Experimental Evidence for a Metallohydrolase Mechanism in Which the Nucleophile Is Not Delivered by a Metal Ion: EPR Spectrokinetic and Structural Studies of Aminopeptidase from \u3cem\u3eVibrio proteolyticus\u3c/em\u3e

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies of reaction products of L-leucyl-L-leucyl-L-leucine and Co(II)-substituted aminopeptidase, and comparison of the EPR data with those from structurally characterized complexes of aminopeptidase with inhibitors, indicated the formation of a catalytically competent post-Michaelis pre-transition state intermediate with a structure analogous to that of the inhibited complex with bestatin. The X-ray crystal structure of an aminopeptidase–L-leucyl-L-leucyl-L-leucine complex was also analogous to that of the bestatin complex. In these structures, no water/hydroxy group was observed bound to the essential metal ion. However, a water/hydroxy group was clearly identified that was bound to the metal-ligating oxygen atom of Glu152. This water/hydroxy group is proposed as a candidate for the active nucleophile in a novel metallohydrolase mechanism that shares features of the catalytic mechanisms of aspartic proteases and of B2 metallo-b-lactamases. Preliminary studies on site-directed variants are consistent with the proposal. Other features of the structure suggest roles for the dinuclear centre in geometrically and electrophilically activating the substrate

Wolf 1130: A Nearby Triple System Containing a Cool, Ultramassive White Dwarf

Following the discovery of the T8 subdwarf WISEJ200520.38+542433.9 (Wolf 1130C), with common proper motion to a binary (Wolf 1130AB) consisting of an M subdwarf and a white dwarf, we set out to learn more about the old binary in the system. We find that the A and B components of Wolf 1130 are tidally locked, which is revealed by the coherence of more than a year of V band photometry phase folded to the derived orbital period of 0.4967 days. Forty new high-resolution, near-infrared spectra obtained with the Immersion Grating Infrared Spectrometer (IGRINS) provide radial velocities and a projected rotational velocity (v sin i) of 14.7 +/- 0.7 km/s for the M subdwarf. In tandem with a Gaia parallax-derived radius and verified tidal-locking, we calculate an inclination of i=29 +/- 2 degrees. From the single-lined orbital solution and the inclination we derive an absolute mass for the unseen primary (1.24+0.19-0.15 Msun). Its non-detection between 0.2 and 2.5 microns implies that it is an old (>3.7 Gyr) and cool (Teff<7000K) ONe white dwarf. This is the first ultramassive white dwarf within 25pc. The evolution of Wolf 1130AB into a cataclysmic variable is inevitable, making it a potential Type Ia supernova progenitor. The formation of a triple system with a primary mass >100 times the tertiary mass and the survival of the system through the common-envelope phase, where ~80% of the system mass was lost, is remarkable. Our analysis of Wolf 1130 allows us to infer its formation and evolutionary history, which has unique implications for understanding low-mass star and brown dwarf formation around intermediate mass stars.Comment: 37 pages, 9 Figures, 5 Table

Fine Mapping of the SCN Resistance Locus \u3ci\u3erhg1-b\u3c/i\u3e from PI 88788

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is the most economically damaging soybean [Glycine max (L.) Merr.] pest in the USA and genetic resistance is a key component for its control. Although SCN resistance is quantitative, the rhg1 locus on chromosome 18 (formerly known as Linkage Group G) confers a high level of resistance. The objective of this study was to fi ne-map the rhg1-b allele that is derived from plant introduction (PI) 88788. F2 and F3 plants and F3:4 lines from crosses between SCN resistant and susceptible genotypes were tested with genetic markers to identify recombination events close to rhg1-b. Lines developed from these recombinant plants were then tested for resistance to the SCN isolate PA3, which originally had an HG type 0 phenotype, and with genetic markers. Analysis of lines carrying key recombination events positioned rhg1-b between the simple sequence repeat (SSR) markers BARCSOYSSR_18_0090 and BARCSOYSSR_18_0094. This places rhg1-b to a 67-kb region of the ‘Williams 82’ genome sequence. The receptor-like kinase gene that has been previously identified as a candidate for the ‘Peking’-derived SCN resistant rhg1 gene is adjacent to, but outside of, the rhg1-b interval defined in the present study

Band Gap Engineering with Ultralarge Biaxial Strains in Suspended Monolayer MoS2

We demonstrate the continuous and reversible tuning of the optical band gap of suspended monolayer MoS2 membranes by as much as 500 meV by applying very large biaxial strains. By using chemical vapor deposition (CVD) to grow crystals that are highly impermeable to gas, we are able to apply a pressure difference across suspended membranes to induce biaxial strains. We observe the effect of strain on the energy and intensity of the peaks in the photoluminescence (PL) spectrum, and find a linear tuning rate of the optical band gap of 99 meV/%. This method is then used to study the PL spectra of bilayer and trilayer devices under strain, and to find the shift rates and Gr\"uneisen parameters of two Raman modes in monolayer MoS2. Finally, we use this result to show that we can apply biaxial strains as large as 5.6% across micron sized areas, and report evidence for the strain tuning of higher level optical transitions.Comment: Nano Lett., Article ASA

Structure and Metal Binding Properties of ZnuA, a Periplasmic Zinc Transporter from \u3cem\u3eEscherichia coli\u3c/em\u3e

ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated K d \u3c 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer

Obesity-Induced Colorectal Cancer Is Driven by Caloric Silencing of the Guanylin-GUCY2C Paracrine Signaling Axis.

Obesity is a well-known risk factor for colorectal cancer but precisely how it influences risks of malignancy remains unclear. During colon cancer development in humans or animals, attenuation of the colonic cell surface receptor guanylyl cyclase C (GUCY2C) that occurs due to loss of its paracrine hormone ligand guanylin contributes universally to malignant progression. In this study, we explored a link between obesity and GUCY2C silencing in colorectal cancer. Using genetically engineered mice on different diets, we found that diet-induced obesity caused a loss of guanylin expression in the colon with subsequent GUCY2C silencing, epithelial dysfunction, and tumorigenesis. Mechanistic investigations revealed that obesity reversibly silenced guanylin expression through calorie-dependent induction of endoplasmic reticulum stress and the unfolded protein response in intestinal epithelial cells. In transgenic mice, enforcing specific expression of guanylin in intestinal epithelial cells restored GUCY2C signaling, eliminating intestinal tumors associated with a high calorie diet. Our findings show how caloric suppression of the guanylin-GUCY2C signaling axis links obesity to negation of a universal tumor suppressor pathway in colorectal cancer, suggesting an opportunity to prevent colorectal cancer in obese patients through hormone replacement with the FDA-approved oral GUCY2C ligand linaclotide

12-h clock regulation of genetic information flow by XBP1s

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pan, Y., Ballance, H., Meng, H., Gonzalez, N., Kim, S., Abdurehman, L., York, B., Chen, X., Schnytzer, Y., Levy, O., Dacso, C. C., McClung, C. A., O'Malley, B. W., Liu, S., & Zhu, B. 12-h clock regulation of genetic information flow by XBP1s. Plos Biology, 18(1), (2020): e3000580, doi:10.1371/journal.pbio.3000580.Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s’s ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.This study was supported by the American Diabetes Association junior faculty development award 1-18-JDF-025 to B.Z., by funding from National Institute of Health HD07879 and 1P01DK113954 to B.W.O, by funding from National Science Foundation award 1703170 to C.C.D. and B.Z., and by funding from Brockman Foundation to C.C.D and B.W.O. This work was further supported by the UPMC Genome Center with funding from UPMC’s Immunotherapy and Transplant Center. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Research reported in this publication was further supported by the National Institute of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health under award number P30DK120531 to Pittsburgh Liver Research Center, in which both S.L. and B.Z. are members. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries

Young female cancer survivors’ use of fertility care after completing cancer treatment

To investigate factors associated with female young adult cancer survivors’ (YCS) use of fertility care (FC), including consultation or fertility treatment, after completing their cancer treatment