Enhanced Social Dominance and Altered Neuronal Excitability in the Prefrontal Cortex of Male KCC2b Mutant Mice.

The K-Cl cotransporter KCC2 is essential in the development of the "GABA switch" that produces a change in neuronal responses to GABA signaling from excitatory to inhibitory early in brain development, and alterations in this progression have previously been hypothesized to play a causal role in autism spectrum disorder (ASD). We investigated the KCC2b (Slc12a5) heterozygous knockout mouse using a battery of rodent behavioral tests relevant to core and comorbid ASD symptoms. Compared to wild-type littermates, KCC2+/- mice were normal in standard measures of locomotor activity, grooming and digging behaviors, and social, vocalization, and anxiety-like behaviors. However, KCC2+/- mice exhibited increased social dominance behaviors and increased amplitude of spontaneous postsynaptic currents in the medial prefrontal cortex (PFC) that were previously implicated in governing social hierarchy and dominance behaviors. Treatment of wild-type mouse brain slices with the KCC2 inhibitor VU0240511 increased the amplitude and frequency of excitatory postsynaptic currents, partially recapitulating the phenotype of KCC2+/- mice. These findings indicate that the activity of KCC2 plays a role in social dominance, in parallel with effects on PFC signaling, further suggesting that KCC2 function has some relevance to social behavior but without the breadth of impact on autism-like behavior suggested by previous studies. Further testing could assess whether KCC2 alters other circuits and whether additional factors such as environmental insults may precipitate autism-related behavioral phenotypes. Autism Research 2019, 12: 732-743. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: A mouse model of altered chloride transporter expression was used to look for a role in behaviors and brain function relevant to autism. There was an imbalance in signaling in the prefrontal cortex, and increased social dominance behavior, although other autism-related behaviors were not changed. These findings indicate that altered chloride transporter function affects prefrontal cortex function and social dominance without a broader impact on autism-like behaviors.

Stress dynamically regulates co-expression networks of glucocorticoid receptor-dependent MDD and SCZ risk genes.

Early-life adversity is an important risk factor for major depressive disorder (MDD) and schizophrenia (SCZ) that interacts with genetic factors to confer disease risk through mechanisms that are still insufficiently understood. One downstream effect of early-life adversity is the activation of glucocorticoid receptor (GR)-dependent gene networks that drive acute and long-term adaptive behavioral and cellular responses to stress. We have previously shown that genetic variants that moderate GR-induced gene transcription (GR-response eSNPs) are significantly enriched among risk variants from genome-wide association studies (GWASs) for MDD and SCZ. Here, we show that the 63 transcripts regulated by these disease-associated functional genetic variants form a tight glucocorticoid-responsive co-expression network (termed GCN). We hypothesized that changes in the correlation structure of this GCN may contribute to early-life adversity-associated disease risk. Therefore, we analyzed the effects of different qualities of social support and stress throughout life on GCN formation across distinct brain regions using a translational mouse model. We observed that different qualities of social experience substantially affect GCN structure in a highly brain region-specific manner. GCN changes were predominantly found in two functionally interconnected regions, the ventral hippocampus and the hypothalamus, two brain regions previously shown to be of relevance for the stress response, as well as psychiatric disorders. Overall, our results support the hypothesis that a subset of genetic variants may contribute to risk for MDD and SCZ by altering circuit-level effects of early and adult social experiences on GCN formation and structure.

Expression and glucocorticoid-dependent regulation of the stress-inducible protein DRR1 in the mouse adult brain.

Identifying molecular targets that are able to buffer the consequences of stress and therefore restore brain homeostasis is essential to develop treatments for stress-related disorders. Down-regulated in renal cell carcinoma 1 (DRR1) is a unique stress-induced protein in the brain and has been recently proposed to modulate stress resilience. Interestingly, DRR1 shows a prominent expression in the limbic system of the adult mouse. Here, we analyzed the neuroanatomical and cellular expression patterns of DRR1 in the adult mouse brain using in situ hybridization, immunofluorescence and Western blot. Abundant expression of DRR1 mRNA and protein was confirmed in the adult mouse brain with pronounced differences between distinct brain regions. The strongest DRR1 signal was detected in the neocortex, the CA3 region of the hippocampus, the lateral septum and the cerebellum. DRR1 was also present in circumventricular organs and its connecting regions. Additionally, DRR1 was present in non-neuronal tissues like the choroid plexus and ependyma. Within cells, DRR1 protein was distributed in a punctate pattern in several subcellular compartments including cytosol, nucleus as well as some pre- and postsynaptic specializations. Glucocorticoid receptor activation (dexamethasone 10 mg/kg s.c.) induced DRR1 expression throughout the brain, with particularly strong induction in white matter and fiber tracts and in membrane-rich structures. This specific expression pattern and stress modulation of DRR1 point to a role of DRR1 in regulating how cells sense and integrate signals from the environment and thus in restoring brain homeostasis after stressful challenges.

Common genes associated with antidepressant response in mouse and man identify key role of glucocorticoid receptor sensitivity.

Response to antidepressant treatment in major depressive disorder (MDD) cannot be predicted currently, leading to uncertainty in medication selection, increasing costs, and prolonged suffering for many patients. Despite tremendous efforts in identifying response-associated genes in large genome-wide association studies, the results have been fairly modest, underlining the need to establish conceptually novel strategies. For the identification of transcriptome signatures that can distinguish between treatment responders and nonresponders, we herein submit a novel animal experimental approach focusing on extreme phenotypes. We utilized the large variance in response to antidepressant treatment occurring in DBA/2J mice, enabling sample stratification into subpopulations of good and poor treatment responders to delineate response-associated signature transcript profiles in peripheral blood samples. As a proof of concept, we translated our murine data to the transcriptome data of a clinically relevant human cohort. A cluster of 259 differentially regulated genes was identified when peripheral transcriptome profiles of good and poor treatment responders were compared in the murine model. Differences in expression profiles from baseline to week 12 of the human orthologues selected on the basis of the murine transcript signature allowed prediction of response status with an accuracy of 76% in the patient population. Finally, we show that glucocorticoid receptor (GR)-regulated genes are significantly enriched in this cluster of antidepressant-response genes. Our findings point to the involvement of GR sensitivity as a potential key mechanism shaping response to antidepressant treatment and support the hypothesis that antidepressants could stimulate resilience-promoting molecular mechanisms. Our data highlight the suitability of an appropriate animal experimental approach for the discovery of treatment response-associated pathways across species.

Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function.

The co-chaperone FKBP5 is a stress-responsive protein-regulating stress reactivity, and its genetic variants are associated with T2D related traits and other stress-related disorders. Here we show that FKBP51 plays a role in energy and glucose homeostasis. Fkbp5 knockout (51KO) mice are protected from high-fat diet-induced weight gain, show improved glucose tolerance and increased insulin signaling in skeletal muscle. Chronic treatment with a novel FKBP51 antagonist, SAFit2, recapitulates the effects of FKBP51 deletion on both body weight regulation and glucose tolerance. Using shorter SAFit2 treatment, we show that glucose tolerance improvement precedes the reduction in body weight. Mechanistically, we identify a novel association between FKBP51 and AS160, a substrate of AKT2 that is involved in glucose uptake. FKBP51 antagonism increases the phosphorylation of AS160, increases glucose transporter 4 expression at the plasma membrane, and ultimately enhances glucose uptake in skeletal myotubes. We propose FKBP51 as a mediator between stress and T2D development, and potential target for therapeutic approaches.

An adverse early life environment can enhance stress resilience in adulthood.

Chronic stress is a major risk factor for depression. Interestingly, not all individuals develop psychopathology after chronic stress exposure. In contrast to the prevailing view that stress effects are cumulative and increase stress vulnerability throughout life, the match/mismatch hypothesis of psychiatric disorders. The match/mismatch hypothesis proposes that individuals who experience moderate levels of early life psychosocial stress can acquire resilience to renewed stress exposure later in life. Here, we have tested this hypothesis by comparing the developmental effects of 2 opposite early life conditions, when followed by 2 opposite adult environments. Male Balb/c mice were exposed to either adverse early life conditions (limited nesting and bedding material) or a supportive rearing environment (early handling). At adulthood, the animals of each group were either housed with an ovariectomized female (supportive environment) or underwent chronic social defeat stress (socially adverse environment) for 3 weeks. At the end of the adult manipulations, all of the animals were returned to standard housing conditions. Then, we compared the neuroendocrine, behavioral and molecular effects of the interaction between early and adult environment. Our study shows that early life adversity does not necessarily result in increased vulnerability to stress. Specific endophenotypes, like hypothalamic-pituitary-adrenal axis activity, anxiety-related behavior and glucocorticoid receptor expression levels in the hippocampus were not significantly altered when adversity is experienced during early life and in adulthood, and are mainly affected by either early life or adult life adversity alone. Overall our data support the notion that being raised in a stressful environment prepares the offspring to better cope with a challenging adult environment and emphasize the role of early life experiences in shaping adult responsiveness to stress.

Prefrontal Cortex Corticotropin-Releasing Factor Receptor 1 Conveys Acute Stress-Induced Executive Dysfunction.

BACKGROUND: The medial prefrontal cortex (mPFC) subserves complex cognition and is impaired by stress. Corticotropin-releasing factor (CRF), through CRF receptor 1 (CRFR1), constitutes a key element of the stress response. However, its contribution to the effects of stress in the mPFC remains unclear. METHODS: Mice were exposed to acute social defeat stress and subsequently to either the temporal order memory (n = 11-12) or reversal learning (n = 9-11) behavioral test. Changes in mPFC Crhr1 messenger RNA levels were measured in acutely stressed mice (n = 12). Crhr1loxP/loxP mice received either intra-mPFC adeno-associated virus-Cre or empty microinjections (n = 17-20) and then were submitted to acute stress and later to the behavioral tests. Co-immunoprecipitation was used to detect activation of the protein kinase A (PKA) signaling pathway in the mPFC of acutely stressed mice (n = 8) or intra-mPFC CRF injected mice (n = 7). Finally, mice received intra-mPFC CRF (n = 11) and/or Rp-isomer cyclic adenosine 3',5' monophosphorothioate (Rp-cAMPS) (n = 12) microinjections and underwent behavioral testing. RESULTS: We report acute stress-induced effects on mPFC-mediated cognition, identify CRF-CRFR1-containing microcircuits within the mPFC, and demonstrate stress-induced changes in Crhr1 messenger RNA expression. Importantly, intra-mPFC CRFR1 deletion abolishes acute stress-induced executive dysfunction, whereas intra-mPFC CRF mimics acute stress-induced mPFC dysfunction. Acute stress and intra-mPFC CRF activate the PKA signaling pathway in the mPFC, leading to cyclic AMP response element binding protein phosphorylation in intra-mPFC CRFR1-expressing neurons. Finally, PKA blockade reverses the intra-mPFC CRF-induced executive dysfunction. CONCLUSIONS: Taken together, these results unravel a molecular mechanism linking acute stress to executive dysfunction via CRFR1. This will aid in the development of novel therapeutic targets for stress-induced cognitive dysfunction.

The amino acid transporter SLC6A15 is a regulator of hippocampal neurochemistry and behavior.

Although mental disorders as major depression are highly prevalent worldwide their underlying causes remain elusive. Despite the high heritability of depression and a clear genetic contribution to the disease, the identification of genetic risk factors for depression has been very difficult. The first published candidate to reach genome-wide significance in depression was SLC6A15, a neuronal amino acid transporter. With a reported 1,42 fold increased risk of suffering from depression associated with a single nucleotide polymorphism (SNP) in a regulatory region of SLC6A15, the polymorphism was also found to affect hippocampal morphology, integrity, and hippocampus-dependent memory. However, the function of SLC6A15 in the brain is so far largely unknown. To address this question, we investigated if alterations in SLC6A15 expression, either using a full knockout or a targeted hippocampal overexpression, affect hippocampal neurochemistry and consequently behavior. We could show that a lack of SLC6A15 reduced hippocampal tissue levels of proline and other neutral amino acids. In parallel, we observed a decreased overall availability of tissue glutamate and glutamine, while at the same time the basal tone of extracellular glutamate in the hippocampus was increased. By contrast, SLC6A15 overexpression increased glutamate/glutamine tissue concentrations. These neurochemical alterations could be linked to behavioral abnormalities in sensorimotor gating, a key translational endophenotype relevant for many psychiatric disorders. Overall, our data supports SLC6A15 as a crucial factor controlling amino acid content in the hippocampus, thereby likely interfering with glutamatergic transmission and behavior. These findings emphasize SLC6A15 as pivotal risk factor for vulnerability to psychiatric diseases.

Homer1/mGluR5 activity moderates vulnerability to chronic social stress.

Stress-induced psychiatric disorders, such as depression, have recently been linked to changes in glutamate transmission in the central nervous system. Glutamate signaling is mediated by a range of receptors, including metabotropic glutamate receptors (mGluRs). In particular, mGluR subtype 5 (mGluR5) is highly implicated in stress-induced psychopathology. The major scaffold protein Homer1 critically interacts with mGluR5 and has also been linked to several psychopathologies. Yet, the specific role of Homer1 in this context remains poorly understood. We used chronic social defeat stress as an established animal model of depression and investigated changes in transcription of Homer1a and Homer1b/c isoforms and functional coupling of Homer1 to mGluR5. Next, we investigated the consequences of Homer1 deletion, overexpression of Homer1a, and chronic administration of the mGluR5 inverse agonist CTEP (2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine) on the effects of chronic stress. In mice exposed to chronic stress, Homer1b/c, but not Homer1a, mRNA was upregulated and, accordingly, Homer1/mGluR5 coupling was disrupted. We found a marked hyperactivity behavior as well as a dysregulated hypothalamic-pituitary-adrenal axis activity in chronically stressed Homer1 knockout (KO) mice. Chronic administration of the selective and orally bioavailable mGluR5 inverse agonist, CTEP, was able to recover behavioral alterations induced by chronic stress, whereas overexpression of Homer1a in the hippocampus led to an increased vulnerability to chronic stress, reflected in an increased physiological response to stress as well as enhanced depression-like behavior. Overall, our results implicate the glutamatergic system in the emergence of stress-induced psychiatric disorders, and support the Homer1/mGluR5 complex as a target for the development of novel antidepressant agents.

Effect of breadmaking process on in vitro gut microbiota parameters in irritable bowel syndrome.

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria-mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small-molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.

The stress-inducible actin-interacting protein DRR1 shapes social behavior.

Understanding the molecular mechanisms by which stress is translated into changes in complex behavior may help to identify novel treatment strategies for stress-associated psychiatric disorders. The tumor suppressor gene down-regulated in renal cell carcinoma 1 (DRR1) was recently characterized as a new molecular link between stress, synaptic efficacy and behavioral performance, most likely through its ability to modulate actin dynamics. The lateral septum is one of the brain regions prominently involved in the stress response. This brain region features high DRR1 expression in adult mice, even under basal conditions. We therefore aimed to characterize and dissect the functional role of septal DRR1 in modulating complex behavior. DRR1 protein expression was shown to be expressed in both neurons and astrocytes of the lateral septum of adult mice. Septal DRR1 mRNA expression increased after acute defeat stress and glucocorticoid receptor activation. To mimic the stress-induced DRR1 increase in the lateral septum of mice, we performed adenovirus-mediated region-specific overexpression of DRR1 and characterized the behavior of these mice. Overexpression of DRR1 in the septal region increased sociability, but did not change cognitive, anxiety-like or anhedonic behavior. The observed changes in social behavior did not involve alterations of the expression of vasopressin or oxytocin receptors, the canonical social neuropeptidergic circuits of the lateral septum. In summary, our data suggest that the stress-induced increase of DRR1 expression in the lateral septum could be a protective mechanism to buffer or counterbalance negative consequences of stress exposure on social behavior.

Interplay between diet-induced obesity and chronic stress in mice: potential role of FKBP51.

While it is known that stress promotes obesity, the effects of stress within an obesogenic context are not so clear and molecular targets at the interface remain elusive. The FK506-binding protein 51 (FKBP51, gene: Fkbp5) has been identified as a target gene implicated in the development of stress-related psychiatric disorders and is a possible candidate for involvement in stress and metabolic regulation. The aims of the current study are to investigate the interaction between chronic stress and an obesogenic context and to additionally examine whether FKBP51 is involved in this interaction. For this purpose, male C57BL/6 mice were exposed to a high-fat diet for 8 weeks before being challenged with chronic social defeat stress. Herein, we demonstrate that chronic stress induces hypophagia and weight loss, ultimately improving features arising from an obesogenic context, including glucose tolerance and levels of insulin and leptin. We show that Fkbp5 expression is responsive to diet and stress in the hypothalamus and hippocampus respectively. Furthermore, under basal conditions, higher levels of hypothalamic Fkbp5 expression were related to increased body weight gain. Our data indicate that Fkbp5 may represent a novel target in metabolic regulation.

Evidence supporting the match/mismatch hypothesis of psychiatric disorders.

Chronic stress is one of the predominant environmental risk factors for a number of psychiatric disorders, particularly for major depression. Different hypotheses have been formulated to address the interaction between early and adult chronic stress in psychiatric disease vulnerability. The match/mismatch hypothesis of psychiatric disease states that the early life environment shapes coping strategies in a manner that enables individuals to optimally face similar environments later in life. We tested this hypothesis in female Balb/c mice that underwent either stress or enrichment early in life and were in adulthood further subdivided in single or group housed, in order to provide aversive or positive adult environments, respectively. We studied the effects of the environmental manipulation on anxiety-like, depressive-like and sociability behaviors and gene expression profiles. We show that continuous exposure to adverse environments (matched condition) is not necessarily resulting in an opposite phenotype compared to a continuous supportive environment (matched condition). Rather, animals with mismatched environmental conditions behaved differently from animals with matched environments on anxious, social and depressive like phenotypes. These results further support the match/mismatch hypothesis and illustrate how mild or moderate aversive conditions during development can shape an individual to be optimally adapted to similar conditions later in life.

Antidepressant treatment outcome depends on the quality of the living environment: a pre-clinical investigation in mice.

Antidepressants represent the standard treatment for major depression. However, their efficacy is variable and incomplete. A growing number of studies suggest that the environment plays a major role in determining the efficacy of these drugs, specifically of selective serotonin reuptake inhibitors (SSRI). A recent hypothesis posits that the increase in serotonin levels induced by SSRI may not affect mood per se, but enhances neural plasticity and, consequently, renders the individual more susceptible to the influence of the environment. Thus, SSRI administration in a favorable environment would lead to a reduction of symptoms, while in a stressful environment might lead to a worse prognosis. To test this hypothesis, we treated C57BL/6 adult male mice with chronic fluoxetine while exposing them to either (i) an enriched environment, after exposure to a chronic stress period aimed at inducing a depression-like phenotype, or (ii) a stressful environment. Anhedonia, brain BDNF and circulating corticosterone levels, considered endophenotypes of depression, were investigated. Mice treated with fluoxetine in an enriched condition improved their depression-like phenotype compared to controls, displaying higher saccharin preference, higher brain BDNF levels and reduced corticosterone levels. By contrast, when chronic fluoxetine administration occurred in a stressful condition, mice showed a more distinct worsening of the depression-like profile, displaying a faster decrease of saccharin preference, lower brain BDNF levels and increased corticosterone levels. Our findings suggest that the effect of SSRI on depression-like phenotypes in mice is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.

Not all stressors are equal: early social enrichment favors resilience to social but not physical stress in male mice.

Early experiences profoundly affect the adult coping response to stress and, consequently, adult vulnerability to psychopathologies triggered by stressing conditions, such as major depression. Though studies in animal models have demonstrated that individuals reared in different conditions are differently vulnerable to a stressor of a specific quality, no information is available as to whether such vulnerability differs when facing stressors of different qualities. To this purpose, we reared C57BL/6 male mice either in standard laboratory rearing condition (SN) or in Communal Nest (CN) condition, the latter consisting of a single nest where three mothers keep their pups together and share care-giving behavior until weaning. We scored the amount of interactions with the mother and with peers and found that CN is a form of social enrichment because both these components are significantly increased. At adulthood, we exposed SN and CN mice, for 4 weeks, to either a physical (forced swim) or a social stress (social instability). Immediately before, at week 1 and at week 4 of the stress procedure, corticosterone levels and the hedonic profile were measured. The results show that CN mice are more resilient to social stress than SN mice since they displayed no anhedonia and lower corticosterone levels. By contrast, both experimental groups were similarly vulnerable to physical stress. Overall, our results show that, in male mice, the adult vulnerability to stress changes according to the quality of the stressor, as a function of early experiences. In addition, the stressor to which CN mice are resilient is qualitatively similar to the stimuli they have experienced early on, both concerning the social domain.

The richness of social stimuli shapes developmental trajectories: Are laboratory mouse pups impoverished?

The early environment is crucial for brain and behavior development. In particular, social experiences involving the mother and the peers are critical in shaping the adult individual. Though animal models of psychiatric disorders have widely investigated the relevance of the mother-offspring interaction, the peer interaction has so far been rarely studied. The communal nest (CN) is an innovative experimental strategy that favors a more comprehensive investigation of the long-term effects of both components. CN is a rearing condition employed by up to 90% of mouse females in naturalistic settings and consists of a single nest where two or more mothers keep their pups together and share care-giving. In a CN, the developing pup is exposed to high levels of both maternal care and interaction with peers. At adulthood, these mice display relevant changes in bran function and behavior, including high levels of neural plasticity markers, such as BDNF, and elaborate adult social competences. Overall, on the one hand, CN is an experimental approach complementary to the ones currently used that allows to investigate how the early environment determines developmental trajectories. On the other, it may represent a strategy to improve the study of animal models of psychiatric disorders characterized by social dysfunction, such as major depression, autism and attention deficit hyperactivity disorder. Indeed, the more elaborate social competences shown by these mice at adulthood may allow to better characterize deficits in the social domain induced by genetic and/or environmental manipulations.

Biochemical traits of Ciauscolo, a spreadable typical Italian dry-cured sausage.

Ciauscolo is a short-ripened fermented sausage manufactured in the Marche region (central Italy) that has recently received a protected geographical indication product classification (PGI). The aim of this study was the exploration of the biochemical traits of this traditional Italian salami, with a special focus on protein and lipid composition. Ciauscolo salami was characterized by pH of 5.1 and 0.91 water activity. A prevalence of lactic acid bacteria in the microbiota was found. The free amino acids and biogenic amines average content was 2657 and 255 mg/kg, respectively. With regards to lipids composition unsaturated fatty acids represented 63% and 72% of total and free fatty acids. Despite these results had wide statistical variability, attributable to differences in the processing parameters and raw matter used, some peculiar traits were found: (1) structural muscular proteins underwent to less proteolysis than sarcoplasmic ones; (2) glycogen phosphorylase, enolase, and aldolase were the most proteolyzed among the sacoplasmic proteins; (3) there was inverse correlation between histamine content and yeasts population, and a direct correlation between the gly-pro content and lactic acid bacteria counts; (4) the content of aspartic acid and methyonine seem to be a possible molecular marker able to distinguish between double and single milling.

Microbiological and technological characterization of sourdoughs destined for bread-making with barley flour.

The aim of the present study was the microbiological and technological characterization of laboratory- made sourdoughs for use in barley-flour-based bread-making. A defined multi-strain starter culture consisting of selected lactic acid bacteria (LAB) and yeasts from wheat sourdoughs was inoculated into three flour-water mixtures, composed of: (i) 100% wheat flour (ii) 50% wheat flour and 50% hull-less barley flour (composite flour); (iii) 100% hull-less barley flour. After two months of continuous propagation, the chemical characteristics of the three sourdoughs were investigated by measuring: pH, total titratable acidity and concentrations of various microbial metabolites by HPLC (i.e. lactic, acetic, phenyllactic and butyric acids and diacetyl). The microbial traits were studied through viable counts, isolation and typing of LAB and yeasts and PCR-DGGE analyses. Only Saccharomyces cerevisiae and Lactobacillus plantarum were detectable in the sourdoughs together with other lactobacilli species which were different depending on the type of flour blend used. The molecular typing of the isolates highlighted that only a few strains among those initially inoculated prevailed. The volume increases of the three types of sourdough were also investigated and a correlation was seen between an increase in the barley flour content and a reduction in the dough volume.

The microbial ecology of a typical Italian salami during its natural fermentation.

We have investigated the bacteria and yeast ecology of the typical Italian Ciauscolo salami that is produced in Central Italy using a polyphasic approach based on culture-dependent and -independent methods. The physico-chemical analyses showed a progressive drop in pH and water activity (aw) during ripening. The viable counts revealed a dominance of lactic acid bacteria (LAB) over coagulase negative cocci (CNC) and yeasts. From the molecular identification of the isolates, the prevalence of Lactobacillus curvatus, Lb. plantarum and Staphylococcus xylosus was shown among the bacteria, while Debaryomyces hansenii was the prevalent species among the yeasts, and it was isolated throughout the whole ripening process. Minority species, namely Rhodotorula mucillaginosa and Trichosporon brassicae, were also recovered from the meat batter. The total microbial community was profiled without cultivation by analyzing the DNA that was directly extracted from the salami samples. Moreover, the cultivable community was profiled by analyzing the DNA recovered from bulk cells that were obtained by harvesting the colonies from serial-dilution agar plates. The 16S rRNA gene V1 and V3 regions were used as targets in the denaturing gradient gel electrophoresis (DGGE) profiling of the LAB and CNC communities, respectively, while the diversity and dynamics of the yeast population were assessed by analyzing a portion of the 28S rRNA gene. Our findings suggest that the microbial diversity of fermented meat products can be successfully investigated by this polyphasic approach that is based on the assessment of both the total and the cultivable community diversity.

Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by culture-dependent techniques and PCR-DGGE.

The microbial ecology of 22 samples of commercially available Ciauscolo salami were investigated using a polyphasic approach, based on culture-dependent and -independent techniques. The viable counts of pathogen and hygiene indicator microorganisms highlighted the adequate application of good manufacturing practices, while the viable counts of the lactic acid bacteria, coagulase negative cocci, and yeasts showed dominance of the first of these microbial groups. Bacterial and fungal DNA were extracted directly from the salami and amplified by PCR, using two primer sets targeting the 16S and 28S rRNA genes, respectively. Denaturing gradient gel electrophoresis (DGGE) and sequencing of selected bands were used to investigate the microbial ecology of these Ciauscolo salami. The most frequently found bacterial species were Lactobacillus sakei and Lb. curvatus, while Debaryomyces hansenii was the prevalent yeast species detected. Cluster analysis of the DGGE profiles and calculation of biodiversity indices allowed the degree of microbial similarity across these salami to be determined.