Determination of optimal experimental conditions for accurate 3D reconstruction of the magnetization vector via XMCD-PEEM.

This work presents a detailed analysis of the performance of X-ray magnetic circular dichroism photoemission electron microscopy (XMCD-PEEM) as a tool for vector reconstruction of magnetization. For this, 360° domain wall ring structures which form in a synthetic antiferromagnet are chosen as the model to conduct the quantitative analysis. An assessment is made of how the quality of the results is affected depending on the number of projections that are involved in the reconstruction process, as well as their angular distribution. For this a self-consistent error metric is developed which allows an estimation of the optimum azimuthal rotation angular range and number of projections. This work thus proposes XMCD-PEEM as a powerful tool for vector imaging of complex 3D magnetic structures.

Controlled evolution of three-dimensional magnetic states in strongly coupled cylindrical nanowire pairs.

Cylindrical magnetic nanowires are promising systems for the development of three-dimensional spintronic devices. Here, we simulate the evolution of magnetic states during fabrication of strongly-coupled cylindrical nanowires with varying degrees of overlap. By varying the separation between wires, the relative strength of exchange and magnetostatic coupling can be tuned. Hence, we observe the formation of six fundamental states as a function of both inter-wire separation and wire height. In particular, two complex three-dimensional magnetic states, a 3D Landau Pattern and a Helical domain wall, are observed to emerge for intermediate overlap. These two emergent states show complex spin configurations, including a modulated domain wall with both Néel and Bloch character. The competition of magnetic interactions and the parallel growth scheme we follow (growing both wires at the same time) favours the formation of these anti-parallel metastable states. This works shows how the engineering of strongly coupled 3D nanostructures with competing interactions can be used to create complex spin textures.

A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as FeH1II(NO2-), where FeH1 represents the ccNiR active site. FeH1II(NO2-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {FeH1NO}7; ccNiR was not reduced beyond the {FeH1NO}7 state. The midpoint potentials for sequential reduction of FeH1III(NO2-) to FeH1II(NO2-) and then to {FeH1NO}7 were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. FeH1II(NO2-) does not accumulate at equilibrium because its reduction to {FeH1NO}7 is so much easier than the reduction of FeH1III(NO2-) to FeH1II(NO2-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {FeH1NO}7 is exceedingly slow (k ∼ 0.001 s-1), but it is somewhat faster (k ∼ 0.050 s-1) while FeH1III(NO2-) is being reduced to {FeH1NO}7; then, the release of NO• from the undetectable transient {FeH1NO}6 can compete with reduction of {FeH1NO}6 to {FeH1NO}7. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {FeH1NO}7 over the more labile {FeH1NO}6.

Diagnostic value of serological biomarkers for detection of non-alcoholic fatty liver disease (NAFLD) and/or advanced liver fibrosis in people living with HIV.

OBJECTIVES: We aimed to evaluate the accuracy of serological biomarkers for non-alcoholic fatty liver disease (NAFLD) and advanced fibrosis (METAVIR-F3F4) in HIV mono-infected individuals. METHODS: In all, 674 participants from the PROSPEC-HIV study (NCT02542020), who had blood sample tests and transient elastography (TE) performed on the same day, were eligible. Exclusion criteria were viral hepatitis co-infection (n = 90), abusive alcohol intake (n = 61), missing data (n = 47) or unreliable TE (n = 39). NAFLD was defined by controlled attenuation parameter ≥ 248 dB/m and advanced fibrosis by liver stiffness measurement ≥ 8.7 kPa with M probe or ≥ 7.2 kPa with XL probe. Biomarkers for NAFLD [Steato-ELSA, Fatty Liver Index (FLI), Hepatic Steatosis Index (HSI), NAFLD-Liver Fat Score (NAFLD-LFS)] and fibrosis [Fibrosis-4 score (FIB-4), Aspartate-to-Platelet Ratio Index (APRI) and NAFLD Fibrosis Score (NFS)] were calculated. RESULTS: A total of 437 patients [57% female, age = 44 (interquartile range: 35-52) years, body mass index (BMI) = 26.1 (23.4-29.3) kg/m2 , CD4 = 660 (427-901) cells/µL] were included. The prevalence [95% confidence interval (CI)] of NAFLD and advanced fibrosis were 38.2% (33.8-42.9) and 10.5% (8.0-13.8), respectively. The areas (95% CI) under the receiver operator curve (AUROCs) for diagnosis of NAFLD were 0.854 (0.818-0.889), 0.840 (0.804-0.877), 0.805 (0.762-0.847) and 0.793 (0.750-0.836) for Steato-ELSA, FLI, HSI and NAFLD-LFS (P < 0.001), respectively. All tests yielded satisfactory sensitivities, specificities and negative predictive values (NPVs). The AUROCs (95% CI) for diagnosis of advanced fibrosis were 0.736 (0.659-0.814), 0.700 (0.614-0.7851) and 0.795 (0.726-0.864) for FIB-4, APRI and NFS (P = 0.077), respectively. These tests yielded high specificities and negative predictive values (NPVs) > 90%. CONCLUSION: Biomarkers for NAFLD had a good accuracy and those for fibrosis had high specificities and NPVs. These tests should be integrated to HIV care to detect NAFLD and to exclude advanced liver fibrosis.

Peripheral microcirculatory alterations are associated with the severity of acute respiratory distress syndrome in COVID-19 patients admitted to intermediate respiratory and intensive care units.

BACKGROUND: COVID-19 is primarily a respiratory disease; however, there is also evidence that it causes endothelial damage in the microvasculature of several organs. The aim of the present study is to characterize in vivo the microvascular reactivity in peripheral skeletal muscle of severe COVID-19 patients. METHODS: This is a prospective observational study carried out in Spain, Mexico and Brazil. Healthy subjects and severe COVID-19 patients admitted to the intermediate respiratory (IRCU) and intensive care units (ICU) due to hypoxemia were studied. Local tissue/blood oxygen saturation (StO2) and local hemoglobin concentration (THC) were non-invasively measured on the forearm by near-infrared spectroscopy (NIRS). A vascular occlusion test (VOT), a three-minute induced ischemia, was performed in order to obtain dynamic StO2 parameters: deoxygenation rate (DeO2), reoxygenation rate (ReO2), and hyperemic response (HAUC). In COVID-19 patients, the severity of ARDS was evaluated by the ratio between peripheral arterial oxygen saturation (SpO2) and the fraction of inspired oxygen (FiO2) (SF ratio). RESULTS: Healthy controls (32) and COVID-19 patients (73) were studied. Baseline StO2 and THC did not differ between the two groups. Dynamic VOT-derived parameters were significantly impaired in COVID-19 patients showing lower metabolic rate (DeO2) and diminished endothelial reactivity. At enrollment, most COVID-19 patients were receiving invasive mechanical ventilation (MV) (53%) or high-flow nasal cannula support (32%). Patients on MV were also receiving sedative agents (100%) and vasopressors (29%). Baseline StO2 and DeO2 negatively correlated with SF ratio, while ReO2 showed a positive correlation with SF ratio. There were significant differences in baseline StO2 and ReO2 among the different ARDS groups according to SF ratio, but not among different respiratory support therapies. CONCLUSION: Patients with severe COVID-19 show systemic microcirculatory alterations suggestive of endothelial dysfunction, and these alterations are associated with the severity of ARDS. Further evaluation is needed to determine whether these observations have prognostic implications. These results represent interim findings of the ongoing HEMOCOVID-19 trial. Trial registration ClinicalTrials.gov NCT04689477 . Retrospectively registered 30 December 2020.

Ovarian stimulation does not influence the uterine immune environment in healthy infertile women.

RESEARCH QUESTION: There is some controversy regarding the impact of ovarian stimulation on immune cells in women undergoing IVF. The study's aim was to determine whether ovarian stimulation affected immune uterine cells in healthy women undergoing IVF. DESIGN: This prospective cohort study included 28 patients undergoing IVF and 47 healthy oocyte donors. Endometrial biopsies were taken in a natural cycle and after ovarian stimulation. All participants had a normal karyotype, pelvic ultrasound and cervical cytology results and thyroid-stimulating hormone concentration, as well as normal glucose and insulin concentrations and inherited and acquired thrombophilia test results. Screening tests including human papillomavirus were normal. Immune cells were analysed using three techniques: fluorescence-activated cell sorting, immunohistochemistry and gene expression. A human leukocyte antigen (HLA)-C tetramer was used as an 'artificial embryo'. The expression of genes including those for tumour necrosis factor (TNF)-α and interleukin-10 (IL-10) was analysed. RESULTS: A comparison was made of the percentage and gene expression of CD56brightCD16- uterine natural killer (uNK), CD56dimCD16+ natural killer cells, CD56-CD16+ natural killer cells and TregCD25+CD4+FoxP3+ cells, uNK binding to the HLA-C tetramer, and TNF-α and IL-10 expression. No between- or within-group differences were observed in natural versus ovarian stimulation cycles. CONCLUSIONS: Ovarian stimulation does not affect the uterine immune cell population or HLA-C binding in healthy women undergoing ovarian stimulation. Further studies are underway to find out if different responses might be seen in women with previous autoimmune disorders.

Corynebacterium godavarianum Jani et al. 2018 and Corynebacterium hadale Wei et al. 2018 are both later heterotypic synonyms of Corynebacterium gottingense Atasayar et al. 2017, proposal of an emended description of Corynebacterium gottingense Atasayar et al. 2017.

Seven strains of an unidentifiable Corynebacterium species recovered from blood cultures, urine or cerebrospinal fluid over 26 years, closest to but differentiated from Corynebacterium imitans by 16S rRNA gene and partial rpoB gene sequencing, were studied. In November 2017, Atasayar et al. described a blood culture isolate as Corynebacterium gottingense sp. nov., which had >99 % similarity by 16S rRNA gene sequencing to the Canadian strains. In January 2018, Jani et al. described Corynebacterium godavarianum sp. nov., recovered from the Godavari River, India, which also had >99 % similarity by 16S/rpoB sequencing to the Canadian strains and C. gottingense. In May 2018, Wei et al. described Corynebacterium hadale recovered from hadopelagic water; this too had >99 % similarity by 16S rRNA gene sequencing to C. gottingense, C. godavarianum and the Canadian strains. C. gottingense DSM 103494T and C. godavarianum LMG 29598T were acquired and whole genome sequencing was performed (not previously done). Results were compared with genomes from C. hadale (GenBank accession NQMQ01) and the Canadian isolates. We found that these ten genomes formed a single taxon when compared using digital DNA-DNAhybridization, average nucleotide identity using blastn and average amino acid identity criteria but exhibited some subtle biochemical and chemotaxonomic differences. Heuristically, we propose that C. godavarianum and C. hadale are later heterotypic synonyms of, and the Canadian isolates are identifiable as, C. gottingense. We provide an emended description of Corynebacterium gottingense Atasayar et al. 2017; genomes ranged from 2.48 to 2.69 Mb (C. gottingense DSM 103494T, 2.62 Mb) with G+C content of 65.1-65.6 mol% (WGS), recovered from clinical and environmental sites.

Emendation of the Genus Auritidibacter Yassin et al. 2011 and Auritidibacter ignavus Yassin et al. 2011 based on features observed from Canadian and Swiss clinical isolates and whole-genome sequencing analysis.

Auritidibacter ignavus is a Gram-stain-positive bacillus derived from otorrhea. Four strains derived from ear discharges in Canada and Switzerland, with features consistent with but distinguishable from Auritidibacter ignavus IMMIB L-1656T (accession number FN554542) by 16S rRNA gene sequencing (97.5 % similarity), were thought to represent a novel species of the genus Auritidibacter. Auritidibacter ignavus DSM 45359T (=IMMIB L-1656T) was acquired to compare with Canadian and Swiss strains by whole-genome sequencing (WGS). Unexpectedly, those isolates were observed to be consistent with A. ignavus DSM 45359T by WGS (ANIb scores >98 %), MALDI-TOF (Bruker), cellular fatty acid analysis and biochemically (some differences were observed). A nearly full 16S rRNA gene sequence could not be readily prepared from A. ignavus DSM 45359T, even after multiple attempts. A 16S rRNA gene chimeric consensus sequence created from the genome assembly of A. ignavus DSM 45359T had only 97.5 % similarity to that of A. ignavus IMMIB L-1656T, implying that 16S rRNA sequence accession number FN554542 could not be replicated. We concluded that our isolates of members of the genus Auritidibacter were consistent with A. ignavus DSM 45359T, did not represent a novel species, and that the sequence corresponding to FN554542 was not reproducible. By WGS, A. ignavus DSM 45359T had genome of 2.53×106 bp with a DNA G+C content of 59.34%, while genomes of Canadian and Swiss isolates ranged from 2.47 to 2.59×106 bp with DNA G+C contents of 59.3-59.52 %. A. ignavus NML 100628 (=NCTC 14178=LMG 30897) did not demonstrate a rodcoccus cycle. Emendation of Auritidibacter ignavus was proposed based on these results.

Flavonoid glycosides and their putative human metabolites as potential inhibitors of the SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp).

BACKGROUND: Since the World Health Organization (WHO) declared Coronavirus disease 2019 (COVID-19) to be a pandemic infection, important severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural proteins (nsp) have been analysed as promising targets in virtual screening approaches. Among these proteins, 3-chymotrypsin-like cysteine protease (3CLpro), also named main protease, and the RNA-dependent RNA polymerase (RdRp), have been identified as fundamental targets due to its importance in the viral replication stages. OBJECTIVES: To investigate, in silico, two of the most abundant flavonoid glycosides from Dysphania ambrosioides; a medicinal plant found in many regions of the world, along with some of the putative derivatives of these flavonoid glycosides in the human organism as potential inhibitors of the SARS-CoV-2 3CLpro and RdRp. METHODS: Using a molecular docking approach, the interactions and the binding affinity with SARS-CoV-2 3CLpro and RdRp were predicted for quercetin-3-O-rutinoside (rutin), kaempferol-3-O-rutinoside (nicotiflorin) and some of their glucuronide and sulfate derivatives. FINDINGS: Docking analysis, based on the crystal structure of 3CLpro and RdRp, indicated rutin, nicotiflorin, and their glucuronide and sulfate derivatives as potential inhibitors for both proteins. Also, the importance of the hydrogen bond and π-based interactions was evidenced for the presumed active sites. MAIN CONCLUSIONS: Overall, these results suggest that both flavonoid glycosides and their putative human metabolites can play a key role as inhibitors of the SARS-CoV-2 3CLpro and RdRp. Obviously, further researches, mainly in vitro and in vivo experiments, are necessary to certify the docking results reported here, as well as the adequate application of these substances. Furthermore, it is necessary to investigate the risks of D. ambrosioides as a phytomedicine for use against COVID-19.

Ketogenic Diet in patients with refractory epilepsy. / Dieta Cetogénica en el paciente con epilepsia refractaria.

INTRODUCTION: Epilepsy affects 0.5 to 1% of the population. 25% of pediatric patients have drug-resistant epilepsy (DRE). Ketogenic Diet (KD) emerges as an effective, non-pharmacological treatment in this group. OBJECTIVE: To describe the effect of KD on seizure control and nutritional status in children whit DRE. PATIENTS AND METHOD: We reviewed the medical records of patients with DRE treated with KD, between 2008 and 2018, evaluating age, diagnosis, number of seizures, number of antiepileptic drugs used, clinical outcomes, and complications. The KD was initiated in all patients hospitalized for a period no longer than seven days, who were evaluated for their nutritional and anthropometric sta tus, with weight and height measurements according to the clinical condition. RESULTS: We analyzed 35 KD in 33 cases. The median age of KD initiation was 4.8 years with an interquartile range (IQR) of 2-3 to 6.8 years. Classical KD was used in 49% of patients, Modified Atkins Diet (MAD) in 37%, and Low-Glycemic Index Treatment (LGIT) in 14% of cases. The average duration was 13 months (SD 11 months). After three months of using KD, we observed at least 50% reduction of seizures in 82% (27/33) of the patients, out of these, 22.8% presented 90% or more reduction of seizures, and 20% ended up seizure-free. Adverse events were observed in 21 patients, mainly gastrointestinal (62%) and dyslipidemia (14%), without effect on height. All side effects resolved with medical ma nagement. CONCLUSIONS: KD is a useful treatment in pediatric patients with DRE without nutritional impact. The adverse events were easily controlled if the patients are evaluated by a multidisciplinary team, according to international guidelines.

Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism.

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

Upon further analysis, neither cytochrome c554 from Nitrosomonas europaea nor its F156A variant display NO reductase activity, though both proteins bind nitric oxide reversibly.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the two- or four-electron-reduced protein will nitrosylate reversibly. The process is first order in C554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 ± 140 M-1s-1, while that for dissociation is 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report (Upadhyay AK, et al. J Am Chem Soc 128:4330, 2006), this study found no evidence of C554-catalyzed NO reduction, either with [Formula: see text] or with [Formula: see text] Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when [Formula: see text] was exposed to an excess of NO, but this is believed to arise from partial intramolecular electron transfer that generates {Fe(NO)}8 at heme II. The vacant heme II coordination site of C554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A2- and F156A4- was about 400× faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C554, the 6-coordinate low-spin hemes of F156A4- oxidized over the course of several minutes after exposure to NO. Concomitant formation of N2O could explain this heme oxidation, though alternative explanations are equally plausible given the available data.

Estimating surface soil moisture from SMAP observations using a Neural Network technique.

A Neural Network (NN) algorithm was developed to estimate global surface soil moisture for April 2015 to March 2017 with a 2-3 day repeat frequency using passive microwave observations from the Soil Moisture Active Passive (SMAP) satellite, surface soil temperatures from the NASA Goddard Earth Observing System Model version 5 (GEOS-5) land modeling system, and Moderate Resolution Imaging Spectroradiometer-based vegetation water content. The NN was trained on GEOS-5 soil moisture target data, making the NN estimates consistent with the GEOS-5 climatology, such that they may ultimately be assimilated into this model without further bias correction. Evaluated against in situ soil moisture measurements, the average unbiased root mean square error (ubRMSE), correlation and anomaly correlation of the NN retrievals were 0.037 m3m-3, 0.70 and 0.66, respectively, against SMAP core validation site measurements and 0.026 m3m-3, 0.58 and 0.48, respectively, against International Soil Moisture Network (ISMN) measurements. At the core validation sites, the NN retrievals have a significantly higher skill than the GEOS-5 model estimates and a slightly lower correlation skill than the SMAP Level-2 Passive (L2P) product. The feasibility of the NN method was reflected by a lower ubRMSE compared to the L2P retrievals as well as a higher skill when ancillary parameters in physically-based retrievals were uncertain. Against ISMN measurements, the skill of the two retrieval products was more comparable. A triple collocation analysis against Advanced Microwave Scanning Radiometer 2 (AMSR2) and Advanced Scatterometer (ASCAT) soil moisture retrievals showed that the NN and L2P retrieval errors have a similar spatial distribution, but the NN retrieval errors are generally lower in densely vegetated regions and transition zones.

Effectiveness of an oral health education seminar for paediatric and family medicine residents.

AIM: To assess the immediate effect of a 60-minute oral health educational seminar for paediatric and family medicine residents in improving their knowledge, attitude, likelihoodtowards incorporating oral health preventive practice in their current practices to well-child visits, and confidence in identifying and referring patients with dental trauma. MATERIALS AND METHODS: Baseline pre- and post-test design was used to evaluate the immediate effect of a 60-minute PowerPoint oral health educational seminar given to the paediatric and family medicine residents. STATISTICS: Multiple-choice items were used and the pre- and post-test data were analysed with McNemar and Wilcoxon signed-rank tests. A p-value <0.05 was considered statistically significant. RESULTS: Sixty-eight residents participated in the oral health educational seminar and completed the questionnaire. The mean age of participants was 29.9 years old (SD ±4.8 yrs.). Immediately following a 60-minute oral health educational seminar, there was an overall significant increase in participants' knowledge, attitudes and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits (p<0.05). More confidence in identifying and referring patients with dental trauma was reported by 100% of participants. CONCLUSIONS: A 60-minute oral health educational seminar was effective in improving paediatric and family medicine residents' immediate knowledge, attitude, and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits. Significantly more residents felt more confident in identifying and referring patients with dental trauma. Key messages: an oral health educational seminar can be effective in improving paediatric and family medicine residents' immediate knowledge, attitude, and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits.

A magnetic shift register with out-of-plane magnetized layers.

Using out-of-plane magnetized layers, a lateral shift register made from discrete elements is demonstrated. By carefully designing the in-plane shape of the elements which make up the shift register, both the position of nucleation of new domains and the coercivity of the element can be controlled. The dipole field from a neighboring element, placed tens of nanometers away, creates a bias field on the nucleation site, which can be used to create a NOT gate. By chaining these NOT gates together, a shift register can be created where data bits consisting of neighboring layers with aligned magnetization are propagated synchronously under a symmetric applied magnetic field. The operation of a 16 element shift register is shown, including field coupled data injection.

Direct Monitoring of the Reaction between Photochemically Generated Nitric Oxide and Mycobacterium tuberculosis Truncated Hemoglobin N Wild Type and Variant Forms: An Assessment of Computational Mechanistic Predictions.

The previously reported nitric oxide precursor [Mn(PaPy2Q)NO]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was used to investigate the interaction between NO and the protein truncated hemoglobin N (trHbN) from the pathogen Mycobacterium tuberculosis. Oxy-trHbN is exceptionally efficient at converting NO to nitrate, with a reported rate constant of 7.45 × 10(8) M(-1) s(-1) [Ouellet, H., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5902] compared to 4 × 10(7) M(-1) s(-1) for oxy-myoglobin [Eich, R. F., et al. (1996) Biochemistry 35, 6976]. This work analyzed the NO dioxygenation kinetics of wild type oxy-trHbN and a set of variants, as well as the nitrosylation kinetics for the reduced (red-trHbN) forms of these proteins. The NO dioxygenation reaction was remarkably insensitive to mutations, even within the active site, while nitrosylation was somewhat more sensitive. Curiously, the most profound change to the rate constant for nitrosylation was effected by deletion of a 12-amino acid dangling N-terminal sequence. The deletion mutant exhibited first-order kinetics with respect to NO but was zero-order with respect to protein concentration; by contrast, all other variants exhibited second-order rate constants of >10(8) M(-1) s(-1). trHbN boasts an extensive tunnel system that connects the protein exterior with the active site, which is likely the main contributor to the protein's impressive NO dioxygenation efficiency. The results herein suggest that N-terminal deletion abolishes a large scale conformational motion, in the absence of which NO can still readily enter the tunnel system but is then prevented from binding to the heme for an extended period of time.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Phenotypic correlations between ovum pick-up in vitro production traits and pregnancy rates in Zebu cows.

The growth of the Gyr breed in Brazil in terms of genetic gain for milk, along with conditions for market, has led to the use of ovum pick-up in vitro production (OPU-IVP) as a leader in biotechnology for the multiplication of genetic material. The aim of this study was to investigate phenotypic correlations between OPU-IVP-linked characteristics and pregnancy rates registered in an embryo transfer program using Gyr cows as oocyte donors. Data collected from 211 OPU sessions and 298 embryo transfers during the years 2012 and 2013 were analyzed and statistical analysis was performed. Estimates of simple Pearson correlations were calculated for NVcoc and PVcoc (number and proportion of viable cumulus-oocyte complexes, respectively); NcleavD4 and PcleavD4 (number and proportion of cleaved embryos on day 4 of culture, respectively); NTembD7 and PTembD7 (number and proportion of transferable embryos on day 7 of culture, respectively); NPrD30 and PPrD30 (number and proportion of pregnancies 30 days after transfer, respectively); and NPrD60 and PPrD60 (number and proportion of pregnancies 60 days after transfer, respectively). Moderate to moderately high correlations were found for all numerical characteristics, suggesting these as the most suitable parameters for selection of oocyte donors in Gyr programs. NVcoc is proposed as a selection trait due to positive correlations with percentage traits and pregnancy rates 30 and 60 days after transfer.

Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.

Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.