Integrated genomic analyses of cutaneous T-cell lymphomas reveal the molecular bases for disease heterogeneity.

Cutaneous T-cell lymphomas (CTCLs) are a clinically heterogeneous collection of lymphomas of the skin-homing T cell. To identify molecular drivers of disease phenotypes, we assembled representative samples of CTCLs from patients with diverse disease subtypes and stages. Via DNA/RNA-sequencing, immunophenotyping, and ex vivo functional assays, we identified the landscape of putative driver genes, elucidated genetic relationships between CTCLs across disease stages, and inferred molecular subtypes in patients with stage-matched leukemic disease. Collectively, our analysis identified 86 putative driver genes, including 19 genes not previously implicated in this disease. Two mutations have never been described in any cancer. Functionally, multiple mutations augment T-cell receptor-dependent proliferation, highlighting the importance of this pathway in lymphomagenesis. To identify putative genetic causes of disease heterogeneity, we examined the distribution of driver genes across clinical cohorts. There are broad similarities across disease stages. Many driver genes are shared by mycosis fungoides (MF) and Sezary syndrome (SS). However, there are significantly more structural variants in leukemic disease, leading to highly recurrent deletions of putative tumor suppressors that are uncommon in early-stage skin-centered MF. For example, TP53 is deleted in 7% and 87% of MF and SS, respectively. In both human and mouse samples, PD1 mutations drive aggressive behavior. PD1 wild-type lymphomas show features of T-cell exhaustion. PD1 deletions are sufficient to reverse the exhaustion phenotype, promote a FOXM1-driven transcriptional signature, and predict significantly worse survival. Collectively, our findings clarify CTCL genetics and provide novel insights into pathways that drive diverse disease phenotypes.

ZEB1 promotes pathogenic Th1 and Th17 cell differentiation in multiple sclerosis.

Inappropriate CD4+ T helper (Th) differentiation can compromise host immunity or promote autoimmune disease. To identify disease-relevant regulators of T cell fate, we examined mutations that modify risk for multiple sclerosis (MS), a canonical organ-specific autoimmune disease. This analysis identified a role for Zinc finger E-box-binding homeobox (ZEB1). Deletion of ZEB1 protects against experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis (MS). Mechanistically, ZEB1 in CD4+ T cells is required for pathogenic Th1 and Th17 differentiation. Genomic analyses of paired human and mouse expression data elucidated an unexpected role for ZEB1 in JAK-STAT signaling. ZEB1 inhibits miR-101-3p that represses JAK2 expression, STAT3/STAT4 phosphorylation, and subsequent expression of interleukin-17 (IL-17) and interferon gamma (IFN-γ). Underscoring its clinical relevance, ZEB1 and JAK2 downregulation decreases pathogenic cytokines expression in T cells from MS patients. Moreover, a Food and Drug Administration (FDA)-approved JAK2 inhibitor is effective in EAE. Collectively, these findings identify a conserved, potentially targetable mechanism regulating disease-relevant inflammation.

Whipworm-Associated Intestinal Microbiome Members Consistent Across Both Human and Mouse Hosts.

The human whipworm Trichuris trichiura infects 289 million people worldwide, resulting in substantial morbidity. Whipworm infections are difficult to treat due to low cure rates and high reinfection rates. Interactions between whipworm and its host's intestinal microbiome present a potential novel target for infection control or prevention but are very complicated and are identified using inconsistent methodology and sample types across the literature, limiting their potential usefulness. Here, we used a combined 16S rRNA gene OTU analysis approach (QIIME2) for samples from humans and mice infected with whipworm (T. trichiura and T. muris, respectively) to identify for the first time, bacterial taxa that were consistently associated with whipworm infection spanning host species and infection status using four independent comparisons (baseline infected vs uninfected and before vs after deworming for both humans and mice). Using these four comparisons, we identified significant positive associations for seven taxa including Escherichia, which has been identified to induce whipworm egg hatching, and Bacteroides, which has previously been identified as a major component of the whipworm internal microbiome. We additionally identified significant negative associations for five taxa including four members of the order Clostridiales, two from the family Lachnospiraceae, including Blautia which was previously identified as positively associated with whipworm in independent human and mouse studies. Using this approach, bacterial taxa of interest for future association and mechanistic studies were identified, and several were validated by RT-qPCR. We demonstrate the applicability of a mouse animal model for comparison to human whipworm infections with respect to whipworm-induced intestinal microbiome disruption and subsequent restoration following deworming. Overall, the novel cross-species analysis approach utilized here provides a valuable research tool for studies of the interaction between whipworm infection and the host intestinal microbiome.

Improving Characterization of Understudied Human Microbiomes Using Targeted Phylogenetics.

Whole-genome bacterial sequences are required to better understand microbial functions, niche-specific bacterial metabolism, and disease states. Although genomic sequences are available for many of the human-associated bacteria from commonly tested body habitats (e.g., feces), as few as 13% of bacterium-derived reads from other sites such as the skin map to known bacterial genomes. To facilitate a better characterization of metagenomic shotgun reads from underrepresented body sites, we collected over 10,000 bacterial isolates originating from 14 human body habitats, identified novel taxonomic groups based on full-length 16S rRNA gene sequences, clustered the sequences to ensure that no individual taxonomic group was overselected for sequencing, prioritized bacteria from underrepresented body sites (such as skin and respiratory and urinary tracts), and sequenced and assembled genomes for 665 new bacterial strains. Here, we show that addition of these genomes improved read mapping rates of Human Microbiome Project (HMP) metagenomic samples by nearly 30% for the previously underrepresented phylum Fusobacteria, and 27.5% of the novel genomes generated here had high representation in at least one of the tested HMP samples, compared to 12.5% of the sequences in the public databases, indicating an enrichment of useful novel genomic sequences resulting from the prioritization procedure. As our understanding of the human microbiome continues to improve and to enter the realm of therapy developments, targeted approaches such as this to improve genomic databases will increase in importance from both an academic and a clinical perspective.IMPORTANCE The human microbiome plays a critically important role in health and disease, but current understanding of the mechanisms underlying the interactions between the varying microbiome and the different host environments is lacking. Having access to a database of fully sequenced bacterial genomes provides invaluable insights into microbial functions, but currently sequenced genomes for the human microbiome have largely come from a limited number of body sites (primarily feces), while other sites such as the skin, respiratory tract, and urinary tract are underrepresented, resulting in as little as 13% of bacterium-derived reads mapping to known bacterial genomes. Here, we sequenced and assembled 665 new bacterial genomes, prioritized from a larger database to select underrepresented body sites and bacterial taxa in the existing databases. As a result, we substantially improve mapping rates for samples from the Human Microbiome Project and provide an important contribution to human bacterial genomic databases for future studies.

Protein lipoylation: an evolutionarily conserved metabolic regulator of health and disease.

Lipoylation is a rare, but highly conserved lysine posttranslational modification. To date, it is known to occur on only four multimeric metabolic enzymes in mammals, yet these proteins are staples in the core metabolic landscape. The dysregulation of these mitochondrial proteins is linked to a range of human metabolic disorders. Perhaps most striking is that lipoylation itself, the proteins that add or remove the modification, as well as the proteins it decorates are all evolutionarily conserved from bacteria to humans, highlighting the importance of this essential cofactor. Here, we discuss the biological significance of protein lipoylation, the importance of understanding its regulation in health and disease states, and the advances in mass spectrometry-based proteomic technologies that can aid these studies.

Assessment of Perioperative Variables That Predict the Need for Surgical Drains Following Robotic Partial Nephrectomy Utilizing Quantitative Drain Creatinine Analysis.

OBJECTIVE: To utilize body fluid creatinine analysis to determine which patients will require a surgical drain following robotic-assisted partial nephrectomy (RAPN). MATERIALS AND METHODS: One hundred fifty consecutive RAPN performed by a single surgeon were reviewed. Postoperative day (POD) 1 drain creatinine was compared to serum creatinine to calculate the drain to serum creatinine ratio (D/S ratio). Elevated D/S ratio was defined as any value >1.2. RESULTS: From February 2008 to April 2015, 140 patients underwent RAPN and had a drain placed (124 had D/S ratio available on POD 1). In the 103 patients with a D/S ratio of <1.2 and the 21 with D/S ratio of >1.2, the mean tumor size was 3.0 and 3.9 cm (P = .001) and mean RENAL score was 7.6 and 8.1 (P = .270), respectively. Collecting system entry occurred in 68.2% of patients with a D/S ratio of <1.2 and 71.4% of patients with a D/S ratio of >1.2. Mean drain time was 2.4 and 4.2 days (P = .001), hospital stay was 2.7 and 3.3 days (P = .036) for the D/S ratio <1.2 and D/S ratio >1.2 groups, respectively. Those with renal mass size of 4-7 cm had increased likelihood of D/S ratio >1.2 (OR 2.78; P = .041). CONCLUSIONS: Most RAPN do not require a surgical drain. A POD 1 elevated D/S ratio is more likely to occur with larger masses (those approaching or greater than 4 cm) and can be associated with prolonged drain time and hospital stay.

Has Sliding-Clip Renorrhaphy Eliminated the Need for Collecting System Repair During Robot-Assisted Partial Nephrectomy?

OBJECTIVE: We evaluated whether single-layer renorrhaphy (SLR) without collecting system (CS) closure is sufficient following robot-assisted partial nephrectomy (RAPN). PATIENTS AND METHODS: One hundred fifty consecutive patients underwent RAPN by a single surgeon and were prospectively labeled with regard to CS entry during surgery. Patients with CS entry were subdivided into two groups: those with classical renorrhaphy (CR) (i.e., two-layer repair) and those with SLR (i.e., without CS repair). Perioperative variables and outcomes were compared between CR and SLR groups. RESULTS: Ninety patients had CS entry during RAPN. Of these 90 patients, 64 had CR, and 26 had SLR, with mean ages of 62 and 59 years (p = 0.22), tumor sizes of 3.4 and 3.3 cm (p = 0.61), Mayo Adhesive Probability scores of 1.8 and 1.8 (p = 0.95), and radius, exophytic/endophytic, nearness to CS, and laterality scores of 8.5 and 8.0 (p = 0.16), respectively. Mean warm ischemia times (WITs) were 19.6 and 17.3 minutes (p = 0.04), hospital stays of 3.0 and 2.8 days (p = 0.62), and drain times of 2.9 and 2.7 days (p = 0.65), for the CR and SLR groups, respectively. Using the Clavien-Dindo classification, there were a total of six grade III or higher complications, with no difference between the CR and SLR subgroups (p = 1.0). Renal function using creatinine or glomerular filtration rate as surrogates showed no difference between groups preoperatively or up until 2 years postoperatively. CONCLUSIONS: Omitting CS repair during RAPN with SLR decreases WIT without altering complications, hospital stay, or drain time. Long-term renal function was not associated with CS repair.

Sirtuin Lipoamidase Activity Is Conserved in Bacteria as a Regulator of Metabolic Enzyme Complexes.

Lipoic acid is an essential metabolic cofactor added as a posttranslational modification on several multimeric enzyme complexes. These protein complexes, evolutionarily conserved from bacteria to humans, are core regulators of cellular metabolism. While the multistep enzymatic process of adding lipoyl modifications has been well characterized in Escherichia coli, the enzyme required for the removal of these lipoyl moieties (i.e., a lipoamidase or delipoylase) has not yet been identified. Here, we describe our discovery of sirtuins as lipoamidases in bacteria and establish their conserved substrates. Specifically, by using a series of knockout, overexpression, biochemical, in vitro, proteomic, and functional assays, we determined the substrates of sirtuin CobB in E. coli as components of the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KDH), and glycine cleavage (GCV) complexes. In vitro assays provided direct evidence for this specific CobB activity and its NAD+ dependence, a signature of all sirtuins. By designing a targeted quantitative mass spectrometry method, we further measured sirtuin-dependent, site-specific lipoylation on these substrates. The biological significance of CobB-modulated lipoylation was next established by its inhibition of both PDH and KDH activities. By restricting the carbon sources available to E. coli, we demonstrated that CobB regulates PDH and KDH under several growth conditions. Additionally, we found that SrtN, the sirtuin homolog in Gram-positive Bacillus subtilis, can also act as a lipoamidase. By demonstrating the evolutionary conservation of lipoamidase activity across sirtuin homologs, along with the conservation of common substrates, this work emphasizes the significance of protein lipoylation in regulating central metabolic processes.IMPORTANCE Here, we demonstrate that sirtuin lipoamidase activity exists in both Gram-positive and Gram-negative bacteria and establishing its conservation from bacteria to humans. Specifically, we discovered that CobB and SrtN act as lipoamidases in E. coli and B. subtilis, respectively. Intriguingly, not only is this sirtuin enzymatic activity conserved, but also the lipoylated substrates and functions are conserved, as bacterial sirtuins negatively regulate the lipoylation levels and activities of PDH and KDH. Considering that PDH and KDH regulate two carbon entry points into the tricarboxylic acid cycle, our finding highlights lipoylation as a conserved molecular toggle that regulates central metabolic pathways. Indeed, our findings from tests in which we limited nutrient availability support this. Furthermore, this study illustrates how the integration of technologies from different disciplines provides avenues to uncover enzymatic activities at the core of cellular metabolism regulation.

Multispecialty retrospective review of the clinical utility of pelvic magnetic resonance imaging in the setting of pelvic pain.

BACKGROUND: Pelvic pain is a common complaint, and management of it is often difficult. We sought to evaluate the utility of magnetic resonance imaging (MRI) in the diagnosis of male pelvic pain. Though MRIs are commonly ordered to evaluate pelvic pain, there are very few studies obtaining the efficacy of pelvic MRI in determining a definitive diagnosis. The primary aim of our study was to evaluate the clinical utility of pelvic MRI for a diagnosis code that included pain. METHODS: After receiving institutional review board approval, a retrospective study was performed of all pelvic MRIs completed at our institution from January 2, 2010 to December 31, 2014. These were further delineated into ordering providers by specialty and urology-specific International Classification of Diseases, Ninth Revision (ICD-9) code diagnoses (male pelvic pain, prostatitis, groin pain, scrotal pain, testicular pain, and penile pain). Clinical utility was defined as positive if MRI findings resulted in a change in management. Subanalysis was performed on patients with an ICD-9 co-diagnosis of previous oncologic concern. RESULTS: A total of 2,643 pelvic MRIs were ordered at our institution over a 5-year period. Of these, 597 pelvic MRIs (23%) were ordered for a diagnosis code that included pain (hip pain, rectal pain, joint pain, penile pain, scrotal pain, male pelvic pain and orchitis). Total utility for MRIs to find anatomic abnormalities potentially responsible for the present pain was 34% (205/597). When ordered by urologic providers, utility was 23%. Oncologists represented the highest positivity rate at 57%. CONCLUSIONS: Chronic pelvic pain is a multispecialty complaint that is difficult to treat. We were surprised to find the large number of both specialists and generalists invested in the management of pelvic pain. The increasing availability of MRI technology makes it a likely candidate to test for a clinically significant anatomic reason for pain. Though MRI is a test with minimal adverse effect and no increased risk of radiation exposure, the cost on the healthcare system should be offset by a clear clinical utility. We found total utility to be 34% across all ordering providers and an increase in positivity with concern of oncologic disease. Therefore, we would recommend pelvic MRIs in the evaluation of patients with refractory pelvic pain.