mobileOG-db: a Manually Curated Database of Protein Families Mediating the Life Cycle of Bacterial Mobile Genetic Elements.

Bacterial mobile genetic elements (MGEs) encode functional modules that perform both core and accessory functions for the element, the latter of which are often only transiently associated with the element. The presence of these accessory genes, which are often close homologs to primarily immobile genes, incur high rates of false positives and, therefore, limits the usability of these databases for MGE annotation. To overcome this limitation, we analyzed 10,776,849 protein sequences derived from eight MGE databases to compile a comprehensive set of 6,140 manually curated protein families that are linked to the "life cycle" (integration/excision, replication/recombination/repair, transfer, stability/transfer/defense, and phage-specific processes) of plasmids, phages, integrative, transposable, and conjugative elements. We overlay experimental information where available to create a tiered annotation scheme of high-quality annotations and annotations inferred exclusively through bioinformatic evidence. We additionally provide an MGE-class label for each entry (e.g., plasmid or integrative element), and assign to each entry a major and minor category. The resulting database, mobileOG-db (for mobile orthologous groups), comprises over 700,000 deduplicated sequences encompassing five major mobileOG categories and more than 50 minor categories, providing a structured language and interpretable basis for an array of MGE-centered analyses. mobileOG-db can be accessed at mobileogdb.flsi.cloud.vt.edu/, where users can select, refine, and analyze custom subsets of the dynamic mobilome. IMPORTANCE The analysis of bacterial mobile genetic elements (MGEs) in genomic data is a critical step toward profiling the root causes of antibiotic resistance, phenotypic or metabolic diversity, and the evolution of bacterial genera. Existing methods for MGE annotation pose high barriers of biological and computational expertise to properly harness. To bridge this gap, we systematically analyzed 10,776,849 proteins derived from eight databases of MGEs to identify 6,140 MGE protein families that can serve as candidate hallmarks, i.e., proteins that can be used as "signatures" of MGEs to aid annotation. The resulting resource, mobileOG-db, provides a multilevel classification scheme that encompasses plasmid, phage, integrative, and transposable element protein families categorized into five major mobileOG categories and more than 50 minor categories. mobileOG-db thus provides a rich resource for simple and intuitive element annotation that can be integrated seamlessly into existing MGE detection pipelines and colocalization analyses.

Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23.

The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood-brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP(+)) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.

Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models.

We hypothesized that in epileptic brains citric acid cycle intermediate levels may be deficient leading to hyperexcitability. Anaplerosis is the metabolic refilling of deficient metabolites. Our goal was to determine the anticonvulsant effects of feeding triheptanoin, the triglyceride of anaplerotic heptanoate. CF1 mice were fed 0-35% calories from triheptanoin. Body weights and dietary intake were similar in mice fed triheptanoin vs. standard diet. Triheptanoin feeding increased blood propionyl-carnitine levels, signifying its metabolism. 35%, but not 20%, triheptanoin delayed development of corneal kindled seizures. After pilocarpine-induced status epilepticus (SE), triheptanoin feeding increased the pentylenetetrazole tonic seizure threshold during the chronically epileptic stage. Mice in the chronically epileptic stage showed various changes in brain metabolite levels, including a reduction in malate. Triheptanoin feeding largely restored a reduction in propionyl-CoA levels and increased methylmalonyl-CoA levels in SE mice. In summary, triheptanoin was anticonvulsant in two chronic mouse models and increased levels of anaplerotic precursor metabolites in epileptic mouse brains. The mechanisms of triheptanoin's effects and its efficacy in humans suffering from epilepsy remain to be determined.

Estrogen effects on high-affinity choline uptake in primary cultures of rat basal forebrain.

Basal forebrain cholinergic neurons (BFCNs) degenerate in aging and Alzheimer's disease. It has been proposed that estrogen can affect the survival and function of BFCNs. This study characterized primary rat BFCN cultures and investigated the effect of estrogen on high-affinity choline uptake (HACU). BFCNs were identified by immunoreactivity to the vesicular acetylcholine transporter (VAChT) and represented up to 5% of total cells. HACU was measured in living BFCN cultures and differentiated from low-affinity choline uptake by hemicholinium-3 (HC-3) inhibition. A HC-3 concentration curve showed that 0.3 muM HC-3, but not higher concentrations that inhibit LACU, could distinguish the two transport activities. 17-beta-Estradiol treatment increased HACU in some culture preparations that contained non-neuronal cells. Elimination of dividing cells using antimitotic treatments resulted in a lack of estrogen effects on HACU. These results suggest that estrogen may have indirect effects on BFCNs that are mediated through non-neuronal cells.

Interbody device endplate engagement effects on motion segment biomechanics.

BACKGROUND CONTEXT: Stand-alone nonbiologic interbody fusion devices for the lumbar spine have been used for interbody fusion since the early 1990s. However, most devices lack the stability found in clinically successful circumferential fusion constructs. Stability results from cage geometry and device/vertebral endplate interface integrity. To date, there has not been a published comparative biomechanical study specifically evaluating the effects of endplate engagement of interbody devices. PURPOSE: Lumbar motion segments implanted with three different interbody devices were tested biomechanically to compare the effects of endplate engagement on motion segment rigidity. The degree of additional effect of supplemental posterior and anterior fixation was also investigated. STUDY DESIGN/SETTING: A cadaveric study of interbody fusion devices with varying degrees of endplate interdigitation. OUTCOME MEASURES: Implanted motion segment range of motion (ROM), neutral zone (NZ), stiffness, and disc height. METHODS: Eighteen human L23 and L45 motion segments were distributed into three interbody groups (n=6 each) receiving a polymeric (polyetheretherketone) interbody spacer with small ridges; a modular interbody device with endplate spikes (InFix, Abbott Spine, Austin, TX, USA); or dual tapered threaded interbody cages (LT [Lordotic tapered] cage; Medtronic, Memphis, TN, USA). Specimens were tested intact using a 7.5-Nm flexion-extension, lateral bending, and axial torsion flexibility protocol. Testing was repeated after implantation of the interbody device, anterior plate fixation, and posterior interpedicular fixation. Radiographic measurements determined changes in disc height and intervertebral lordosis. ROM and NZ were calculated and compared using analysis of variance. RESULTS: The interbody cages with endplate spikes or threads provided a statistically greater increase in disc height versus the polymer spacer (p=.01). Relative to intact, all stand-alone devices significantly reduced ROM in lateral bending by a mean 37% to 61% (p< or =.001). The cages with endplate spikes or threads reduced ROM by approximately 50% and NZ by approximately 60% in flexion-extension (p< or =.02). Only the cage with endplate spikes provided a statistically significant reduction in axial torsion ROM compared with the intact state (50% decrease, p<.001). Posterior fixation provided a significant reduction in ROM in all directions versus the interbody device alone (p<.001). Anterior plating decreased ROM over interbody device alone in flexion-extension and torsion but did not have additional effect on lateral bending ROM. CONCLUSION: The cages with endplate spikes or threads provide substantial motion segment rigidity compared with intact in bending modes. Only the cages with endplate spikes were more rigid than intact in torsion. All devices experienced increased rigidity with anterior plating and even greater rigidity with posterior fixation. It appears that the endplate engagement with spikes may be beneficial in limiting torsion, which is generally difficult with other "stand-alone" devices tested in the current and prior reports.

NGP1-01 is a brain-permeable dual blocker of neuronal voltage- and ligand-operated calcium channels.

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels. NGP1-01 (1 microM) inhibited depolarization-induced calcium influx by 78% in cortical neurons preloaded with fura-2 AM, with a potency similar to nimodipine. NGP1-01 (1 microM) also inhibited N-methyl-D: -aspartate (NMDA)-induced (1 mM) calcium influx by 52%, only slightly less potent than memantine. Using in vivo-microdialysis, we monitored choline release during NMDA infusion as a measure of excitotoxic membrane breakdown. Intraperitoneal injection of NGP1-01 (40 mg/kg) reduced NMDA-induced membrane breakdown by 31% (P < 0.01) while memantine (10 mg/kg) reduced choline release by 40%. Our results demonstrate that NGP1-01 simultaneously blocks both major neuronal calcium channels and is sufficiently brain-permeable. We conclude that NGP1-01 is a promising lead structure for a new class of dual-mechanism neuroprotective agents.