Uridine Bisphosphonates Differentiate Phosphoglycosyl Transferase Superfamilies.

Complex bacterial glycoconjugates drive interactions between pathogens, symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphosphosugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). The two distinct superfamilies of PGT enzymes (polytopic and monotopic) show striking differences in their structure and mechanism. We designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate of the UDP and UDP-sugar is replaced by a substituted methylene bisphosphonate (CXY-BPs; X/Y = F/F, Cl/Cl, (S)-H/F, (R)-H/F, H/H, CH3/CH3). UBPs and UBPs incorporating an N-acetylglucosamine (GlcNAc) substituent at the ß-phosphonate were evaluated as inhibitors of a polytopic PGT (WecA from Thermotoga maritima) and a monotopic PGT (PglC from Campylobacter jejuni). Although CHF-BP most closely mimics diphosphate with respect to its acid/base properties, the less basic CF2-BP conjugate more strongly inhibited PglC, whereas the more basic CH2-BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies, implicating a modified P-O- interaction with the structural Mg2+. For the monoPGT enzyme, the two diastereomeric CHF-BP conjugates, which feature a chiral center at the Pα-CHF-Pß carbon, also exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native α-anomer configuration significantly improved binding affinity. UBP-sugars are thus revealed as informative new mechanistic probes of PGTs that may aid development of novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily.

Characterization of PglJ, a Glycosyltransferase in the Campylobacter concisus N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The Campylobacter genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As Campylobacter concisus is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of C. concisus strains have been reported to diverge from other Campylobacter in steps after the biosynthesis of N-acetylgalactosamine-α1,3-N,N'-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of C. concisus. Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the Pseudomonas aeruginosa WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the Campylobacter genus. These studies now allow detailed characterization of the later steps in the pgl pathway in C. concisus strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.

Uridine Bisphosphonates Differentiate Phosphoglycosyl Transferase Superfamilies.

Complex bacterial glycoconjugates are essential for bacterial survival, and drive interactions between pathogens and symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphospho-sugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). Two distinct superfamilies of PGT enzymes, denoted as polytopic and monotopic, carry out this reaction but show striking differences in structure and mechanism. With the goal of creating non-hydrolyzable mimics (UBP-sugars) of the UDP-sugar substrates as chemical probes to interrogate critical aspects of these essential enzymes, we designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate bridging oxygen of the UDP and UDP-sugar is replaced by a substituted methylene group (CXY; X/Y = F/F, Cl/Cl, (S)-H/F, (R)-H/F, H/H, CH3/CH3). These compounds, which incorporated as the conjugating sugar an N-acetylglucosamine (GlcNAc) substituent at the ß-phosphonate, were evaluated as inhibitors of a representative polytopic PGT (WecA from Thermotoga maritima) and a monotopic PGT (PglC from Campylobacter jejuni). Although CHF-BP most closely mimics pyrophosphate with respect to its acid/base properties, the less basic CF2-BP conjugate most strongly inhibited PglC, whereas the more basic CH2-BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies implicating a modified P-O- interaction with the structural Mg2+, consistent with their catalytic divergence. Furthermore, at least for the monoPGT superfamily example, this was not the sole determinant of ligand binding: the two diastereomeric CHF-BP conjugates, which feature a chiral center at the Pα-CHF-Pß carbon, exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native α-anomer configuration significantly improved binding affinity. UBP-sugars are a valuable tool for elucidating the structures and mechanisms of the distinct PGT superfamilies and offer a promising scaffold to develop novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily.

Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

Defining early steps in Bacillus subtilis biofilm biosynthesis.

IMPORTANCE: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here, we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide (EPS) synthesis pathway. Together, our studies and approaches provide the foundation for the sequential characterization of the steps in EPS biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenyl diphosphate-linked glycan substrates.

User-centered design of feedback regarding health-related behaviors derived from wearables: An approach targeting older adults and persons living with neurodegenerative disease.

Objective: There has been tremendous growth in wearable technologies for health monitoring but limited efforts to optimize methods for sharing wearables-derived information with older adults and clinical cohorts. This study aimed to co-develop, design and evaluate a personalized approach for information-sharing regarding daily health-related behaviors captured with wearables. Methods: A participatory research approach was adopted with: (a) iterative stakeholder, and evidence-led development of feedback reporting; and (b) evaluation in a sample of older adults (n = 15) and persons living with neurodegenerative disease (NDD) (n = 25). Stakeholders included persons with lived experience, healthcare providers, health charity representatives and individuals involved in aging/NDD research. Feedback report information was custom-derived from two limb-mounted inertial measurement units and a mobile electrocardiography device worn by participants for 7-10 days. Mixed methods were used to evaluate reporting 2 weeks following delivery. Data were summarized using descriptive statistics for the group and stratified by cohort and cognitive status. Results: Participants (n = 40) were 60% female (median 72 (60-87) years). A total of 82.5% found the report easy to read or understand, 80% reported the right amount of information was shared, 90% found the information helpful, 92% shared the information with a family member or friend and 57.5% made a behavior change. Differences emerged in sub-group comparisons. A range of participant profiles existed in terms of interest, uptake and utility. Conclusions: The reporting approach was generally well-received with perceived value that translated into enhanced self-awareness and self-management of daily health-related behaviors. Future work should examine potential for scale, and the capacity for wearables-derived feedback to influence longer-term behavior change.

A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles.

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Defining Early Steps in B. subtilis Biofilm Biosynthesis.

The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the exopolysaccharide of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the exopolysaccharide biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the B. subtilis biofilm exopolysaccharide biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using UDP-di- N -acetyl bacillosamine as phospho-sugar donor. EpsD is a GT-B fold glycosyl transferase that facilitates the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP- N -acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing exopolysaccharide unit. In doing so we provide the first evidence of the presence of bacillosamine in an exopolysaccharide synthesized by a Gram-positive bacterium. IMPORTANCE: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide synthesis pathway. Together our studies and approaches provide the foundation for the sequential characterization of the steps in exopolysaccharide biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenol diphosphate-linked glycan substrates.

Incidence and implications of atrial fibrillation in patients hospitalized for COVID compared to non-COVID pneumonia: A multicenter cohort study.

Background: Atrial fibrillation (AF) has been reported to occur with coronavirus disease 2019 (COVID-19), but whether it is related to myocarditis or lung injury is unclear. Objectives: The purpose of this study was to compare incident AF in patients with pneumonia/adult respiratory distress syndrome (ARDS) with and without COVID. Methods: This retrospective multicenter cohort study from 17 hospitals (March 2020 to December 2021) utilizing the University of California COVID Research Data Set (CORDS) included patients aged ≥18 years with primary diagnosis of pneumonia or ARDS during hospitalization. Patients with a history of AF were excluded. All subjects had documented COVID test results. Cohorts were compared using the χ2 test for categorical variables and the Wilcoxon rank test for continuous variables. Multivariable logistic regression models were used to investigate the association between COVID and development of new AF. Results: Of the 39,415 subjects, 12.2% had COVID. The COVID+ cohort consisted predominantly of younger males with more comorbidities. Incident AF was lower in the COVID+ group than in the non-COVID group (523 [10.85%] vs 4899 [14.16%]; odds ratio [OR] 0.74; P <.001), which remained significant after adjustment for demographics and comorbidities (OR 0.71; P <.001). Patients had normal cardiac troponin levels. AF was related to intensive care unit care, pressor support, and mechanical ventilation, and was associated with higher mortality (26.2% vs 10.21%; P <.001) and longer hospitalization (22.5 vs 15.1 days; P <.001) in the COVID+ group compared to the controls. Conclusion: Incident AF is lower in COVID+ compared to non-COVID pneumonia/ARDS patients and seems to be related to severity of illness rather cardiac injury. AF was associated with higher mortality and prolonged hospitalization.

Noninvasive Stereotactic Radiation for Refractory Ventricular Tachycardia After Failure of Cardiac Sympathetic Denervation.

Stereotactic body radiation therapy is a novel treatment option for refractory ventricular tachycardia. We present a case of ventricular tachycardia, with epicardial origin located in large inferior infarct scar, that recurred despite treatment with multiple antiarrhythmic drugs, catheter ablation, and cardiac sympathetic denervation. Stereotactic body radiation therapy safely and effectively terminated the arrhythmia. (Level of Difficulty: Advanced.).

Catheter Ablation for Atrial Fibrillation in Adult Congenital Heart Disease: An International Multicenter Registry Study.

BACKGROUND: Data on atrial fibrillation (AF) ablation and outcomes are limited in patients with congenital heart disease (CHD). We aimed to investigate the characteristics of patients with CHD presenting for AF ablation and their outcomes. METHODS: A multicenter, retrospective analysis was performed of patients with CHD undergoing AF ablation between 2004 and 2020 at 13 participating centers. The severity of CHD was classified using 2014 Pediatric and Congenital Electrophysiology Society/Heart Rhythm Society guidelines. Clinical data were collected. One-year complete procedural success was defined as freedom from atrial tachycardia or AF in the absence of antiarrhythmic drugs or including previously failed antiarrhythmic drugs (partial success). RESULTS: Of 240 patients, 127 (53.4%) had persistent AF, 62.5% were male, and mean age was 55.2±13.3 years. CHD complexity categories included 147 (61.3%) simple, 68 (28.3%) intermediate, and 25 (10.4%) severe. The most common CHD type was atrial septal defect (n=78). More complex CHD conditions included transposition of the great arteries (n=14), anomalous pulmonary veins (n=13), tetralogy of Fallot (n=8), cor triatriatum (n=7), single ventricle physiology (n=2), among others. The majority (71.3%) of patients had trialed at least one antiarrhythmic drug. Forty-six patients (22.1%) had reduced systemic ventricular ejection fraction <50%, and mean left atrial diameter was 44.1±8.2 mm. Pulmonary vein isolation was performed in 227 patients (94.6%); additional ablation included left atrial linear ablations (40%), complex fractionated atrial electrogram (19.2%), and cavotricuspid isthmus ablation (40.8%). One-year complete and partial success rates were 45.0% and 20.5%, respectively, with no significant difference in the rate of complete success between complexity groups. Overall, 38 patients (15.8%) required more than one ablation procedure. There were 3 (1.3%) major and 13 (5.4%) minor procedural complications. CONCLUSIONS: AF ablation in CHD was safe and resulted in AF control in a majority of patients, regardless of complexity. Future work should address the most appropriate ablation targets in this challenging population.

Iatrogenic Atrial Septal Defect Closure Through the Steerable Guide Catheter: Description of Technique and Single-Center Experience.

OBJECTIVES: To introduce a novel method of direct iatrogenic atrial septal defect (iASD) closure through the MitraClip steerable guide catheter (SGC). BACKGROUND: MitraClip implantation requires transseptal puncture and the creation of an iASD. There are relatively rare instances, such as hemodynamically significant shunting or concerns for embolus, where iASD must be closed during index procedure. In these instances, it may be beneficial to not give up access to left atrium. METHODS: We retrospectively reviewed all iASD closures during MitraClip implantation at our institution from 2015 to 2020. Cases where an ASD occluder was deployed directly through SGC were included. RESULTS: Eleven patients had immediate iASD closure through the SGC. Indications for using this method included concern for paradoxical embolus, large defect size and/or significant shunting. Closure device sizes ranged from 8 to 22 mm. Mean time from removal of clip delivery system to occlusion of iASD was 14.6 minutes. There were no procedural complications related to iASD closure using this method. CONCLUSION: Closure of iASD intra-procedurally directly through transseptal guide sheath via the method described was safe and allowed for continuous left atrium access.

A 70-Year-Old Man With Relapsed CNS Lymphoma Has Incidental Finding of Right Atrial Mass.

CASE PRESENTATION: A 70-year-old man was admitted to the hospital for planned chemotherapy for recently diagnosed CNS lymphoma. His medical history included follicular lymphoma (achieved remission 1 year prior with chemotherapy) and tonic-clonic seizure 1 month prior to admission, which led to his eventual biopsy-confirmed diagnosis of CNS lymphoma. Physical examination revealed temperature 36.4 °C, heart rate of 60 beats/min, BP of 160/81 mm Hg, and 98% oxygen saturation on room air. Neurologic condition, including mental status examination, was normal. His cardiac examination revealed regular rate and rhythm with normal first and second heart sounds without murmurs, rubs, or gallops. The remainder of the examination was unremarkable. Review of systems noted progressive and intermittent confusion prior to his seizure. He denied any shortness of breath, dyspnea on exertion, orthopnea, lower extremity edema, palpitations, or syncope. Laboratory data were unremarkable.

The surprising structural and mechanistic dichotomy of membrane-associated phosphoglycosyl transferases.

Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol phosphate. Studies on the PGTs have been hampered because they are integral membrane proteins, and often prove to be recalcitrant to expression, purification and analysis. However, in recent years exciting new information has been derived on the structures and the mechanisms of PGTs, revealing the existence of two unique superfamilies of PGT enzymes that enact catalysis at the membrane interface. Genome neighborhood analysis shows that these superfamilies, the polytopic PGT (polyPGT) and monotopic PGT (monoPGT), may initiate different pathways within the same organism. Moreover, the same fundamental two-substrate reaction is enacted through two different chemical mechanisms with distinct modes of catalysis. This review highlights the structural and mechanistic divergence between the PGT enzyme superfamilies and how this is reflected in differences in regulation in their varied glycoconjugate biosynthesis pathways.

An Excitatory and Epileptogenic Effect of Dentate Gyrus Mossy Cells in a Mouse Model of Epilepsy.

The sparse activity of hippocampal dentate gyrus (DG) granule cells (GCs) is thought to be critical for cognition and behavior, whereas excessive DG activity may contribute to disorders such as temporal lobe epilepsy (TLE). Glutamatergic mossy cells (MCs) of the DG are potentially critical to normal and pathological functions of the DG because they can regulate GC activity through innervation of GCs or indirectly through GABAergic neurons. Here, we test the hypothesis that MC excitation of GCs is normally weak, but under pathological conditions, MC excitation of GCs is dramatically strengthened. We show that selectively inhibiting MCs during severe seizures reduced manifestations of those seizures, hippocampal injury, and chronic epilepsy. In contrast, selectively activating MCs was pro-convulsant. Mechanistic in vitro studies using optogenetics further demonstrated the unanticipated ability of MC axons to excite GCs under pathological conditions. These results demonstrate an excitatory and epileptogenic effect of MCs in the DG.

Novelty and Novel Objects Increase c-Fos Immunoreactivity in Mossy Cells in the Mouse Dentate Gyrus.

The dentate gyrus (DG) and its primary cell type, the granule cell (GC), are thought to be critical to many cognitive functions. A major neuronal subtype of the DG is the hilar mossy cell (MC). MCs have been considered to play an important role in cognition, but in vivo studies to understand the activity of MCs during cognitive tasks are challenging because the experiments usually involve trauma to the overlying hippocampus or DG, which kills hilar neurons. In addition, restraint typically occurs, and MC activity is reduced by brief restraint stress. Social isolation often occurs and is potentially confounding. Therefore, we used c-fos protein expression to understand when MCs are active in vivo in socially housed adult C57BL/6 mice in their home cage. We focused on c-fos protein expression after animals explored novel objects, based on previous work which showed that MCs express c-fos protein readily in response to a novel housing location. Also, MCs are required for the training component of the novel object location task and novelty-encoding during a food-related task. GluR2/3 was used as a marker of MCs. The results showed that MC c-fos protein is greatly increased after exposure to novel objects, especially in ventral DG. We also found that novel objects produced higher c-fos levels than familiar objects. Interestingly, a small subset of neurons that did not express GluR2/3 also increased c-fos protein after novel object exposure. In contrast, GCs appeared relatively insensitive. The results support a growing appreciation of the role of the DG in novelty detection and novel object recognition, where hilar neurons and especially MCs are very sensitive.

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis.

Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.

Effective Modulation of Male Aggression through Lateral Septum to Medial Hypothalamus Projection.

Aggression is a prevalent behavior in the animal kingdom that is used to settle competition for limited resources. Given the high risk associated with fighting, the central nervous system has evolved an active mechanism to modulate its expression. Lesioning the lateral septum (LS) is known to cause "septal rage," a phenotype characterized by a dramatic increase in the frequency of attacks. To understand the circuit mechanism of LS-mediated modulation of aggression, we examined the influence of LS input on the cells in and around the ventrolateral part of the ventromedial hypothalamus (VMHvl)-a region required for male mouse aggression. We found that the inputs from the LS inhibited the attack-excited cells but surprisingly increased the overall activity of attack-inhibited cells. Furthermore, optogenetic activation of the projection from LS cells to the VMHvl terminated ongoing attacks immediately but had little effect on mounting. Thus, LS projection to the ventromedial hypothalamic area represents an effective pathway for suppressing male aggression.

Potential implications of a monosynaptic pathway from mossy cells to adult-born granule cells of the dentate gyrus.

The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs - and therefore the output of the DG - is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory 'gate' functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the "dormant basket cell hypothesis." Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the "dormant immature granule cell hypothesis."

Targeting cells with single vectors using multiple-feature Boolean logic.

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.