Two bovine hepacivirus genome sequences from U.S. cattle.

Bovine hepacivirus (BoHV) is closely related to the hepatitis C virus (HCV) in humans and can cause both acute and chronic liver infections in cattle. BoHV was first identified in Ghana and Germany in 2015 and since then it has been detected and characterized in other countries around the world, but no strains have been sequenced from U.S. cattle. To date, BoHV has been classified into 2 genotypes (1 and 2), with genotype 1 being further divided into 11 subtypes (A-K). However, the true genetic diversity of BoHV is likely underestimated given limited surveillance and a lack of published genome sequences. Here, we sequenced 2 nearly complete BoHV genomes from serum samples collected in 2019 from beef cattle in Missouri. Sequence comparisons and phylogenetic analysis showed that isolate MARC/2019/60 had high sequence homology with genotype 1, subtype E isolates from China. In contrast, isolate MARC/2019/50 represented a novel BoHV subtype within genotype 2. Thus, we report the first genomic characterization of BoHV isolates from U.S. cattle, and the second complete BoHV2 genome worldwide. This work increases our knowledge of the global genetic diversity of BoHV and demonstrates the co-circulation of divergent BoHV strains in U.S. cattle.

Structure and Function of Alkane Monooxygenase (AlkB).

ConspectusEvery year, perhaps as much as 800 million tons of hydrocarbons enters the environment; alkanes make up a large percentage of it. Most are transformed by organisms that utilize these molecules as sources of energy and carbon. Both aerobic and anaerobic alkane transformation chemistries exist, capitalizing on the presence of alkanes in both oxic and anoxic environments. Over the past 40 years, tremendous progress has been made in understanding the structure and mechanism of enzymes that catalyze the transformation of methane. By contrast, progress involving enzymes that transform liquid alkanes has been slower with the first structures of AlkB, the predominant aerobic alkane hydroxylase in the environment, appearing in 2023. Because of the fundamental importance of C-H bond activation chemistries, interest in understanding how biology activates and transforms alkanes is high.In this Account, we focus on steps we have taken to understand the mechanism and structure of alkane monooxygenase (AlkB), the metalloenzyme that dominates the transformation of liquid alkanes in the environment (not to be confused with another AlkB that is an α-ketogluturate-dependent enzyme involved in DNA repair). First, we briefly describe what is known about the prevalence of AlkB in the environment and its role in the carbon cycle. Then we review the key findings from our recent high-resolution cryoEM structure of AlkB and highlight important similarities and differences in the structures of members of class III diiron enzymes. Functional studies, which we summarize, from a number of single residue variants enable us to say a great deal about how the structure of AlkB facilitates its function. Next, we overview work from our laboratories using mechanistically diagnostic radical clock substrates to characterize the mechanism of AlkB and contextualize the results we have obtained on AlkB with results we have obtained on other alkane-oxidizing enzymes and explain these results in light of the enzyme's structure. Finally, we integrate recent work in our laboratories with information from prior studies of AlkB, and relevant model systems, to create a holistic picture of the enzyme. We end by pointing to critical questions that still need to be answered, questions about the electronic structure of the active site of the enzyme throughout the reaction cycle and about whether and to what extent the enzyme plays functional roles in biology beyond simply initiating the degradation of alkanes.

Assessing the Role of Systems Thinking for Stocker Cattle Operations.

Bovine respiratory disease (BRD) is recognized as a complex multifactorial disease often resulting in significant economic losses for the stocker industry through reduced health and performance of feeder calves. Conventional approaches to manage BRD in stocker production systems can be challenged with a restricted view of the system, most importantly the structure, which drives the behavior of the system and fails to anticipate unintended consequences. The translation and implementation of systems thinking into veterinary medicine can offer an alternative method to problem-solving. Fundamental to the success of the systems thinker is the conceptualization of the Iceberg Diagram intended to identify root causes of complex problems such as BRD. Furthermore, veterinary and animal health professionals are well-positioned to serve as facilitators to establish creative tension, the positive energy necessary to identify high-leverage strategies. The interrelationships and interconnected behaviors of complex stocker systems warrant an understanding of various archetypes. Archetypes provide the systems thinker with a decision-making tool to explore tactics in a nonlinear fashion for the purpose of recognizing short- and long-term outcomes. Developing literacy in the discipline of systems thinking will further equip professionals with the skillset necessary to address the multitude of challenges ingrained in complex stocker cattle systems.

Energy Landscape for the Electrocatalytic Oxidation of Water by a Single-Site Oxomanganese(V) Porphyrin.

A cationic manganese porphyrin, MnIII-TDMImP, is an efficient, homogeneous, single-site water oxidation electrocatalyst at neutral pH. The measured turnover frequency for oxygen production is 32 s-1. Mechanistic analyses indicate that MnV(O)(OH2), the protonated form of the corresponding trans-MnV(O)2 species, is generated from the MnIII(OH2)2 precursor in a 2-e- two-proton process and is responsible for O-O bond formation with a H2O molecule. Chloride ion is a competitive substrate with H2O for the MnV(O)(OH2) oxidant, forming hypochlorous acid with a rate constant that is 3 orders of magnitude larger than that of water oxidation. The data allow the construction of an experimental energy landscape for this water oxidation catalysis process.

Clinical and microbiological effects in high-risk beef calves administered intranasal or parenteral modified-live virus vaccines.

Experimental bovine respiratory syncytial virus (BRSV) infection can enhance Histophilus somni (Hs) disease in calves; we thus hypothesized that modified-live virus (MLV) vaccines containing BRSV may alter Hs carriage. Our objective was to determine the effects of an intranasal (IN) trivalent (infectious bovine rhinotracheitis virus [IBRV], parainfluenza-3 virus [PI3V], and BRSV) respiratory vaccine with parenteral (PT) bivalent bovine viral diarrhea virus (BVDV) type I + II vaccine, or a PT pentavalent (BVDV type I and II, IBRV, BRSV, and PI3V) respiratory vaccine, on health, growth, immunity, and nasal pathogen colonization in high-risk beef calves. Calves (n = 525) were received in five truckload blocks and stratified by body weight (213 ± 18.4 kg), sex, and presence of a pre-existing ear-tag. Pens were spatially arranged in sets of three within a block and randomly assigned to treatment with an empty pen between treatment groups consisting of: 1) no MLV respiratory vaccination (CON), 2) IN trivalent MLV respiratory vaccine with PT BVDV type I + II vaccine (INT), or 3) PT pentavalent, MLV respiratory vaccine (INJ). The pen was the experimental unit, with 15 pens/treatment and 11 to 12 calves/pen in this 70-d receiving study. Health, performance, and BRSV, Hs, Mycoplasma bovis (Mb), Mannheimia haemolytica (Mh), and Pasteurella multocida (Pm) level in nasal swabs via rtPCR was determined on days 0, 7, 14, and 28, and BRSV-specific serum neutralizing antibody titer, and serum IFN-γ concentration via ELISA, were evaluated on days 0, 14, 28, 42, 56, and 70. Morbidity (P = 0.83), mortality (P = 0.68) and average daily gain (P ≥ 0.82) did not differ. Serum antibodies against BRSV increased with time (P < 0.01). There was a treatment × time interaction (P < 0.01) for Hs detection; on days 14 and 28, INT (21.1% and 57.1%) were more frequently (P < 0.01) Hs positive than CON (3.6% and 25.3%) or INJ (3.4 % and 8.4%). Also, INT had reduced (P = 0.03) cycle time of Hs positive samples on day 28. No difference (P ≥ 0.17) was found for IFN-γ concentration and Mb, Mh, or Pm detection. The proportion of Mh positive culture from lung specimens differed (P < 0.01); INT had fewer (0.0%; 0 of 9) Mh positive lungs than INJ (45.5%; 6 of 13) or CON (74.0%; 14 of 19). Vaccination of high-risk calves with MLV did not clearly impact health or growth during the receiving period. However, INT was associated with an altered upper respiratory microbial community in cattle resulting in increased detection and level of Hs.

Our objective was to determine the safety, efficiency, and effects on immunity and nasal shedding of respiratory pathogens for high-risk cattle administered an intranasal (IN), trivalent (infectious bovine rhinotracheitis virus [IBRV], parainfluenza-3 virus [PI3V], and bovine respiratory syncytial virus [BRSV]) respiratory vaccine with parenteral, bivalent bovine viral diarrhea virus (BVDV), or a parenteral, pentavalent (BVDV type I and II, IBRV, BRSV, and PI3V) respiratory vaccine, compared to an unvaccinated negative control. The results of this study indicate that modified-live virus (MLV) vaccination of high-risk calves upon arrival, either parenterally or intranasally, did not clearly impact health or growth during the feedlot receiving period. However, cattle that were intranasally vaccinated had increased carriage of Histophilus somni in the naris, greater amount of H. somni in nasal swabs indicated by reduced PCR cycle time, and less frequent culture of Mannheimia haemolytica from lung tissue samples upon necropsy. Therefore, intranasal administration of MLV vaccines may alter the microbial community and balance of opportunistic pathogens in the respiratory tract of cattle.

How Forces of a Complex Adaptive System Affect Ability to Control Bovine Respiratory Disease in Feeder Cattle.

This chapter provides an introductory look into the practical application of the principals of systems thinking as a methodology to gain deeper understanding of the nature of bovine respiratory disease (BRD) in current North American beef production models. The "limits to success" archetype is used to explore the dynamic relationship between technological BRD mitigation improvements and the resultant adaptive changes made by the system. The chapter concludes, by using the tragedy of the common archetype, with an investigation into how the common shared resource of antimicrobials can be damaged and depleted over time.

Current Reality of Beef Cattle Veterinary Practice in North America: A Systems Thinking Perspective.

Beef cattle veterinarians provide services to the increasingly complex beef industry system. Systems thinking offers pathways to better understand and communicate ranges of issues such as prevailing mental models, importance of match quality relative to clientele needs, and identification of leverage to better adapt and continually improve. Thinking in systems identifies and helps us to understand patterns or structures that are organized and interconnected that result in the outcomes observed and experienced in the practice of beef cattle veterinary medicine.

Preparing for the Second Surge: Preventing Posttraumatic Stress Disorder and Building Resilience for Health Care Workers in the Face of COVID-19.

The global community needs to be aware of the potential psychosocial consequences that may be experienced by health care workers who are actively managing patients with coronavirus disease (COVID-19). These health care workers are at increased risk for experiencing mood and trauma-related disorders, including posttraumatic stress disorder (PTSD). In this concept article, strategies are recommended for individual health care workers and hospital leadership to aid in mitigating the risk of PTSD, as well as to build resilience in light of a potential second surge of COVID-19.

Aerobic Partial Oxidation of Alkanes Using Photodriven Iron Catalysis.

Photodriven oxidations of alkanes in trifluoroacetic acid using commercial and synthesized Fe(III) sources as catalyst precursors and dioxygen (O2) as the terminal oxidant are reported. The reactions produce alkyl esters and occur at ambient temperature in the presence of air, and catalytic turnover is observed for the oxidation of methane in a pure O2 atmosphere. Under optimized conditions, approximately 17% conversion of methane to methyl trifluoroacetate at more than 50% selectivity is observed. It is demonstrated that methyl trifluoroacetate is stable under catalytic conditions, and thus overoxidized products are not formed through secondary oxidation of methyl trifluoroacetate.

Toxicity of the iron siderophore mycobactin J in mouse macrophages: Evidence for a hypoxia response.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an obligate intracellular pathogen that lives within the phagosome of macrophages. Here we demonstrate that the siderophore mycobactin J, produced by the closely related intracellular pathogen Mycobacterium paratuberculosis, is toxic to murine macrophage cells. Its median lethal dose, 10 µM, is lower than that of the iron chelators desferrioxamine B and TrenCAM, an enterobactin analog. To determine the source of this toxicity, we conducted microarray, ELISA, and metabolite profiling experiments. The primary response is hypoxia-like, which implies iron starvation as the underlying cause of the toxicity. This observation is consistent with our recent finding that mycobactin J is a stronger iron chelator than had been inferred from previous studies. Mycobactin J is known to partition into cell membranes and hydrophobic organelles indicating that enhanced membrane penetration is also a likely factor. Thus, mycobactin J is shown to be toxic, eliciting a hypoxia-like response under physiological conditions.

Concise Modular Synthesis and NMR Structural Determination of Gallium Mycobactin T.

A modular synthesis of mycobactin T and its N-acetyl analogue is reported in a route that facilitates permutation of the lipid tails. A key feature is the generation of N(α)-Cbz-N(ε)-benzyloxy-N(ε)-Boc-lysine (A4) with methyl(trifluoromethyl)dioxirane in 59% yield. Selective hydroxamate N-acylation was achieved with acyl fluorides, enabling installation of lipids tails in the final step. O-Benzyl-dehydrocobactin T (B4) was prepared by modifying a known five-step sequence with an overall yield of 49%. 2-Hydroxyphenyl-4-carboxyloxazoline (C3) was prepared from 2-hydroxybenzoic acid and l-serine methyl ester in three steps with an overall yield of 55%. Ester coupling of A4 and B4 with EDCI afforded MbI-1 in 73% yield. Catalytic hydrogenation with Pd/BaSO4 and 50 psi of H2 simultaneously effected alkene reduction and debenzylation to afford MbI-2 in 96% yield. Fragment C3 was converted into acyl fluoride C4, which coupled with MbI-2 to afford MbI-3 in 51% yield. Finally, Boc-removal with HCl/EtOAc and treatment of the resultant hydroxylamine with stearyl fluoride furnished mycobactin T in 65% yield. Overall, the yield is 4% over 14 steps. The gallium mycobactin T-N-acetyl derivative (GaMbT-NAc) structure was determined by 1H NMR. The structure shows an octahedral Ga and two internal hydrogen bonds between peptidic N-Hs and two of the oxygen atoms coordinating Ga.

Macroinvertebrate Communities and Body Condition of Larval Eastern Hellbender Salamanders (Cryptobranchus alleganiensis) in North Carolina.

Salamanders are important biological indicators of freshwater aquatic ecosystems. The Eastern Hellbender, Cryptobranchus alleganiensis, is found primarily in streams across the southeastern, midwestern, and eastern portions of the United States. However, this unique large aquatic salamander is facing numerous threats and declines across its geographic range, including in Appalachia. Moreover, little is known regarding the early life history stages (gilled larvae), particularly regarding food availability (aquatic insects present in streams) and body condition. In this study, we assessed the macroinvertebrate communities of streams sampled for larval C. alleganiensis and report on the body condition index within western North Carolina streams. We found varying levels of diversity across sample locations for macroinvertebrates (total sampled = 3,619, representing over 30 genera), with the most prevalent insects from Trichoptera order, with an overall high percent of Ephemeroptera, Plecoptera, and Trichoptera (EPT) in all streams, ranging from 68.5% to 90.7%. Functional Feeding Groups included 40.5% filterers, 24.3% predators, 17.6% gatherers, 10.7% shredders, and 6.9% scrapers. Shannon Diversity Index in sample streams ranged from 2.101 to 2.698. Body condition or SMI (scaled mass index) ranged from 1.5 to 3.3 2 across sites with a largely consistent and strong linear relationship between log mass and total length (r = 0.910). Our results add to the body of knowledge on the larval ecology of this North American salamander and may aid in future management of hellbender stream habitats.

Practical Solutions for Healthcare Worker Protection During the COVID-19 Pandemic Response in the Ambulatory, Emergency, and Inpatient Settings.

OBJECTIVE: Protecting healthcare workers is an essential component of a successful response to the COVID-19 pandemic. The resource intensive nature of infectious disease protection, budgetary constraints, and global shortages of personal protective equipment (PPE) make this a daunting task. Practical, easily implemented strategies for healthcare workers (HCW) protection are needed. METHODS: We cross-reference the "Systems, Space, Staff, and Stuff" paradigm from disaster management and the "Hierarchy of Controls" approach to infection prevention from the Center for Disease Control and Prevention (CDC) to generate a narrative overview of worker protection strategies relevant to COVID-19. RESULTS: Alternative types of PPE, management of hazards, and reorganizing how people work can optimize HCWs protection. CONCLUSIONS: A comprehensive PPE strategy can utilize the "systems, space, staff, stuff" paradigm of disaster management to identify new or underutilized solutions to HCWs protection.

Details to Attend to When Managing High-Risk Cattle.

This article provides insights into the management of bovine respiratory disease in high-risk cattle populations. Biocontainment strategies, records, procurement, transport, arrival/receiving management, vaccination, and treatment protocols are discussed from practical and systems-thinking perspectives regarding their impact on health in high-risk cattle. Arrival management considerations, such as facilities, nutritional management, metaphylaxis, bovine viral diarrhea virus persistent infection testing, parasite control, and castration, are also addressed. Caretaker morale and job satisfaction are suggested as important factors to consider when managing high-risk cattle. The inter-relationships of variables within the system are explored as contributing causative factors to bovine respiratory disease in high-risk cattle.

Photoinduced charge flow inside an iron porphyrazine complex.

Tracking inorganic photochemistry with high resolution poses considerable challenges. Here, sub-picosecond electronic and structural motions and MLCT/d-d intersystem crossing in a cationic iron-porphyrazine are probed using ultrafast transient absorption, stimulated Raman spectroscopy, and quantum calculations. By delineating photoinduced energy relaxation, strategies for extending the lifetime of MLCT state are discussed.

Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides.

Indoleamine 2,3-dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of the indole ring of tryptophan to afford N-formylkynurenine. This activity significantly suppresses the immune response, mediating inflammation and autoimmune reactions. These consequential effects are regulated through redox changes in the heme cofactor of IDO1, which autoxidizes to the inactive ferric state during turnover. This change in redox status increases the lability of the heme cofactor leading to further suppression of activity. The cell can thus regulate IDO1 activity through the supply of heme and reducing agents. We show here that polysulfides bind to inactive ferric IDO1 and reduce it to the oxygen-binding ferrous state, thus activating IDO1 to maximal turnover even at low, physiologically significant concentrations. The on-rate for hydrogen disulfide binding to ferric IDO1 was found to be >106 M-1 s-1 at pH 7 using stopped-flow spectrometry. Fe K-edge XANES and EPR spectroscopy indicated initial formation of a low-spin ferric sulfur-bound species followed by reduction to the ferrous state. The µM affinity of polysulfides for IDO1 implicates these polysulfides as important signaling factors in immune regulation through the kynurenine pathway. Tryptophan significantly enhanced the relatively lower-affinity binding of hydrogen sulfide to IDO1, inspiring the use of the small molecule 3-mercaptoindole (3MI), which selectively binds to and activates ferric IDO1. 3MI sustains turnover by catalytically transferring reducing equivalents from glutathione to IDO1, representing a novel strategy of upregulating innate immunosuppression for treatment of autoimmune disorders. Reactive sulfur species are thus likely unrecognized immune-mediators with potential as therapeutic agents through these interactions with IDO1.

A reevaluation of iron binding by Mycobactin J.

The complex stability constant (log ß110) and the free iron concentration (pM) are used to compare the relative strength of iron binding by siderophores. Direct measurements of these thermodynamic parameters are often not possible for siderophores due to very large log ß110 values ranging from 30 to 50. Instead, siderophore iron(III)-binding constants are determined by competitive experiments with other strong chelators with known values, such as EDTA. Iron(III) binding constants of water-insoluble siderophores, such as the mycobactins produced by the mycobacterium family, have never been directly measured. Since mycobactins contain two hydroxamic acid binding motifs, their log ß110 values have been assumed to be comparable to those of other hydroxamate-based siderophores like desferrioxamine B, at ~ 30. However, exochelin MN, another mycobacterial siderophore that contains two hydroxamic acid moieties, has a log ß110 of 39.1 and a pM of 31.1, which makes it among the strongest siderophores known. We have found that mycobactin J, the amphiphilic siderophore of Mycobacterium paratuberculosis, can remove iron(III) from TrenCAM (log ß110 = 43.6) within 1 min in methanol. This surprising result indicates that log ß110 for mycobactin J is ~ 43 and the ligand exchange kinetics in methanol is fast. The results imply that mycobactins are capable of removing iron quickly from very strongly binding siderophores in a cellular milieu. We propose a model mechanism for iron acquisition by pathogenic mycobacteria in vivo. This model explains how the host iron captured by siderophores can be returned to the invading pathogen even in the absence of active uptake mechanisms.

Site-selective 18F fluorination of unactivated C-H bonds mediated by a manganese porphyrin.

The first direct C-H 18F fluorination reaction of unactivated aliphatic sites using no-carrier-added [18F]fluoride is reported. Under the influence of a manganese porphyrin/iodosylbenzene system, a variety of unactivated aliphatic C-H bonds can be selectively converted to C-18F bonds. The mild conditions, broad substrate scope and generally inaccessible regiochemistry make this radio-fluorination a powerful alternate to established nucleophilic substitution for the preparation of 18F labeled radio tracers.

Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form.

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to N-formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function. The importance of this immunosuppressive mechanism has spurred intense interest in the development of clinical IDO1 inhibitors. Herein, we describe the mechanism by which a class of compounds effectively and specifically inhibits IDO1 by targeting its apo-form. We show that the in vitro kinetics of inhibition coincide with an unusually high rate of intrinsic enzyme-heme dissociation, especially in the ferric form. X-ray crystal structures of the inhibitor-enzyme complexes show that heme is displaced from the enzyme and blocked from rebinding by these compounds. The results reveal that apo-IDO1 serves as a unique target for inhibition and that heme lability plays an important role in posttranslational regulation.