Functional imaging localization of complex organic hallucinations

Background: fMRI of mental phenomena is quite difficult to perform because lack of patient’s cooperation or because the symptoms are stable. In some exceptional cases, however, fMRI and DTI are capable to provide insights on the anatomy of organic hallucinations. Methods: In this report we describe a 14-year-old boy with a left fronto-dorsal tumor who experienced chronic complex brief, frequent and repetitive complex visual and auditory hallucinations. His clinical picture included multiple and severe social and mood problems. During a presurgical fMRI mapping the patient complained of having the visual and auditory hallucinations. A block-design FMRI paradigm was obtained from the event timecourse. Deterministic DTI of the brain was obtained seeding the lesion as ROI. The patient underwent surgery and electrocorticography of the lesional area. Results: The fMRI of the hallucinations showed activation in the left inferior frontal gyrus (IFG) and the peri-lesional area. The tractography of the tumor revealed structural aberrant connectivity to occipital and temporal areas in addition to the expected connectivity with the IFG via the aslant fasciculus and homotopic contralateral areas. Intraoperative EEG demonstrated epileptic discharges in the tumor and neighboring areas. After resection, the patient’s hallucinations stopped completely. He regained his normal social life and recover his normal mood. He remained asymptomatic for 90 days. Afterwards, hallucinations reappeared but with less intensity. Conclusions: To our knowledge, this is the first reported case of combined functional and structural connectivity imaging demonstrating brain regions participating in a network involved in the generation of complex auditory and visual hallucinations.     

An evaluation of empagliflozin and it’s applicability to hypertension as a therapeutic option

ABSTRACT

Introduction

Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as Empagliflozin are novel antihyperglycemic drugs approved for the treatment of type 2 diabetes (T2D). In addition to its glucose-lowering effects, Empagliflozin promotes weight loss, blood pressure reduction, and other beneficial metabolic benefits.     

X-ray crystallographic and EPR spectroscopic analysis of HydG,a maturase in [FeFe]-hydrogenase H-cluster assembly

Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN⁻ ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)_x(CN)_y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)₈ triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ₂-sulfide ion to a mononuclear Fe²⁺ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)_x(CN)_y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.The assembly of the [FeFe]-hydrogenase diiron subcluster (1, 2) requires three maturase proteins, HydE, HydF, and HydG (3), and in vitro, they can assemble an active hydrogenase (4). The sequence and structure of the maturase HydE (5) indicates that it is a member of the radical S-adenosyl methionine (SAM) superfamily, although the biochemical function of HydE has not been experimentally determined. The GTPase HydF (6, 7) has been shown to transfer synthetic (8) or biologically derived (7, 9) diiron subclusters into apo-hydrogenase, suggesting that HydF functions as a template for diiron subcluster assembly. The tyrosine lyase HydG is also a member of the radical SAM superfamily and uses SAM and a reductant (such as dithionite) to cleave the Cα–Cβ bond of tyrosine, yielding p-cresol as the side chain-derived byproduct (10) and fragmenting the amino acid moiety into cyanide (CN⁻) (11) and carbon monoxide (CO) (12), which are ultimately incorporated as ligands in the H cluster of the [FeFe]-hydrogenase HydA (4). Two site-differentiated [4Fe-4S] clusters in HydG have been identified using a combination of spectroscopy and site-directed mutagenesis (12–16). The cluster bound close to the N terminus ([4Fe-4S]_RS) by the CX₃CX₂C cysteine triad motif (SI Appendix, Fig. S1) is typical of the radical SAM superfamily (17, 18) and has been shown to catalyze the reductive cleavage of SAM (11, 13). The resultant highly reactive 5′-deoxyadenosyl radical is thought to abstract a hydrogen atom from tyrosine, thereby inducing Cα–Cβ-bond homolysis with release of dehydroglycine (DHG) and the spectroscopically characterized 4-oxidobenzyl radical anion (16), which is quenched to yield p-cresol (Fig. 1A, step A). The second (auxiliary) Fe-S cluster is proposed to promote the conversion of DHG into CO and CN⁻ (Fig. 1A, step B) (13, 16). Two intermediates have been observed by stopped-flow IR spectroscopic analysis (19): an enzyme-bound organometallic species (complex A) (Fig. 1A, 4) that converts to a species that features an Fe(CO)₂(CN) moiety (complex B) (Fig. 1A, 5). These results, combined with ⁵⁷Fe electron-nuclear double resonance (ENDOR) studies that showed that iron from HydG is incorporated into mature hydrogenase, led to the proposal that an organometallic synthon with a minimum stoichiometry of [Fe(CO)₂CN] is synthesized at the auxiliary cluster of HydG and eventually transferred to apo-hydrogenase (19).Open in a separate windowFig. 1.Overall [FeFe]-hydrogenase H-cluster assembly and structure of TiHydG. (A) Formation of the Fe(CO)₂CN synthon is proposed to occur at the auxiliary cluster of HydG (square brackets). (B) Overall fold of HydG with an end-on view of the TIM barrel showing the radical SAM core (green), the N-terminal extension (pink), and the C-terminal extension (blue). Monomer A is shown and contains a [4Fe-4S] cluster to catalyze the formation of the 5′-deoxyadenosyl radical from SAM and a [5Fe-5S] auxiliary cluster proposed to promote the conversion of DHG into cyanide and carbon monoxide. (C) The position of the two Fe-S clusters in TiHydG. The strands of the TIM barrel are shown. The orientation is rotated 90° from B.Herein, we report the crystal structure of Thermoanaerobacter italicus HydG (TiHydG) complexed with SAM (the Protein Data Bank ID code for the structure of HydG is 4WCX). The structure, which contains two HydG monomers per asymmetric unit, reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a structurally unprecedented [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ₂-sulfide to a mononuclear Fe(II) center (which we term the labile iron). To supplement the crystallographic studies of TiHydG, we also report EPR spectroscopic studies of Shewanella oneidensis HydG (SoHydG) that provide solution-state characterization of the [5Fe-5S] cluster and show its conversion to a [4Fe-4S] cluster in the presences of exogenous cyanide. Taken together, these results support a proposed mechanism for [FeFe]-hydrogenase maturation in which the labile iron of the [5Fe-5S] cluster is the site for Fe(CO)_x(CN)_y synthon assembly.     

Effect of Ki‐67 on Immunohistochemical Classification of Luminal A to Luminal B Subtypes of Breast Carcinoma

It has recently been proposed to include an immunohistochemical marker of cell proliferation, Ki‐67, as an element with which to classify the molecular subtypes of breast cancer. The objective of this study was to evaluate the effect of the introduction of the Ki‐67 marker on the molecular classification of breast cancer by immunohistochemistry. This study was performed on 234 cases of invasive ductal carcinoma of the breast submitted to two immunohistochemical classification panels, one including Ki‐67 and the other not. The data obtained with the two classifications were correlated with well‐established prognostic factors such as histologic grade, the number of lymph nodes affected and tumor size. The molecular classification without Ki‐67 identified: 136 cases of luminal A (58.1%), 19 cases of luminal B (8.1%), 27 cases of human epidermal growth‐factor receptor 2 overexpressing (11.5%), 27 cases of basal‐like (11.5%), and 25 cases of nonbasal‐like triple‐negative tumors (10.7%). When Ki‐67 was included, this situation changed significantly, with the following cases being identified: 72 cases of luminal A (30.8%) and 83 cases of luminal B tumors (35.5%), resulting in a Kappa score of 0.216. Evaluation of correlations between the luminal A and luminal B tumor subtypes and the selected prognostic factors showed a statistically significant difference only when Ki‐67 was included and only with respect to histologic grade (p < 0.001). The new classification with Ki‐67 significantly altered the prevalence of the luminal A and luminal B subtypes and improved correlation with the histologic grade.