An integrative systems genetic analysis of mammalian lipid metabolism.

Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity.

Suicide Rates and Methods in Active Duty Military Personnel, 2005 to 2011: A Cohort Study.

BACKGROUND: Suicide prevention programs have become ubiquitous among military units; identifying temporal trends and nonclinical factors associated with the chosen suicide methods may help improve suicide prevention strategies. OBJECTIVE: To calculate suicide rates of active duty military personnel and identify those who are at risk for firearm-specific suicide. DESIGN: Retrospective cohort study. SETTING: Military units in the United States. PATIENTS: All active duty enlisted U.S. military personnel from 2005 to 2011. MEASUREMENTS: Suicide rates per 100 000 were calculated for each branch. Adjusted odds ratios for firearm-specific suicide were calculated with 95% CIs. RESULTS: 1455 military personnel committed suicide from 2005 to 2011. From 2006 to 2011, the rates were highest among army personnel (19.13 to 29.44 cases per 100 000). Among suicides with a known cause of death, 62% were attributed to firearms. The results of this study also suggest that among army personnel or marines who committed suicide, those with infantry or special operations job classifications were more likely than those in noninfantry positions to use a firearm. LIMITATIONS: Results are generalizable only to enlisted personnel and reflect only stateside suicides. Data regarding previous psychiatric illness, deployment history, and firearms ownership were lacking. CONCLUSION: These results may help inform policymakers and advisors about differences in risks of suicide and violent suicide among the armed services and may help guide efforts to prevent self-harm within the military. PRIMARY FUNDING SOURCE: None.

Enhancing tumor-infiltrating T cells with an exclusive fuel source.

Solid tumors harbor immunosuppressive microenvironments that inhibit tumor infiltrating lymphocytes (TILs) through the voracious consumption of glucose. We sought to restore TIL function by providing them with an exclusive fuel source. The glucose disaccharide cellobiose, which is a building block of cellulose, contains a ß-1,4-glycosidic bond that cannot be hydrolyzed by animals (or their tumors), but fungal and bacterial organisms have evolved enzymes to catabolize cellobiose and use the resulting glucose. By equipping T cells with two proteins that enable import and hydrolysis of cellobiose, we demonstrate that supplementation of cellobiose during glucose withdrawal restores T cell cytokine production and cellular proliferation. Murine tumor growth is suppressed and survival is prolonged. Offering exclusive access to a natural disaccharide is a new tool that augments cancer immunotherapies. Beyond cancer, this approach could be used to answer questions about the regulation of glucose metabolism across many cell types, biological processes, and diseases.

ALK upregulates POSTN and WNT signaling to drive neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.

Association of lower urinary tract symptoms and diuretic adherence.

OBJECTIVE: To assess whether more severe urinary symptoms and poorer quality of life among patients on diuretic therapy are associated with decreased adherence to the diuretic regimen. METHODS: Participants were recruited via ResearchMatch.org and sent a REDCap survey. The Overactive Bladder Questionnaire-Short Form (OAB-q SF) was used to assess urinary symptom bother and health-related quality of life (HRQL). Participants were asked if they skip diuretic doses due to urinary symptoms with a bivariate (yes or no) outcome. Subgroup analyses of loop vs non-loop diuretic and those taking the diuretic for a cardiovascular indication (hypertension or heart failure) were performed. RESULTS: A total of 4029 surveys were sent, 285 were returned (7.1% response rate), and 279 were included in the study. Fifty-three participants admitted to skipping diuretic doses due to urinary symptoms. Lower HRQL scores were significantly associated with poorer adherence scores among all participants (P < .001), among participants taking a loop diuretic (P < .001), and among participants with hypertension and heart failure (P < .039). Association between symptoms and adherence remained significant after adjustment in the multivariate model for the whole cohort and loop diuretic subgroup but lost significance in the hypertension and heart failure subgroup. CONCLUSIONS: Worsening quality of life due to urinary symptoms may be associated with poorer adherence to diuretics, particularly loop diuretics.

Augmenting T-cell responses to tumors by in situ nanomanufacturing.

Recent innovations in immunoregulatory treatments have demonstrated both the impressive potential and vital role of T cells in fighting cancer. These treatments come at a cost, with systemic side effects including life-threatening autoimmunity and immune dysregulation the norm. Here, we developed an approach to locally synthesize immune therapies and in this way, avoid systemic toxicity. Rather than just encapsulating cytokines, we endowed our nanoparticles with transcriptional and translational machinery to make cytokines locally, in situ, and on demand (activated by light). We demonstrated the capabilities of these particles in vitro and in vivo, in a mouse model of melanoma, and showed that tumor-infiltrating T cells were more highly activated in the context of these "microfactory" particles that make the synthetic cytokine.

Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.

Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.

Generating trunk neural crest from human pluripotent stem cells.

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior "cranial" NCC form craniofacial bone, whereas solely posterior "trunk" NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.

Aluminum water treatment residuals as permeable reactive barrier sorbents to reduce phosphorus losses.

Two aluminum water treatment residuals (Al-WTRs) from water treatment plants in Manatee County, FL and Punta Gorda, FL were evaluated as potential permeable reactive barrier (PRB) media to reduce groundwater phosphorus (P) losses. Short-term (<24h) P sorption kinetics and long-term P sorption capacity were determined using batch equilibration studies. Phosphorus desorption was characterized following P loadings of 10, 20, 30, 40 and >70 g kg(-1). Sorption and desorption studies were conducted on the <2.0mm material and three size fractions within the <2.0mm material. The effect of dissolved organic carbon (DOC) on P retention was determined by reacting Al-WTRs with P-spiked groundwater samples of varying initial DOC concentrations. Phosphorus sorption kinetics were rapid for all size fractions of both Al-WTRs (>98% P sorption effectiveness at shaking times ≥2 h). The effect of DOC was minimal at <150 mg DOCL(-1), but modest reductions (<22%) in P sorption effectiveness occurred at 587 mg DOC L(-1). The P sorption capacities of the Manatee and Punta Gorda Al-WTRs (<2.0mm) are ∼44 g kg(-1) and >75 g kg(-1), respectively, and the lifespan of an Al-WTR PRB is likely many decades. Desorption was minimal (<2% of the P sorbed) for cumulative P loadings <40 g kg(-l), but increased (<9% of the P sorbed) at cumulative P loads >70 g kg(-1). The <2.0mm Manatee and Punta Gorda Al-WTRs are regarded as ideal PRB media for P remediation.

Accuracy of ICD-9-CM coding of cervical spine fractures: implications for research using administrative databases.

OBJECTIVE: ICD-9-CM codes are often used for trauma research due to their ready availability in administrative databases. They are also used to classify injury severity in trauma patients. However, errors in coding may limit the use of these codes. Prior studies have found coding accuracy ranging from 20 to 100%, casting doubt on the reliability of studies utilizing these codes. The goal of this study was to determine the accuracy of ICD-9-CM coding for cervical spine fractures. METHODS: We used ICD-9-CM codes to identify trauma admissions and cervical spine fractures at a Level I trauma center in 2006. Cervical spine CT or CTA reports were reviewed by two independent observers. Data were compared to ICD-9-CM codes to determine accuracy. RESULTS: Of 1620 trauma admissions, 174 (11%) included a cervical spine fracture defined by ICD-9-CM codes. A cervical spine fracture was the primary diagnosis in 79 admissions and a secondary diagnosis in 63 admissions. Of the 142 cervical spine fractures defined by ICD-9-CM code, there were 133 (94%) cervical fractures by radiology report. Accuracy varied by primary diagnosis (97%) versus secondary diagnosis (89%). By cervical level, there were 230 fractures by CT report. Of these, 7% of ICD-9-CM codes documented a fractured level not noted in the CT report. Conversely, 14% of fractured levels noted by CT report did not have a corresponding ICD-9-CM code. IMPLICATIONS: We found an overall 94% accuracy of ICD-9-CM coding compared to radiology reports. Inaccuracy of coding fracture level ranged from 7 to 14%. Researchers using these codes should refer back to the medical record or perform a sensitivity analysis to improve reliability.

Modified osteoplastic orbitozygomatic craniotomy in the pediatric population.

OBJECT: Anterior and anterolateral skull base approaches offer the advantages of improved visualization and minimal brain retraction for lesions involving the orbital apex, parasellar regions, and anterior and middle fossa floors. These approaches are seldom used in the pediatric population due to the perceived increase in morbidity and surgical complexity. We report the application of the previously described modified osteoplastic orbitozygomatic (OZ) craniotomy to pediatric neurosurgical cases. This approach offers a number of advantages and is technically straightforward. MATERIALS AND METHODS: The results from six pediatric cases are reported. Age ranged from 26 months to 15 years, with a follow-up period of 5 to 22 months. Pathology included craniopharyngioma (three), frontal epidural abscess-subdural empyema with intraorbital extension (one), hypothalamic hamartoma (one), and optic pathway glioma (one). No complications related to the surgical approach were noted. In all cases, good postoperative cosmesis was achieved with excellent realignment of the orbital rim. Temporalis muscle bulk was preserved and symmetric in all cases. CONCLUSION: The modified osteoplastic OZ craniotomy can be safely and effectively applied to the pediatric population. Advantages include: (1) ease of use; (2) superior exposure and therefore less brain retraction; (3) an easily replaced one-piece bone flap which obviates the need for plating-suturing at the orbital rim; (4) a vascularized bone flap less susceptible to infection; and (5) maintenance of normal temporalis muscle anatomy for improved cosmesis and function.

Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

Adamantane-like cluster complexes of mixed-valent copper-copper and nickel-copper thiolates.

Square-planar copper(II) and nickel(II) derivatives of the cis-dithiolate N(2)S(2) ligand bis(N,N'-2-mercapto-2-methylpropyl)-1,5-diazocyclooctane, (bme*daco)M, nucleate four Cu(I)Cl moieties, forming M(II)(2)Cu(I)(4)S(4) clusters with unusual triply bridging thiolates, mu(3)-SR, in the topological form of adamantane. As determined by X-ray crystallography, the (bme*daco)M (M = Cu or Ni) metallothiolate serves as a bidentate ligand that bridges four Cu(I) ions, utilizing all lone pairs on sulfurs. Further characterization by electrochemical and electronic spectral measurements suggests greater electron delocalization in the all-copper complex as compared to the NiCu heterometallic complex. Mass spectral data imply that the mixed-metal Ni(II)(2)Cu(I)(4)S(4) is more stable toward CuCl loss than Cu(II)(2)Cu(I)(4)S(4), a result that is corroborated by extraction of Cu(I) by 1,2-bis(diphenylphosphino)ethane in the latter but not the former.

Bismercaptoethanediazacyclooctane as a N2S2 chelating agent and Cys-X-Cys mimic for Fe(NO) and Fe(NO)2.

The N-protonated bismercaptoethanediazacyclooctane serves as a bidentate dithiolate ligand to oxidized Fe(NO)(2) of Enemark-Feltam notation, E-F [Fe(NO)(2)],(9) mimicking Cys-X-Cys binding of Fe(NO)(2) to proteins or thio-biomolecules. The neutral compound is characterized by the well-known g = 2.03 EPR signal which is a hallmark of dinitrosyl iron complexes, DNIC's. The Fe(NO)(2) unit can be removed from the chelate by excess PhS(-), producing (PhS)(2)Fe(NO)(2)(-). Transfer of NO from Fe(H(+)bme-daco)(NO)(2) (nu(NO) = 1740, 1696 cm(-)(1)) to Fe(II) of [(bme-daco)Fe](2) yields the five-coordinate, square-pyramidal N(2)S(2)Fe(NO) (nu(NO) = 1649 cm(-)(1)), where NO is in the apical position. Its isotropic EPR signal at g = 2.05 is consistent with E-F [Fe(NO)](7) formulation. In excess NO, Roussin's red ester-type molecules are formed as dinuclear or tetranuclear species, [(micro-SRS)[Fe(2)(NO)(4)]](n)() (n =1, 2). These well-characterized molecules furnish reference points for positions and patterns in nu(NO) vibrational spectroscopy expected to be useful for in vivo studies of NO degradation of iron-sulfur clusters in ferredoxins.

Activation of alkenes and H2 by [Fe]-H2ase model complexes.

The established ability of the Fe(II) bridging hydride species (micro-H)(micro-pdt)[Fe(CO)2(PMe3)]2+, 1-H+, to take-up and heterolytically activate dihydrogen, resulting in H/D scrambling of H2/D2 and H2/D2O mixtures (Zhao et al. Inorg. Chem. 2002, 41, 3917) has prompted a study of simultaneous alkene/H2 activation by such [Fe]H2ase model complexes. That the required photolysis produced an open site was substantiated by substitution of CO in 1-H+ by CH3CN with formation of structurally characterized [(micro-H)(micro-pdt)[Fe(CO)2(PMe3)][Fe(CO)(CH3CN)(PMe3)]]+[PF6]-. Under similar photolytic conditions, H/D exchange reactions between D2 and terminal alkenes (ethylene, propene and 1-butene), but not bulkier alkenes such as 2-butene or cyclohexene, were catalyzed by 1-H+ and the edt (SCH2CH2S) analogue, 2-H+. Substantial regioselectivity for H/D exchange at the internal vinylic hydrogen was observed. The extent to which the olefins were deuterium enriched vs deuterated was catalyst dependent. The stabilizing effect of the binuclear chelating ligands, SCH2CH2CH2S, pdt, and SCH2CH2S, edt, is required for the activity of binuclear catalysts, as the mono-dentate micro-SEt analogue decomposed to inactive products under the photolytic conditions of the catalysis. Reactions of 1 and 2 with EtOSO2CF3 yielded the S-alkylated products, [(micro-SCH2CH2CH2SEt)[Fe(CO)2(PMe3)]2]+[SO3CF3]- (1-Et+), and 2-Et+, rather than micro-C2H5 analogues to the micro-H of 1-H+. The stability and lack of reactivity toward H2 of 1-Et+ and 2-Et+, indicates they are not on the reaction path of the olefin/D2 H/D exchange process. A mechanism with olefin binding to an open site created by CO loss and formation of an Fe-(CH2CHDR) intermediate is indicated. A likely role of a binuclear chelate effect is implicated for the unique S-XXX-S cofactor in the active site of [Fe]H2ase.

Catalysis of H(2)/D(2) scrambling and other H/D exchange processes by [Fe]-hydrogenase model complexes.

Protonation of the [Fe]-hydrogenase model complex (mu-pdt)[Fe(CO)(2)(PMe(3))](2) (pdt = SCH(2)CH(2)CH(2)S) produces a species with a high field (1)H NMR resonance, isolated as the stable [(mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+)[PF(6)](-) salt. Structural characterization found little difference in the 2Fe2S butterfly cores, with Fe.Fe distances of 2.555(2) and 2.578(1) A for the Fe-Fe bonded neutral species and the bridging hydride species, respectively (Zhao, X.; Georgakaki, I. P.; Miller, M. L.; Yarbrough, J. C.; Darensbourg, M. Y. J. Am. Chem. Soc. 2001, 123, 9710). Both are similar to the average Fe.Fe distance found in structures of three Fe-only hydrogenase active site 2Fe2S clusters: 2.6 A. A series of similar complexes (mu-edt)-, (mu-o-xyldt)-, and (mu-SEt)(2)[Fe(CO)(2)(PMe(3))](2) (edt = SCH(2)CH(2)S; o-xyldt = SCH(2)C(6)H(4)CH(2)S), (mu-pdt)[Fe(CO)(2)(PMe(2)Ph)](2), and their protonated derivatives likewise show uniformity in the Fe-Fe bond lengths of the neutral complexes and Fe.Fe distances in the cationic bridging hydrides. The positions of the PMe(3) and PMe(2)Ph ligands are dictated by the orientation of the S-C bonds in the (mu-SRS) or (mu-SR)(2) bridges and the subsequent steric hindrance of R. The Fe(II)(mu-H)Fe(II) complexes were compared for their ability to facilitate H/D exchange reactions, as have been used as assays of H(2)ase activity. In a reaction that is promoted by light but inhibited by CO, the [(mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+) complex shows H/D exchange activity with D(2), producing [(mu-D)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+) in CH(2)Cl(2) and in acetone, but not in CH(3)CN. In the presence of light, H/D scrambling between D(2)O and H(2) is also promoted by the Fe(II)(mu-H)Fe(II) catalyst. The requirement of an open site suggests that the key step in the reactions involves D(2) or H(2) binding to Fe(II) followed by deprotonation by the internal hydride base, or by external water. As indicated by similar catalytic efficiencies of members of the series, the nature of the bridging thiolates has little influence on the reactions. Comparison to [Fe]H(2)ase enzyme active site redox levels suggests that at least one Fe(II) must be available for H(2) uptake while a reduced or an electron-rich Fe(I)Fe(I) metal-metal bonded redox level is required for proton uptake.