Regionally Distinct Alterations in the Composition of the Gut Microbiota in Rats with Streptozotocin-Induced Diabetes

The aim of this study was to map the microbiota distribution along the gut and establish whether colon/faecal samples from diabetic rats adequately reflect the diabetic alterations in the microbiome. Streptozotocin-treated rats were used to model type 1 diabetes mellitus (T1D). Segments of the duodenum, ileum and colon were dissected, and the microbiome of the lumen material was analysed by using next-generation DNA sequencing, from phylum to genus level. The intestinal luminal contents were compared between diabetic, insulin-treated diabetic and healthy control rats. No significant differences in bacterial composition were found in the luminal contents from the duodenum of the experimental animal groups, whereas distinct patterns were seen in the ileum and colon, depending on the history of the luminal samples. Ileal samples from diabetic rats exhibited particularly striking alterations, while the richness and diversity obscured some of the modifications in the colon. Characteristic rearrangements in microbiome composition and diversity were detected after insulin treatment, though the normal gut flora was not restored. The Proteobacteria displayed more pronounced shifts than those of the predominant phyla (Firmicutes and Bacteroidetes) in the rat model of T1D. Diabetes and insulin replacement affect the composition of the gut microbiota in different, gut region-specific manners. The luminal samples from the ileum appear more suitable for diagnostic purposes than the colon/faeces. The Proteobacteria should be at the focus of diagnosis and potential therapy. Klebsiella are recommended as biomarkers of T1D.     

Altered Community Flammability in Florida’s Apalachicola Ravines and Implications for the Persistence of the Endangered Conifer Torreya taxifolia

Plant species and communities often reflect historic fire regimes via ecological and evolutionary responses to recurrent fires. Plant communities of the southeastern USA experience a wide array of fire regimes, perhaps nowhere more marked than the juxtaposition of fire-prone uplands and adjacent mesic ravines along Florida’s Apalachicola River. The ravines contain many endemic and disjunct species, most notably the endangered endemic conifer Torreya taxifolia. A rapid decline in T. taxifolia over the past 60 years has been associated with widespread replacement by other tree species. To understand the changes accompanying the shift in ravine composition, we compared leaf litter flammability of nine historic and contemporary species. We measured maximum flame height, flame duration, smoldering duration, mass loss, absorptive capacity, and drying rate. Ordination and perMANOVA suggest the nine species segregated into three distinct groups: the fire-impeding T. taxifolia and Taxus floridana; an intermediate group of three deciduous angiosperms; and a mixed cluster of four flammable species. Results suggest T. taxifolia and T. floridana were fire-impeding species in these communities, while contemporary dominants burn similarly to the upslope pyric species. The increasing presence of fire-facilitating species may portend a shifting fire regime that further imperils T. taxifolia and other rare species in the formerly fire-safe ravines.     

Cut‐off values and significance of Oil Red O‐positive cells in bronchoalveolar lavage fluid

C. Basset‐Léobon, L. Lacoste‐Collin, J. Aziza, J.C. Bes, S. Jozan and M. Courtade‐Saïdi
Cut‐off values and significance of Oil Red O‐positive cells in bronchoalveolar lavage fluid Objective: To evaluate the percentage and predictive value of Oil Red O‐positive macrophages (ORO‐PM) to identify lipid‐laden macrophages in bronchoalveolar lavage fluids (BALF) from patients with different pathologies. Methods: The percentage and absolute numbers of ORO‐PM were evaluated in 305 BALF. The patients were separated into ten groups: corticosteroid treatment (n = 18), amiodarone treatment (n = 8), interstitial fibrosis (n = 11), human immunodeficiency virus (HIV)‐positive (n = 25), infectious pneumonia (n = 43), severe haematological disorder (n = 25), interstitial syndrome (n = 109), suspicion of cancer (n = 17), transplant recipients (n = 50) and controls (n = 43). The total and differential cell counts in BALF were recorded. The presence of specific pathogens was also noted. Parametric and non‐parametric tests were used to compare the values between groups. Receiver–operating characteristics (ROC) curves were established in order to determine a cut‐off value. Results: The percentages of ORO‐PM were (mean ± standard deviation) 21.67 ± 29.12 in the corticosteroid group, 10.00 ± 12.49 in the amiodarone group, 19.45 ± 20.72 in the interstitial fibrosis group, 47.80 ± 30.46 in the HIV group, 19.72 ± 26.26 in the infectious pneumonia group, 27.42 ± 30.04 in the severe haematological disorder group, 25.18 ± 30.63 in the interstitial syndrome group, 17.64 ± 27.76 in the suspicion of cancer group, 22.50 ± 27.27 in the transplanted recipients group and 2.63 ± 3.48 in the control group. Significantly higher values were found in all groups when compared with the control group (P < 0.001). Only the HIV group showed higher numbers of ORO‐PM when compared with the interstitial syndrome group (P < 0.01). According to ROC curves, > 6% ORO‐PM was suggested as the positive cut‐off value. Conclusion: Significantly increased numbers of ORO‐PM were associated with various lung pathologies. However, the higher numbers observed in HIV patients require further investigations.     

3-Desamino-3α-hydroxysolanocapsine—a steroidal alkaloid from Solanum aculeatum

A new steroidal alkaloid has been obtained from roots of Solanum aculeatum and its structure elucidated by IR, NMR and mass spectral studies as 3-desamino-3β-hydroxysolanocapsine.     

Synthesis of the C-terminal domain of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

According to recent investigations, the C-terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N-terminal domain of the molecule. The C-terminal domain has been prepared for structure-biological activity investigations. After the chemical synthesis and the folding of the linear peptide. LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N-terminal domain no entirely correct folding of the C-terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated--using disulphide bonded TIMP-1(Cys145-Cys166) as a model--that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid.     

Elevated atmospheric CO2 decreases oxidative stress and increases essential oil yield in leaves of Thymus vulgaris grown in a mini-FACE system

Potted one-year-old plants of Thymus vulgaris L. (thyme, Lamiaceae, C3 metabolism), were grown for three months (10 June–10 September, 2004) in a “mini-free-air-CO₂-enrichment” (“mini-FACE”) system, under 500 μmol mol^?1 and ambient concentrations of atmospheric carbon dioxide (CO₂). Compared to ambient CO₂, elevated CO₂ stimulated leaf superoxide dismutase (SOD, EC 1.15.1.1) activity only at the first sampling-time (July), followed by no variation or even a trend of decreased activity on the other two sampling-times (August and September). Under high CO₂, guaiacol peroxidase (GPX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) leaf activities showed no variation or drop throughout the duration of the experiment. By contrast, under elevated CO₂, leaf glutathione reductase (GR, EC 1.6.4.2) activity increased on all the sampling-times, and also a duration-dependent upward trend of glutathione (GSH) content was recorded, with this increase becoming significant – compared with ambient CO₂ – at the third sampling-time (September). Simultaneously, leaves from plants grown under high CO₂ showed a marked increase in essential oil yield, with slight increments in phenolic component and decrements in mono- and sesquiterpene components. Also, a drop in thiobarbituric acid reactive substances (TBARS) content under elevated CO₂ was displayed. Thus, in general, the results reported here point to a downregulation of leaf antioxidant enzymes under elevated CO₂, supporting the notion of reduced reactive oxygen species (ROS) formation under these circumstances. Relying instead on antioxidant-regenerating enzymes, namely GR, fairly high GSH content and essential oils, might be a ‘low cost’ life strategy for growth under elevated CO₂, not requiring synthesis/activation of energy-intensive and expensive metabolic processes.     

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK''s pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen–glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.In many disorders of the nervous system, overactivation of N-methyl-D-aspartate (NMDA) receptors leads to neuronal death and consequent neurological impairment. NMDA-induced neuronal death, that is, excitotoxicity, has been implicated in many neurodegenerative diseases such as stroke, epilepsy, Alzheimer disease, spinal cord injury, traumatic brain injury, hearing loss, Parkinson''s and Huntington diseases.¹ However, the molecular mechanisms underlying excitotoxic neuronal death remain only partially understood.Excitotoxicity triggers complex signal transduction events that induce the neuronal death program. Among them, activation of the c-Jun N-terminal kinase (JNK) pathway has a key role.^{2, 3, 4, 5} There are only two direct upstream activators of JNK: mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7).^{6, 7} In some cell types, MKK4 activates JNK primarily in response to stress stimuli, whereas MKK7 signaling is triggered by release of inflammatory cytokines.^{8, 9, 10} In neurons, however, we showed that MKK7 is mainly responsible for JNK overactivation during excitotoxicity both in vitro³ and in vivo following middle cerebral artery occlusion (MCAo).⁴ Conversely, MKK4 controls JNK physiological role and its activation is not affected by excitotoxic stimuli.³Inhibition of the JNK pathway by the specific JNK inhibitor peptide, D-JNKI1, has been proposed for the treatment of ischemia.² D-JNKI1 induces powerful neuroprotection in in vitro models of excitoxicity^{2, 11} and leads to a 93% reduction in the infarct size in rodent models of ischemia.^{2, 4, 12} Despite the potent and long-lasting neuroprotective effect of D-JNKI1, total inhibition of JNK is not deprived of negative side effects, as it regulates a variety of physiological events¹³ such as cell proliferation, survival and differentiation.¹³ For these reasons, MKK7 may represent a more attractive target for clinical application, as it activates JNK specifically after toxic stimuli. Thus, by targeting MKK7 the physiological role of JNK, regulated by MKK4, will be preserved.Here we designed a set of new cell-permeable inhibitor peptides able to block MKK7 activity and protect against excitotoxic death.We took advantage of the growth arrest and DNA damage-inducible 45β (GADD45β) ability to bind MKK7.^{9, 14, 15} GADD45β is involved in the control of cell stress responses in cell cycle, DNA repair and oncogenesis.^{9, 16} GADD45β binds tightly to MKK7 and inhibits its enzymatic activity¹⁵ by interacting with its catalytic domain.⁹ More importantly, GADD45β inhibition is MKK7-specific and has no effect on MKK4, MKK3/6 and MEK1/2 activity.⁹ The minimal essential domain of interaction between MKK7 and GADD45β has already been defined (GADD45β_60–86 and _69–86 sequences).¹⁵ We here used in silico approaches to design an effector peptide, based on the domain of GADD45β, and optimize its affinity for MKK7. We then linked the effector peptide to a TAT-cargo in order to penetrate neuronal plasma membrane.¹⁷ The selected cell-permeable MKK7 inhibitor peptide (GADD45β-l) confers neuroprotection in vitro against NMDA and oxygen–glucose deprivation (OGD) toxicity, as well as in vivo in two models of MCAo with a clinically relevant post-ischemic temporal window (6 h) at both 24 h and 1 week after lesion. These data shed light on a new approach for the treatment of ischemia.     

Familiar Face Detection in 180ms

The visual system is tuned for rapid detection of faces, with the fastest choice saccade to a face at 100ms. Familiar faces have a more robust representation than do unfamiliar faces, and are detected faster in the absence of awareness and with reduced attentional resources. Faces of family and close friends become familiar over a protracted period involving learning the unique visual appearance, including a view-invariant representation, as well as person knowledge. We investigated the effect of personal familiarity on the earliest stages of face processing by using a saccadic-choice task to measure how fast familiar face detection can happen. Subjects made correct and reliable saccades to familiar faces when unfamiliar faces were distractors at 180ms—very rapid saccades that are 30 to 70ms earlier than the earliest evoked potential modulated by familiarity. By contrast, accuracy of saccades to unfamiliar faces with familiar faces as distractors did not exceed chance. Saccades to faces with object distractors were even faster (110 to 120 ms) and equivalent for familiar and unfamiliar faces, indicating that familiarity does not affect ultra-rapid saccades. We propose that detectors of diagnostic facial features for familiar faces develop in visual cortices through learning and allow rapid detection that precedes explicit recognition of identity.