Pathogenic and Commensal Gut Bacteria Harboring Glycerol/Diol Dehydratase Metabolize Glycerol and Produce DNA-Reactive Acrolein.

Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes pduCDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein's high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Salmonella Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of S. Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a pduC-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of Limosilactobacillus reuteri, but not a pduCDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 108 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.

Impact of manipulation of glycerol/diol dehydratase activity on intestinal microbiota ecology and metabolism.

Glycerol/diol dehydratases (GDH) are enzymes that catalyse the production of propionate from 1,2-propanediol, and acrolein from glycerol. Acrolein reacts with dietary carcinogenic heterocyclic amines (HCA), reducing HCA mutagenicity, but is itself also an antimicrobial agent and toxicant. Gut microbial GDH activity has been suggested as an endogenous acrolein source; however, there is limited information on the potential of the intestinal microbiota to have GDH activity, and what impact it can have on the intestinal ecosystem and host health. We hypothesized that GDH activity of gut microbiota is determined by the abundance and distribution of GDH-active taxa and can be enhanced by supplementation of the GDH active Anaerobutyricum hallii, and tested this hypothesis combining quantitative profiling of gdh, model batch fermentations, microbiota manipulation, and kinetic modelling of acrolein formation. Our results suggest that GDH activity is a common trait of intestinal microbiota shared by a few taxa, which was dependent on overall gdh abundance. Anaerobutyricum hallii was identified as a key taxon in GDH metabolism, and its supplementation increased the rate of GDH activity and acrolein release, which enhanced the transformation of HCA and reduced fermentation activity. The findings of this first systematic study on acrolein release by intestinal microbiota indicate that dietary and microbial modulation might impact GDH activity, which may influence host health.

Safety and Effectiveness of Field Nitroglycerin in Patients with Suspected ST Elevation Myocardial Infarction.

Objective: While widely used in the treatment of cardiac conditions, only limited data characterize out-of-hospital nitroglycerin (NTG) use. We sought to determine the safety of out-of-hospital sublingual NTG administered for suspected ST-segment elevation myocardial infarction (STEMI) and its effect on the patient's pain score. Methods: We prospectively identified adult patients with suspected STEMI transported by paramedics to three percutaneous Coronary Intervention PCI-capable hospitals in a large urban-suburban emergency medical services (EMS) system. We compared patients who received field NTG to those who did not. The primary outcome was the change in systolic blood pressure (SBP) between initial EMS measurement and emergency department (ED) triage vital signs. Secondary outcomes included the frequency of hypotension (SBP < 100 mmHg) and bradycardia (HR < 60) on ED arrival, drop in SBP ≥ 30 mmHg, out-of-hospital cardiac arrest (OHCA), and the change in pain score compared to an a priori threshold of -1.39. Results: Among 940 EMS transports for suspected STEMI, we excluded 160 for initial SBP < 100 mmHg, leaving 780 subjects for the analysis. Median age was 67 with 61% male. NTG was administered to 340 (44%) patients. The median change in SBP was -10 mmHg (IQR -27, 2) and -3 mmHg (IQR -20, 9) in patients treated with and without NTG, respectively. The median difference in the decrease in SBP was 6 mmHg (95% CI 3, 9 mmHg). The frequencies of ED hypotension and bradycardia, the drop in SBP ≥ 30 mmHg, and the OHCA did not differ between groups. For patients with an initial pain score > 0, the average change in pain score for patients treated with NTG was -2.6 (95% CI -3.0, -2.2), while patients who did not receive NTG had a change in pain score of -1.4 (95% CI -1.8, -1.0). Conclusion: In this cohort, field NTG did not result in a clinically significant decrease in blood pressure when compared with patients who did not receive NTG. However, NTG did cause a clinically significant reduction in pain.

5-Alkyloxytryptamines are membrane-targeting, broad-spectrum antibiotics.

Antibiotic adjuvant therapy represents an exciting opportunity to enhance the activity of clinical antibiotics by co-dosing with a secondary small molecule. Successful adjuvants decrease the concentration of antibiotics used to defeat bacteria, increase activity (in some cases introducing activity against organisms that are drug resistant), and reduce the frequency at which drug-resistant bacteria emerge. We report that 5-alkyloxytryptamines are a new class of broad-spectrum antibacterial agents with exciting activity as antibiotic adjuvants. We synthesized 5-alkyloxytryptamine analogs and found that an alkyl chain length of 6-12 carbons and a primary ammonium group are necessary for the antibacterial activity of the compounds, and an alkyl chain length of 6-10 carbons increased the membrane permeability of Gram-positive and Gram-negative bacteria. Although several of the most potent analogs also have activity against the membranes of human embryonic kidney cells, we demonstrate that below the minimum inhibitory concentration (MIC)-where mammalian cell toxicity is low-these compounds may be successfully used as adjuvants for chloramphenicol, tetracycline, ciprofloxacin, and rifampicin against clinical strains of Salmonella typhimurium, Acinetobacter baumannii and Staphylococcus aureus, reducing MIC values by as much as several logs.

Aphids alter host-plant nitrogen isotope fractionation.

Plant sap-feeding insects and blood-feeding parasites are frequently depleted in (15)N relative to their diet. Unfortunately, most fluid-feeder/host nitrogen stable-isotope studies simply report stable-isotope signatures, but few attempt to elucidate the mechanism of isotopic trophic depletion. Here we address this deficit by investigating the nitrogen stable-isotope dynamics of a fluid-feeding herbivore-host plant system: the green peach aphid, Myzus persicae, feeding on multiple brassicaceous host plants. M. persicae was consistently more than 6‰ depleted in (15)N relative to their hosts, although aphid colonized plants were 1.5‰ to 2.0‰ enriched in (15)N relative to uncolonized control plants. Isotopic depletion of aphids relative to hosts was strongly related to host nitrogen content. We tested whether the concomitant aphid (15)N depletion and host (15)N enrichment was coupled by isotopic mass balance and determined that aphid (15)N depletion and host (15)N enrichment are uncoupled processes. We hypothesized that colonized plants would have higher nitrate reductase activity than uncolonized plants because previous studies had demonstrated that high nitrate reductase activity under substrate-limiting conditions can result in increased plant δ(15)N values. Consistent with our hypothesis, nitrate reductase activity in colonized plants was twice that of uncolonized plants. This study offers two important insights that are likely applicable to understanding nitrogen dynamics in fluid-feeder/host systems. First, isotopic separation of aphid and host depends on nitrogen availability. Second, aphid colonization alters host nitrogen metabolism and subsequently host nitrogen stable-isotope signature. Notably, this work establishes a metabolic framework for future hypothesis-driven studies focused on aphid manipulation of host nitrogen metabolism.

Facile chemoenzymatic strategies for the synthesis and utilization of S-adenosyl-(L)-methionine analogues.

A chemoenzymatic platform for the synthesis of S-adenosyl-L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product "alkylrandomization", a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4'-O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.

Double jeopardy: Intimate partner violence vulnerability among emerging adult women through lenses of race and sexual orientation.

Objective: Intimate partner violence (IPV) remains a public health concern for women age 18-25. While much is known about the IPV risk and experiences of heterosexual women, little is known about the IPV risk and experiences of their LGBTQ + counterparts and any contributions of multiple marginalization in such risk. This study examines the emotional, physical, and sexual intimate partner violence (IPV) vulnerability of emerging adult college women with and without multiple minority statuses (e.g., women with both racial/ethnic and sexual minority identities). Participants: Participants were 9,435 women ages 18-25 from the National College Health Assessment (NCHA). Results: Findings demonstrate that being a sexual minority increases risk vulnerability for all forms of IPV, regardless of race. Conclusions: The risk for college women with multiple marginalized identities is exponentially greater than either their White or heterosexual counterparts. Implications for colleges/universities, university counseling centers and professionals, and future research directions are discussed.

Minimally Invasive Skin Transcriptome Extraction Using a Dermal Biomarker Patch.

INTRODUCTION: Advances in the scientific understanding of the skin and characteristic genomic dermal signatures continue to develop rapidly. Nonetheless, skin diagnosis remains predicated on a subjective visual examination, frequently followed by biopsy and histology. These procedures often are not sufficiently sensitive, and in the case of many inflammatory diseases, biopsies are not justified, creating a situation where high-quality samples can be difficult to obtain. The wealth of molecular information available and the pace at which new data are acquired suggest that methods for minimally invasive biomarker collection could dramatically alter our understanding of skin disease and positively impact treatment paradigms. METHODS: A chemical method was optimized to covalently modify custom dermal patches with single-stranded DNA that could bind to messenger RNA. These patches were applied to ex vivo skin samples and penetration evaluated by histological methods. Patches were then applied to both the skin of normal human subjects (lower arm) as well as lesional skin of psoriasis patients, and the transcriptome captured (N = 7; 33 unique samples). Standard RNA-Seq processing was performed to assess the gene detection rate and assessments made of the reproducibility of the extraction procedure as well as the overlap with matched punch biopsy samples from the same patient. RESULTS: We have developed a dermal biomarker patch (DBP) designed to be minimally invasive and extract the dermal transcriptome. Using this platform, we have demonstrated successful molecular analysis from healthy human skin and psoriatic lesions, replicating the molecular information captured with punch biopsy. CONCLUSION: This DBP enables an unprecedented ability to monitor the molecular "fingerprint" of the skin over time or with various interventions, and generate previously inaccessible rich datasets. Furthermore, use of the DBP could be favored by patients relative to biopsy by limiting pain resulting from biopsy procedures. Given the large dynamic range observed in psoriatic skin, analysis of complex phenotypes is now possible, and the power of machine-learning methods can be brought to bear on dermatologic disease.

Evaluation of the in vitro effects of aqueous black walnut extract on equine mononuclear cells.

OBJECTIVE: To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. SAMPLE: Mononuclear cells separated from blood samples from 8 horses. PROCEDURES: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference. RESULTS: BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.

Evaluation of the ability of aqueous black walnut extracts to induce the production of reactive oxygen species.

OBJECTIVE: To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum. SAMPLE: 3 BWEs. PROCEDURES: Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity. RESULTS: BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG. CONCLUSIONS AND CLINICAL RELEVANCE: The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.

Measurement of plasma renin concentration in cats by use of a fluorescence resonance energy transfer peptide substrate of renin.

OBJECTIVE: To evaluate the use of a commercially available 5-carboxyfluorescein-based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats. SAMPLE POPULATION: Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin). PROCEDURES: Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration. RESULTS: Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R(2) = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly. CONCLUSIONS AND CLINICAL RELEVANCE: The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.

Direct Brain Stimulation Modulates Encoding States and Memory Performance in Humans.

People often forget information because they fail to effectively encode it. Here, we test the hypothesis that targeted electrical stimulation can modulate neural encoding states and subsequent memory outcomes. Using recordings from neurosurgical epilepsy patients with intracranially implanted electrodes, we trained multivariate classifiers to discriminate spectral activity during learning that predicted remembering from forgetting, then decoded neural activity in later sessions in which we applied stimulation during learning. Stimulation increased encoding-state estimates and recall if delivered when the classifier indicated low encoding efficiency but had the reverse effect if stimulation was delivered when the classifier indicated high encoding efficiency. Higher encoding-state estimates from stimulation were associated with greater evidence of neural activity linked to contextual memory encoding. In identifying the conditions under which stimulation modulates memory, the data suggest strategies for therapeutically treating memory dysfunction.

Targeting quinolone- and aminocoumarin-resistant bacteria with new gyramide analogs that inhibit DNA gyrase.

Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs-from a library of 183 derivatives-that are potent inhibitors of DNA gyrase and are active against clinical strains of gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC50s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.

Targeting the Bacterial Division Protein FtsZ.

Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.

Membrane-Targeting DCAP Analogues with Broad-Spectrum Antibiotic Activity against Pathogenic Bacteria.

We performed a structure-activity relationship study of 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), which is an antibacterial agent that disrupts the membrane potential and permeability of bacteria. The stereochemistry of DCAP had no effect on the biological activity of DCAP. The aromaticity and electronegativity of the chlorine-substituted carbazole was required for activity, suggesting that its planar and dipolar characteristics orient DCAP in membranes. Increasing the hydrophobicity of the tail region of DCAP enhanced its antibiotic activity. Two DCAP analogues displayed promising antibacterial activity against the BSL-3 pathogens Bacillus anthracis and Francisella tularensis. Codosing DCAP analogues with ampicillin or kanamycin increased their potency. These studies demonstrate that DCAP and its analogues may be a promising scaffold for developing chemotherapeutic agents that bind to bacterial membranes and kill strains of slow-growing or dormant bacteria that cause persistent infections.

Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.

Understanding molecular recognition of promiscuity of thermophilic methionine adenosyltransferase sMAT from Sulfolobus solfataricus.

UNLABELLED: Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoEth) bound and unbound forms. Notably, among these structures, the AdoEth bound form offers the first MAT structure containing a non-native product, and cumulatively these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in Escherichia coli and human. As a thermostable MAT structure from archaea, the structures herein also provide a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed 'alkylrandomization' and/or the study of methylation in the context of biological processes. DATABASES: PDB IDs: 4HPV, 4L7I, 4K0B and 4L2Z. EC 2.5.1.6 STRUCTURED DIGITAL ABSTRACT: • sMAT and sMAT bind by x-ray crystallography (View interaction).

Effect of calf age and administration route of initial multivalent modified-live virus vaccine on humoral and cell-mediated immune responses following subsequent administration of a booster vaccination at weaning in beef calves.

OBJECTIVE: To compare immune responses following modified-live virus (MLV) vaccination at weaning after intranasal or SC administration of an MLV vaccine to beef calves at 2 or 70 days of age. ANIMALS: 184 calves. PROCEDURES: Calves were allocated to 1 of 5 groups. The IN2 (n = 37) and IN70 (37) groups received an MLV vaccine containing bovine herpesvirus 1 (BHV1), bovine viral diarrhea virus (BVDV) types 1 and 2, bovine respiratory syncytial virus (BRSV), and parainfluenza 3 virus intranasally and a Mannheimia haemolytica and Pasteurella multocida bacterin SC at median ages of 2 and 70 days, respectively. The SC2 (n = 36) and SC70 (37) groups received a 7-way MLV vaccine containing BHV1, BVDV1, BVDV2, BRSV, parainfluenza 3 virus, M haemolytica, and P multocida SC at median ages of 2 and 70 days, respectively; the control group (37) remained unvaccinated until weaning. All calves received the 7-way MLV vaccine SC at median ages of 217 (weaning) and 231 days. Serum neutralizing antibody (SNA) titers against BHV1, BVDV1, and BRSV and intranasal IgA concentrations were determined at median ages of 2, 70, 140, 217, and 262 days. Cell-mediated immunity (CMI) against BHV1, BRSV, BVDV1, and P multocida was determined for 16 calves/group. RESULTS: At median ages of 140 and 217 days, BVDV1 SNA titers were significantly higher for the SC70 group than those for the other groups. Intranasal IgA concentrations and CMI increased over time for all groups. Vaccination at weaning increased SNA titers and CMI in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: SC administration of an MLV vaccine to 70-day-old calves significantly increased BVDV1 antibody titers before weaning.

Synthesis of a γ-lactam library via formal cycloaddition of imines and substituted succinic anhydrides.

Formal cycloaddition reactions between imines and cyclic anhydrides serve as starting point for the synthesis of diverse libraries of small molecules. The synthesis of succinic anhydrides substituted with electron-withdrawing groups is facilitated by new mild conditions for alkylation of aryl-substituted acetyl esters with ethyl bromoacetate. These anhydrides are then used in formal cycloaddition reactions with imines to produce γ-lactams. 2-Fluoro-5-nitrophenylsuccinic anhydride reacts efficiently with imines to provide lactams that are further diversified by conversion of the nitro group to either an aniline and an azide for subsequent reactions with acylating agents and alkynes, respectively. The synthesis of cyanosuccinic anhydride is reported for the first time, and the use of this compound in reactions with imines and subsequent functionalization of the resultant lactams is demonstrated.

Partial characterization of the Sox2+ cell population in an adult murine model of digit amputation.

Tissue regeneration in response to injury in adult mammals is generally limited to select tissues. Nonmammalian species such as newts and axolotls undergo regeneration of complex tissues such as limbs and digits via recruitment and accumulation of local and circulating multipotent progenitors preprogrammed to recapitulate the missing tissue. Directed recruitment and activation of progenitor cells at a site of injury in adult mammals may alter the default wound-healing response from scar tissue toward regeneration. Bioactive molecules derived from proteolytic degradation of extracellular matrix (ECM) proteins have been shown to recruit a variety of progenitor cells in vitro and in vivo to the site of injury. The present study further characterized the population of cells accumulating at the site of injury after treatment with ECM degradation products in a well-established model of murine digit amputation. After a mid-second phalanx digit amputation in 6-8-week-old adult mice, treatment with ECM degradation products resulted in the accumulation of a heterogeneous population of cells, a subset of which expressed the transcription factor Sox2, a marker of pluripotent and adult progenitor cells. Sox2+ cells were localized lateral to the amputated P2 bone and coexpressed progenitor cell markers CD90 and Sca1. Transgenic Sox2 eGFP/+ and bone marrow chimeric mice showed that the bone marrow and blood circulation did not contribute to the Sox2+ cell population. The present study showed that, in addition to circulating progenitor cells, resident tissue-derived cells also populate at the site of injury after treatment with ECM degradation products. Although future work is necessary to determine the contribution of Sox2+ cells to functional tissue at the site of injury, recruitment and/or activation of local tissue-derived cells may be a viable approach to tissue engineering of more complex tissues in adult mammals.