Pediatric Right Ventricular Cardiac Steatosis following Immunosuppressive Treatment.

Background Right ventricular myocardial fatty infiltration has been observed in pediatric cardiac allografts with an associated decrease in graft life expectancy. A possible explanation included an immunosuppressant drug effect which prompted us to examine common factors between non-cardiac transplanted immunosuppressed patients and postmortem hearts of pediatric patients who died of non-cardiac causes.Materials and Methods Sixty-one of 1,030 pediatric autopsies were from immunosuppressed children who were treated with corticosteroids for malignant tumors, non-cardiac transplantations, or other chronic clinical conditions. 62 children who died for non-medical reasons served as controls. Cardiac H&E autopsy slides were examined for right ventricular fatty infiltration.Results There was a significant increase in right ventricular fatty infiltration in patients that were non-cardiac transplanted and immunosuppressed compared to controls. None of the index patients had other features of arrhythmogenic right ventricular dysplasia.Conclusions Immunosuppression may lead to right ventricular fatty infiltration in childhood.

Synthesis and Speciation-Dependent Properties of a Multimetallic Model Complex of NiSOD That Exhibits Unique Hydrogen-Bonding.

The complex Na3[{NiII(nmp)}3S3BTAalk)] (1) (nmp2- = deprotonated form of N-(2-mercaptoethyl)picolinamide; H3S3BTAalk = N1,N3,N5-tris(2-mercaptoethyl)benzene-1,3,5-tricarboxamide, where H = dissociable protons), supported by the thiolate-benzenetricarboxamide scaffold (S3BTAalk), has been synthesized as a trimetallic model of nickel-containing superoxide dismutase (NiSOD). X-ray absorption spectroscopy (XAS) and 1H NMR measurements on 1 indicate that the NiII centers are square-planar with N2S2 coordination, and Ni-N and Ni-S distances of 1.95 and 2.16 Å, respectively. Additional evidence from IR indicates the presence of H-bonds in 1 from the approximately -200 cm-1 shift in νNH from free ligand. The presence of H-bonds allows for speciation that is temperature-, concentration-, and solvent-dependent. In unbuffered water and at low temperature, a dimeric complex (1A; λ = 410 nm) that aggregates through intermolecular NH···O═C bonds of BTA units is observed. Dissolution of 1 in pH 7.4 buffer or in unbuffered water at temperatures above 50 °C results in monomeric complex (1M; λ = 367 nm) linked through intramolecular NH···S bonds. DFT computations indicate a low energy barrier between 1A and 1M with nearly identical frontier MOs and Ni-ligand metrics. Notably, 1A and 1M exhibit remarkable stability in protic solvents such as MeOH and H2O, in stark contrast to monometallic [NiII(nmp)(SR)]- complexes. The reactivity of 1 with excess O2, H2O2, and O2•- is species-dependent. IR and UV-vis reveal that 1A in MeOH reacts with excess O2 to yield an S-bound sulfinate, but does not react with O2•-. In contrast, 1M is stable to O2 in pH 7.4 buffer, but reacts with O2•- to yield a putative [NiII(nmp)(O2)]- complex from release of the BTA-thiolate based on EPR.

Steric Enforcement about One Thiolate Donor Leads to New Oxidation Chemistry in a NiSOD Model Complex.

Ni-containing superoxide dismutase (NiSOD) represents an unusual member of the SOD family due to the presence of oxygen-sensitive Ni-SCys bonds at its active site. Reported in this account is the synthesis and properties of the NiII complex of the N3S2 ligand [N3S2Me2]3- ([N3S2Me2]3- = deprotonated form of 2-((2-mercapto-2-methylpropyl)(pyridin-2-ylmethyl)amino)-N-(2-mercaptoethyl)acetamide), namely Na[Ni(N3S2Me2)] (2), as a NiSOD model that features sterically robust gem-(CH3)2 groups on the thiolate α-C positioned trans to the carboxamide. The crystal structure of 2, coupled with spectroscopic measurements from 1H NMR, X-ray absorption, IR, UV-vis, and mass spectrometry (MS), reveal a planar NiII (S = 0) ion coordinated by only the N2S2 basal donors of the N3S2 ligand. While the structure and spectroscopic properties of 2 resemble those of NiSODred and other models, the asymmetric S ligands open up new reaction paths upon chemical oxidation. One unusual oxidation product is the planar NiII-N3S complex [Ni(Lox)] (5; Lox = 2-(5,5-dimethyl-2-(pyridin-2-yl)thiazolidin-3-yl)-N-(2-mercaptoethyl)acetamide), where two-electron oxidation takes place at the substituted thiolate and py-CH2 carbon to generate a thiazolidine heterocycle. Electrochemical measurements of 2 reveal irreversible events wholly consistent with thiolate redox, which were identified by comparison to the ZnII complex Na[Zn(N3S2Me2)] (3). Although no reaction is observed between 2 and azide, reaction of 2 with superoxide produces multiple products on the basis of UV-vis and MS data, one of which is 5. Density functional theory (DFT) computations suggest that the HOMO in 2 is π* with primary contributions from Ni-dπ/S-pπ orbitals. These contributions can be modulated and biased toward Ni when electron-withdrawing groups are placed on the thiolate α-C. Analysis of the oxidized five-coordinate species 2ox* by DFT reveal a singly occupied spin-up (α) MO that is largely thiolate based, which supports the proposed NiIII-thiolate/NiII-thiyl radical intermediates that ultimately yield 5 and other products.

Synthesis of Co(II)-NO(-) Complexes and Their Reactivity as a Source of Nitroxyl.

Metal-nitroxyl (M-HNO/M-NO(-)) coordination units are found in denitrification enzymes of the global nitrogen cycle, and free HNO exhibits pharmacological properties related to cardiovascular physiology that are distinct from nitric oxide (NO). To elucidate the properties that control the binding and release of coordinated nitroxyl or its anion at these biological metal sites, we synthesized {CoNO}(8) (1, 2) and {CoNO}(9) (3, 4) complexes that contain diimine-dipyrrolide supporting ligands. Experimental (NMR, IR, MS, EPR, XAS, XRD) and computational data (DFT) support an oxidation state assignment for 3 and 4 of high spin Co(II) (SCo = 3/2) coordinated to (3)NO(-) (SNO = 1) for Stot = 1/2. As suggested by DFT, upon protonation, a spin transition occurs to generate a putative low spin Co(II)-(1)HNO (SCo = Stot = 1/2); the Co-NO bond is ∼0.2 Šlonger, more labile, and facilitates the release of HNO. This property was confirmed experimentally through the detection and quantification of N2O (∼70% yield), a byproduct of the established HNO self-reaction (2HNO → N2O + H2O). Additionally, 3 and 4 function as HNO donors in aqueous media at pH 7.4 and react with known HNO targets, such as a water-soluble Mn(III)-porphyrin ([Mn(III)(TPPS)](3-); TPPS = meso-tetrakis(4-sulfonatophenyl)porphyrinate) and ferric myoglobin (metMb) to quantitatively yield [Mn(TPPS)(NO)](4-) and MbNO, respectively.

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways.

Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals.

De novo protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in de novo protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α3DIV at pH 7.0, a de novo designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ1). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α3D, the substitution of Leu residues to Cys enhanced the α-helical content of α3DIV while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg(II), Pb(II), or Cd(II) bound α3DIV by 18-24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)-S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR.

Adenocarcinoma involving the urinary bladder.

A man in his 70s previously diagnosed with an adenocarcinoma of the prostate, received external beam radiation therapy (EBRT) and brachytherapy 11 years ago. Ten years later, he developed urinary symptoms and a cystoscopy identified a bladder neck tumour. A transurethral resection of a bladder tumour was performed, and pathology revealed a high-grade adenocarcinoma consistent with a colorectal primary. A colonoscopy was unremarkable, and imaging studies showed tumour involving the bladder and prostate. Tumour markers and a CARIS genomic prevalence score also favoured a colorectal cancer primary.The patient refused surgery and underwent chemoradiation with a combination of EBRT and brachytherapy with concurrent capecitabine. Imaging studies obtained 6 months after reirradiation revealed an enlarged left-sided mesorectal lymph node concerning for disease recurrence. The lymph node was treated with Stereotactic Body Radiation Therapy and his post-treatment imaging revealed a response to treatment with no other evidence of disease.

Metastatic spread of serous ovarian carcinoma to the bilateral breasts: a rare presentation.

A woman presented with a mass in her right breast. She had previously been treated with carboplatin, paclitaxel and bevacizumab for serous ovarian carcinoma diagnosed 5 years previously and was currently on maintenance olaparib. A right breast mammogram demonstrated periareolar skin thickening and the physical examination revealed an erythematous, non-blanching cutaneous lesion. A punch biopsy revealed high-grade serous carcinoma of ovarian origin, positive for PAX-8, WT-1 and p53. Positron emission tomogram-CT scan showed diffusely increased fluorodeoxyglucose uptake in the right breast. She was treated with external beam radiation therapy to the right breast and regional lymphatics and received 5200 cGy in 20 fractions to the right breast and supraclavicular region with good response. Two weeks after completing radiation therapy, she presented with a new lesion inferior to her left areola, concerning for metastasis to the contralateral breast. Subsequent biopsy of the left breast identified metastatic serous ovarian carcinoma for which she received an additional 5200 cGy in 20 fractions to the breast.

Radiation induced mucoepidermoid carcinoma of the parotid gland following post-operative radiotherapy to the earlobe for keloid prophylaxis.

Radiation-induced malignancies (RIMs) are rare but well-documented late toxicities associated with exposure to radiation or radiotherapy. A keloid scar is a common benign proliferation of scar tissue which commonly develops at the site of an injury, such as on the earlobe after ear-piercing. While typically reserved for management of malignancies, radiotherapy is often utilized in the management of some benign conditions, including keloids. Given the benign nature of keloids, any theoretical late toxicity from radiotherapy, particularly a life-threatening toxicity such as a RIM, is particularly concerning. Here, we report a case of a 34-year-old male who presented with a radiation induced mucoepidermoid carcinoma of the parotid gland which developed in a previously irradiated field ten years after the patient received electron radiotherapy for a keloid of the earlobe. Using available literature, we estimate the risk of a RIM of the parotid gland from a typical course of radiotherapy to the earlobe as 0.007% per year.

Predictors of Acute Radiation Dermatitis and Esophagitis in African American Patients Receiving Whole-Breast Radiation Therapy.

PURPOSE: Adjuvant whole breast radiation therapy after breast-conserving surgery is the standard of care in the management of early-stage breast cancer. Two of the most common acute toxicities of breast radiation therapy are radiation esophagitis (RE) and radiation dermatitis (RD). African American individuals are at higher risk for experiencing treatment-related toxic effects and are often underrepresented in clinical trials. METHODS AND MATERIALS: An institutional database was developed to include all African American patients with a history of breast cancer or ductal carcinoma in situ undergoing adjuvant radiation therapy at a single institution from 2013 to 2019. Records were reviewed to identify patient age, body mass index (BMI), radiation dose, prone versus supine position, inclusion of boost, and inclusion of regional nodal irradiation. Radiation treatment plans were reviewed to identify breast size as well as dosimetric parameters to the breast and esophagus. Medical records were reviewed to identify which patients were prescribed Silvadene or Mylanta-lidocaine during or immediately after their course of radiation therapy, which was used as a surrogate for grade 2 (G2) or higher RD or RE, respectively. RESULTS: A total of 272 patients were included in the final analysis. On univariable analysis, patients with morbid obesity were more likely to develop G2RD, whereas hypofractionated radiation therapy was associated with lower rates of G2RD. On multivariable analysis, greater breast volume was associated with higher rates of G2RD. In the subset of patients receiving regional node irradiation, 19% of the patients experienced G2RE, and the best predictor on multivariable analysis was the mean radiation dose (Dmean) to the esophagus. CONCLUSIONS: Radiation dermatitis and esophagitis are common toxic effects in African American patients undergoing adjuvant breast radiation therapy. In this study, greater breast size, irrespective of the patient's BMI, was associated with a higher rate of dermatitis. Prone positioning during treatment and hypofractionated radiation reduced rates of G2RD. The Dmean to the esophagus was the dosimetric parameter most correlated with G2RE. These results may be used to help select patients at higher risk for toxic effects of G2 or greater during radiation therapy.

Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures.

We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.

Prognostic value of changes in neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and lymphocyte-to-monocyte ratio (LMR) for patients with cervical cancer undergoing definitive chemoradiotherapy (dCRT).

BACKGROUND: Peripheral NLR, PLR, and LMR have prognostic value in various malignancies as they are surrogates for inflammation. Recent studies have identified NLR, PLR, and LMR correlate with patient outcomes in cervical cancer patients however there remains uncertainty regarding the optimal time point for assessing these markers. METHODS: We retrospectively reviewed cervical cancer patients underoing definitive chemoradiation therapy (dCRT). NLR, PLR, and LMR values were identified before, during, and after dCRT and both relative and absolute changes in these values were calculated and compared with patient outcmoes. RESULTS: Ninety-nine patients who met the includsion criteria were identified. NLR values before, during, and after dCRT correlated with progression free survival (PFS) and overall survival (OS). In addition, increasing NLR after treatment was associated with worse PFS and OS. LMR before and after treatment had a positive correlation with PFS however increasing LMR during dCRT was found to have a negative correlation with PFS and OS. CONCLUSIONS: NLR serves as a prognostic indicator irrespective of timing with response to dCRT. While higher LMR before treatment was a positive prognostic indicator, increasing LMR was found to negatively correlate with PFS and OS.

Simultaneous nitrosylation and N-nitrosation of a Ni-thiolate model complex of Ni-containing SOD.

Nitric oxide (NO) is used as a substrate analogue/spectroscopic probe of metal sites that bind and activate oxygen and its derivatives. To assess the interaction of superoxide with the Ni center in Ni-containing superoxide dismutase (NiSOD), we studied the reaction of NO+ and NO with the model complex, Et4N[Ni(nmp)(SPh-o-NH2-p-CF3)] (1; nmp2- = dianion of N-(2-mercaptoethyl)picolinamide; -SPh-o-NH2-p-CF3 = 2-amino-4-(trifluoromethyl)benzenethiolate) and its oxidized analogue 1ox , respectively. The ultimate products of these reactions are the disulfide of -SPh-o-NH2-p-CF3 and the S,S-bridged tetrameric complex [Ni4(nmp)4], a result of S-based redox activity. However, introduction of NO to 1 affords the green dimeric {NiNO}10 complex (Et4N)2[{Ni(κ2-SPh-o-NNO-p-CF3)(NO)}2] (2) via NO-induced loss of nmp2- as the disulfide and N-nitrosation of the aromatic thiolate. Complex 2 was characterized by X-ray crystallography and several spectroscopies. These measurements are in-line with other tetrahedral complexes in the {NiNO}10 classification. In contrast to the established stability of this metal-nitrosyl class, the Ni-NO bond of 2 is labile and release of NO from this unit was quantified by trapping the NO with a CoII-porphyrin (70-80% yield). In the process, the Ni ends up coordinated by two o-nitrosaminobenzenethiolato ligands to result in the structurally characterized trans-(Et4N)2[Ni(SPh-o-NNO-p-CF3)2] (3), likely by a disproportionation mechanism. The isolation and characterization of 2 and 3 suggest that: (i) the strongly donating thiolates dominate the electronic structure of Ni-nitrosyls that result in less covalent Ni-NO bonds, and (ii) superoxide undergoes disproportionation via an outer-sphere mechanism in NiSOD as complexes in the {NiNO}9/8 state have yet to be isolated.

Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli.

Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains.

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation.

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein "Isu1". Sulfur for cluster assembly is provided by cysteine desulfurase "Nfs1", a protein that works in union with its accessory protein "Isd11". Frataxin "Yfh1" helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(ii) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(ii) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain.

The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4. Previously, it has been postulated that this domain regulates hZIP4 levels in the plasma membrane in a zinc-dependent manner. The objective of this research was to examine the zinc binding properties of the large intracellular loop of hZIP4. Therefore, we have recombinantly expressed and purified M3M4 and showed that this domain binds two zinc ions. Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn(2+) ions bind sequentially, with the first Zn(2+) binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn(2+) binding to a His4 site with a weaker affinity. Circular dichroism spectroscopy revealed that the M3M4 domain is intrinsically disordered, with only a small structural change induced upon Zn(2+) coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn(2+) concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn(2+) binding.