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Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

A chemically programmed bispecific antibody (cp-bsAb) that targeted Cysteine protease legumain and alpha v beta 3 integrin has been prepared using the aldolase antibody chemical programming (AACP) strategy. In vitro evaluation of the anti-legumain, anti-integrin cp-bsAb and its comparison with cpAbs targeting either integrin or leg-umain have shown that the former possesses superior functions, including receptor binding and inhibitory effects on cell proliferation as well as capillary tube formation, among all three cpAbs. The anti-legumain, anti-integrin cp-bsAb also inhibited growth of primary tumor more effectively than either anti-legumain or anti-integrin cpAb as observed in the MDA-MB-231 human breast cancer mouse model. The AACP-based cp-bsAb, which contains a generic aldolase antibody, can also serve as a suitable platform for combination therapy, where two equally potent compounds are used to target extracellular receptors.

Purpose: To evaluate the feasibility of focal intraluminal irreversible electroporation (IRE) in the ureter with a novel electrode catheter and to study the treatment effects in response to increasing pulse strength. Materials and Methods: Five IRE treatment settings were each evaluated twice for the ablation of normal ureter in 5 Yorkshire pigs (n = 1-4 ablations per animal; total of 10 ablations) with the use of a prototype device under ultrasound and fluoroscopic guidance. Animals received unilateral or bilateral treatment, limited to a maximum of 2 ablations in any 1 ureter. Treatment was delivered with increasing pulse strength (from 1,000 V to 3,000 V in increments of 500 V) while keeping the pulse duration (100 mu s) and number of pulses (n = 90) constant. Ureter patency was assessed with antegrade ureteropyelography immediately following treatment. Animals were euthanized within 4 hours after treatment, and treated urinary tract was harvested for histopathologic analysis with hematoxylin and eosin and Masson trichrome stains. Results: IRE was successfully performed in all animals, without evidence of ureteral perforation. Hematoxylin and eosin analysis of IRE treatments demonstrated full-thickness ablation at higher field strengths (mucosa to the adventitia). Masson trichrome stains showed preservation of connective tissue at all field strengths. Conclusions: Intraluminal catheter-directed IRE ablation is feasible and produces full-thickness ablation of normal ureters. There was no evidence of lumen perforation even at the maximum voltages evaluated.

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.

Bacteria secrete peptides and proteins to communicate, to poison competitors, and to manipulate host cells. Among the various protein-translocation machineries, the peptidase-containing ATP-binding cassette transporters (PCATs) are appealingly simple. Each PCAT contains two peptidase domains that cleave the secretion signal from the substrate, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that hydrolyse ATP. In Gram-positive bacteria, PCATs function both as maturation proteases and exporters for quorum-sensing or antimicrobial polypeptides. In Gram-negative bacteria, PCATs interact with two other membrane proteins to form the type 1 secretion system. Here we present crystal structures of PCAT1 from Clostridium thermocellum in two different conformations. These structures, accompanied by biochemical data, show that the translocation pathway is a large a-helical barrel sufficient to accommodate small folded proteins. ATP binding alternates access to the transmembrane pathway and also regulates the protease activity, thereby coupling substrate processing to translocation.

Numerous epidemiological studies link vascular disorders, such as hypertension, diabetes mellitus, and stroke, with Alzheimer's disease (AD). Hypertension, specifically, is an important modifiable risk factor for late-onset AD. To examine the link between midlife hypertension and the onset of AD later in life, we chemically induced chronic hypertension in the TgSwDI mouse model of AD in early adulthood. Hypertension accelerated cognitive deficits in the Barnes maze test (P<0.05 after 3 months of treatment; P<0.001 after 6 months), microvascular deposition of -amyloid (P<0.001 after 3 months of treatment; P<0.05 after 6 months), vascular inflammation (P<0.05 in the dentate gyrus and P<0.001 in the dorsal subiculum after 6 months of treatment), blood-brain barrier leakage (P<0.05 after 3 and 6 months of treatment), and pericyte loss (P<0.05 in the dentate gyrus and P<0.01 in the dorsal subiculum after 6 months of treatment) in these mice. In addition, hypertension induced hippocampal neurodegeneration at an early age in this mouse line (43% reduction in the dorsal subiculum; P<0.05), establishing this as a useful research model of AD with mixed vascular and amyloid pathologies.

During metastatic progression, circulating cancer cells become lodged within the microvasculature of end organs, where most die from mechanical deformation. Although this phenomenon was first described over a half-century ago, the mechanisms enabling certain cells to survive this metastasis-suppressive barrier remain unknown. By applying whole-transcriptome RNA-sequencing technology to isogenic cancer cells of differing metastatic capacities, we identified a mutation encoding a truncated form of the pannexin-1 (PANX1) channel, PANX1(1-89), as recurrently enriched in highly metastatic breast cancer cells. PANX(1-89) functions to permit metastatic cell survival during traumatic deformation in the microvasculature by augmenting ATP release from mechanosensitive PANX1 channels activated by membrane stretch. PANX1-mediated ATP release acts as an autocrine suppressor of deformation-induced apoptosis through P2Y-purinergic receptors. Finally, small-molecule therapeutic inhibition of PANX1 channels is found to reduce the efficiency of breast cancer metastasis. These data suggest a molecular basis for metastatic cell survival on microvasculature-induced biomechanical trauma.

Large-scale sequencing of prokaryotic (meta) genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

The study of the spin-parity and tensor structure of the interactions of the recently discovered Higgs boson is performed using the H -> ZZ, Z gamma*, gamma*gamma* -> 4l, H -> WW -> l(nu)l(nu), and H -> gamma gamma decay modes. The full data set recorded by the CMS experiment during the LHC run 1 is used, corresponding to an integrated luminosity of up to 5.1 fb(-1) at a center-of-mass energy of 7 TeV and up to 19.7 fb(-1) at 8 TeV. A wide range of spin-two models is excluded at a 99% confidence level or higher, or at a 99.87% confidence level for the minimal gravitylike couplings, regardless of whether assumptions are made on the production mechanism. Any mixed-parity spin-one state is excluded in the ZZ and WW modes at a greater than 99.999% confidence level. Under the hypothesis that the resonance is a spin-zero boson, the tensor structure of the interactions of the Higgs boson with two vector bosons ZZ, Z gamma, gamma gamma, and WW is investigated and limits on eleven anomalous contributions are set. Tighter constraints on anomalous HVV interactions are obtained by combining the HZZ and HWW measurements. All observations are consistent with the expectations for the standard model Higgs boson with the quantum numbers J(PC) = 0(++).

A search for pair production of third-generation scalar leptoquarks decaying to top quark and tau lepton pairs is presented using proton-proton collision data at a center-of-mass energy of root s=8TeV collected with the CMS detector at the LHC and corresponding to an integrated luminosity of 19.7 fb(-1). The search is performed using events that contain an electron or a muon, a hadronically decaying tau lepton, and two or more jets. The observations are found to be consistent with the standard model predictions. Assuming that all leptoquarks decay to a top quark and a tau lepton, the existence of pair produced, charge -1/3, third-generation leptoquarks up to a mass of 685 GeV is excluded at 95% confidence level. This result constitutes the first direct limit for leptoquarks decaying into a top quark and a tau lepton, and may also be applied directly to the pair production of bottom squarks decaying predominantly via the R-parity violating coupling lambda'(333) .