Conducting inclusive research in genetics for transgender, gender-diverse, and sex-diverse individuals: Case analyses and recommendations from a clinical genomics study.

A person's phenotypic sex (i.e., endogenous expression of primary, secondary, and endocrinological sex characteristics) can impact crucial aspects of genetic assessment and resulting clinical care recommendations. In studies with genetics components, it is critical to collect phenotypic sex, information about current organ/tissue inventory and hormonal milieu, and gender identity. If researchers do not carefully construct data models, transgender, gender diverse, and sex diverse (TGSD) individuals may be given inappropriate care recommendations and/or be subjected to misgendering, inflicting medical and psychosocial harms. The recognized need for an inclusive care experience should not be limited to clinical practice but should extend to the research setting, where researchers must build an inclusive experience for TGSD participants. Here, we review three TGSD participants in the Family History and Cancer Risk Study (FOREST) to critically evaluate sex- and gender-related survey measures and associated data models in a study seeking to identify patients at risk for hereditary cancer syndromes. Furthermore, we leverage these participants' responses to sex- and gender identity-related questions in FOREST to inform needed changes to the FOREST data model and to make recommendations for TGSD-inclusive genetics research design, data models, and processes.

Analysis of virulence phenotypes and antibiotic resistance in clinical strains of Acinetobacter baumannii isolated in Nashville, Tennessee.

BACKGROUND: Acinetobacter baumannii is a gram-negative bacterium which causes opportunistic infections in immunocompromised hosts. Genome plasticity has given rise to a wide range of strain variation with respect to antimicrobial resistance profiles and expression of virulence factors which lead to altered phenotypes associated with pathogenesis. The purpose of this study was to analyze clinical strains of A. baumannii for phenotypic variation that might correlate with virulence phenotypes, antimicrobial resistance patterns, or strain isolation source. We hypothesized that individual strain virulence phenotypes might be associated with anatomical site of isolation or alterations in susceptibility to antimicrobial interventions. METHODOLOGY: A cohort of 17 clinical isolates of A. baumannii isolated from diverse anatomical sites were evaluated to ascertain phenotypic patterns including biofilm formation, hemolysis, motility, and antimicrobial resistance. Antibiotic susceptibility/resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin was determined. RESULTS: Antibiotic resistance was prevalent in many strains including resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin. All strains tested induced hemolysis on agar plate detection assays. Wound-isolated strains of A. baumannii exhibited higher motility than strains isolated from blood, urine or Foley catheter, or sputum/bronchial wash. A. baumannii strains isolated from patient blood samples formed significantly more biofilm than isolates from wounds, sputum or bronchial wash samples. An inverse relationship between motility and biofilm formation was observed in the cohort of 17 clinical isolates of A. baumannii tested in this study. Motility was also inversely correlated with induction of hemolysis. An inverse correlation was observed between hemolysis and resistance to ticarcillin-k clavulanate, meropenem, and piperacillin. An inverse correlation was also observed between motility and resistance to ampicillin-sulbactam, ceftriaxone, ceftoxamine, ceftazidime, ciprofloxacin, or levofloxacin. CONCLUSIONS: Strain dependent variations in biofilm and motility are associated with anatomical site of isolation. Biofilm and hemolysis production both have an inverse association with motility in the cohort of strains utilized in this study, and motility and hemolysis were inversely correlated with resistance to numerous antibiotics.

Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells.

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) modulates signaling pathways in head and neck squamous cell carcinoma cell line SQ20B.

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-ß was examined to determine whether levels of the TGF-ß binding AHSG influenced the effect of TGF-ß on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-ß influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

Longitudinal Education and Career Outcomes of a Cancer Research Training Program for Underrepresented Students: The Meharry-Vanderbilt-Tennessee State University Cancer Partnership.

This study examined longitudinal education and career outcomes of the Meharry-Vanderbilt-Tennessee State University Cancer Partnership, the longest-running National Cancer Institute (NCI) Comprehensive Partnerships in Advancing Cancer Health Equity (CPACHE) program site in the United States. Degree completion rates were calculated and progression along the entire postsecondary "pipeline" was quantified for 204 participants recruited between 2011 and 2020. For participants who had entered the workforce, career outcomes were also analyzed. Relative to comparison data, participants completed degrees and progressed through the higher education "pipeline" to earn advanced degrees at remarkably high rates; the majority entered careers in which they support or conduct cancer research. The latter is important, because most participants identify with demographic categories currently underrepresented in the cancer research workforce. This article makes two contributions to knowledge on research training programs for underrepresented students: 1) it quantifies participants' progression along the entire postsecondary education pipeline as well as into the workforce, and 2) it identifies points where participants are most prone to exit the pipeline rather than progress. We identify two types of exits-permanent and temporary-and offer empirically supported operational definitions for both. Evaluators may find the quantitative model and/or definitions useful for analyzing similar programs.

Antibiofilm Activity of Human Milk Oligosaccharides against Multidrug Resistant and Susceptible Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is a serious threat to human health, per the Centers for Disease Control and Prevention's latest threat assessment. A. baumannii is a Gram-negative opportunistic bacterial pathogen that causes severe community and nosocomial infections in immunocompromised patients. Treatment of these infections is confounded by the emergence of multi- and pan-drug resistant strains of A. baumannii. A. baumannii colonizes abiotic and biotic surfaces and evades antimicrobial challenges by forming biofilms, which are three-dimensional architectural structures of cells adhered to a substrate and encased in an extracellular matrix comprised of polymeric substances such as polysaccharides, proteins, and DNA. Biofilm-inhibiting compounds have recently gained attention as a chemotherapeutic strategy to prevent or disperse A. baumannii biofilms and restore the utility of traditional antimicrobial strategies. Recent work indicates that human milk oligosaccharides (HMOs) have potent antibacterial and biofilm-inhibiting properties. We sought to test the utility of HMOs against a bank of clinical isolates of A. baumannii to ascertain changes in bacterial growth or biofilm formation. Our results indicate that out of 18 strains tested, 14 were susceptible to the antibiofilm activities of HMOs, and that the potent antibiofilm activity was observed in strains isolated from diverse anatomical sites, disease manifestations, and across antibiotic-resistant and susceptible strains.

Analysis of Antimicrobial and Antibiofilm Activity of Human Milk Lactoferrin Compared to Bovine Lactoferrin against Multidrug Resistant and Susceptible Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised patients. The emergence of multi- and pan-drug resistant strains of A. baumannii from clinical sources has confounded treatment and enhanced morbidity and mortality associated with these infections. One way that A. baumannii circumnavigates environmental and antimicrobial challenge is by forming tertiary architectural structures of cells known as biofilms. Biofilm-inhibiting molecules could be deployed as a potential chemotherapeutic strategy to inhibit or disrupt A. baumannii biofilms and mitigate adverse outcomes due to infection. Lactoferrin is an innate immune glycoprotein produced in high concentrations in both human and bovine milk which has previously been shown to have antibacterial and antibiofilm activities. We sought to test lactoferrin against a bank of clinical isolates of A. baumannii to determine changes in bacterial growth or biofilm formation. Our results indicate that human lactoferrin has slightly more potent antibacterial activities than bovine lactoferrin against certain strains of A. baumannii and that these effects are associated with anatomical site of isolation. Additionally, we have shown that both bovine and human lactoferrin can inhibit A. baumannii biofilm formation and that these effects are associated with anatomical site of isolation and whether the strain forms robust or weak biofilms.

Multidrug resistant Acinetobacter baumannii: a descriptive study in a city hospital.

BACKGROUND: Multidrug resistant Acinetobacter baumannii, (MRAB) is an important cause of hospital acquired infection. The purpose of this study is to determine the risk factors for MRAB in a city hospital patient population. METHODS: This study is a retrospective review of a city hospital epidemiology data base and includes 247 isolates of Acinetobacter baumannii (AB) from 164 patients. Multidrug resistant Acinetobacter baumannii was defined as resistance to more than three classes of antibiotics. Using the non-MRAB isolates as the control group, the risk factors for the acquisition of MRAB were determined. RESULTS: Of the 247 AB isolates 72% (177) were multidrug resistant. Fifty-eight percent (143/247) of isolates were highly resistant (resistant to imipenem, amikacin, and ampicillin-sulbactam). Of the 37 patients who died with Acinetobacter colonization/infection, 32 (86%) patients had the organism recovered from the respiratory tract. The factors which were found to be significantly associated (p < or = 0.05) with multidrug resistance include the recovery of AB from multiple sites, mechanical ventilation, previous antibiotic exposure, and the presence of neurologic impairment. Multidrug resistant Acinetobacter was associated with significant mortality when compared with sensitive strains (p < or = 0.01). When surgical patients (N = 75) were considered separately, mechanical ventilation and multiple isolates remained the factors significantly associated with the development of multidrug resistant Acinetobacter. Among surgical patients 46/75 (61%) grew a multidrug resistant strain of AB and 37/75 (40%) were resistant to all commonly used antibiotics including aminoglycosides, cephalosporins, carbepenems, extended spectrum penicillins, and quinolones. Thirty-five percent of the surgical patients had AB cultured from multiple sites and 57% of the Acinetobacter isolates were associated with a co-infecting organism, usually a Staphylococcus or Pseudomonas. As in medical patients, the isolation of Acinetobacter from multiple sites and the need for mechanical ventilation were significantly associated with the development of MRAB. CONCLUSIONS: The factors significantly associated with MRAB in both the general patient population and surgical patients were mechanical ventilation and the recovery of Acinetobacter from multiple anatomic sites. Previous antibiotic use and neurologic impairment were significant factors in medical patients. Colonization or infection with MRAB is associated with increased mortality.

A rapidly growing human papillomavirus-positive oral tongue squamous cell carcinoma in a 21-year old female: A case report.

Oral tongue squamous cell carcinoma (OTSCC) has a median age at diagnosis of 62 years. The incidence of OTSCC in young adults has been increasing, and the reason is unclear. The present study describes a case, and molecular analysis, of OTSCC in a 21-year-old female. Clinical and pathological information were collected from medical records. Formalin-fixed paraffin-embedded biopsy tissue from the patient was reassessed using standard hematoxylin & eosin staining, and immunohistochemistry was used to assess the expression of cellular p16, MutL homolog (MLH)1, MLH2, MutS homolog 6 (MSH6) and PMS1 homolog 2 (PMS2). The human papilloma virus (HPV) genome was detected by PCR analysis of the extracted DNA. The young age of the patient with OTSCC was unusual. The original pathology report indicated koilocytotic atypia, a cellular abnormality associated with HPV. Although HPV-positive oral cancer tends to occur in 'younger' individuals, 21 years is unusual. The confirmation of biologically active HPV in the tumor was obtained via the observation of strong positive staining for cellular p16. The patient described a maternal family cluster of rare cancer types, thus the possibility that this rapidly growing cancer resulted from HPV infection combined with an underlying genetic mutation causing decreased DNA-mismatch repair was explored. However, MSH1, MSH2, MSH6 and PSM2, proteins that are associated with Lynch Syndrome, were expressed at normal levels. A rapidly growing OTSCC of a 21-year-old female was determined to be HPV-positive. The patient underwent combination chemotherapy and radiation and has experienced long-term survival without recurrence. The reason this tumor grew so quickly in such a young individual remains unknown. These types of cases warrant additional genomic and proteomic studies to improve understanding of this phenomenon.

Draft Genome Sequence of Multidrug-Resistant Acinetobacter baumannii Strain MMC4, Isolated from a Patient in Tennessee.

Acinetobacter baumannii multidrug-resistant strain MMC4 was isolated from a bronchoalveolar lavage fluid sample from a patient in Nashville, TN, USA. Here, we report a draft genome sequence with a size of 3,985,367 bp, an average G+C content of 39.8%, and 3,863 predicted protein-coding sequences.

Structural mass spectrometry of tissue extracts to distinguish cancerous and non-cancerous breast diseases.

Aberrant metabolism in breast cancer tumors has been widely studied by both targeted and untargeted analyses to characterize the affected metabolic pathways. In this work, we utilize ultra-performance liquid chromatography (UPLC) in tandem with ion mobility-mass spectrometry (IM-MS), which provides chromatographic, structural, and mass information, to characterize the aberrant metabolism associated with breast diseases such as cancer. In a double-blind analysis of matched control (n = 3) and disease tissues (n = 3), samples were homogenized, polar metabolites were extracted, and the extracts were characterized by UPLC-IM-MS/MS. Principle component analysis revealed a strong separation between disease tissues, with one diseased tissue clustering with the control tissues along PC1 and two others separated along PC2. Using post-ion mobility MS/MS spectra acquired by data-independent acquisition, the features giving rise to the observed grouping were determined to be biomolecules associated with aggressive breast cancer tumors, including glutathione, oxidized glutathione, thymosins ß4 and ß10, and choline-containing species. Pathology reports revealed the outlier of the disease tissues to be a benign fibroadenoma, whereas the other disease tissues represented highly metabolic benign and aggressive tumors. This IM-MS-based workflow bridges the transition from untargeted metabolomic profiling to tentative identifications of key descriptive molecular features using data acquired in one analysis, with additional experiments performed only for validation. The ability to resolve cancerous and non-cancerous tissues at the biomolecular level demonstrates UPLC-IM-MS/MS as a robust and sensitive platform for metabolomic profiling of tissues.

Molecular phenotyping of immune cells from young NOD mice reveals abnormal metabolic pathways in the early induction phase of autoimmune diabetes.

Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse--a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (~90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes.

Challenges unique to the design of a comprehensive questionnaire assessing breast cancer risk factors among women in sub-Saharan Africa.

We assessed the efficacy of a pilot questionnaire designed to elicit information about external risk factors for breast cancer in sub-Saharan African women. Preliminary analysis identified areas of the questionnaire and interviewing process that required modification, as well as socioeconomic factors that contribute to reduced participation among these understudied populations.

Bridging the gap between systems biology and medicine.

Systems biology has matured considerably as a discipline over the last decade, yet some of the key challenges separating current research efforts in systems biology and clinically useful results are only now becoming apparent. As these gaps are better defined, the new discipline of systems medicine is emerging as a translational extension of systems biology. How is systems medicine defined? What are relevant ontologies for systems medicine? What are the key theoretic and methodologic challenges facing computational disease modeling? How are inaccurate and incomplete data, and uncertain biologic knowledge best synthesized in useful computational models? Does network analysis provide clinically useful insight? We discuss the outstanding difficulties in translating a rapidly growing body of data into knowledge usable at the bedside. Although core-specific challenges are best met by specialized groups, it appears fundamental that such efforts should be guided by a roadmap for systems medicine drafted by a coalition of scientists from the clinical, experimental, computational, and theoretic domains.

New data analysis and mining approaches identify unique proteome and transcriptome markers of susceptibility to autoimmune diabetes.

Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta cells leading to insulin-dependent diabetes. In this study we developed and used new data analysis and mining approaches on combined proteome and transcriptome (molecular phenotype) data to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age, 2-4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by two-dimensional gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the two-dimensional gel proteome data that included two-way analysis of variance, cluster analysis, and principal component analysis. Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis-related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant over-representation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points toward abnormalities in regulation of the opposing processes of cell renewal and cell death even before there are any clear signatures of immune system activation.

Effector CD8+ T cells recovered from an influenza pneumonia differentiate to a state of focused gene expression.

The restriction of influenza A virus replication to mouse respiratory epithelium means that this host response is anatomically compartmentalized, on the one hand, to sites of T cell stimulation and proliferation in the secondary lymphoid tissue and, on the other hand, to the site of effector T cell function and pathology in the pneumonic lung. Thus, it is hardly surprising that virus-specific CD8(+) T cells recovered by bronchoalveolar lavage (BAL) from the infected respiratory tract seem more "activated" in terms of both cytolytic activity and cytokine production than those cells isolated from the spleen. The present analysis uses Affymetrix microarray technology to compare profiles of gene expression in these two lineage-related, yet anatomically separate, lymphocyte populations. Ninety differentially expressed genes were identified for influenza-specific CD8(+)D(b)NP(366)(+) T cells obtained directly ex vivo by BAL or spleen disruption, with nine genes being further analyzed by quantitative, real-time PCR at the population level. Integrin alphaE, for example, was shown by Affymetrix and real-time mRNA analyses and then by single-cell PCR and protein staining to be present at a much higher prevalence on the BAL CD8(+)D(b)NP(366)(+) set. The unpredicted finding, however, was that mRNA expression for 75% of the 90 genes was lower in T cells from the BAL than from the spleen. Apparently, the localization of virus-specific CD8(+) T cells to the site of virus-induced pathology is associated with a narrowing, or "focusing," of gene expression that favors enhanced effector function in the damaged, "high-antigen load" environment of the pneumonic lung.