ABSTRACT

Production of metallo-β-lactamases (MBLs), which hydrolyze carbapenems, is a cause of carbapenem resistance in Enterobacteriaceae. Development of effective inhibitors for MBLs is one approach to restore carbapenem efficacy in carbapenem-resistant Enterobacteriaceae (CRE). We report here that sulfamoyl heteroarylcarboxylic acids (SHCs) can competitively inhibit the globally spreading and clinically relevant MBLs (i.e., IMP-, NDM-, and VIM-type MBLs) at nanomolar to micromolar orders of magnitude. Addition of SHCs restored meropenem efficacy against 17/19 IMP-type and 7/14 NDM-type MBL-producing Enterobacteriaceae to satisfactory clinical levels. SHCs were also effective against IMP-type MBL-producing Acinetobacter spp. and engineered Escherichia coli strains overproducing individual minor MBLs (i.e., TMB-2, SPM-1, DIM-1, SIM-1, and KHM-1). However, SHCs were less effective against MBL-producing Pseudomonas aeruginosa. Combination therapy with meropenem and SHCs successfully cured mice infected with IMP-1-producing E. coli and dually NDM-1/VIM-1-producing Klebsiella pneumoniae clinical isolates. X-ray crystallographic analyses revealed the inhibition mode of SHCs against MBLs; the sulfamoyl group of SHCs coordinated to two zinc ions, and the carboxylate group coordinated to one zinc ion and bound to positively charged amino acids Lys224/Arg228 conserved in MBLs. Preclinical testing revealed that the SHCs showed low toxicity in cell lines and mice and high stability in human liver microsomes. Our results indicate that SHCs are promising lead compounds for inhibitors of MBLs to combat MBL-producing CRE.

IMPORTANCE Carbapenem antibiotics are the last resort for control of severe infectious diseases, bloodstream infections, and pneumonia caused by Gram-negative bacteria, including Enterobacteriaceae. However, carbapenem-resistant Enterobacteriaceae (CRE) strains have spread globally and are a critical concern in clinical settings because CRE infections are recognized as a leading cause of increased mortality among hospitalized patients. Most CRE produce certain kinds of serine carbapenemases (e.g., KPC- and GES-type β-lactamases) or metallo-β-lactamases (MBLs), which can hydrolyze carbapenems. Although effective MBL inhibitors are expected to restore carbapenem efficacy against MBL-producing CRE, no MBL inhibitor is currently clinically available. Here, we synthesized 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), which is a potent inhibitor of MBLs. SPC is a remarkable lead compound for clinically useful MBL inhibitors and can potentially provide a considerable benefit to patients receiving treatment for lethal infectious diseases caused by MBL-producing CRE.

ABSTRACT

Microbes adapt their metabolism to take advantage of nutrients in their environment. Such adaptations control specific metabolic pathways to match energetic demands with nutrient availability. Upon depletion of nutrients, rapid pathway recovery is key to release cellular resources required for survival under the new nutritional conditions. Yet, little is known about the regulatory strategies that microbes employ to accelerate pathway recovery in response to nutrient depletion. Using the fatty acid catabolic pathway in Escherichia coli, here, we show that fast recovery can be achieved by rapid release of a transcriptional regulator from a metabolite-sequestered complex. With a combination of mathematical modeling and experiments, we show that recovery dynamics depend critically on the rate of metabolite consumption and the exposure time to nutrients. We constructed strains with rewired transcriptional regulatory architectures that highlight the metabolic benefits of negative autoregulation over constitutive and positive autoregulation. Our results have wide-ranging implications for our understanding of metabolic adaptations, as well as for guiding the design of gene circuitry for synthetic biology and metabolic engineering.

IMPORTANCE Rapid metabolic recovery during nutrient shift is critical to microbial survival, cell fitness, and competition among microbiota, yet little is known about the regulatory mechanisms of rapid metabolic recovery. This work demonstrates a previously unknown mechanism where rapid release of a transcriptional regulator from a metabolite-sequestered complex enables fast recovery to nutrient depletion. The work identified key regulatory architectures and parameters that control the speed of recovery, with wide-ranging implications for the understanding of metabolic adaptations as well as synthetic biology and metabolic engineering.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized "EDEMP cycle" (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each.

IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer ("fluxomic") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed ("fluxed") through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this "blueprint" is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

ABSTRACT

Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized.

IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

ABSTRACT

Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. While both Ruegeria pomeroyi and Ruegeria lacuscaerulensis possessed genes encoding the DMSP demethylation and cleavage pathways, their responses to DMSP differed. A glucose-fed, chemostat culture of R. pomeroyi consumed 99% of the DMSP even when fed a high concentration of 5 mM. At the same time, cultures released 19% and 7.1% of the DMSP as dimethylsulfide (DMS) and methanethiol, respectively. Under the same conditions, R. lacuscaerulensis consumed only 28% of the DMSP and formed one-third of the amount of gases. To examine the pathways of sulfur and methyl C assimilation, glucose-fed chemostats of both species were fed 100 μM mixtures of unlabeled and doubly labeled [dimethyl-¹³C, ³⁴S]DMSP. Both species derived nearly all of their sulfur from DMSP despite high sulfate availability. In addition, only 33% and 50% of the methionine was biosynthesized from the direct capture of methanethiol in R. pomeroyi and R. lacuscaerulensis, respectively. The remaining methionine was biosynthesized by the random assembly of free sulfide and methyl-tetrahydrofolate derived from DMSP. Thus, although the two species possessed similar genes encoding DMSP metabolism, their growth responses were very different.

IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. DMSP is the precursor for the majority of atmospheric dimethylsulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Although research into the assimilation of DMSP has been conducted for over 20 years, the fate of DMSP in microbial biomass is not well understood. In particular, the biosynthesis of methionine from DMSP has been a focal point, and it has been widely believed that most methionine was synthesized via the direct capture of methanethiol. Using an isotopic labeling strategy, we have demonstrated that the direct capture of methanethiol is not the primary pathway used for methionine biosynthesis in two Ruegeria species, a genus comprised primarily of globally abundant marine bacteria. Furthermore, although the catabolism of DMSP by these species varied greatly, the anabolic pathways were highly conserved.

ABSTRACT

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum, must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro. Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo. However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo, indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages.

IMPORTANCE Histoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment.

ABSTRACT

Reversible repression of HIV-1 5' long terminal repeat (5'-LTR)-mediated transcription represents the main mechanism for HIV-1 to maintain latency. Identification of host factors that modulate LTR activity and viral latency may help develop new antiretroviral therapies. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are known to regulate gene expression and possess multiple physiological functions. hnRNP family members have recently been identified as the sensors for viral nucleic acids to induce antiviral responses, highlighting the crucial roles of hnRNPs in regulating viral infection. A member of the hnRNP family, X-linked RNA-binding motif protein (RBMX), has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells. Mechanistically, RBMX binds to HIV-1 proviral DNA at the LTR downstream region and maintains the repressive trimethylation of histone H3 lysine 9 (H3K9me3), leading to a blockage of the recruitment of the positive transcription factor phosphorylated RNA polymerase II (RNA pol II) and consequential impediment of transcription elongation. This RBMX-mediated modulation of HIV-1 transcription maintains viral latency by inhibiting viral reactivation from an integrated proviral DNA. Our findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

IMPORTANCE HIV-1 latency featuring silence of transcription from HIV-1 proviral DNA represents a major obstacle for HIV-1 eradication. Reversible repression of HIV-1 5'-LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency. The 5'-LTR-driven HIV gene transcription can be modulated by multiple host factors and mechanisms. The hnRNPs are known to regulate gene expression. A member of the hnRNP family, RBMX, has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells and maintains viral latency. These findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

ABSTRACT

The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ~75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.

IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

ABSTRACT

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca²⁺-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La³⁺) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.

IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La³⁺) is mostly substrate specific and that La³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

For decades, government, health systems, universities, foundations, exceptional individuals, and thought leaders across North Carolina have been testing, implementing, modifying, and just plain trying new ways of improving the way we seek, receive, and experience health care.

More recently, North Carolina has been striving to not simply address the cost, efficiency, and value that are so frustratingly elusive in health care, but also recognizing that we simply need to improve the health of our residents. We have looked to interventions both compatible with and beyond health care to do this.

The National Academy of Medicine, formerly the National Institute of Medicine, since 2016 has boldly laid out Vital Directions in Health Care, focusing on 19 priority issues and recommendations for health policy to better achieve health and well-being for all Americans. They have taken their show on the road, beyond the halls of Congress and think tanks and universities to the people on the ground in states across the country to present, discuss, listen, and learn how policy recommendations come to life.

This issue of the journal highlights the National Academy of Medicine bringing its spotlight to North Carolina last November, an acknowledgment that states are often where policy is put into action, and that North Carolina has been a leader in innovating, planning, implementing, and evaluating again and again to get better and better results for our residents. Pull your chair up to the edge of the stage for a good read in the glow of the spotlight.

Recent earthquakes involving complex multi-fault rupture have increased our appreciation of the variety of rupture geometries and fault interactions that occur within the short duration of coseismic slip. Geometrical complexities are intrinsically linked with spatially heterogeneous slip and stress drop distributions, and hence need incorporating into seismic hazard analysis. Studies of exhumed ancient fault zones facilitate investigation of rupture processes in the context of lithology and structure at seismogenic depths. In the Gairloch Shear Zone, NW Scotland, foliated amphibolites host pseudotachylytes that record rupture geometries of ancient low-magnitude (≤M_W 3) seismicity. Pseudotachylyte faults are commonly foliation parallel, indicating exploitation of foliation planes as weak interfaces for seismic rupture. Discordance and complexity are introduced by fault segmentation, stepovers, branching and brecciated dilational volumes. Pseudotachylyte geometries indicate that slip nucleation initiated simultaneously across several parallel foliation planes with millimetre and centimetre separations, leading to progressive interaction and ultimately linkage of adjacent segments and branches within a single earthquake. Interacting with this structural control, a lithological influence of abundant low disequilibrium melting-point amphibole facilitated coseismic melting, with relatively high coseismic melt pressure encouraging transient dilational sites. These faults elucidate controls and processes that may upscale to large active fault zones hosting major earthquake activity.

Supplementary material: Supplementary Figures 1 and 2, unannotated versions of field photographs displayed in Figures 4a and 5 respectively, are available at https://doi.org/10.6084/m9.figshare.c.4573256

Thematic collection: This article is part of the SJG Collection on Early-Career Research available at: https://www.lyellcollection.org/cc/SJG-early-career-research

Photorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO₂ assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activity.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

The polysaccharide pectin is a major component of the plant cell wall. The pectic glycan homogalacturonan (HG) is a proportionally small but important component of a specialized seed cell wall called mucilage. HG is synthesized in a highly methylesterified form, and, following secretion, is de-methylesterified by pectin methylesterases (PMEs). The degree of methylesterification of HG determines the structural and functional properties of pectin, but how methylesterification is regulated remains largely unknown. Here, we identified two BEL1-Like homeodomain (BLH) transcription factors, BLH2 and BLH4, as positive regulators of HG de-methylesterification in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. BLH2 and BLH4 were significantly expressed in mucilage secretory cells during seed mucilage production. BLH2 and BLH4 single mutants exhibited no obvious mucilage phenotype, but the blh2 blh4 double mutant displayed significantly reduced mucilage adherence to the seed. Reduced mucilage adherence in blh2 blh4 was caused by decreased PME activity in the seed coat, which increased the degree of methylesterification of HG in mucilage. The expression of several PME metabolism-related genes, including PME58, PECTIN METHYLESTERASE INHIBITOR6, SEEDSTICK, and MYB52 was significantly altered in blh2 blh4 seeds. BLH2 and BLH4 directly activated PME58 expression by binding to its TGACAGGT cis-element. Moreover, pme58 mutants exhibited reduced mucilage adherence similar to that of blh2 blh4, and the blh2 blh4 pme58 triple mutant exhibited no additional mucilage adherence defects. Furthermore, overexpression of PME58 in blh2 blh4 rescued the mucilage adherence defect. Together, these results demonstrate that BLH2 and BLH4 redundantly regulate de-methylesterification of HG in seed mucilage by directly activating PME58.

The treatment for obstructive sleep apnoea (OSA) with continuous positive airway pressure (CPAP) or mandibular advancement devices (MADs) is associated with blood pressure (BP) reduction; however, the overall effect is modest. The aim of this systematic review and meta-analysis of randomised controlled trials (RCTs) comparing the effect of such treatments on BP was to identify subgroups of patients who respond best to treatment.

The article search was performed in three different databases with specific search terms and selection criteria. From 2289 articles, we included 68 RCTs that compared CPAP or MADs with either passive or active treatment. When all the studies were pooled together, CPAP and MADs were associated with a mean BP reduction of –2.09 (95% CI –2.78– –1.40) mmHg for systolic BP and –1.92 (95% CI –2.40– –1.43) mmHg for diastolic BP and –1.27 (95% CI –2.34– –0.20) mmHg for systolic BP and –1.11 (95% CI –1.82– –0.41) mmHg for diastolic BP, respectively. The subgroups of patients who showed a greater response were those aged <60 years (systolic BP –2.93 mmHg), with uncontrolled BP at baseline (systolic BP –4.14 mmHg) and with severe oxygen desaturations (minimum arterial oxygen saturation measured by pulse oximetry <77%) at baseline (24-h systolic BP –7.57 mmHg).

Although this meta-analysis shows that the expected reduction of BP by CPAP/MADs is modest, it identifies specific characteristics that may predict a pronounced benefit from CPAP in terms of BP control. These findings should be interpreted with caution; however, they are particularly important in identifying potential phenotypes associated with BP reduction in patients treated for OSA.

There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g., CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license.

Key Points

Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium...

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell...

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking...

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire–mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled...

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously...

The biological carbon pump (BCP) comprises wide-ranging processes that set carbon supply, consumption, and storage in the oceans’ interior. It is becoming increasingly evident that small changes in the efficiency of the BCP can significantly alter ocean carbon sequestration and, thus, atmospheric CO2 and climate, as well as the functioning...

Ayelen M. Blanco, Juan I. Bertucci, Jose L. Soengas, and Suraj Unniappan

This research assessed the direct effects of insulin on nutrient-sensing mechanisms in the brain of rainbow trout (Oncorhynchus mykiss) using an in vitro approach. Cultured hypothalamus and hindbrain were exposed to 1 µmol l^–1 insulin for 3 h, and signals involved in appetite regulation and nutrient-sensing mechanisms were measured. Additionally, the involvement of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the actions of insulin was studied by using the inhibitor wortmannin. Treatment with insulin alone did not elicit many changes in the appetite regulators and nutrient-sensing-related genes and enzymes tested in the hypothalamus and hindbrain. However, we found that, when insulin and nutrients were added together, insulin reversed most of the effects exerted by nutrients alone, suggesting that insulin changes responsiveness to nutrients at the central level. Effects reversed by insulin included expression levels of genes related to the sensing of both glucose (slc2a2, slc5a1, gck, pck1, pklr, g6pcb, gys1, tas1r3 and nr1h3 in the hindbrain, and slc2a2, pklr and pck1 in the hypothalamus) and fatty acid (cd36 in the hindbrain, and cd36 and acly in the hypothalamus). Nutrient-induced changes in the activity of Acly and Cpt-1 in the hindbrain and of Pepck, Acly, Fas and Hoad in the hypothalamus were also reversed by insulin. Most of the insulin effects disappeared in the presence of wortmannin, suggesting the PI3K/Akt pathway is a mediator of the effects of insulin reported here. This study adds new information to our knowledge of the mechanisms regulating nutrient sensing in fish.

Christopher T. Richards and Enrico A. Eberhard

Muscle force-length dynamics are governed by intrinsic contractile properties, motor stimulation and mechanical load. Although intrinsic properties are well-characterised, physiologists lack in vitro instrumentation accounting for combined effects of limb inertia, musculoskeletal architecture and contractile dynamics. We introduce in vitro virtual-reality (in vitro-VR) which enables in vitro muscle tissue to drive a musculoskeletal jumping simulation. In hardware, muscle force from a frog plantaris was transmitted to a software model where joint torques, inertia and ground reaction forces were computed to advance the simulation at 1 kHz. To close the loop, simulated muscle strain was returned to update in vitro length. We manipulated 1) stimulation timing and, 2) the virtual muscle's anatomical origin. This influenced interactions among muscular, inertial, gravitational and contact forces dictating limb kinematics and jump performance. We propose that in vitro-VR can be used to illustrate how neuromuscular control and musculoskeletal anatomy influence muscle dynamics and biomechanical performance.

Qingxiang Guo, Yang Liu, Yanhua Zhai, and Zemao Gu

Disassembling the parasitic spores and acquiring the main subunits is a prerequisite for deep understanding of the basic biology of parasites. Herein we present a fast and efficient method to dissect the myxospores in a few steps, which mainly involved sonication, sucrose density gradient and Percoll density gradient. We tested our method on three myxozoans species and demonstrated this method allows the dismembering of myxospores, isolation of intact and clean nematocysts and shell valves within 2h by low-cost. This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.

Marc E. Deetjen, Diana D. Chin, and David Lentink

Animal flight requires aerodynamic power, which is challenging to determine accurately in vivo. Existing methods rely on approximate calculations based on wake flow field measurements, inverse dynamics approaches, or invasive muscle physiological recordings. In contrast, the external mechanical work required for terrestrial locomotion can be determined more directly by using a force platform as an ergometer. Based on an extension of the recent invention of the aerodynamic force platform, we now present a more direct method to determine the in vivo aerodynamic power by taking the dot product of the aerodynamic force vector on the wing with the representative wing velocity vector based on kinematics and morphology. We demonstrate this new method by studying a slowly flying dove, but it can be applied more generally across flying and swimming animals as well as animals that locomote over water surfaces. Finally, our mathematical framework also works for power analyses based on flow field measurements.

C. M. Lind, J. Agugliaro, and T. M. Farrell

Mounting an immune response may be energetically costly and require the diversion of resources away from other physiological processes. Yet, both the metabolic cost of immune responses and the factors that impact investment priorities remain poorly described in many vertebrate groups. For example, although viviparity has evolved many times in vertebrates, the relationship between immune function and pregnancy has been disproportionately studied in placental mammals. To examine the energetic costs of immune activation and the modulation of immune function during pregnancy in a non-mammalian vertebrate, we elicited an immune response in pregnant and non-pregnant pygmy rattlesnakes, Sistrurus miliarius, using lipopolysaccharide (LPS). Resting metabolic rate (RMR) was measured using flow-through respirometry. Immune function was examined using bactericidal assays and leukocyte counts. The RMR of pygmy rattlesnakes increased significantly in response to LPS injection. There was no statistically significant difference in the metabolic response of non-reproductive and pregnant snakes to LPS. Mean metabolic increments for pregnant females, non-reproductive females, and males were 13%, 18%, and 26%, respectively. The ratio of heterophils to lymphocytes was elevated in response to LPS across reproductive categories; however, LPS did not impact plasma bactericidal ability in non-reproductive snakes. Although pregnant females had significantly higher plasma bactericidal ability compared to non-reproductive snakes prior to manipulation, their bactericidal ability declined in response to LPS. LPS administration also significantly reduced several litter characteristics, particularly when administrated relatively early in pregnancy. Our results indicate that immune performance is energetically costly, altered during pregnancy, and that immune activation during pregnancy may result in tradeoffs that affect offspring in a viviparous reptile.

Jing Wen, Tingbei Bo, Xueying Zhang, Zuoxin Wang, and Dehua Wang

Ambient temperature and food composition can affect energy metabolism of the host. Thermal transient receptor potential (thermo-TRPs) ion channels can detect temperature signals and are involved in the regulation of thermogenesis and energy homeostasis. Further, the gut microbiota has also been implicated in thermogenesis and obesity. In the present study, we tested the hypothesis that thermo-TRPs and gut microbiota are involved in reducing diet-induced obesity (DIO) during low temperature exposure. C57BL/6J mice in obese (body mass gain >45%), lean (body mass gain <15%), and control (body mass gain<1%) groups were exposed to high (23±1°C) or low (4±1°C) ambient temperature for 28 days. Our data showed that low temperature exposure attenuated DIO, but enhanced brown adipose tissue (BAT) thermogenesis. Low temperature exposure also resulted in increased norepinephrine (NE) concentrations in the hypothalamus, decreased TRP melastatin 8 (TRPM8) expression in the small intestine, and altered composition and diversity of gut microbiota. In DIO mice, there was a decrease in overall energy intake along with a reduction in TRP ankyrin 1 (TRPA1) expression and an increase in NE concentration in the small intestine. DIO mice also showed increases in Oscillospira, [Ruminococcus], Lactococcus, and Christensenella and decreases in Prevotella, Odoribacter, and Lactobacillus at the genus level in fecal samples. Together, our data suggest that thermos-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure in DIO mice.

Understanding fructose metabolism might provide insights to renal pathophysiology. To support systemic glucose concentration, the proximal tubular cells reabsorb fructose as a substrate for gluconeogenesis. However, in instances when fructose intake is excessive, fructose metabolism is costly, resulting in energy depletion, uric acid generation, inflammation, and fibrosis in the kidney. A recent scientific advance is the discovery that fructose can be endogenously produced from glucose under pathologic conditions, not only in kidney diseases, but also in diabetes, in cardiac hypertrophy, and with dehydration. Why humans have such a deleterious mechanism to produce fructose is unknown, but it may relate to an evolutionary benefit in the past. In this article, we aim to illuminate the roles of fructose as it relates to gluconeogenesis and fructoneogenesis in the kidney.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

BACKGROUND:Ventilator-associated pneumonia (VAP) is a common and serious complication of mechanical ventilation. We conducted a meta-analysis of published randomized controlled trials to evaluate the efficacy and safety of probiotics for VAP prevention in patients who received mechanical ventilation.METHODS:We searched a number of medical literature databases to identify randomized controlled trials that compared probiotics with controls for VAP prevention. The results were expressed as odds ratios (OR) or mean differences with accompanying 95% CIs. Study-level data were pooled by using a random-effects model. Data syntheses were accomplished by using statistical software.RESULTS:Fourteen studies that involved 1,975 subjects met our inclusion criteria. Probiotic administration was associated with a reduction in VAP incidence among all 13 studies included in the meta-analysis (OR 0.62, 95% CI 0.45–0.85; P = .003; I2 = 43%) but not among the 6 double-blinded studies (OR 0.72, 95% CI 0.44–1.19; P = .20; I2 = 55%). We found a shorter duration of antibiotic use for VAP (mean difference −1.44, 95% CI −2.88 to −0.01; P = .048, I2 = 30%) in the probiotics group than in the control group, and the finding comes from just 2 studies. No statistically significant differences were found between the groups in terms of ICU mortality (OR 0.95, 95% CI 0.67–1.34; P = .77; I2 = 0%), ICU stay (mean difference –0.77, 95% CI –2.58 to 1.04; P = .40; I2 = 43%), duration of mechanical ventilation (mean difference –0.91, 95% CI –2.20 to 0.38; P = .17; I2 = 25%), or occurrence of diarrhea (OR 0.72, 95% CI 0.45–1.15; P = .17; I2 = 41%).CONCLUSIONS:The meta-analysis results indicated that the administration of probiotics significantly reduced the incidence of VAP. Furthermore, our findings need to be verified in large-scale, well-designed, randomized, multi-center trials.

BACKGROUND:In this study, we aimed to validate the agreement between pulmonary function measurements obtained with a portable spirometer and measurements obtained with conventional spirometry in Chinese pediatric and adult populations.METHODS:Pulmonary function testing was performed to evaluate subjects enrolled at Shanghai Zhongshan Hospital (n = 104) and Shanghai Children's Medical Center (n = 103). The portable spirometers and conventional devices were applied to each subject with a 20-min quiescent period between each measurement. Pulmonary function parameters of FVC, FEV1, peak expiratory flow, maximum expiratory flow at 25%, 50%, and 75% of FVC (MEF25, MEF50, and MEF75, respectively), and FEV1/FVC% were compared with intraclass correlation and Bland-Altman methods.RESULTS:A satisfactory concordance of pulmonary function was observed between spirometry measurements obtained with portable versus conventional spirometers. Intraclass correlation indicated excellent reliability (>0.75) for all pulmonary function indicators in pediatric and adult subjects. Significant positive correlations of all variables measured with different spirometers were observed (all P < .001). No significant bias was observed in either group, although limits of agreement varied. Funnel effects were observed for peak expiratory flow in pediatric subjects and for FVC, FEV1, MEF50, and MEF25 in adult subjects.CONCLUSIONS:The portable spirometer is an alternative to the conventional device for the measurement of pulmonary function. Compared with the conventional device, the portable spirometer is expected to provide convenient, operational, and financial advantages.

BACKGROUND:Most children are exposed to human metapneumovirus (HMPV) by the age of 5 y. This study aimed to describe the morbidity associated with HMPV infections in a cohort of children in the Midwest of the United States.METHODS:This was a retrospective 2-center cohort study including children (0–17 y old) hospitalized with HMPV infections at 2 tertiary care pediatric hospitals from 2009 to 2013. Demographics, chronic medical conditions, viral coinfections, and hospitalization characteristics, including the need for respiratory support, high-flow nasal cannula, CPAP, bi-level positive airway pressure, invasive mechanical ventilation, pediatric ICU admission, acute kidney injury (AKI), use of extracorporeal membrane oxygenation, and length of stay, were collected.RESULTS:In total, 131 subjects were included. Those with one or more comorbidities were older than their otherwise healthy counterparts, with a median age of 2.8 y (interquartile range [IQR] 1.1–7.0) compared to 1.3 y (IQR 0.6–2.0, P < .001), respectively. Ninety-nine (75.6%) subjects required respiratory support; 72 (55.0%) subjects required nasal cannula, simple face mask, or tracheostomy mask as their maximum support. Additionally, 1 (0.8%) subject required high-flow nasal cannula, 1 (0.8%) subject required CPAP, 2 (1.5%) subjects required bi-level positive airway pressure, 15 (11.5%) subjects required invasive mechanical ventilation, 4 (3.1%) subjects required high-frequency oscillatory or jet ventilation, and 4 (3.1%) subjects required extracorporeal membrane oxygenation. Fifty-one (38.9%) subjects required pediatric ICU admission, and 16 (12.2%) subjects developed AKI. Subjects with AKI were significantly older than those without AKI at 5.4 y old (IQR 1.6–11.7) versus 1.9 y old (IQR 0.7–3.5, P = .003). After controlling for the presence of at least one comorbidity and cystic fibrosis, each year increase in age led to a 16% increase in the odds of AKI (P = .01). The median length of stay for the entire cohort was 4.0 d (IQR 2.7–7.0).CONCLUSIONS:Children hospitalized with HMPV may be at risk for AKI. Risk of HMPV-associated AKI appears to increase with age regardless of severity of respiratory illness or presence of comorbidities.

Dependence of drug metabolism on dosing time has long been recognized. However, only recently are the underlying mechanisms for circadian drug metabolism being clarified. Diurnal rhythmicity in expression of drug-metabolizing enzymes is believed to be a key factor determining circadian metabolism. Supporting the notion that biological rhythms are generated and maintained by the circadian clock, a number of diurnal enzymes are under the control of the circadian clock. In general, circadian clock genes generate and regulate diurnal rhythmicity in drug-metabolizing enzymes via transcriptional actions on one or two of three cis-elements (i.e., E-box, D-box, and Rev-erb response element or RAR-related orphan receptor response element). Additionally, cycling or clock-controlled nuclear receptors such as hepatocyte nuclear factor 4α and peroxisome proliferator–activated receptor are contributors to diurnal enzyme expression. These newly discovered mechanisms for each of the rhythmic enzymes are reviewed in this article. We also discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics. Our discussion is also extended to two diurnal transporters (P-glycoprotein and multidrug resistance-associated protein 2) that have an important role in drug absorption. Although the experimental evidence is lacking in metabolism-based chronoefficacy, circadian genes (e.g., Rev-erbα) as drug targets are shown to account for diurnal variability in drug efficacy.

The kidneys play an important role in many processes, including urine formation, water conservation, acid-base equilibrium, and elimination of waste. The anatomic and functional development of the kidney has different maturation time points in humans versus animals, with critical differences between species in maturation before and after birth. Absorption, distribution, metabolism, and excretion (ADME) of drugs vary depending on age and maturation, which will lead to differences in toxicity and efficacy. When neonate/juvenile laboratory animal studies are designed, a thorough knowledge of the differences in kidney development between newborns/children and laboratory animals is essential. The human and laboratory animal data must be combined to obtain a more complete picture of the development in the kidneys around the neonatal period and the complexity of ADME in newborns and children. This review examines the ontogeny and cross-species differences in ADME processes in the developing kidney in preterm and term laboratory animals and children. It provides an overview of insights into ADME functionality in the kidney by identifying what is currently known and which gaps still exist. Currently important renal function properties such as glomerular filtration rate, renal blood flow, and ability to concentrate are generally well known, while detailed knowledge about transporter and metabolism maturation is growing but is still lacking. Preclinical data in those properties is limited to rodents and generally covers only the expression levels of transporter or enzyme-encoding genes. More knowledge on a functional level is needed to predict the kinetics and toxicity in neonate/juvenile toxicity and efficacy studies.

Doxophylline (DOXO) and theophylline are widely used as bronchodilators for treating asthma and chronic obstructive pulmonary disease, and DOXO has a better safety profile than theophylline. How DOXO’s metabolism and disposition affect its antiasthmatic efficacy and safety remains to be explored. In this study, the metabolites of DOXO were characterized. A total of nine metabolites of DOXO were identified in vitro using liver microsomes from human and four other animal species. Among them, six metabolites were reported for the first time. The top three metabolites were theophylline acetaldehyde (M1), theophylline-7-acetic acid (M2), and etophylline (M4). A comparative analysis of DOXO metabolism in human using liver microsomes, S9 fraction, and plasma samples demonstrated the following: 1) The metabolism of DOXO began with a cytochrome P450 (P450)–mediated, rate-limiting step at the C ring and produced M1, the most abundant metabolite in human liver microsomes. However, in human plasma, the M1 production was rather low. 2) M1 was further converted to M2 and M4, the end products of DOXO metabolism in vivo, by non-P450 dismutase in the cytosol. This dismutation process also relied on the ratio of NADP⁺/NADPH in the cell. These findings for the first time elucidated the metabolic sites and routes of DOXO metabolism in human.

VOLUME 287 (2012) PAGES 44508–44517In Fig. 1A, the wrong image for the control group was presented. The authors inadvertently cropped the control images in Fig. 1, A and E, from the same raw image. Fig. 1A has now been corrected and does not affect the results or conclusions of the work. The authors sincerely apologize for their mistake during figure preparation and for any inconvenience this may have caused readers.jbc;295/19/6781/F1F1F1Figure 1A.

Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18–labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Objective

To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4⁺ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs).

Most British railway embankments were constructed between 120 and 180 years ago without the benefit of modern design and construction methods. This can result in undesirable load-deformation characteristics and consequent disruption to present-day railway operations, for which there is unprecedented demand. Annual rail passenger kilometres have approximately doubled in the last 20 years and freight has increased by 60% over the same period. Whereas elements such as rails or bridges can be refurbished or replaced to meet increasing demand, the same is not usually feasible for embankments. Development of techniques to assess embankment performance risks posed by operational capacity enhancements is therefore of increasing significance to railway geotechnical asset management. The two case studies presented in this paper demonstrate how geospatial analysis and data management techniques may be applied to this challenge at both strategic (regional or national) and tactical (site-specific) scales for embankments incorporating plastic clay fill. The case studies also demonstrate, in a world of ever more abundant data, the growing need for engineering geologists and geotechnical engineers to augment their traditional knowledge with comprehensive data management and geospatial analysis skills, these being essential for modern infrastructure asset management.

Thematic collection: This article is part of the ‘Ground-related risk to transportation infrastructure’ collection available at: https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template.

A time course experiment is a widely used design in the study of cellular processes such as differentiation or response to stimuli. In this paper, we propose time course regulatory analysis (TimeReg) as a method for the analysis of gene regulatory networks based on paired gene expression and chromatin accessibility data from a time course. TimeReg can be used to prioritize regulatory elements, to extract core regulatory modules at each time point, to identify key regulators driving changes of the cellular state, and to causally connect the modules across different time points. We applied the method to analyze paired chromatin accessibility and gene expression data from a retinoic acid (RA)–induced mouse embryonic stem cells (mESCs) differentiation experiment. The analysis identified 57,048 novel regulatory elements regulating cerebellar development, synapse assembly, and hindbrain morphogenesis, which substantially extended our knowledge of cis-regulatory elements during differentiation. Using single-cell RNA-seq data, we showed that the core regulatory modules can reflect the properties of different subpopulations of cells. Finally, the driver regulators are shown to be important in clarifying the relations between modules across adjacent time points. As a second example, our method on Ascl1-induced direct reprogramming from fibroblast to neuron time course data identified Id1/2 as driver regulators of early stage of reprogramming.