Critical evaluation of the potential energy surface of the CH3 + HO2 reaction system.

The CH3 + HO2 reaction system was studied theoretically by a newly developed, HEAT345-(Q) method based CHEAT1 protocol and includes the combined singlet and triplet potential energy surfaces. The main simplification is based on the CCSDT(Q)/cc-pVDZ calculation which is computationally inexpensive. Despite the economic and black-box treatment of higher excitations, the results are within 0.6 kcal/mol of the highly accurate literature values. Furthermore, the CHEAT1 surpassed the popular standard composite methods such as CBS-4M, CBS-QB3, CBS-APNO, G2, G3, G3MP2B3, G4, W1U, and W1BD mainly due to their poor performance in characterizing transition states (TS). For TS structures, various standard DFT and MP2 method have also been tested against the resulting CCSD/cc-pVTZ geometry of our protocol. A fairly good agreement was only found in the cases of the B2PLYP and BHandHLYP functionals, which were able to reproduce the structures of all TS studied within a maximum absolute deviation of 7%. The complex reaction mechanism was extended by three new low lying reaction channels. These are indirect water elimination from CH3OOH resulted formaldehyde, H2 elimination yielded methylene peroxide, and methanol and reactive triplet oxygen were formed via H-shift in the third channel. CHEAT1 protocol based on HEAT345-(Q) method is a robust, general, and cheap alternative for high accurate kinetic calculations.

The effect of oxidative stress on the bursopentin peptide structure: a theoretical study.

Bursopentin (BP(5), H-Cys(1)-Lys(2)-Arg(3)-Val(4)-Tyr(5)-OH), found in the bursa Fabricius of the chicken, is a pentapeptide that protects the organism from oxidative stress by reducing the intracellular generation of reactive oxygen species. Hydrogen abstraction, a common oxidative reaction occurring in proteins, often results in the formation of d amino acid residues. To study the effect of this phenomenon on the structure of bursopentin, each of its residues were converted from the l configuration to the d configuration, and the structures of these peptide epimers were compared to that of the wild-type bursopentin. The conformations, secondary structures, compactness and hydrogen bonding of bursopentin were compared to its epimers using molecular dynamics simulations and first principles quantum chemical computations. It was discovered that the repulsion between the side chains of Lys(2) and Arg(3) influenced the conformation of the peptide regardless of the configuration of these residues. Epimerisation of the Val(4) and Tyr(5) caused a reduction in the compactness of bursopentin. In all cases, the occurrence of a turn structure was relatively high, especially when Arg(3) was in the d configuration. Thermodynamic analysis of the epimerisation process showed that the formation of d amino acid residues is favourable.

Corrigendum: Physiology of the Nitrite-Oxidizing Bacterium Candidatus Nitrotoga sp. CP45 Enriched From a Colorado River.

[This corrects the article DOI: 10.3389/fmicb.2021.709371.].

Physiology of the Nitrite-Oxidizing Bacterium Candidatus Nitrotoga sp. CP45 Enriched From a Colorado River.

Nitrogen cycling microbes, including nitrite-oxidizing bacteria (NOB), perform critical ecosystem functions that help mitigate anthropogenic stresses and maintain ecosystem health. Activity of these beneficial nitrogen cycling microbes is dictated in part by the microorganisms' response to physicochemical conditions, such as temperature, pH, and nutrient availability. NOB from the newly described Candidatus Nitrotoga genus have been detected in a wide range of habitats across the globe, yet only a few organisms within the genus have been physiologically characterized. For freshwater systems where NOB are critical for supporting aquatic life, Ca. Nitrotoga have been previously detected but little is known about the physiological potential of these organisms or their response to changing environmental conditions. Here, we determined functional response to environmental change for a representative freshwater species of Ca. Nitrotoga (Ca. Nitrotoga sp. CP45, enriched from a Colorado river). The physiological findings demonstrated that CP45 maintained nitrite oxidation at pH levels of 5-8, at temperatures from 4 to 28°C, and when incubated in the dark. Light exposure and elevated temperature (30°C) completely halted nitrite oxidation. Ca. Nitrotoga sp. CP45 maintained nitrite oxidation upon exposure to four different antibiotics, and potential rates of nitrite oxidation by river sediment communities were also resilient to antibiotic stress. We explored the Ca. Nitrotoga sp. CP45 genome to make predictions about adaptations to enable survival under specific conditions. Overall, these results contribute to our understanding of the versatility of a representative freshwater Ca. Nitrotoga sp. Identifying the specific environmental conditions that maximize NOB metabolic rates may ultimately direct future management decisions aimed at restoring impacted systems.

Proposed heparin binding site in antithrombin based on arginine 47. A new variant Rouen-II, 47 Arg to Ser.

Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.

Circulating proalbumin associated with a variant proteinase inhibitor.

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity.

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase. Implications for the heparin activation mechanism.

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted at [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

A sensitive technique by differential scanning for detecting haemoglobins of fetal origin.

A simple modification is made to the ultraviolet scanning technique for the detection of the tryptophan notch which is characteristic of haemoglobins of fetal origin. This involves a differential scan using Hb A solution in the reference compartment. This technique has all the advantages of the standard scan method but extends the scope of it by being much more sensitive and decisive.

Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.

The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.

A competitive ELISA for lipoprotein(a).

A competitive ELISA for lipoprotein(a) (Lp(a)) is described. The method uses a commercially available polyclonal anti-Lp(a) antibody and an IgG biotinstreptavidin-horseradish peroxidase detection system. The method is simple and robust with an assay sensitivity of 0.7 ng/well (1.4 micrograms/l). The antibody cross-reactivity was 0.14% against LDL and 0.70% against plasminogen. The coefficients of variation obtained with control sera of 266 and 552 mg/l were: 5.0% and 4.6% (n = 6), respectively for the intraassay; and 10.8% and 9.5% (n = 16), respectively for the interassay. The method showed an excellent correlation with a commercial immunoradiometric assay (IRMA), y (ELISA) = 0.94x (IRMA) - 8, (r = 0.98). A recovery study in which a 200 mg/L standard and four plasma samples were diluted with different proportions of a low plasma sample, gave linear relationships and also confirmed the specificity of the antibody.

Macro-alkaline phosphatase due to IgG kappa complex: demonstration with polyethylene glycol precipitation and immunofixation.

An otherwise unexplained, persistently elevated plasma alkaline phosphatase concentration in a 71-year-old woman was found to be attributable to the presence of macro-alkaline phosphatase using polyethylene glycol precipitation. Gel filtration showed two high MW peaks with masses of about 330 kDa and 560 kDa. The alkaline phosphatase (ALP) complex was characterized by immunoelectrophoresis as a complex with IgG with kappa light chains.

Facial swelling and discharging lesions associated with abnormalities of the mandible in kunekune pigs.

CASE HISTORIES: Four adult kunekune pigs developed facial swelling at the base of the right ear that ruptured and discharged food material. A further six pigs that had similar clinical signs were reported by members of the New Zealand Kunekune Association who responded to an email survey, one of which was confirmed by post-mortem examination. CLINICAL FINDINGS: Inside the mouth of each pig there was an opening at the junction of the body and ramus of the mandible just lateral to the most caudal visible molar that was impacted with masticated feed. The food packed into the mandible resulted in infection and progressive erosion of the medullary cavity of the bone until it reached the ramus where it eroded through the lateral cortex. The feed material then tracked through the soft tissues to form a subcutaneous abscess, which eventually ruptured resulting in a draining lesion. In Case 2, which had had the lesion for 2 years, the cavity in the mandible was lined with mucosa that had healed to the skin to produce a fistula. In all four pigs there was also a lesion in the left side of the mandible that was not as developed as that on the right side. DIAGNOSIS: The facial swellings were produced by feed material that had impacted into the mandible through an opening immediately caudal to the cheek teeth and then emerged through one or more lesions in the lateral aspect of the ramus of the mandible. CLINICAL RELEVANCE: Although it has not been previously reported, anecdotal reports and our survey suggest that this condition may occur relatively frequently in kunekune pigs. It should be considered as a differential diagnosis for facial swellings and discharging lesions in these animals.

Bone volume and regional density of the central tarsal bone detected using computed tomography in a cross-sectional study of adult racing greyhounds.

AIMS: To determine whether left-to-right asymmetry of the central tarsal bone (CTB) of racing greyhounds was detectable using computed tomography (CT) in live dogs; to quantify the asymmetry in terms of average bone volume, volumetric bone mineral density (vBMD), and dorsal cortical shape, and to determine if age, gender, bodyweight, number of starts or history of tarsal injury were significant covariates. METHODS: One trainer supplied 11 male and seven female, unrelated, skeletally mature, actively racing greyhounds, including dogs with a history of tarsal injury diagnosed by the trainer and/or track veterinarian (n=8), and dogs without a history of tarsal injury (n=10). Using CT, standardised parameters of the CTB were measured including volume and average vBMD of the left and right CTB, vBMD of regions within the CTB, and bone shape. RESULTS: There was no difference in the volumes of the left and right CTB and no association with number of racing starts. Volume of CTB in dogs with a history of tarsal injury was greater than in dogs with no history of injury (p<0.001). Mean vBMD of the right was greater than the left CTB (p=0.004), and was independent of history of injury, gender, bodyweight, and number of starts. Males with a history of injury weighed more than those with no history of injury (p=0.004). The region of greatest difference in vBMD between right and left limbs was the centrodorsomedial aspect of the CTB. Middle plantar ligament enthesiopathies and fractures of tarsal bones other than the central tarsal bone were identified in dogs with and without a history of tarsal injury. CONCLUSIONS: Significant left-to-right asymmetry of the CTB in racing greyhounds was detected using CT. Contrary to previous suggestions, the asymmetry was not associated with the number of racing starts. We propose that the majority of the adaptive modelling of the CTB occurred rapidly following the onset of counter-clockwise training, with little further modelling throughout the racing career of the dog, however further investigation is warranted. CLINICAL RELEVANCE: This study described a technique using CT for imaging the CTB in live dogs, which opens the way for a longitudinal study of bone modelling of the CTB in response to training and racing in a counter-clockwise direction.

Idiopathic canine juvenile cranial hyperostosis in a Pit Bull Terrier.

CASE HISTORY: An 8-month-old male Pit Bull Terrier was presented with bilateral firm swellings of the cranium in the region of the frontal sinuses. The dog was aggressive when approached and had also been anorexic during the 3.5 days prior to presentation. CLINICAL FINDINGS: Using computed tomography (CT) and radiology, periosteal new bone formation and hyperostosis were detected on the frontal, parietal and mandibular bones bilaterally. Histopathology of the calvarial and mandibular bones was similar, comprised of thick trabeculae of immature woven and mature lamellar bone separated by cementing lines. Sites of prominent osteoblastic and osteoclastic activity were also present. DIAGNOSIS: Craniomandibular osteopathy with clinical similarities to calvarial hyperostosis. CLINICAL RELEVANCE: Pit Bull Terriers are not predisposed to either craniomandibular osteopathy or calvarial hyperostosis. This observation, in addition to the clinical and pathological similarities between the two diseases, suggests that craniomandibular osteopathy and calvarial hyperostosis may represent a single disorder, with predilection sites that vary between breeds of dog. We propose a new term, idiopathic canine juvenile cranial hyperostosis, for this disorder, to include both craniomandibular osteopathy and calvarial hyperostosis.