Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans

Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Modulation of neurotransmission in locus coeruleus by metabotropic glutamate receptors.

The locus coeruleus, a small group of pontine neurons, is the most prominent noradrenaline-containing nucleus in the central nervous system. Activity of this nucleus is thought to modulate the function throughout the brain through its widespread afferent network. Here, the effects of selective activation of metabotropic glutamate receptors on synaptic transmission in intracellularly recorded locus coeruleus neurons in brain slice preparations are described. Perfusion of either t-ACPD (0.1-500 $\mu$M) or L-AP4 (0.1-500 $\mu$M) caused a depression of excitatory postsynaptic potentials in a dope-dependent fashion to about 70% inhibition. Both agonists exerted their effects with estimated EC\sb{50}s of 2.6 $\mu$M and 11.5 $\mu$M for L-AP4 and t-ACPD respectively. Both t-ACPD and L-AP4 produced an increase in paired-pulse facilitation, and failed to change the response of locus coeruleus neurons to focally-applied glutamate, indicating a presynaptic locus of action. Both group II and III metabotropic glutamate receptors have been shown to be coupled to a G\rm\sb{i/o}-protein and, in certain systems, activation of these receptors results in a decrease of forskolin-stimulated cAMP levels. However, many all G\rm\sb{i/o}-coupled receptors have also been shown to block calcium channels in different cell types. Thus, several tests were carried out in an attempt to define the signal transduction pathway involved in the t-ACPD and L-AP4 effects in locus coeruleus. In a first set of experiments, N-ethylmaleimide, a compound known to disrupt the interaction between G\rm\sb{i/o} proteins and calcium channels, was tested. Pretreatment of slices with N-ethylmaleimide (50-100 $\mu$M, 15 min.) resulted in an almost complete block of the effects of t-ACPD and L-AP4. In a second set of experiments, perfusion of a "cAMP cocktail" (200$\mu$M 8-bromo-cAMP, 20$\mu$M forskolin and 1mM IBMX) was tested on the response of locus coeruleus to t-ACPD and LAP4. In the last part of this study, the functional role of synaptically-released excitatory amino acid on metabotropic glutamate receptors in locus coeruleus was investigated. When single stimuli were applied to the afferents at intervals greater than 200ms, the amplitude of the second (test (T)) excitatory postsynaptic potential was identical in amplitude to the first (control(C)). However, when a train of stimulation was delivered prior to T, the amplitude of T was consistently smaller than C. The depression was dependent on the frequency and duration of the train and the interval between the train and T. In most experiments, optimal inhibition was observed with a 300ms, 70 Hz train delivered 600ms prior to the test EPSP. This activity-dependent depression of excitatory postsynaptic potentials was enhanced in the presence of an excitatory amino acid uptake inhibitor L-trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC, 100 $\mu$M) from a T/C ratio of 0.80 $\pm$ 0.03 (mean $\pm$ SEM) in control to 0.68 $\pm$ 0.05 in t-PDC. Together, the studies described above provide evidence that activation of either of two different presynaptic metabotropic glutamate receptors belonging to group II and m, respectively, produce a decrease in excitatory synaptic transmission in locus coeruleus through an undetermined mechanism but in a cAMP-independent manner. (Abstract shortened by UMI.

Toward Understanding How Early-Life Stress Reprograms Cognitive and Emotional Brain Networks

Vulnerability to emotional disorders including depression derives from interactions between genes and environment, especially during sensitive developmental periods. Adverse early-life experiences provoke the release and modify the expression of several stress mediators and neurotransmitters within specific brain regions. The interaction of these mediators with developing neurons and neuronal networks may lead to long-lasting structural and functional alterations associated with cognitive and emotional consequences. Although a vast body of work has linked quantitative and qualitative aspects of stress to adolescent and adult outcomes, a number of questions are unclear. What distinguishes ‘normal' from pathologic or toxic stress? How are the effects of stress transformed into structural and functional changes in individual neurons and neuronal networks? Which ones are affected? We review these questions in the context of established and emerging studies. We introduce a novel concept regarding the origin of toxic early-life stress, stating that it may derive from specific patterns of environmental signals, especially those derived from the mother or caretaker. Fragmented and unpredictable patterns of maternal care behaviors induce a profound chronic stress. The aberrant patterns and rhythms of early-life sensory input might also directly and adversely influence the maturation of cognitive and emotional brain circuits, in analogy to visual and auditory brain systems. Thus, unpredictable, stress-provoking early-life experiences may influence adolescent cognitive and emotional outcomes by disrupting the maturation of the underlying brain networks. Comprehensive approaches and multiple levels of analysis are required to probe the protean consequences of early-life adversity on the developing brain. These involve integrated human and animal-model studies, and approaches ranging from in vivo imaging to novel neuroanatomical, molecular, epigenomic, and computational methodologies. Because early-life adversity is a powerful determinant of subsequent vulnerabilities to emotional and cognitive pathologies, understanding the underlying processes will have profound implications for the world's current and future children