Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Measurement of CP observables in B-+/- -> DK +/- and B-+/- -> D pi(+/-) with D -> KS0<mml:msup>K +/-</mml:msup><mml:msup>pi -/+</mml:msup> decays

Measurements of $CP$ observables in $B^\pm \to D K^\pm$ and $B^\pm \to D \pi^\pm$ decays are presented, where $D$ represents a superposition of $D^0$ and $\bar{D}^0$ states. The $D$ meson is reconstructed in the three-body final state $K_{\rm{S}}^0K^\pm \pi^\mp$. The analysis uses samples of $B$ mesons produced in proton-proton collisions, corresponding to an integrated luminosity of 1.0, 2.0, and 6.0 fb$^{-1}$ collected with the LHCb detector at centre-of-mass energies of $\sqrt{s} =$ 7, 8, and 13 TeV, respectively. These measurements are the most precise to date, and provide important input for the determination of the CKM angle $\gamma$

Intradialytic exercise increases cardiac power index

Introduction: Mortality rates are high in end-stage renal disease due to cardiovascular complications. Perfusion of the myocardium declines during and after hemodialysis sessions with the potential for aerobic exercise to mitigate these during hemodialysis. Objectives: The purpose of this study was to investigate acute changes in hemodynamics in subjects with end-stage renal disease (ESRD) during exercise. Patients and Methods: Subjects (n = 10) were monitored for 1.5 hours during hemodialysis treatment during a control (CON) and an exercise (EX) session. Subjects cycled using an ergometer strapped to the reclining dialysis chair at an RPE of 11-13 for 30 minutes during the EX session beginning at 30 min into dialysis and ending at 60 minutes. Data for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were collected using an automated blood pressure cuff attached to the hemodialysis machine. Data for cardiac output (Q̇ ), cardiac power index (CPI), stroke volume (SV), systemic vascular resistance (SVR), and heart rate (HR) were collected using the NICaS bioelectrical impedance device. Results: During the EX session, CPI, Q̇ , SV, and HR were significantly greater (P<0.05) than the CON session. Additionally, Q̇ was significantly (P< 0.05) greater at 45 minutes and 60 minutes compared to 15 minutes. HR was significantly (P<0.05) greater at 45 minutes compared to 90 minutes. No significant interactions were found for MAP, CPI, Q̇ , HR, SV, SBP, DBP, or SVR. Conclusion: In conclusion, exercise during dialysis may decrease the likelihood of experiencing ischemic or hypotensive events by enhancing myocardial perfusion through increasing CPI and Q̇

Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment

Increased formate overflow is a hallmark of oxidative cancer

Formate overflow coupled to mitochondrial oxidative metabolism\ has been observed in cancer cell lines, but whether that takes place in the tumor microenvironment is not known. Here we report the observation of serine catabolism to formate in normal murine tissues, with a relative rate correlating with serine levels and the tissue oxidative state. Yet, serine catabolism to formate is increased in the transformed tissue of in vivo models of intestinal adenomas and mammary carcinomas. The increased serine catabolism to formate is associated with increased serum formate levels. Finally, we show that inhibition of formate production by genetic interference reduces cancer cell invasion and this phenotype can be rescued by exogenous formate. We conclude that increased formate overflow is a hallmark of oxidative cancers and that high formate levels promote invasion via a yet unknown mechanism

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions