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Aims: Biological soil crusts (biocrusts) are microbial communities commonly found in the upper layer of arid soils. These microorganisms release exopolysaccharides (EPS), which form the exopolymeric matrix (EPM), allowing them to bond soil particles together and survive long periods of dryness. The aim of this work is to develop methods for measuring metabolite retention by biocrust EPM and EPS. Methods: We report new methods for the investigation of metabolite sorption on biocrusts compared to the underlying unconsolidated subcrust fraction. A 13C–labeled bacterial lysate metabolite mixture was incubated with biocrust, subcrust and biocrust-extracted EPS. Non-sorbed metabolites were extracted and analyzed by liquid chromatography/mass spectrometry. Results: This simple and rapid approach enabled the comparison of metabolite sorption on the biocrust EPM or EPS versus mineral sorption on the underlying soils. Our results suggest that the biocrust (and its extracted EPS) sorb more metabolites, especially amino acids and organic acids, than the underlying subcrust. Conclusions: This study demonstrates a useful method to highlight the essential role of biocrust (especially the EPM), which acts as a passive nutrient filter, sequestering metabolites released by microbes during wetting events. This may facilitate recovery of the community upon wetting and further enhance biocrust survival and nutrient retention.

Desert biological soil crusts (BSCs) are cyanobacteria-dominated surface soil microbial communities common to plant interspaces in arid environments. The capability to significantly dampen their metabolism allows them to exist for extended periods in a desiccated dormant state that is highly robust to environmental stresses. However, within minutes of wetting, metabolic functions reboot, maximizing activity during infrequent permissive periods. iMicrocoleus vaginatus/i, a primary producer within the crust ecosystem and an early colonizer, initiates crust formation by binding particles in the upper layer of soil via exopolysaccharides, making microbial dominated biological soil crusts highly dependent on the viability of this organism. Previous studies have suggested that biopolymers play a central role in the survival of this organism by powering resuscitation, rapidly forming compatible solutes, and fueling metabolic activity in dark, hydrated conditions. To elucidate the mechanism of this phenomenon and provide a basis for future modeling of BSCs, we developed a manually curated, genome-scale metabolic model of iMicrocoleus vaginatus/i (iNJ1153). To validate this model, gas chromatography-mass spectroscopy (GC-MS) and liquid chromatography-mass spectroscopy (LC-MS) were used to characterize the rate of biopolymer accumulation and depletion in in hydrated iMicrocoleus vaginatus/i under light and dark conditions. Constraint-based flux balance analysis showed agreement between model predictions and experimental reaction fluxes. A significant amount of consumed carbon and light energy is invested into storage molecules glycogen and polyphosphate, while iβ/i-polyhydroxybutyrate may function as a secondary resource. Pseudo-steady-state modeling suggests that glycogen, the primary carbon source with the fastest depletion rate, will be exhausted if iM. vaginatus /i experiences dark wetting events 4 times longer than light wetting events.

Objective: To evaluate the extent to which personal well-being may be associated with empathy, while controlling for potential confounders. Settings/Location: Residency programs throughout the United States. Subjects: A total of 407 medical residents from residencies including general medicine, surgery, specialized and diagnostic medicine participated in this study. Outcome Measures: Well-being was measured using the modified existential well-being subscale of the spiritual well-being scale. Empathy was measured using the Jefferson Scale of Empathy. Results: Well-being was found to be positively correlated with empathy when adjusted for possible confounders ( p  < 0.001). In addition to well-being, other factors noted to be statistically significant contributors to higher empathy scores while controlling for the others included age, gender, year in residency, specialty, and work-hours ( p  < 0.05 for each). After controlling for these factors, a resident's year in residency was not found to be a statistically significant contributor to empathy score. Conclusions: In this study, well-being was associated with empathy in medical and surgical residents. Empathy is a fundamental component of physician competency, and its development is an essential aspect of medical training. These findings suggest that efforts to increase well-being may promote empathy among medical residents.

Abstract: Musculoskeletal ultrasound has become a fundamental diagnostic and treatment tool in the field of physical medicine and rehabilitation. However, there is no standardized curriculum for teaching and practicing musculoskeletal ultrasound during physical medicine and rehabilitation residency. The objective of this study was to describe a longitudinal curriculum using unembalmed fresh frozen cadavers to teach physical medicine and rehabilitation residents ultrasound-guided procedures. This protocol can help guide residents to begin learning how to independently identify important musculoskeletal structures and perform some of the most common musculoskeletal procedures relevant to clinical practice. Residents performed a procedure on average 6.99 times per block, and residents' self-reported confidence in various aspects of ultrasound practice significantly improved after this curriculum ( P < 0.005). Hence, a cadaver-based training curriculum may be a worthwhile tool for preparing physical medicine and rehabilitation residents to perform musculoskeletal ultrasound-guided procedures in the clinical setting., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Tracking the metabolic activity of whole soil communities can improve our understanding of the transformation and fate of carbon in soils. We used stable isotope metabolomics to trace 13 C from nine labeled carbon sources into the water-soluble metabolite pool of an agricultural soil over time. Soil was amended with a mixture of all nine sources, with one source isotopically labeled in each treatment. We compared changes in the 13 C enrichment of metabolites with respect to carbon source and time over a 48-day incubation and contrasted differences between soluble sources (glucose, xylose, amino acids, etc.) and insoluble sources (cellulose and palmitic acid). Whole soil metabolite profiles varied singularly by time, while the composition of 13 C-labeled metabolites differed primarily by carbon source ( R 2 = 0.68) rather than time ( R 2 = 0.07), with source-specific differences persisting throughout incubations. The 13 C labeling of metabolites from insoluble carbon sources occurred slower than that from soluble sources but yielded a higher average atom percent (atom%) 13 C in metabolite markers of biomass (amino acids and nucleic acids). The 13 C enrichment of metabolite markers of biomass stabilized between 5 and 15 atom% 13 C by the end of incubations. Temporal patterns in the 13 C enrichment of tricarboxylic acid cycle intermediates, nucleobases (uracil and thymine), and by-products of DNA salvage (allantoin) closely tracked microbial activity. Our results demonstrate that metabolite production in soils is driven by the carbon source supplied to the community and that the fate of carbon in metabolites do not generally converge over time as a result of ongoing microbial processing and recycling. IMPORTANCE Carbon metabolism in soil remains poorly described due to the inherent difficulty of obtaining information on the microbial metabolites produced by complex soil communities. Our study demonstrates the use of stable isotope probing (SIP) to study carbon metabolism in soil by tracking 13 C from supplied carbon sources into metabolite pools and biomass. We show that differences in the metabolism of sources influence the fate of carbon in soils. Heterogeneity in 13 C-labeled metabolite profiles corresponded with compositional differences in the metabolically active populations, providing a basis for how microbial community composition correlates with the quality of soil carbon. Our study demonstrates the application of SIP-metabolomics in studying soils and identifies several metabolite markers of growth, activity, and other aspects of microbial function.

Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article.

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon ( rrn ) copy number, as a proxy for life-history strategies, with pairs of "high" and "low" rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Wilhelm, Swenson, Silver, Andeer, Golini, Kosina, Bowen, Buckley and Northen.)

Background: Modic change grading is heterogeneous, inconsistent, and lacks a single nomenclature across the published literature. A new method of Modic change classification has been established by Dr. Peter Udby which hopes to unify how Modic changes are classified while also adding grading of the cranial/caudal extent of the Modic change across the vertebral body from the respective endplate involved to best capture the clinically relevant information of Modic changes., Methods: Twenty magnetic resonance images of potential basivertebral nerve ablation candidates were independently reviewed by two board-certified and fellowship trained neuroradiologist and two board-certified and fellowship-trained interventional spine physiatrists for the presence and characterization of Modic changes using the newly described Udby classification. 100% agreement of all four reviewers of Modic change presence, Type, and Udby classification was required to be classified as agreement. There were 480 total data points each with 10 unique choices to compare across the four independent reviewers., Results: The kappa value of their agreement was 0.5899 (95% CI 0.4860-0.6939)., Conclusion: This study, requiring unanimous agreement between 4 physicians in application of the Udby classification, demonstrated an interrater reliability score of 0.5899 (95% CI 0.4860-0.6939). While this figure provides a first estimate, larger scale research is necessary before definitive claims regarding the interrater agreement validity of the Udby characterization system may be made., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article.

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts., (© 2021. The Author(s).)

Abstract: Previous surveys have demonstrated an increasing trend among graduating physiatry residents who desired to pursue a subspecialty fellowship. There has been sparse information on whether residents start their training with a subspecialty interest in mind and what factors influenced them to choose a fellowship. This article describes a prospective survey in the 2019-2020 academic year in which 175 responses were collected representing 65 (78.3%) of the 83 physical medicine and rehabilitation programs with graduating residents. Nearly 3 in 4 (73.7%, 129/175) reported matching into a fellowship, and among those, 79.8% (103/129) had matched into a pain, spine, or sports medicine fellowship. At the start of residency, 62.3% (109/175) were planning to pursue a fellowship, with 54.9% (96/175) planning to focus on either pain, sports, or spine medicine. Most respondents (72.2%) did not change their initial subspecialty focus during their residency training. Forty-six percent agreed that their anticipated subspecialty influenced their choice of residency program. The results of this survey demonstrate that most graduating residents are matriculating into fellowship training with pain, spine, and/or sports medicine being among the top choices. These results underscore the importance of subspecialty interests of trainees at the start of their residency and how training may influence their subspecialty interest., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Biological soil crusts (biocrusts) are photosynthetic "hot spots" in deserts and cover ∼12% of the Earth's terrestrial surface, and yet they face an uncertain future given expected shifts in rainfall events. Laboratory wetting of biocrust communities is known to cause a bloom of Firmicutes which rapidly become dominant community members within 2 days after emerging from a sporulated state. We hypothesized that their bacteriophages (phages) would respond to such a dramatic increase in their host's abundance. In our experiment, wetting caused Firmicutes to bloom and triggered a significant depletion of cyanobacterial diversity. We used genome-resolved metagenomics to link phage to their hosts and found that the bloom of the genus Bacillus correlated with a dramatic increase in the number of Caudovirales phages targeting these diverse spore-formers ( r  = 0.762). After 2 days, we observed dramatic reductions in the relative abundances of Bacillus , while the number of Bacillus phages continued to increase, suggestive of a predator-prey relationship. We found predicted auxiliary metabolic genes (AMGs) associated with sporulation in several Caudovirales genomes, suggesting that phages may influence and even benefit from sporulation dynamics in biocrusts. Prophage elements and CRISPR-Cas repeats in Firmicutes metagenome-assembled genomes (MAGs) provide evidence of recent infection events by phages, which were corroborated by mapping viral contigs to their host MAGs. Combined, these findings suggest that the blooming Firmicutes become primary targets for biocrust Caudovirales phages, consistent with the classical "kill-the-winner" hypothesis. IMPORTANCE This work forms part of an overarching research theme studying the effects of a changing climate on biological soil crust (biocrust) in the Southwestern United States. To our knowledge, this study was the first to characterize bacteriophages in biocrust and offers a view into the ecology of phages in response to a laboratory wetting experiment. The phages identified here represent lineages of Caudovirales , and we found that the dynamics of their interactions with their Firmicutes hosts explain the collapse of a bacterial bloom that was induced by wetting. Moreover, we show that phages carried host-altering metabolic genes and found evidence of proviral infection and CRISPR-Cas repeats within host genomes. Our results suggest that phages exert controls on population density by lysing dominant bacterial hosts and that they further impact biocrust by acquiring host genes for sporulation. Future research should explore how dominant these phages are in other biocrust communities and quantify how much the control and lysis of blooming populations contributes to nutrient cycling in biocrusts.

Integrating multiomics datasets is critical for microbiome research; however, inferring interactions across omics datasets has multiple statistical challenges. We solve this problem by using neural networks (https://github.com/biocore/mmvec) to estimate the conditional probability that each molecule is present given the presence of a specific microorganism. We show with known environmental (desert soil biocrust wetting) and clinical (cystic fibrosis lung) examples, our ability to recover microbe-metabolite relationships, and demonstrate how the method can discover relationships between microbially produced metabolites and inflammatory bowel disease.

The molecular composition of soil organic matter (SOM) sets the foundation for terrestrial microbial community structures and carbon cycling dynamics. However, the specific chemical constituents of SOM are underexplored. In this chapter we present a protocol for the extraction of small molecule metabolites from soil followed by compound detection and identification using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. There are options within the protocol to assess either the extracellular pool of metabolites or the total pool (including intracellular) and either polar or nonpolar metabolites, depending on the reader's research interests. These methods can be followed individually for a more targeted analysis or all methods can be combined to obtain a more comprehensive understanding of SOM metabolite composition (such as amino acids, nucleobases, organic acids, fatty acids, carbohydrates, secondary metabolites, and antibiotics).

Metagenomic sequencing provides a window into microbial community structure and metabolic potential; however, linking these data to exogenous metabolites that microorganisms process and produce (the exometabolome) remains challenging. Previously, we observed strong exometabolite niche partitioning among bacterial isolates from biological soil crust (biocrust). Here we examine native biocrust to determine if these patterns are reproduced in the environment. Overall, most soil metabolites display the expected relationship (positive or negative correlation) with four dominant bacteria following a wetting event and across biocrust developmental stages. For metabolites that were previously found to be consumed by an isolate, 70% are negatively correlated with the abundance of the isolate's closest matching environmental relative in situ, whereas for released metabolites, 67% were positively correlated. Our results demonstrate that metabolite profiling, shotgun sequencing and exometabolomics may be successfully integrated to functionally link microbial community structure with environmental chemistry in biocrust.

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the <1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.

Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

Aldehyde oxidase (AOX) metabolizes many xenobiotics in vitro, but its importance in vivo is usually unknown relative to cytochrome P450s (CYPs) and other detoxification systems. Currently, the most important insecticides are neonicotinoids, which are metabolized in vitro by AOX on reduction of the nitroimino group and by CYPs via oxidation reactions. The goal of this study was to establish the relative importance of AOX and CYPs in vivo using the mouse model. The procedure was to reduce liver AOX activity by providing tungsten or hydralazine in the drinking water or to use the AOX-deficient DBA/2 mouse strain. None of these approaches reduced CYP activity measured in vitro with an isozyme nonspecific substrate. Liver AOX activity was reduced by 45% with tungsten and 61% with hydralazine and 81% in AOX-deficient mice relative to controls. When mice were treated ip with the major neonicotinoid imidacloprid (IMI), metabolism by CYP oxidation reactions was not appreciably affected, whereas the AOX-generated nitrosoguanidine metabolite was decreased by 30% with tungsten and 56% with hydralazine and 86% in the AOX-deficient mice. The other IMI nitroreduction metabolite, desnitro-IMI, was decreased by 55%, 65%, and 81% with tungsten, hydralazine, and in the AOX-deficient mice, respectively. Thus, decreasing liver AOX activity by three quite different procedures gave a corresponding decrease for in vivo reductive metabolites in the liver of IMI-treated mice. Possible AOX involvement in IMI metabolism in insects was evaluated using AOX-expressing and AOX-deficient Drosophila, but no differences were found in IMI nitroreduction or sensitivity between the two strains. This is the first study to establish the in vivo relevance of AOX in neonicotinoid metabolism in mammals and one of the first for xenobiotics in general.

Thiamethoxam (TMX), an important insecticide, is hepatotoxic and hepatocarcinogenic in mice but not rats. Studies of Syngenta Central Toxicology Laboratory on species specificity in metabolism established that TMX is a much better substrate for mouse liver microsomal CYPs than the corresponding rat or human enzymes in forming desmethyl-TMX (dm-TMX), which is also hepatotoxic, and clothianidin (CLO), which is not hepatotoxic or hepatocarcinogenic. They proposed that TMX hepatotoxicity/hepatocarcinogencity is due to dm-TMX and a further metabolite desmethyl-CLO (dm-CLO) (structurally analogous to a standard inducible nitric oxide synthase inhibitor) acting synergistically. The present study considers formation of formaldehyde (HCHO) and N-methylol intermediates as an alternative mechanism of TMX hepatotoxicity/hepatocarcinogenicity. Comparison of neonicotinoid metabolism by mouse, rat and human microsomes with NADPH showed two important points. First, TMX and dm-TMX yield more HCHO than any other commercial neonicotinoid. Second, mouse microsomes give much higher conversion than rat or human microsomes. These observations provide an alternative hypothesis of HCHO and N-methylol intermediates from CYP-mediated oxidative oxadiazinane ring cleavage as the bioactivated hepatotoxicants. However, the proposed mono-N-methylol CYP metabolites are not observed, possibly further reacting in situ., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Neonicotinoids not only control insect pests but also sometimes independently alter plant growth and response to stress. We find that imidacloprid, thiacloprid, acetamiprid, thiamethoxam, and clothianidin but not nitenpyram and dinotefuran induce foliar lesions and peroxidative damage in soybean ( Glycine max ) seedlings assayed with the 3,3'-diaminobenzidine stain. The chloropyridinyl-carboxylic acid (COOH) but not the -carboxaldehyde (CHO) metabolites induce peroxidative damage but in a different pattern. Surprisingly, the chlorothiazolyl -CHO and -COOH metabolites induce chlorosis but no clear superimposable peroxidative damage or cell death. Four metallo-oxidases known to modulate reactive oxygen species were not sensitive in vitro to the parent neonicotinoid itself but were to several CHO and COOH metabolites and related compounds, with a sensitivity order of CHO > COOH and tyrosinase > xanthine oxidase and aldehyde oxidase > catalase. Although metallo-oxidase inhibition does not correlate overall with lesion formation, it may play an as yet unknown role in plant response to neonicotinoids.

Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo.

Prominent features of atheromata include smooth muscle cells, cholesteryl ester-loaded macrophage foam cells, extracellular matrix, extracellularly trapped and aggregated lipoproteins, and various enzymes including lipoprotein lipase (LpL) and sphingomyelinase (SMase). The interplay of these factors was investigated in cell culture. Incubation of bovine aortic smooth muscle cells for 18 h at 37 degrees C with low density lipoprotein (LDL) in the presence of LpL and SMase led to massive aggregation of LDL on the surface of the cells as viewed by phase, fluorescence (using 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-LDL), and electron microscopy. This aggregation required both enzymes. Studies with 125I-LDL confirmed these observations: 125I-LDL cell association in the presence of LpL plus SMase was 50-100-fold greater than in the absence of the two enzymes and was 10-fold greater than in the presence of either enzyme alone. A similar effect (68-fold enhancement) was seen with 125I-labeled lipoprotein(a) (Lp(a)), another atherogenic lipoprotein. In all cases, 125I-lipoprotein degradation was relatively low (< 5% of cell-associated material). LpL/SMase-mediated association of 125I-LDL with smooth muscle cells was still observed when enzymatically inactive LpL was used. The effect was markedly diminished when the smooth muscle cells were treated with a combination of chondroitin ABC lyase and heparitinase or when mutant Chinese hamster ovary cells that lack cell-surface proteoglycans were used, indicating a specific role for cellular proteoglycans. When smooth muscle cells with 125I-LDL or 125I-Lp(a) aggregates were rinsed and then coincubated with mouse peritoneal macrophages for a further 24 h, visible aggregates disappeared, and there was marked 125I-lipoprotein degradation. Electron micrographs after 24 h of co-culture showed lipid-laden, foamy macrophages situated on top of smooth muscle cells, suggesting that the macrophages phagocytosed and metabolized the smooth muscle cell-associated LDL aggregates. Last, 125I-LDL association with smooth muscle cell extracellular matrix was also synergistically enhanced by LpL and SMase, to a level that was 19-fold greater than in the absence of the two enzymes. Thus, the interaction of LDL and Lp(a) with four atheroma components, namely, smooth muscle cells, extracellular matrix, LpL, and SMase, represents a physiologically plausible mechanism for massive, focal retention and aggregation of atherogenic lipoproteins in the arterial wall with subsequent macrophage foam cell formation.

We sought to investigate effects of lipoprotein lipase (LpL) on cellular catabolism of lipoproteins rich in apolipoprotein B-100. LpL increased cellular degradation of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) by 277% +/- 3.8% and 32.5% +/- 4.1%, respectively, and cell association by 509% +/- 8.7% and 83.9% +/- 4.0%. The enhanced degradation was entirely lysosomal. Enhanced degradation of Lp(a) had at least two components, one LDL receptor-dependent and unaffected by heparitinase digestion of the cells, and the other LDL receptor-independent and heparitinase-sensitive. The effect of LpL on LDL degradation was entirely LDL receptor-independent, heparitinase-sensitive, and essentially absent from mutant Chinese hamster ovary cells that lack cell surface heparan sulfate proteoglycans. Enhanced cell association of Lp(a) and LDL was largely LDL receptor-independent and heparitinase-sensitive. The ability of LpL to reduce net secretion of apolipoprotein B-100 by HepG2 cells by enhancing cellular reuptake of nascent lipoproteins was also LDL receptor-independent and heparitinase-sensitive. None of these effects on Lp(a), LDL, or nascent lipoproteins required LpL enzymatic activity. We conclude that LpL promotes binding of apolipoprotein B-100-rich lipoproteins to cell surface heparan sulfate proteoglycans. LpL also enhanced the otherwise weak binding of Lp(a) to LDL receptors. The heparan sulfate proteoglycan pathway represents a novel catabolic mechanism that may allow substantial cellular and interstitial accumulation of cholesteryl ester-rich lipoproteins, independent of feedback inhibition by cellular sterol content.

We showed previously that net secretory output of apolipoprotein B (apo B) from cultured human hepatoma cells (HepG2) is regulated by rapid reuptake of nascent lipoproteins before they have diffused away from the vicinity of the cells. We now sought to determine if the nascent lipoproteins could be remodeled to enhance or impede reuptake. We found that lipoprotein lipase (LpL), an enzyme that hydrolyzes lipoprotein triglyceride, reduced HepG2 output of apo B to one-quarter to one-half of control. The reduction was apparent during co-incubations as short as 2 h and as long as 24 h. Heparin, which blocks receptor-mediated binding of lipoproteins, abolished the effect of LpL on apo B output, without causing enzyme inhibition. To assess uptake directly, we prepared labeled nascent lipoproteins. LpL tripled the cellular uptake of labeled nascent lipoproteins, from 15.2% +/- 0.7% to 48.7% +/- 0.3% of the total applied to the cells. Cellular uptake of 125I-labeled anti-LDL receptor IgG was unaffected by LpL; thus, LpL enhanced reuptake by altering lipoproteins, not receptors. Because LpL is present in the space of Disse in the liver, we conclude that LpL may act on newly secreted lipoproteins to enhance reuptake in vivo. LpL deficiency would reduce local reuptake of apo B, which would appear as overproduction, thereby providing a mechanistic link between partial LpL deficiency and familial combined hyperlipidemia.

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Three monoclonal neutralizing antibodies to the CETP were obtained by immunizing mice with purified CETP. The antibodies, each recognizing a similar epitope on CETP, caused parallel and complete immunotitration of plasma cholesteryl ester and triglyceride transfer activities but only partial inhibition of phospholipid transfer activity. Monoclonal immunoaffinity chromatography of plasma or its fractions showed complete removal of cholesteryl ester and triglyceride transfer activities but incomplete removal of phospholipid transfer activity. Sodium dodecyl sulfate gel electrophoresis and immunoblotting of the immunoaffinity-retained fractions showed that only the Mr 74,000 protein was immunoreactive. The results suggest that the previously characterized CETP accounts for all of the cholesteryl ester and triglyceride transfer activity in human plasma but only part of the phospholipid transfer activity.

The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon.

The plasma cholesteryl ester-transfer protein (CETP, Mr 74,000) promotes exchange of both neutral lipids and phospholipids (phosphatidylcholine, PC) between lipoproteins. To investigate the mechanism of facilitated lipid transfer, CETP was incubated with unilamellar egg PC vesicles containing small amounts of cholesteryl ester (CE) or triglyceride, and then analyzed by gel filtration chromatography. There was rapid transfer of radiolabeled CE or triglyceride and PC from vesicles to CETP. The CETP with bound lipids was isolated and incubated with low density lipoproteins (LDL), resulting in transfer of the lipids to LDL. The CETP bound up to 0.9 mol of CE or 0.2 mol of triglyceride and 11 mol of PC/mol of CETP. para-Chloromercuriphenylsulfonate, an inhibitor of CE and triglyceride transfer, was found to decrease the binding of radiolabeled CE and triglyceride by CETP. Under various conditions the CETP eluted either as an apparent monomer with bound lipid (Mr 75,000-93,000), or in complexes with vesicles. The distribution of CETP between these two states was influenced by the presence of apoA-I or albumin, incubation time, vesicle/CETP ratio, and buffer pH and ionic strength. The results indicate that the CETP has binding sites for CE, triglyceride, and PC which readily equilibrate with lipoprotein lipids and suggest that CETP can act as a carrier of lipid between lipoproteins.

The effect of inhibiting cholesteryl ester transfer protein (CETP) on the in vitro redistribution of apolipoproteins(apo) A-IV and apoE among lipoproteins in whole plasma was studied in seven normal male subjects. Plasmas were incubated in the presence of a purified monoclonal antibody TP2 (Mab TP2) that neutralizes the activity of CETP. Mab TP2 had no effect on lecithin:cholesterol acyltransferase (LCAT) activity. Prior to and following a 6-h incubation at 37 degrees C in the presence of Mab TP2 or a control mouse myeloma immunoglobulin (IgG), plasmas were gel-filtered on Sephacryl S-300 and the distribution of apoA-IV and apoE among lipoproteins was determined by radioimmunoassay. Incubation (i.e., with active LCAT and CETP) increased the amount of apoA-IV associated with lipoproteins by 240%. When CETP activity was inhibited during incubation, the amount of apoA-IV that became lipoprotein-associated was significantly increased (315% of basal). Plasma incubation also caused a redistribution of apoE from high density lipoproteins (HDL) to larger lipoproteins (131% of basal); however, when CETP was inhibited, significantly greater amounts of apoE became associated with the larger particles (155% of basal). These effects were observed in all seven subjects. Increased movement of apoE from HDL to triglyceride-rich particles was not due to displacement by apoA-IV since loss of apoE from HDL was still observed when no movement of apoA-IV onto HDL occurred, such as during LCAT or combined LCAT and CETP inhibition. We speculate that low CETP activity (e.g., in species such as rats) may lead to an increased content of HDL apoA-IV and also to apoE enrichment of triglyceride-rich lipoproteins, augmenting their clearance.

Cholesteryl ester transfer protein (CETP) promotes in vitro transfer of cholesteryl ester (CE) and triglyceride (TG) between lipoproteins. We studied the function of CETP in vivo in rabbit lipoprotein metabolism using a neutralizing monoclonal antibody (MAb, TP1) to CETP. Rabbits were injected with TP1 (n = 8), or irrelevant MAb or saline (control, n = 8), resulting in an initial 71% inhibition of CETP, which fell to 45% after 48 h. HDL CE rose in the inhibited animals, reaching levels that doubled initial and control values at 48 h (P less than 0.001). HDL TG fell reciprocally, but HDL protein did not change, suggesting a CE for TG exchange. VLDL CE/TG decreased. Rabbits were also given [3H]cholesteryl ether HDL (a CE analogue). CETP inhibition delayed the initial clearance of radioactivity from HDL (control 6.8 vs. TP1 4.1 pools/d) and plasma (7.8 vs. 5.2 pools/d). We conclude that CETP plays a quantitatively important role in HDL CE catabolism in the rabbit, promoting the exchange of TG for CE and the clearance of CE from plasma.

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.

Current studies on the synthesis of long-chain fatty acids by isolated rat liver cells are largely concerned with the regulation of the activity of previously existing acetyl-CoA carboxylase and fatty acid synthetase, and with the regulation of the quantity of these enzymes. These studies have required the development of methods for obtaining high yields of viable hepatocytes that respond to hormonal treatment. Such methods have been developed over the past 10-15 years through the efforts of several laboratories. These studies have also required the development of a method to determine whether a change in the activity of an enzyme is due to a modification of preexisting enzyme or to a change in quantity of that enzyme. The most satisfactory method to use for such studies is immunotitration of enzyme activity. In recent years studies on the regulation of acetyl-CoA carboxylase have largely centered upon the effect of phosphorylation-dephosphorylation on the activity of this enzyme and whether glucagon inhibits the activity of this enzyme through this process. Much data from a number of laboratories have suggested that glucagon regulates the activity of this enzyme through phosphorylation-dephosphorylation. However, several of these studies involved the use of crude systems in which competing enzymes and substrates that can significantly interfere with acetyl-CoA carboxylase activity measurements were still present. Hence, a confirmation of these studies needs to be carried out under conditions in which the effects of competing enzymes and substrates are eliminated. Studies on changes in quantity of acetyl-CoA carboxylase and fatty acid synthetase have shown that these enzymes are induced by the fasting and refeeding of animals. They have also shown that insulin stimulates (10- to 30-fold) the induction of these enzymes. This induction appears to be due to a change in the quantity of translatable mRNA which may, in turn, be due to a change in the rate of transcription of the genes coding for these enzymes.

A soluble protein that binds malonyl-CoA without requiring cofactors has been purified from rat liver. Until saturated, it competes with fatty acid synthetase for free malonyl-CoA, temporarily reducing the rate of fatty acid synthesis at low levels of malonyl-CoA, as in fatty acid synthetase--coupled assays for acetyl-CoA carboxylase. These assays yield low estimates for carboxylase activity with crude and partially purified homogenates containing the malonyl-CoA-binding protein. The protein does not inhibit assays for carboxylase activity that measure nonvolatile radioactivity incorporated from bicarbonate or NADH oxidation coupled to ADP formation. It has an Mr of 180,000 and a subunit of 90,000. It has a lower affinity for ATP, ADP, and acetyl-CoA and none for CO2 or fatty acid synthetase. No enzymatic function has been identified. The protein may regulate malonyl-CoA-binding enzymes.