Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

Structure, aggregation dynamics and crystallization of superfolder green fluorescent protein: Effect of long alkyl chain imidazolium ionic liquids.

Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-Cn-3-methylimidazolium chloride ionic liquid (IL) series ([Cnmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore fluorescence, particularly in long alkyl chain ILs ([C10mim]Cl and [C12mim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [C10mim]Cl and [C12mim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [C10mim]Cl and [C12mim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [C2mim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.

Small angle X-ray scattering investigation of ionic liquid effect on the aggregation behavior of globular proteins.

Globular proteins are well-folded model proteins, where ions can substantially influence their structure and aggregation. Ionic liquids (ILs) are salts in the liquid state with versatile ion combinations. Understanding the IL effect on protein behavior remains a major challenge. Here, we employed small angle X-ray scattering to investigate the effect of aqueous ILs on the structure and aggregation of globular proteins, namely, hen egg white lysozyme (Lys), human lysozyme (HLys), myoglobin (Mb), ß-lactoglobulin (ßLg), trypsin (Tryp) and superfolder green fluorescent protein (sfGFP). The ILs contain ammonium-based cations paired with the mesylate, acetate or nitrate anion. Results showed that only Lys was monomeric, whereas the other proteins formed small or large aggregates in buffer. Solutions with over 17 mol% IL resulted in significant changes in the protein structure and aggregation. The Lys structure was expanded at 1 mol% but compact at 17 mol% with structural changes in loop regions. HLys formed small aggregates, with the IL effect similar to Lys. Mb and ßLg mostly had distinct monomer and dimer distributions depending on IL type and IL concentration. Complex aggregation was noted for Tryp and sfGFP. While the anion had the largest ion effect, changing the cation also induced the structural expansion and protein aggregation.

Neurobehavioral deficits and a progressive ictogenesis in the tetrodotoxin model of epileptic spasms.

OBJECTIVE: Our goal was to determine whether animals with a history of epileptic spasms have learning and memory deficits. We also used continuous (24/7) long-term electroencephalographic (EEG) recordings to evaluate the evolution of epileptiform activity in the same animals over time. METHODS: Object recognition memory and object location memory tests were undertaken, as well as a matching to place water maze test that evaluated working memory. A retrospective analysis was undertaken of long-term video/EEG recordings from rats with epileptic spasms. The frequency and duration of the ictal events of spasms were quantified. RESULTS: Rats with a history of epileptic spasms showed impairment on the three behavioral tests, and their scores on the object recognition memory and matching to place water maze tests indicated neocortical involvement in the observed impaired cognition. Analysis of EEG recordings unexpectedly showed that the ictal events of spasms and their accompanying behaviors progressively increased in duration over a 2-week period soon after onset, after which spasm duration plateaued. At the same time, spasm frequency remained unchanged. Soon after spasm onset, ictal events were variable in wave form but became more stereotyped as the syndrome evolved. SIGNIFICANCE: Our EEG findings are the first to demonstrate progressive ictogenesis for epileptic spasms. Furthermore, in demonstrating cognitive deficits in the tetrodotoxin model, we have met a criterion for an animal model of West syndrome. Animal models will allow in-depth studies of spasm progression's potential role in cognitive regression and may elucidate why early treatment is considered essential for improved neurodevelopmental outcomes in children.

Circulating Soluble ACE2 Plays an Independent Role to Protect against Vascular Damage in Diabetic Mice.

Circulating levels of soluble ACE2 are increased by diabetes. Although this increase is associated with the presence and severity of cardiovascular disease, the specific role of soluble ACE2 in atherogenesis is unclear. Previous studies suggested that, like circulating ACE, soluble ACE2 plays a limited role in vascular homeostasis. To challenge this hypothesis, we aimed to selectively increase circulating ACE2 and measure its effects on angiotensin II dependent atherogenesis. Firstly, in Ace2/ApoE DKO mice, restoration of circulating ACE2 with recombinant murine soluble (rmACE219-613; 1 mg/kg/alternate day IP) reduced plaque accumulation in the aortic arch, suggesting that the phenotype may be driven as much by loss of soluble ACE2 as the reduction in local ACE2. Secondly, in diabetic ApoE KO mice, where activation of the renin angiotensin system drives accelerated atherosclerosis, rmACE219-613 also reduced plaque accumulation in the aorta after 6 weeks. Thirdly, to ensure consistent long-term delivery of soluble ACE2, an intramuscular injection was used to deliver a DNA minicircle encoding ACE219-613. This strategy efficiently increased circulating soluble ACE2 and reduced atherogenesis and albuminuria in diabetic ApoE KO mice followed for 10 weeks. We propose that soluble ACE2 has independent vasculoprotective effects. Future strategies that increase soluble ACE2 may reduce accelerated atherosclerosis in diabetes and other states in which the renin angiotensin system is upregulated.

A Role for Insulin-like Growth Factor 1 in the Generation of Epileptic Spasms in a murine model.

OBJECTIVE: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms. METHODS: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1, acts through the IGF-1 receptor to abolish spasms. RESULTS: Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3)IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals. INTERPRETATION: Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder. ANN NEUROL 2022;92:45-60.

Severe acute respiratory syndrome coronavirus 2 as a potential cause of type 1 diabetes facilitated by spike protein receptor binding domain attachment to human islet cells: An illustrative case study and experimental data.

AIMS: Aim of this study is to report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, responsible for coronavirus disease 2019 (COVID-19), as a possible cause for type 1 diabetes by providing an illustrative clinical case of a man aged 45 years presenting with antibody-negative diabetic ketoacidosis post-recovery from COVID-19 pneumonia and to explore the potential for SARS-CoV-2 to adhere to human islet cells. METHODS: Explanted human islet cells from three independent solid organ donors were incubated with the SARS-CoV-2 spike protein receptor biding domain (RBD) fused to a green fluorescent protein (GFP) or a control-GFP, with differential adherence established by flow cytometry. RESULTS: Flow cytometry revealed dose-dependent specific binding of RBD-GFP to islet cells when compared to control-GFP. CONCLUSIONS: Although a causal basis remains to be established, our case and in vitro data highlight a potential mechanism by which SARS-CoV-2 infection may result in antibody-negative type 1 diabetes.

Effect of ionic liquids on the fluorescence properties and aggregation of superfolder green fluorescence protein.

Proteins generally tend to aggregate with less desirable properties in numerous solvents, which is one of the major challenges in the development of solvents for functional proteins. This work aims to utilize fluorescence spectroscopy and small angle X-ray scattering (SAXS) to understand the effects of ionic liquids (ILs) on the fluorescence and aggregation behavior of superfolder green fluorescent protein (sfGFP). The studied ILs consisted of four different anions coupled with primary, tertiary and quaternary ammonium cations. The results show that the chromophore fluorescence was generally maintained in 1 mol% IL-water mixtures, then decreased with increasing IL concentration. We primarily employed the pseudo-radius of gyration (pseudo-Rg) to evaluate sfGFP aggregation. The sfGFP was less aggregated with nitrate-based ILs compared to in buffer, and more aggregated in the mesylate-based ILs. Further, we show that the polyol additives of glycerol and glucose in IL-water mixtures slightly decreased the sfGFP propensity to aggregate. Size-exclusion chromatography (SEC)-SAXS was used to characterize the monomeric sfGFP in ethylammonium nitrate (EAN) and triethylammonium mesylate (TEAMs)-water mixtures. The presence of 1 mol% TEAMs maintained the sfGFP fluorescence, promoted the compact structure, but slightly increased the amount of large aggregates, which contrasted with that of EAN.

Neocortical Slow Oscillations Implicated in the Generation of Epileptic Spasms.

OBJECTIVE: Epileptic spasms are a hallmark of severe seizure disorders. The neurophysiological mechanisms and the neuronal circuit(s) that generate these seizures are unresolved and are the focus of studies reported here. METHODS: In the tetrodotoxin model, we used 16-channel microarrays and microwires to record electrophysiological activity in neocortex and thalamus during spasms. Chemogenetic activation was used to examine the role of neocortical pyramidal cells in generating spasms. Comparisons were made to recordings from infantile spasm patients. RESULTS: Current source density and simultaneous multiunit activity analyses indicate that the ictal events of spasms are initiated in infragranular cortical layers. A dramatic pause of neuronal activity was recorded immediately prior to the onset of spasms. This preictal pause is shown to share many features with the down states of slow wave sleep. In addition, the ensuing interictal up states of slow wave rhythms are more intense in epileptic than control animals and occasionally appear sufficient to initiate spasms. Chemogenetic activation of neocortical pyramidal cells supported these observations, as it increased slow oscillations and spasm numbers and clustering. Recordings also revealed a ramp-up in the number of neocortical slow oscillations preceding spasms, which was also observed in infantile spasm patients. INTERPRETATION: Our findings provide evidence that epileptic spasms can arise from the neocortex and reveal a previously unappreciated interplay between brain state physiology and spasm generation. The identification of neocortical up states as a mechanism capable of initiating epileptic spasms will likely provide new targets for interventional therapies. ANN NEUROL 2021;89:226-241.

Dicarbonyl-mediated AGEing and diabetic kidney disease.

Increased glycolytic flux into the diabetic kidney, combined with glycolytic inefficiencies introduced by oxidative stress, acts to increase the generation of triose-phosphate intermediates, which spontaneously degrade to form methylglyoxal. At the same time, the glyoxalase-catalysed pathway that degrades excess methylglyoxal is impaired. The resulting dicarbonyl stress increases the accumulation of Advanced Glycation End-products (AGEs), as highly reactive dicarbonyls modify proteins, DNA, phospholipids and even small molecules like glutathione and nitric oxide. The resulting molecular dysfunction, contributes to the development and progression of kidney disease in diabetes. The importance of the dicarbonyls in diabetic kidney disease is clearly demonstrated by the reno-protective benefits of structurally-disparate dicarbonyl scavengers in experimental studies. Equally, modulating the glyoxalase pathway is able to alter both dicarbonyl generation and renal dysfunction in the presence and absence of hyperglycaemia. However, beyond improving glycemia control and reducing oxidative stress, an effective way to attenuate dicarbonyl-mediated damage in patients with diabetic kidney disease remains an elusive goal.

mGlu5 in GABAergic neurons modulates spontaneous and psychostimulant-induced locomotor activity.

RATIONALE: A role of group I metabotropic glutamate receptor 5 (mGlu5) in regulating spontaneous locomotion and psychostimulant-induced hyperactivity has been proposed. OBJECTIVES: This study aims to determine if mGlu5 in GABAergic neurons regulates spontaneous or psychostimulant-induced locomotion. METHODS: We generated mice specifically lacking mGlu5 in forebrain GABAergic neuron by crossing DLX-Cre mice with mGlu5flox/flox mice to generate DLX-mGlu5 KO mice. The locomotion of adult mice was examined in the open-field assay (OFA) and home cage setting. The effects of the mGlu5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP), cocaine, and methylphenidate on acute motor behaviors in DLX-mGlu5 KO and littermate control mice were assessed in OFA. Striatal synaptic plasticity of these mice was examined with field potential electrophysiological recordings. RESULTS: Deleting mGlu5 from forebrain GABAergic neurons results in failure to induce long-term depression (LTD) in the dorsal striatum and absence of habituated locomotion in both novel and familiar settings. In a familiar environment (home cage), DLX-mGlu5 KO mice were hyperactive. In the OFA, DLX-mGlu5 KO mice exhibited initial hypo-activity, and then gradually increased their locomotion with time, resulting in no habituation response. DLX-mGlu5 KO mice exhibited almost no locomotor response to MPEP (40 mg/kg), while the same dose elicited hyperlocomotion in control mice. The DLX-mGlu5 KO mice also showed reduced hyperactivity response to cocaine, while they retained normal hyperactivity response to methylphenidate, albeit with delayed onset. CONCLUSION: mGlu5 in forebrain GABAergic neurons is critical to trigger habituation upon the initiation of locomotion as well as to mediate MPEP-induced hyperlocomotion and modulate psychostimulant-induced hyperactivity.

Transactivation of RAGE mediates angiotensin-induced inflammation and atherogenesis.

Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB-driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a target for vasculoprotective interventions. As proof of concept, we showed that treatment with the mutant RAGE peptide S391A-RAGE362-404 was able to inhibit transactivation of RAGE and attenuate Ang II-dependent inflammation and atherogenesis. Furthermore, treatment with WT RAGE362-404 restored Ang II-dependent atherogenesis in Ager/Apoe-KO mice, without restoring ligand-mediated signaling via RAGE, suggesting that the major effector of RAGE activation was its transactivation.

A novel synthetic small molecule DMFO targets Nrf2 in modulating proinflammatory/antioxidant mediators to ameliorate inflammation.

Inflammation is a protective immune response against invading pathogens, however, dysregulated inflammation is detrimental. As the complex inflammatory response involves multiple mediators, including the involvement of reactive oxygen species, concomitantly targeting proinflammatory and antioxidant check-points may be a more rational strategy. We report the synthesis and anti-inflammatory/antioxidant activity of a novel indanedione derivative DMFO. DMFO scavenged reactive oxygen species (ROS) in in-vitro radical scavenging assays and in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. In acute models of inflammation (carrageenan-induced inflammation in rat paw and air pouch), DMFO effectively reduced paw oedema and leucocyte infiltration with an activity comparable to diclofenac. DMFO stabilised mast cells (MCs) in in-vitro A23187 and compound 48/80-induced assays. Additionally, DMFO stabilised MCs in an antigen (ovalbumin)-induced MC degranulation model in-vivo, without affecting serum IgE levels. In a model of chronic immune-mediated inflammation, Freund's adjuvant-induced arthritis, DMFO reduced arthritic score and contralateral paw oedema, and increased the pain threshold with an efficacy comparable to diclofenac but without being ulcerogenic. Additionally, DMFO significantly reduced serum TNFα levels. Mechanistic studies revealed that DMFO reduced proinflammatory genes (IL1ß, TNFα, IL6) and protein levels (COX2, MCP1), with a concurrent increase in antioxidant genes (NQO1, haem oxygenase 1 (HO-1), Glo1, Nrf2) and protein (HO-1) in LPS-stimulated macrophages. Importantly, the anti-inflammatory/antioxidant effect on gene expression was absent in primary macrophages isolated from Nrf2 KO mice suggesting an Nrf2-targeted activity, which was subsequently confirmed using siRNA transfection studies in RAW macrophages. Therefore, DMFO is a novel, orally-active, safe (even at 2 g/kg p.o.), a small molecule which targets Nrf2 in ameliorating inflammation.

Recent novel approaches to limit oxidative stress and inflammation in diabetic complications.

Diabetes is considered a major burden on the healthcare system of Western and non-Western societies with the disease reaching epidemic proportions globally. Diabetic patients are highly susceptible to developing micro- and macrovascular complications, which contribute significantly to morbidity and mortality rates. Over the past decade, a plethora of research has demonstrated that oxidative stress and inflammation are intricately linked and significant drivers of these diabetic complications. Thus, the focus now has been towards specific mechanism-based strategies that can target both oxidative stress and inflammatory pathways to improve the outcome of disease burden. This review will focus on the mechanisms that drive these diabetic complications and the feasibility of emerging new therapies to combat oxidative stress and inflammation in the diabetic milieu.

mGluR5 Exerts Cell-Autonomous Influences on the Functional and Anatomical Development of Layer IV Cortical Neurons in the Mouse Primary Somatosensory Cortex.

UNLABELLED: Glutamate neurotransmission refines synaptic connections to establish the precise neural circuits underlying sensory processing. Deleting metabotropic glutamate receptor 5 (mGluR5) in mice perturbs cortical somatosensory map formation in the primary somatosensory (S1) cortex at both functional and anatomical levels. To examine the cell-autonomous influences of mGluR5 signaling in the morphological and functional development of layer IV spiny stellate glutamatergic neurons receiving sensory input, mGluR5 genetic mosaic mice were generated through in utero electroporation. In the S1 cortex of these mosaic brains, we found that most wild-type neurons were located in barrel rings encircling thalamocortical axon (TCA) clusters while mGluR5 knock-out (KO) neurons were placed in the septal area, the cell-sparse region separating barrels. These KO neurons often displayed a symmetrical dendritic morphology with increased dendritic complexity, in contrast to the polarized pattern of wild-type neurons. The dendritic spine density of mGluR5 KO spiny stellate neurons was significantly higher than in wild-type neurons. Whole-cell electrophysiological recordings detected a significant increase in the frequencies of spontaneous and miniature excitatory postsynaptic events in mGluR5 KO neurons compared with neighboring wild-type neurons. Our mosaic analysis provides strong evidence supporting the cell-autonomous influence of mGluR5 signaling on the functional and anatomical development of cortical glutamatergic neurons. Specifically, mGluR5 is required in cortical glutamatergic neurons for the following processes: (1) the placement of cortical glutamatergic neurons close to TCA clusters; (2) the regulation of dendritic complexity and outgrowth toward TCA clusters; (3) spinogenesis; and (4) tuning of excitatory inputs. SIGNIFICANCE STATEMENT: Glutamatergic transmission plays a critical role in cortical circuit formation. Its dysfunction has been proposed as a core factor in the etiology of many neurological diseases. Here we conducted mosaic analysis to reveal the cell-autonomous role of the metabotropic glutamate receptor 5 (mGluR5). We found that mGluR5 is required for several key steps in wiring up the thalamocortical connections to form the cortical somatosensory map. mGluR5-dependent processes during early postnatal brain development affect the following: (1) placement of activity-directed cortical neurons; (2) regulation of polarized dendritic outgrowth toward thalamocortical axons relaying sensory information, (3) synaptogenesis; and (4) development of functional connectivity in spiny stellate neurons. Perturbing mGluR5 expression could lead to abnormal neuronal circuits, which may contribute to neurological and psychiatric disease.

Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

Imbalances in GABA (γ-aminobutyric acid) homoeostasis underlie psychiatric and movement disorders. The ability of the 65 kDa isoform of GAD (glutamic acid decarboxylase), GAD65, to control synaptic GABA levels is influenced through its capacity to auto-inactivate. In contrast, the GAD67 isoform is constitutively active. Previous structural insights suggest that flexibility in the GAD65 catalytic loop drives enzyme inactivation. To test this idea, we constructed a panel of GAD65/67 chimaeras and compared the ability of these molecules to auto-inactivate. Together, our data reveal the important finding that the C-terminal domain of GAD plays a key role in controlling GAD65 auto-inactivation. In support of these findings, we determined the X-ray crystal structure of a GAD65/67 chimaera that reveals that the conformation of the catalytic loop is intimately linked to the C-terminal domain.

The conjugation protein TcpC from Clostridium perfringens is structurally related to the type IV secretion system protein VirB8 from Gram-negative bacteria.

Bacterial conjugation is important for the acquisition of virulence and antibiotic resistance genes. We investigated the mechanism of conjugation in Gram-positive pathogens using a model plasmid pCW3 from Clostridium perfringens. pCW3 encodes tetracycline resistance and contains the tcp locus, which is essential for conjugation. We showed that the unique TcpC protein (359 amino acids, 41 kDa) was required for efficient conjugative transfer, localized to the cell membrane independently of other conjugation proteins, and that membrane localization was important for its function, oligomerization and interaction with the conjugation proteins TcpA, TcpH and TcpG. The crystal structure of the C-terminal component of TcpC (TcpC(99-359)) was determined to 1.8-Å resolution. TcpC(99-359) contained two NTF2-like domains separated by a short linker. Unexpectedly, comparative structural analysis showed that each of these domains was structurally homologous to the periplasmic region of VirB8, a component of the type IV secretion system from Agrobacterium tumefaciens. Bacterial two-hybrid studies revealed that the C-terminal domain was critical for interactions with other conjugation proteins. The N-terminal region of TcpC was required for efficient conjugation, oligomerization and protein-protein interactions. We conclude that by forming oligomeric complexes, TcpC contributes to the stability and integrity of the conjugation apparatus, facilitating efficient pCW3 transfer.

What can we get from 'barrels': the rodent barrel cortex as a model for studying the establishment of neural circuits.

Sensory inputs triggered by external stimuli are projected into discrete arrays of neuronal modules in the primary sensory cortex. This whisker-to-barrel pathway has gained in popularity as a model system for studying the development of cortical circuits and sensory processing because its clear patterns facilitate the identification of genetically modified mice with whisker map deficits and make possible coordinated in vitro and in vivo electrophysiological studies. Numerous whisker map determinants have been identified in the past two decades. In this review, we summarize what have we learned from the detailed studies conducted in various mutant mice with cortical whisker map deficits. We will specifically focus on the anatomical and functional establishment of the somatosensory thalamocortical circuits.

mGluR5 in cortical excitatory neurons exerts both cell-autonomous and -nonautonomous influences on cortical somatosensory circuit formation.

Glutamatergic neurotransmission plays important roles in sensory map formation. The absence of the group I metabotropic glutamate receptor 5 (mGluR5) leads to abnormal sensory map formation throughout the mouse somatosensory pathway. To examine the role of cortical mGluR5 expression on barrel map formation, we generated cortex-specific mGluR5 knock-out (KO) mice. Eliminating mGluR5 function solely in cortical excitatory neurons affects, not only the whisker-related organization of cortical neurons (barrels), but also the patterning of their presynaptic partners, the thalamocortical axons (TCAs). In contrast, subcortical whisker maps develop normally in cortical-mGluR5 KO mice. In the S1 cortex of cortical-mGluR5 KO, layer IV neurons are homogenously distributed and have no clear relationship to the location of TCA clusters. The altered dendritic morphology of cortical layer IV spiny stellate neurons in cortical-mGluR5 KO mice argues for a cell-autonomous role of mGluR5 in dendritic patterning. Furthermore, morphometric analysis of single TCAs in both cortical- and global-mGluR5 KO mice demonstrated that in these mice, the complexity of axonal arbors is reduced, while the area covered by TCA arbors is enlarged. Using voltage-clamp whole-cell recordings in acute thalamocortical brain slices, we found that KO of mGluR5 from cortical excitatory neurons reduced inhibitory but not excitatory inputs onto layer IV neurons. This suggests that mGluR5 signaling in cortical excitatory neurons nonautonomously modulates the functional development of GABAergic circuits. Together, our data provide strong evidence that mGluR5 signaling in cortical principal neurons exerts both cell-autonomous and -nonautonomous influences to modulate the formation of cortical sensory circuits.

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.