Complete elucidation of the late steps of bafilomycin biosynthesis in Streptomyces lohii.

Bafilomycins are an important subgroup of polyketides with diverse biological activities and possible applications as specific inhibitors of vacuolar H+-ATPase. However, the general toxicity and structural complexity of bafilomycins present formidable challenges to drug design via chemical modification, prompting interests in improving bafilomycin activities via biosynthetic approaches. Two bafilomycin biosynthetic gene clusters have been identified, but their post-polyketide synthase (PKS) tailoring steps for structural diversification and bioactivity improvement remain largely unknown. In this study, the post-PKS tailoring pathway from bafilomycin A1 (1)→C1 (2)→B1 (3) in the marine microorganism Streptomyces lohii was elucidated for the first time by in vivo gene inactivation and in vitro biochemical characterization. We found that fumarate is first adenylated by a novel fumarate adenylyltransferase Orf3. Then, the fumaryl transferase Orf2 is responsible for transferring the fumarate moiety from fumaryl-AMP to the 21-hydroxyl group of 1 to generate 2. Last, the ATP-dependent amide synthetase BafY catalyzes the condensation of 2 and 2-amino-3-hydroxycyclopent-2-enone (C5N) produced by the 5-aminolevulinic acid synthase BafZ and the acyl-CoA ligase BafX, giving rise to the final product 3. The elucidation of fumarate incorporation mechanism represents the first paradigm for biosynthesis of natural products containing the fumarate moiety. Moreover, the bafilomycin post-PKS tailoring pathway features an interesting cross-talk between primary and secondary metabolisms for natural product biosynthesis. Taken together, this work provides significant insights into bafilomycin biosynthesis to inform future pharmacological development of these compounds.

Enhancing Terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate.

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

Characterization of the bafilomycin biosynthetic gene cluster from Streptomyces lohii.

New hope for old bones: The plecomacrolide bafilomycin has been explored for decades as an anti-osteoporotic. However, its structural complexity has limited the synthesis of analogues. The cloning of the bafilomycin biosynthetic gene cluster from the environmental isolate Streptomyces lohii opens the door to the production of new analogues through bioengineering.

COSPAR Sample Safety Assessment Framework (SSAF).

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Evaluation of Effects of Laboratory Disinfectants on Mouse Gut Microbiota.

Disturbances in the gut microbiota are known to be associated with numerous human diseases. Mice have proven to be an invaluable tool for investigating the role of the gut microbiota in disease processes. Nonexperimental factors related to maintaining mice in the laboratory environment are increasingly being shown to have inadvertent effects on the gut microbiota and may function as confounding variables. Microisolation technique is a term used to describe the common biosecurity practice of spraying gloved hands with disinfectant before handling research mice. This practice prevents contamination with pathogenic microorganisms. To investigate if exposure to disinfectants can affect the mouse gut microbiota, C57BL/6 mice were exposed daily for 27 consecutive days to commonly used laboratory disinfectants through microisolation technique. The effects of 70% ethanol and disinfectant products containing chlorine dioxide, hydrogen peroxide, or potassium peroxymonosulfate were each evaluated. Fecal pellets were collected after 7, 14, 21, and 28 d of disinfectant exposure, and cecal contents were collected at day 28. DNA extractions were performed on all cecal and fecal samples, and microbial community structure was characterized using 16S ribosomal RNA amplicon sequencing. Alpha and ß diversity metrics and taxon-level analyses were used to evaluate differences in microbial communities. Disinfectant had a small but significant effect on fecal microbial communities compared with sham-exposed controls, and effects varied by disinfectant type. In general, longer exposure times resulted in greater changes in the fecal microbiota. Effects on the cecal microbiota were less pronounced and only seen with the hydrogen peroxide and potassium peroxymonosulfate disinfectants. These results indicate that laboratory disinfectant use should be considered as a potential factor that can affect the mouse gut microbiota.

Reference Intervals for Total T4 and Free T4 in Cynomolgus Macaques (Macaca fascicularis) and Rhesus Macaques (Macaca mulatta).

Thyroid diseases, associated with either increased or decreased concentrations of circulating thyroid hormones, are prevalent in both human and veterinary populations. Hypothyroidism is a differential diagnosis for many medical problems as the disease presents with nonspecific clinical signs that can include lethargy, weight gain, cold intolerance, and dermatologic manifestations such as alopecia. Alopecia is a frequently reported problem in captive nonhuman primates (NHP), and hypothyroidism is considered to be a differential diagnosis. However, thyroid function test results in NHP using total T4 (TT4) and free T4 (FT4) assays are difficult to interpret without accurate reference intervals (RI) for comparison. As a consequence, hypothyroidism may be underdiagnosed in these species. The objective of this study was to establish RI for TT4 and FT4 in healthy populations of cynomolgus macaques ( n = 133; age range 2.6 to 24.7 y) and rhesus macaques ( n = 172; age range 0.8 to 31.0 y). Serum samples were collected across a 14-y period during routine anesthetic events in clinically healthy animals, and TT4 and FT4 concentrations were measured using commercially available immunoassays. The RI established for TT4 and FT4 were 5.1 to 14.9 ug/dL and 0.48 to 1.17 ng/dL for cynomolgus macaques, and 3.9 to 14.7 ug/dL and 0.36 to 1.12 ng/dL for rhesus macaques. Significant differences in thyroid hormone concentrations were found between Indian and Chinese origin rhesus, and between Mauritian and other origin cynomolgus. In addition, juvenile and subadult rhesus exhibited significantly higher FT4 and TT4 concentrations than did older animals. Individual RI were established for subgroups with adequately different thyroid hormone concentrations. These results will allow a more thorough diagnostic evaluation of cynomolgus and rhesus macaques with clinical signs consistent with thyroid disease and will ultimately be a refinement in NHP medicine.

The Effects of Water Volume and Bacterial Concentration on the Water Filtration Assay Used in Zebrafish Health Surveillance.

The number of zebrafish in biomedical research has increased exponentially over the past decades, leading to pressure on the laboratory animal community to develop and refine techniques to monitor zebrafish health so that suitable stocks can be maintained for research. The water filtration assay is a promising technique in which water from a zebrafish system is filtered, and the filter analyzed by PCR. In the present report, we studied how the volume of water tested and the concentration of bacterial pathogens affected test results. To do so, we used stock solutions of 3 zebrafish pathogens: Edwardsiella ictaluri, Aeromonas hydrophila, and Mycobacterium marinum. We used these stocks to create solutions with known concentrations of each pathogen, ranging between 10² and 107 Colony Forming Units (CFU) per ml. One, 2, and 3 L of each solution was filtered using positive pressure, and the filters were submitted to a commercial lab for PCR testing. Results were fit with a logistic regression model, and the probability of obtaining a positive result were calculated. Test sensitivity varied by organism, but in general, test results were positively correlated with the volume of the water filtered and with the concentration of bacteria in solution. We conclude that a positive result can be expected for E. ictaluri at 105 CFU per mL, A. hydrophila at 106 CFU per ml, and M. marinum at 106 CFU per mL, when 3 L of solution are filtered.

Guiding Ethical Principles in Engineering Biology Research.

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development.

Behavioral Effects of Cage Size and Environmental Enrichment in New Zealand White Rabbits.

One of the goals of environmental enrichment is to encourage species-typical behaviors, while discouraging abnormal behaviors or stereotypies. Assessing the effectiveness of various enrichment modalities can be challenging, particularly for prey species such as rabbits that exhibit freezing responses in the presence of people. In this study, we housed rabbits in 3 different sized cages and observed their behaviors. The 3 cage sizes were our standard rabbit housing cage, a medium sized cage, and a large run. Based on analysis of the recordings, ethograms were constructed and behaviors were quantified. The rabbits in large runs spent more time performing active, exploratory behaviors (431 ± 74 s) than rabbits in the standard cages(184 ± 55 s). However, space constraints inside research facilities often make it impractical to house rabbits in large runs.Therefore, we decided to explore if enrichment devices could promote the expression of active behaviors, similar to those displayed by rabbits housed in the large runs. We selected 3 devices: a hanging toy, a destructible device, and a dig bin. All 3 enrichment devices promoted more time spent performing active, exploratory behaviors (389 ± 48, 463 ± 50, and 420 ± 44 s,respectively), compared with control rabbits housed without an enrichment device (226 ± 53 s). We also analyzed the fecal glucocorticoids of rabbits after shipping or surgery to determine if enrichment devices could mitigate the physiologic impact of these stressors. We found no significant differences in fecal glucocorticoid levels between rabbits that experienced the stressor and rabbits that did not, or between rabbits with or without enrichment devices. Overall, the provision of largercaging and/or addition of enrichment devices encouraged a broad spectrum of active, species-typical rabbit behaviors, suggestiveof improved animal welfare.

Assessment of Pain Associated with the Injection of Sodium Pentobarbital in Laboratory Mice (Mus musculus).

The AVMA Guidelines for the Euthanasia of Animals considers injection of barbiturates to be an acceptable method of euthanasia in rodents but states there is a potential for pain when administered intraperitoneally. This study examined the potential for pain in mice by assessing visceral pain after intraperitoneal administration and acute pain by using a paw-lick test. Male and female mice (n = 160) intraperitoneally received a euthanizing dose of sodium pentobarbital at a concentration of 5, 50, or 390 mg/mL and were observed for writhing, peritoneum-directed behaviors (PDB), loss of righting reflex, and time to death. Writhing was not observed in any animal. There was no significant difference in the number of mice exhibiting PDB or in the rate of PDB for responders receiving either saline or the 390-mg/mL solution. There was a significant treatment effect on time, with greater concentration and dose resulting in more rapid loss of righting reflex and death. In the second set of experiments, the same solutions were injected subcutaneously into the plantar hindpaw of male and female mice (n = 84). The number of responders, latency until the first lick, and the number of licks per responder were recorded. The number of responders was increased in the 50-mg/mL group; however, there was no difference in latency or the number of licks per responder. These results show that intraperitoneal injection of sodium pentobarbital for euthanasia in mice did not result in increased behavioral signs of pain, and animals lose consciousness more rapidly than the onset of pain seen in the pawlick test. Therefore, although sodium pentobarbital is capable of inducing inflammation, euthanasia through intraperitoneal administration is rapid and does not result in overt signs of pain when compared with injection of saline.

Evaluation of Analgesic Patches in Cynomolgus Macaques (Macaca fascicularis).

Opioids are widely used in veterinary and human medicine to manage pain. However, there is a paucity of information in the literature regarding the pharmacokinetics of opioid transdermal patches (TDP) in NHP. Therefore, to determine whether opioid TDP attain therapeutic concentrations in NHP, the pharmacokinetics of fentanyl (25 µg/h) and buprenorphine (10 and 20 µg/h) TDP were evaluated in naïve, adult, male cynomolgus macaques (n = 4) in a crossover study. Plasma opioid levels were determined by tandem liquid chromatography-mass spectrometry. The AUC0-inf for fentanyl and the low and high dose buprenorphine patches were 115 ± 14, 462 ± 74, and 778 ± 344 ng× h/mL, and the plasma half-lifes were 22 ± 4, 77 ± 27, and 42 ± 11 h, respectively. No adverse effects were noted throughout the study. Minimal therapeutic concentrations for fentanyl (0.2 ng/mL) and buprenorphine (0.1 ng/mL) were achieved in all macaques within 8 h of fentanyl and 24 h of buprenorphine TDP application. Therapeutic levels for the fentanyl and low- and high-dose buprenorphine patches were maintained for 96, 120, and 144 h, respectively. These findings suggest that 25-µg/h fentanyl patches should be replaced every 4 d, and the low- and high-dose buprenorphine patches should be replaced every 5 and 6 d, respectively. The results of this study show that fentanyl and buprenorphine patches achieve minimal therapeutic levels for clinically relevant periods of time and should be considered viable options for pain management in cynomolgus macaques.

Carriage of Methicillin-resistant Staphylococcus aureus in a Colony of Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques.

Methicillin-resistant Staphylococcus aureus (MRSA) carriage and infection are well documented in the human and veterinary literature; however only limited information is available regarding MRSA carriage and infection in laboratory NHP populations. The objective of this study was to characterize MRSA carriage in a representative research colony of rhesus and cynomolgus macaques through a cross-sectional analysis of 300 animals. MRSA carriage was determined by using nasal culture. Demographic characteristics of carriers and noncarriers were compared to determine factors linked to increased risk of carriage, and MRSA isolates were analyzed to determine antimicrobial susceptibility patterns, staphylococcal chromosome cassette mec (SCCmec) type, and multilocus sequence type (ST). Culture results demonstrated MRSA carriage in 6.3% of the study population. Animals with greater numbers of veterinary or experimental interventions including antibiotic administration, steroid administration, dental procedures, and surgery were more likely to carry MRSA. Susceptibility results indicated that MRSA isolates were resistant to ß-lactams, and all isolates were resistant to between 1 and 4 non ß-lactam antibiotics. In addition, 73.7% of MRSA isolates were identified as ST188-SCCmec IV, an isolate previously observed in an unrelated population of macaques and 15.8% were ST3268-SCCmec V, which has only been described in macaques. A single isolate had a novel sequence type, ST3478, and carried SCCmec V. These results suggest that NHP-adapted strains of MRSA exist and highlight the emergence of antimicrobial resistance in laboratory NHP populations.

Isolation and Characterization of Bacterial Cellulase Producers for Biomass Deconstruction: A Microbiology Laboratory Course.

The conversion of biomass to biofuels presents a solution to one of the largest global challenges of our era, climate change. A critical part of this pipeline is the process of breaking down cellulosic sugars from plant matter to be used by microbes containing biosynthetic pathways that produce biofuels or bioproducts. In this inquiry-based course, students complete a research project that isolates cellulase-producing bacteria from samples collected from the environment. After obtaining isolates, the students characterize the production of cellulases. Students then amplify and sequence the 16S rRNA genes of confirmed cellulase producers and use bioinformatic methods to identify the bacterial isolates. Throughout the course, students learn about the process of generating biofuels and bioproducts through the deconstruction of cellulosic biomass to form monosaccharides from the biopolymers in plant matter. The program relies heavily on active learning and enables students to connect microbiology with issues of sustainability. In addition, it provides exposure to basic microbiology, molecular biology, and biotechnology laboratory techniques and concepts. The described activity was initially developed for the Introductory College Level Experience in Microbiology (iCLEM) program, a research-based immersive laboratory course at the US Department of Energy Joint BioEnergy Institute. Originally designed as an accelerated program for high-potential, low-income, high school students (11th-12th grade), this curriculum could also be implemented for undergraduate coursework in a research-intensive laboratory course at a two- or four-year college or university.

Effects of Intracage Ammonia on Markers of Pulmonary Endothelial Integrity in Mice Housed in Static Microisolation Cages.

Time-weighted exposure limits to ammonia are established for humans; however similar guidelines have not been defined for laboratory rodents. The Guide recommends maintaining air pollutants at concentrations below levels irritating to mucous membranes but does not provide specific values. Numerous studies have examined ammonia and its effects on animal health, yet none have assessed the effects of naturally occurring intracage ammonia on the lower pulmonary tree and pulmonary endothelial and epithelial integrity in mice. We performed several assays commonly used in mouse acute lung-injury studies (bronchoalveolar lavage fluid [BAL] cell counts and protein concentration, excess lung water content [ELW], Evans blue permeability assay [EBA], lung tissue myeloperoxidase assay [MPO], and lung histopathology) to evaluate the effects of exposure to cyclical, naturally occurring ammonia levels on pulmonary integrity and inflammation. C57BL/6 mice were maintained in static microisolation or open-top cages. Cages were changed weekly, and ammonia levels were measured for 6 wk on days 0, 1, 3, 5, and 7 of each cage-change cycle. Ammonia levels in static microisolation cages began to increase on day 3 and peaked at a mean of 141.3 ppm on day 7. Ammonia levels in open-top cages never exceeded 5 ppm. Neither BAL cell counts, protein concentration, ELW, EBA, nor MPO differed significantly between groups. Lung histopathology showed minimal, incidental changes in all mice. Our findings indicate that the ammonia concentrations in the static microisolation cages we used did not alter the integrity of the lower pulmonary tract nor influence key indicators used to assess acute lung injury.

Evaluation of Analgesic Efficacy of Meloxicam and 2 Formulations of Buprenorphine after Laparotomy in Female Sprague-Dawley Rats.

Managing postoperative pain in rodents is an important part of any animal care and use program, and identifying an optimal analgesic plan for a surgical procedure is critical to providing for animal welfare. Opioids and NSAID are commonly used in rodents, but few studies have evaluated their efficacy in surgical models. The current study aimed to evaluate the therapeutic efficacy of clinically relevant doses of buprenorphine (2 formulations) or meloxicam used in combination with ketamine and xylazine anesthesia in a Sprague-Dawley rat ovariohysterectomy surgical model. Rats received either subcutaneous saline once daily for 3 d, low-dose (0.05 mg/kg SC) or high-dose (0.1 mg/kg SC) buprenorphine twice daily for 3 d, a single injection of sustained-release buprenorphine (1.2 mg/kg SC), or low-dose (1 mg/kg SC) or high-dose (2 mg/kg SC) meloxicam once daily for 3 d. Clinical analgesic efficacy was assessed over 8 d according to cageside observation scoring, body weight, and behavioral testing. Ovariohysterectomy was associated with 2 d of postoperative pain, and all 3 buprenorphine dosing strategies and both doses of meloxicam demonstrated varying amounts of analgesia. Given the results of the current study, we recommend 0.05 mg/kg SC buprenorphine at least twice daily or a single dose of 1.2 mg/kg SC of sustained-release buprenorphine for rats undergoing midline laparotomy with ovariohysterectomy. Alternatively, meloxicam at 1 to 2 mg/kg SC once daily could be used for this indication.

Cross-Foster Rederivation Compared with Antibiotic Administration in the Drinking Water to Eradicate Bordetella pseudohinzii.

Bordetella pseudohinzii is a microbial agent of potential importance in mice and has confounded pulmonary research at our institution. The purpose of this study was to evaluate cross-foster rederivation and antibiotic administration in the drinking water as methods to eradicate B. pseudohinzii. To evaluate the efficacy of cross-foster rederivation, 29 litters representing 16 strains of mice were cross-fostered from cages positive for B. pseudohinzii to B. pseudohinzii-negative Crl:CD1-Elite surrogate dams. To evaluate antibiotic administration, sulfamethoxazole and trimethoprim (TMS; 0.66 and 0.13 mg/mL, respectively) and tetracycline (4.5 mg/mL) were administered in the drinking water. We assessed 3 antibiotic treatment groups with 12 B. pseudohinzii-positive cages per group (6 cages of CD1 and 6 cages of C57BL/6 mice): TMS for 4 wk, TMS for 6 wk, and tetracycline for 6 wk. Of the 29 litters that underwent cross-foster rederivation, 24 were negative for B. pseudohinzii. Five of the 12 cages treated with TMS for 4 wk and 1 of the 12 cages treated with TMS for 6 wk were negative for B. pseudohinzii at 2 wk after treatment. Three of the 12 cages treated with tetracycline were negative for B. pseudohinzii at 2 wk after treatment. Pearson χ2 analysis revealed significant association between the method of eradication (cross-foster rederivation compared with antibiotic administration) and B. pseudohinzii infection, and an odds-ratio estimate from a logistic regression demonstrated that cross-foster rederivation was more successful. Whereas antibiotic administration in the drinking water failed to eradicate B. pseudohinzii, cross-foster rederivation was successful and has been used to establish a B. pseudohinzii-negative barrier.

Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (∼30%-40%). Health disparities exist with African descent (AD) subjects exhibiting greater mortality than European descent (ED) individuals. Myosin light chain kinase is encoded by MYLK, whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, that is, cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK cytosine-guanine dinucleotides (CpGs) between ARDS patients and intensive care unit (ICU) controls overall and by ethnicity in a nested case-control study of 39 ARDS cases and 75 non-ARDS ICU controls. Two MYLK CpG sites (cg03892735 and cg23344121) were differentially modified between ARDS subjects and controls (P < 0.05; q < 0.25) in a logistic regression model, where no effect modification by ethnicity or age was found. One CpG site was associated with ARDS in patients aged <58 years, cg19611163 (intron 19, 20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2, 3) and CpG (cg16212219, intron 31, 32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting modified cytosine quantitative trait loci (mQTL) were identified using linear regression between local genetic variants and modification levels for 2 ARDS-associated CpGs (cg23344121 and cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS.

The Future of Antibiotics: Emerging Technologies and Stewardship.

Antibiotic resistance is on the rise while the number of antibiotics being brought to market continues to drop. While this is a dire situation, a number of emerging technologies have the potential to reverse this trend. These, and supporting legislative initiatives, promise to stave off the post-antibiotic era.

Bleeding the laboratory mouse: Not all methods are equal.

The laboratory mouse is the model most frequently used in hematologic studies and assessment of blood parameters across a broad range of disciplines. Often, analysis of blood occurs in a nonterminal manner. However, the small body size of the mouse limits collection based on volume, frequency, and accessible sites. Commonly used sites in the mouse include the retro-orbital sinus, facial vein, tail vein, saphenous vein, and heart. The method of blood acquisition varies considerably across laboratories and is often not reported in detail. In this study, we report significant alterations in blood parameters, particularly of total white blood cells, specific populations of dendritic cells and myeloid-derived suppressor cells, and hematopoietic progenitor cells, as a result of site and manner of sampling. Intriguingly, warming of mice prior to tail bleeding was found to significantly alter blood values. Our findings suggest that the same method should be used across an entire study, that mice should be warmed prior to tail bleeds to make levels uniform, and that accurate description of bleeding methods in publications should be provided to allow for interpretation of comparative reports and inter- and intralaboratory experimental variability.

Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.