Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to "turn down" neuronal circuits controlling locomotion.     

Mice with selective elimination of striatal acetylcholine release are lean,show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)^{D2‐Cre‐flox/flox} mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT^{D2‐Cre‐flox/flox} mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT^{D2‐Cre‐flox/flox} mice showed increased physical activity. Interestingly, VAChT^{D2‐Cre‐flox/flox} mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.     

Metallothioneins I/II are involved in the neuroprotective effect of sildenafil in focal brain injury

We recently reported that administration of the non-selective cyclic GMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast to cortically cryoinjured rats results three days post-lesion in reduced neuronal cell death that was associated to decreased macrophage/microglial activation and oxidative stress and increased astrogliosis and angiogenesis. Similar effects have been observed in cryoinjured animals overexpressing metallothioneins I/II (MT-I/II), metal-binding cysteine-rich proteins that are up-regulated in response to injury. In this work we have examined the effect of administration of the selective PDE5 inhibitor sildenafil (10 mg/kg, sc) 2 h before and 24 and 48 h after induction of cortical cryolesion in wild-type and MT-I/II-deficient mice. Our results show that in wild-type animals sildenafil induces similar changes in glial reactivity, angiogenesis and antioxidant and antiapoptotic effects in the cryolesioned cortex as those observed in rats with zaprinast, indicating that inhibition of PDE5 is responsible for the neuroprotective actions. However, these effects were not observed in mice deficient in MT-I/II. We further show that sildenafil significantly increases MT-I/II protein levels in homogenates of lesioned cortex and MT-I/II immunostaining in glial cells around the lesion. Taken together these results indicate that cGMP-mediated pathways regulate expression of MT-I/II and support the involvement of these proteins in the neuroprotective effects of sildenafil in focal brain lesion.     

Genetic markers for the identification of hybrids among catfish species of the family Pimelodidae

Fish hybrids provide genetically manipulated products of excellent value for the commercial aquaculture industry. However, if handled or marketed incorrectly, they can cause great financial loss to producers as well as threaten the native species. Herein, molecular markers are established to identify hybrid lineages of pimelodids and characterize them in relation to their parental species, Pseudoplatystoma corruscans, Pseudoplatystoma reticulatum, Phractocephalus hemioliopterus and Leiarius marmoratus. The results show that the mitochondrial genes are useful for identification of the cross‐direction through the characterization of the maternal lineage. The nuclear genes allow identification of the interspecific hybrids. Use of genetic markers can avoid misidentification of hybrids that occur in simple morphological analysis. Thus, the present results allow the routine monitoring of pimelodid hybrids for their correct management and trade in aquaculture.     

P2X7 receptor deletion attenuates oxidative stress and liver damage in sepsis

Purinergic Signalling - Sepsis is a severe disease characterized by an uncontrolled systemic inflammation and consequent organ dysfunction generated in response to an infection. Extracellular ATP...     

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.     

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs,Direct Stool and Stool Culture

Background

We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs.

Methodology/Principal Findings

The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10⁶ CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%–98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%–88.6%) and 100 %, respectively.

Conclusion

This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.     

Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells

Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3′ kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3′ kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3′ kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.     

GluK1 antagonists from 6-(tetrazolyl)phenyl decahydroisoquinoline derivatives: In vitro profile and in vivo analgesic efficacy

We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.     

Successful Treatment of Invasive Fungal Infection Due to Highly Resistant Aspergillus calidoustus in an Allogeneic Hematopoietic Cell Transplant Recipient

Invasive aspergillosis (IA) is the most common invasive fungal infection following a hematopoietic cell transplant, with emerging cryptic species exhibiting resistance to commonly used antifungals such as azoles. These species have been increasingly found after the introduction of anti-mold prophylaxis. We report a case of a 56-year-old female with primary myelofibrosis whose allogeneic hematopoietic cell transplant was complicated by disseminated fungal infection (skin, lung) due to Aspergillus calidoustus, a cryptic specie. Treatment of Aspergillus species remains challenging as these cryptic species are usually resistant to azoles including voriconazole which is the first line of treatment of IA. Infection was successfully treated with surgical excision and combination antifungal therapy based on in vitro susceptibility and synergy testing. Therapy included isavuconazole, a drug that has been shown to be non-inferior to voriconazole in the treatment of invasive mold infections.