susceptible, making an oral macrolide an important treatment component and increasing the likelihood of long-term cure of the infection.

This review will utilize essential questions about nontuberculous mycobacterial (NTM) lung disease to succinctly address important new developments in the pathogenesis, diagnosis and management of NTM lung disease with a focus on practical information and "bottom line" answers. 1) What do I tell my patients who ask, “where did I get this infection” and, “should I take showers”? 2) What is the connection between bronchiectasis and the acquisition of NTM lung infection? 3) What other factors are important in the pathogenesis of NTM lung disease? 4) Why does it seem that am I seeing more new NTM lung disease patients? 5) Why is the diagnosis of NTM lung disease so complicated and does the diagnosis of NTM lung infection obligate specific treatment? 6) Unlike traditional tuberculosis, what is behind the irrelevance of most in vitro susceptibility testing reports for NTM infections? 7) Is there anything new for the management of patients with Mycobacterium avium complex lung disease? How does the radiographic appearance influence treatment? 8) Is there anything new for the management of patients with Mycobacterium abscessus lung disease? 9) What about the management of other NTM respiratory pathogens? 10) Is there a role for the use of macrolide monotherapy for non-cystic fibrosis bronchiectasis?