The association between time-series milk ß-hydroxybutyrate dynamics and early reproductive performance of dairy cows.

The DeLaval Herd Navigator is an on-farm sensor system that measures on a frequent basis milk progesterone (P4) and ß-hydroxybutyrate (BHB) in individual cows to closely monitor reproductive performance and energy balance. This information provides the opportunity to investigate the dynamics of BHB measured in milk (mBHB) and study the association between mBHB and early reproductive performance. The objectives of the study were (1) to describe mBHB dynamics within the first 20 d in milk (DIM), and (2) to evaluate the association between mBHB dynamics and early reproductive performance at cow-level. Two-year time-series data from 4,133 dairy cows in 38 Dutch dairy farms were available for analysis. Data included information on mBHB, daily milk yield and the indicators of early reproductive performance, days from calving to resumption of cyclicity, days from calving to first estrus, and days from calving to first insemination. The following mBHB dynamic parameters were defined based on the first 20 DIM for each individual cow: average mBHB (AvgBHB), DIM when mBHB was for the first time ≥80 µmol/L (OnsetKeto), the total number of consecutive days a cow had mBHB concentration ≥80 µmol/L, and the number of measurements mBHB concentration was ≥80 µmol/L. Three Cox proportional hazard regression models with random herd effect were developed to evaluate the association between cow level mBHB dynamics and days from calving to resumption of cyclicity, first estrus, and first insemination. Results showed that the mean AvgBHB within 20 DIM among all cows was 73 µmol/L. The mean OnsetKeto within 20 DIM, was 8 DIM. Among all cows having hyperketolactia, 55.8% (1,350/2,419) had OnsetKeto in the first week of lactation. In total, 41.5% (1,714/4,133) of the cows did not have OnsetKeto in the first 20 DIM. An early onset of hyperketolactia was associated with delayed fertility events. Cows with higher AvgBHB have a prolonged time interval from calving to resumption of cyclicity and first estrus. Information on mBHB dynamics and the association with early reproductive performance provides insights that might be helpful to improve reproductive performance of individual dairy cows.

Behavioral as well as hippocampal transcriptomic and microglial responses differ across sexes in adult mouse offspring exposed to a dual genetic and environmental challenge.

INTRODUCTION: A wide range of positive, negative, and cognitive symptoms compose the clinical presentation of schizophrenia. Schizophrenia is a multifactorial disorder in which genetic and environmental risk factors interact for a full emergence of the disorder. Infectious challenges during pregnancy are a well-known environmental risk factor for schizophrenia. Also, genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia. Translational animal models recapitulating these complex gene-environment associations have a great potential to untangle schizophrenia neurobiology and propose new therapeutic strategies. METHODS: Given that genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia, we compared the outcomes of a well-characterized model of maternal immune activation induced using the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) in wild-type versus fractalkine receptor knockout mice. Possible behavioral and immune alterations were assessed in male and female offspring during adulthood. Considering the role of the hippocampus in schizophrenia, microglial analyses and bulk RNA sequencing were performed within this region to assess the neuroimmune dynamics at play. Males and females were examined separately. RESULTS: Offspring exposed to the dual challenge paradigm exhibited symptoms relevant to schizophrenia and unpredictably to mood disorders. Males displayed social and cognitive deficits related to schizophrenia, while females mainly presented anxiety-like behaviors related to mood disorders. Hippocampal microglia in females exposed to the dual challenge were hypertrophic, indicative of an increased surveillance, whereas those in males showed on the other end of the spectrum blunted morphologies with a reduced phagocytosis. Hippocampal bulk-RNA sequencing further revealed a downregulation in females of genes related to GABAergic transmission, which represents one of the main proposed causes of mood disorders. CONCLUSIONS: Building on previous results, we identified in the current study distinctive behavioral phenotypes in female mice exposed to a dual genetic and environmental challenge, thus proposing a new model of neurodevelopmentally-associated mood and affective symptoms. This paves the way to future sex-specific investigations into the susceptibility to developmental challenges using animal models based on genetic and immune vulnerability as presented here.

Interactive molecular dynamics in virtual reality for modelling materials and catalysts.

Interactive molecular dynamics simulation in virtual reality (iMD-VR) is emerging as a promising technique in molecular science. Here, we demonstrate its use in a range of fifteen applications in materials science and heterogeneous catalysis. In this work, the iMD-VR package Narupa is used with the MD package, DL_POLY [1]. We show how iMD-VR can be used to: (i) investigate the mechanism of lithium fast ion conduction by directing the formation of defects showing that vacancy transport is favoured over interstitialcy mechanisms, and (ii) guide a molecule through a zeolite pore to explore diffusion within zeolites, examining in detail the motion of methyl n-hexanoate in H-ZSM-5 zeolite and identifying bottlenecks restricting diffusion. iMD-VR allows users to manipulate these systems intuitively, to drive changes in them and observe the resulting changes in structure and dynamics. We make these simulations available, as a resource for both teaching and research. All simulation files, with videos, can be found online (https://doi.org/10.5281/zenodo.8252314) and are provided as open-source material.

Data on the intermolecular interactions of 1,1,1,2-tetrafluoroethane liquids from molecular dynamics simulations.

Detailed atomistic interactions of 1,1,1,2-tetrafluoroethane (HFA-134a) liquid were presented in a data format, namely, DL_ANALYSER Notation for Atomic Interactions (DANAI), that annotates precisely the nature of interactions that is discoverable and searchable without having to resolve to diagrammatic illustrations. The datasets were obtained from raw atomic trajectory files of HFA-134a pure liquid models produced by using DL_POLY molecular dynamics software package. The trajectory datafiles contain expressions of atomic species in a natural chemical sense, and hence, provide localized key interactions, 'at a glance', of the liquid model on otherwise a typically disordered system consists of complex network of intermolecular interactions. The data provide insights to detailed structural behavior of molecules in liquid phase, and can be used as cheminformatics comparative investigations, linking to other molecular system models that contain similar interaction types and chemical species. This can form the foundation of investigations into the role of HFA-134a plays within different applications. For example, it can be used to compare structural and atomic interaction differences with alternative refrigerants, or as liquid propellants in pharmaceutical devices when solvating formulation ingredients.

Cellulose Iß microfibril interaction with pristine graphene in water: Effects of amphiphilicity by molecular simulation.

Graphene-cellulose interactions have considerable potential in the development of new materials. In previous computational work (Biomacromolecules2016, 16, 1771), we predicted that the model 100 hydrophobic surface of cellulose interacted favourably with pristine graphene in aqueous solution molecular dynamics simulations; conversely, a model of the hydrophilic 010 surface of cellulose exhibited progressive rearrangement to present a more hydrophobic face with the graphene, with weakened hydrogen bonds between cellulose chains and partial permeation of water. Here, we extend this work by simulating the interaction in aqueous solution of the amphiphilic 110 surface of a cellulose Iß microfibril model, comprising 36 chains of 40 glucosyl residues, with an infinite sheet of pristine graphene. This face of the microfibril is of intermediate hydrophilicity and progressively associates with graphene over replicate simulations. As cellulose chains adhere to the graphene surface, forming interactions via its CH and OH groups, we observe a degree of local and global untwisting of the microfibril. Complementary rippling of the graphene surface is also observed, as it adapts to interaction with the microfibril. This adsorption process is accompanied by increased exclusion of water between cellulose and graphene although some water localises between chains at the immediate interface. The predicted propensity of a cellulose microfibril to adsorb spontaneously on the graphene surface, with mutual structural accommodation, highlights the amphiphilic nature of cellulose and the types of interactions that can be harnessed to design new graphene-carbohydrate biopolymer materials.

Polyguluronate simulations shed light onto the therapeutic action of OligoG CF-5/20.

Chronic mucoid P. aeruginosa cystic fibrosis (CF) lung infections are associated with the development of a biofilm composed of anionic acetylated exopolysaccharide (EPS) alginate, electrostatically stabilised by extracellular Ca2+ ions. OligoG CF-5/20, a low molecular weight guluronate rich oligomer, is emerging as a novel therapeutic capable of disrupting mature P. aeruginosa biofilms. However, its method of therapeutic action on the mucoid biofilm EPS is not definitively known at a molecular level. This work, utilising molecular dynamics (MD) and Density-Functional Theory (DFT), has revealed that OligoG CF-5/20 interaction with the EPS is facilitated solely through bridging Ca2+ ions, which are not liberated from their native EPS binding sites upon OligoG CF-5/20 dispersal, suggesting that OligoG CF-5/20 does not cause disruptions to mature P. aeruginosa biofilms through breaking EPS-Ca2+-EPS ionic cross-links. Rather it is likely that the therapeutic activity arises from sequestering free Ca2+ ions and preventing further Ca2+ induced EPS aggregation.

Atomic-scale interactions between quorum sensing autoinducer molecules and the mucoid P. aeruginosa exopolysaccharide matrix.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung coloniser whose chronicity is associated with the formation of cation cross-linked exopolysaccharide (EPS) matrices, which form a biofilm that acts as a diffusion barrier, sequestering cationic and neutral antimicrobials, and making it extremely resistant to pharmacological challenge. Biofilm chronicity and virulence of the colony is regulated by quorum sensing autoinducers (QSAIs), small signalling metabolites that pass between bacteria, through the biofilm matrix, regulating genetic responses on a population-wide scale. The nature of how these molecules interact with the EPS is poorly understood, despite the fact that they must pass through EPS matrix to reach neighbouring bacteria. Interactions at the atomic-scale between two QSAI molecules, C4-HSL and PQS-both utilised by mucoid P. aeruginosa in the CF lung-and the EPS, have been studied for the first time using a combined molecular dynamics (MD) and density functional theory (DFT) approach. A large-scale, calcium cross-linked, multi-chain EPS molecular model was developed and MD used to sample modes of interaction between QSAI molecules and the EPS that occur at physiological equilibrium. The thermodynamic stability of the QSAI-EPS adducts were calculated using DFT. These simulations provide a thermodynamic rationale for the apparent free movement of C4-HSL, highlight key molecular functionality responsible for EPS binding and, based on its significantly reduced mobility, suggest PQS as a viable target for quorum quenching.

The structural pathway from its solvated molecular state to the solution crystallisation of the α- and ß-polymorphic forms of para amino benzoic acid.

Para amino benzoic acid (PABA) has two well-characterised α- and ß-polymorphic forms and, whilst both crystallise in the monoclinic space group P21/n, they have quite different crystal chemistry and crystallisability behaviour. Previous work has shown that the molecular conformation deformation energy in the crystalline state is higher for the ß-form than for the α-form and that the lattice energy for the former converges more slowly than for the latter overall. This suggests that not only is there a higher barrier to crystallisation for the ß-form but also that low solution supersaturations might be needed for it to preferentially nucleate. Additionally, solute cluster propensity and solute solvation energetic analysis highlight the importance of an aqueous solvation environment in inhibiting the α-form's strong OH⋯O carboxylic acid hydrogen bond (H-bond) dimer. Despite this, the detailed molecular-scale pathway from solvated molecules to 3D crystallographic structure still remains unclear, most notably regarding how the nucleation process is activated and how, as a result, this mediates the preferential formation of either of the two polymorphic forms. Molecular dynamics (MD) simulations coupled with FTIR studies and intermolecular synthon analysis address this issue through characterisation of the propensity of the incipient bulk synthons that are important in the crystallisation of the two polymorphic forms within the solution state. MD molecular trajectory analysis within crystallisation solutions reveals a greater propensity for OH⋯O synthons (both single H-bonds and homodimers) typical of the α-form and NH⋯O synthons found in both the α- and ß-forms when compared to aqueous solution but much lower propensities for the ß-form's "fingerprinting" OH⋯N and π-π stacking synthons. In contrast, data from the aqueous solution environment reveals a much greater propensity for the ß-form's π-π interaction synthons. IR dilution studies in acetonitrile in the carbonyl region reveal the presence of two CO vibrational stretching bands, whose relative intensities vary as a function of solution dilution. These were assigned to the solvated PABA monomer and a COOH dimer of PABA. Similar data in ethanol shows a main CO stretching band with a shoulder peak suggesting a similar monomer vs. dimer speciation may exist in this solvent. The IR data is consistent with the organic solvent MD data, albeit the corresponding analysis for the aqueous solution was precluded due to the latter's strong OH vibrational mode which restricted validation in aqueous solutions.

Triglycine (GGG) Adopts a Polyproline II (pPII) Conformation in Its Hydrated Crystal Form: Revealing the Role of Water in Peptide Crystallization.

Polyproline II (pPII) is a left-handed 31-helix conformation, which has been observed to be the most abundant secondary structure in unfolded peptides and proteins compared to α-helix and ß-sheet. Although pPII has been reported as the most stable conformation for several unfolded short chain peptides in aqueous solution, it is rarely observed in their solid state. Here, we show for the first time a glycine homopeptide (gly-gly-gly) adopting the pPII conformation in its crystalline dihydrate structure. The single crystal X-ray structure with molecular dynamic simulation suggests that a network of water and the charged carboxylate group is critical in stabilizing the pPII conformation in solid state, offering an insight into the structures of unfolded regions of proteins and the role of water in peptide crystallization.

An online survey of Malaysian long-term e-cigarette user perceptions.

INTRODUCTION: The use of e-cigarettes (EC) has reached alarming proportions among Malaysians. On a national level, little is known about the profile and perceptions of Malaysian EC users. This study aimed to explore the prevalence of long-term EC usage and its associated factors among EC users in Malaysia. METHODS: This nationwide online questionnaire survey was administered among 694 EC users across 13 states and 1 Federal Territory in Malaysia, between January and April 2018. A survey link was e-mailed to EC users that were recruited from an official national vape entity through their Facebook association page. We obtained information on respondents' sociodemographic characteristics, smoking habits, long-term e-cigarette usage and perceptions of EC use. We estimated long-term EC user prevalence and fitted multivariate regression models to predict factors associated with long-term EC usage. Statistical significance was set at p<0.05. RESULTS: Respondents were predominantly Malays (87.6%), aged >30 years (68.1%) and tertiary educated (71%). The majority were employed (93.1%) with a monthly household income of MYR 4000 or less (56.6%). About 84% were former smokers, while 10% were current smokers. The prevalence of long-term EC usage in this study was 82.3%. Most users believed that EC had helped them to cut down tobacco smoking (94.8%), reduced the urge to smoke (88.3%) and ultimately helped them to quit smoking (87.2%). Respondents aged >30 years and those who perceived that EC has helped them stop smoking were significantly more likely to be long-term EC users. CONCLUSIONS: Most respondents engaged in EC use to quit smoking. They were more likely to be long-term EC users if they were older and perceived that EC has helped them to quit smoking. This information is valuable for targeted prevention, health promotion and policy regulations.

Infection due to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) presenting as ischemic colitis.

Infections due to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) are often mild and self-limiting, but more severe cases can develop into hemolytic uremic syndrome (HUS) and hemorrhagic colitis. This case report documents a sporadic case of ischemic colitis likely triggered by EHEC but without manifestations of hemolytic uremic syndrome. From our literature review, we identified only one other case of an EHEC infection presenting as ischemic colitis in the absence of HUS. To our knowledge, this is the first case presentation of EHEC-induced ischemic colitis which did not lead to significant morbidity.

A rare cause of back pain and radiculopathy - spinal tophi: a case report.

BACKGROUND: Gout is a monosodium urate deposition disease which is prevalent worldwide. The usual manifestations are crystal arthropathy and tophi deposition in the soft tissues. Spinal tophi may also occur and are rarely reported, resulting in various clinical manifestations such as back pain, spinal cord compression, radiculopathy, and even mimicking epidural abscess and spondylodiscitis. CASE PRESENTATION: We report a case of a 42-year-old Chinese man with underlying gout who presented with back pain and radiculopathy. The diagnosis of spinal tophi was unsuspected and he was initially treated for epidural abscess and spondylodiscitis. He underwent a laminectomy and posterolateral fusion during which tophus material was discovered. He recovered and medications for gout were started. CONCLUSION: Spinal tophi are rare. The diagnosis is difficult and spinal tophi may be mistaken for epidural abscess, spondylodiscitis, or neoplasm.

A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase.

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2-) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2- to the T2Cu centre, but very little is known about how NO2- binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2- may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2- with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Prenatal Immune Challenge in Mice Leads to Partly Sex-Dependent Behavioral, Microglial, and Molecular Abnormalities Associated with Schizophrenia.

Epidemiological studies revealed that environmental factors comprising prenatal infection are strongly linked to risk for later development of neuropsychiatric disorders such as schizophrenia. Considering strong sex differences in schizophrenia and its increased prevalence in males, we designed a methodological approach to investigate possible sex differences in pathophysiological mechanisms. Prenatal immune challenge was modeled by systemic administration of the viral mimic polyinosinic-polycytidylic acid (Poly I:C) to C57BL/6 mice at embryonic day 9.5. The consequences on behavior, gene expression, and microglia-brain immune cells that are critical for normal development-were characterized in male vs. female offspring at adulthood. The cerebral cortex, hippocampus, and cerebellum, regions where structural and functional alterations were mainly described in schizophrenia patients, were selected for cellular and molecular analyses. Confocal and electron microscopy revealed most pronounced differences in microglial distribution, arborization, cellular stress, and synaptic interactions in the hippocampus of male vs. female offspring exposed to Poly I:C. Sex differences in microglia were also measured under both steady-state and Poly I:C conditions. These microglial alterations were accompanied by behavioral impairment, affecting for instance sensorimotor gating, in males. Consistent with these results, increased expression of genes related to inflammation was measured in cerebral cortex and hippocampus of males challenged with Poly I:C. Overall, these findings suggest that schizophrenia's higher incidence in males might be associated, among other mechanisms, with an increased microglial reactivity to prenatal immune challenges, hence determining disease outcomes into adulthood.

Relation between HbA1c and incident cardiovascular disease over a period of 6 years in the Hong Kong population.

AIM: The current trend on diabetes management advocates replacing the paradigm from a uniform to an individualized patient-centered haemoglobin A1c (HbA1c) target, but there is no consensus on the optimal HbA1c level. The study aimed at examining the association between HbA1c and the risk of cardiovascular diseases (CVD) for diabetic patients with different characteristics, in order to identify patient-centered treatment targets. METHODS: A retrospective cohort study was conducted on 115,782 Chinese adult primary care patients with type 2 diabetes mellitus (DM) but no known CVD history, who were prescribed antidiabetic medications in 2010-2011. The cumulative mean HbA1c over a median follow-up period of 5.8 years was used to evaluate the relationship between HbA1c and CVD incidence using Cox analysis. Subgroup analyses were conducted by stratifying different baseline characteristics including gender, age, smoking status, diabetes duration, body mass index, Charlson's comorbidity index and DM treatment modalities. RESULTS: For patients with a DM duration of<2years, an exponential relationship between HbA1c and risk of CVD was identified, suggesting that there was no threshold HbA1c level for CVD risk. For other diabetic patients, an HbA1c level of 6.8-7.2% was associated with a minimum risk for CVD and a J-shaped curvilinear association between HbA1c. The risk of CVD increased in patients with HbA1c<6.5% or ≥7.5%. CONCLUSION: Among Chinese primary care patients at the early (<2years) disease stage, lower HbA1c targets (<6.5%) may be warranted to prevent CVD events whilst for all others, excessively lower HbA1c levels may not necessarily better and can potentially be harmful.

Leukotriene receptor antagonism may not be effective in atopic dermatitis treatment after all.

WHAT IS KNOWN AND OBJECTIVE: Literature evidence suggests leukotriene involvement in the pathogenesis of atopic dermatitis. This article aimed to discuss whether the off-label use of montelukast, a leukotriene receptor antagonist, is justifiable for the treatment of atopic dermatitis. COMMENT: Most non-randomized studies supported the use of montelukast for atopic dermatitis treatment. However, evidence from these studies should be interpreted with caution as it is relatively weak due to the absence of randomization, control groups and blinding processes, subjecting the results to high risk of selection and reporting biases. The inconsistent findings across RCTs may be related to the limited number of patients, nuances in study designs, varying severity of disease and the concomitant use of steroids in some of the studies. WHAT IS NEW AND CONCLUSION: Current literature evidence is limited to rationally support the use of montelukast in atopic dermatitis treatment. For now, the conventional treatments should be preferred in the clinical setting.

The Impact of Tumour Distance From the Anal Verge on Clinical Management and Outcomes in Patients Having a Curative Resection for Rectal Cancer.

AIM: The clinico-oncological significance of the distance of rectal cancer from the anal verge is unclear and not well reported. The aim of this study is to assess the influence of the rectal cancer distance from the anal verge on clinical management and long-term outcomes after curative resection in a specialised colorectal cancer unit. METHODS: Prospectively collected data on patients who underwent primary rectal cancer treatment at our unit between January 2005 and December 2010 were analysed. Low rectal cancer (LRC) was defined as tumour < 5 cm from the anal verge on MRI scan. Recurrent cancer, palliative resections, perforated tumours and those requiring total pelvic exenteration were excluded. RESULTS: Three hundred fifty-nine patients underwent surgery for rectal cancer (226 male/133 female). Of these, 149 (41.5%) patients had low rectal cancer (LRC). Compared to patients with mid/upper rectal cancer (M/URC), patients with low rectal cancers were significantly more likely to receive neo-adjuvant therapy (75.2 vs 38%; p < 0.001), to be associated with lower rate of restorative surgery (15.4 vs 79%; p < 0.001) and to have higher rates of pathological positive circumferential resection margin involvement (14.1 vs 7.1%; p = 0.047). There were however no significant difference in the rates of recurrent disease or survival among the two groups. CONCLUSION: Distance of rectal cancer from the anal verge does influence the use of neo-adjuvant treatment and ultimate R0 resection rate. It does not influence loco-regional or systemic recurrence rates.

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.