Metabarcoding approach to identify bacterial community profiling related to nosocomial infection and bacterial trafficking-routes in hospital environments.

Nosocomial infections (NIs) appear in patients under medical care in the hospital. The surveillance of the bacterial communities employing high-resolution 16S rRNA profiling, known as metabarcoding, represents a reliable method to establish factors that may influence the composition of the bacterial population during NIs. The present study aimed to utilize high-resolution 16S rRNA profiling to identify high bacterial diversity by analyzing 11 inside and 10 outside environments from the General Hospital of Ribeirão Preto Medical School, Brazil. Our results identified a high bacterial diversity, and among these, the most abundant bacterial genera linked to NIs were Cutibacterium, Streptococcus, Staphylococcus, and Corynebacterium. A Acinetobacter was detected in cafeterias, bus stops, and adult and pediatric intensive care units (ICUs). Data suggest an association between transport and alimentation areas proximal to the hospital ICU environment. Interestingly, the correlation and clusterization analysis showed the potential of the external areas to directly influence the ICU pediatric department microbial community, including the outpatient's clinic, visitor halls, patient reception, and the closest cafeterias. Our results demonstrate that high-resolution 16S rRNA profiling is a robust and reliable tool for bacterial genomic surveillance. In addition, the metabarcoding approach might help elaborate decontamination policies, and consequently reduce NIs.

Wnt signaling is involved in crotalphine-induced analgesia in a rat model of neuropathic pain

The aberrant activation of Wnt/β-catenin and atypical Wnt/Ryk signaling pathways in the spinal cord is critical for the development and maintenance of neuropathic pain. Crotalphine is a structural analog to a peptide first identified in Crotalus durissus terrificus snake venom, which induces antinociception by activating kappa-opioid and CB2 cannabinoid receptors. Consistent with previous data, we showed that the protein levels of the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways are increased in neuropathic rats. Importantly, the administration of crotalphine downregulates these protein levels, including its downstream cascades, such as TCF4 from the canonical pathway and NR2B glutamatergic receptor and Ca2+-dependent signals, via the Ryk receptor. The CB2 receptor antagonist, AM630, abolished the crotalphine-induced atypical Wnt/Ryk signaling pathway activation. However, the selective CB2 agonist affects both canonical and non-canonical Wnt signaling in the spinal cord. Next, we showed that crotalphine blocked hypersensitivity and significantly decreased the concentration of IL-1ɑ, IL-1β, IL-6, IL-10, IL-18, TNF-ɑ, MIP-1ɑ and MIP-2 induced by intrathecal injection of exogenous Wnt-3a agonist. Taken together, our findings show that crotalphine induces analgesia in a neuropathic pain model by down-regulating the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways and, consequently controlling neuroinflammation. This effect is, at least in part, mediated by CB2 receptor activation. These results open a perspective for new approaches that can be used to target Wnt signaling in the context of chronic pain.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice / A ativação do eixo PKCε-ALDH2 previne a dor induzida por 4-HNE em camundongos

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Participação da aldeído desidrogenase-2 na neuroinflamação induzida pela constrição do nervo isquiático

Background: Aldehyde dehydrogenase-2 (ALDH2) is a mitochondrial enzyme responsible for detoxifying aldehydes generated from the lipid peroxidation. We previously showed that Alda-1, a small molecule that activates ALDH2, has a potent antinociceptive effect in a chronic neuropathic pain model induced by the chronic constriction injury of the sciatic nerve (CCI) in mice, by decreasing reactive aldehydes, such as 4-hydroxynonenal (4-HNE) at the injured site. One of the most important mechanisms involved in the neuropathic pain onset and chronification is the activation of immune and glial cells. Considering that reactive aldehydes are pro- inflammatory and pro-nociceptive, we hypothesized that 4-HNE is involved in the central sensitization in CCI-induced neuropathy model. Methods: The nociceptive threshold was assessed by the up and down method using von Frey filaments, before (0), 7-, and 14-days post-injury (DPI). The ALDH2 function was modulated by Alda-1 (16 mg/Kg/day, s.c. osmotic pump) or by using a transgenic mice model with an inactivating mutation found commonly in Asiatic populations that impairs ~95% in ALDH2 activity (ALDH2*2). The 4-HNE protein adducts and the activation of glial cells in the dorsal horn of the spinal cord was assessed by using immunofluorescence techniques, followed by microglia morphology, and co- localization analysis. Moreover, axonal degeneration was assessed by evaluating the cell infiltration and fragmentation of myelin in histological preparation of sciatic nerve. Results: CCI decreases the nociceptive threshold in 7 and 14DPI, in wildtype (WT) and ALDH2*2 animals, and Alda-1 prevents CCI-induced hypernociception in both periods and genotypes. Also, CCI increases the immunostaining for 4-HNE adducts, GFAP (astrocyte), Iba-1 (microglia) along with a change in microglia morphology to pro-inflammatory profile in wild type mice. Alda-1 prevents 4-HNE adducts formation and glial cells activation, as measured in microglia morphology. Interestingly, the impairment in ALDH2 activity contributes to changes in glial activation dynamic. Alda- 1 reduces 4-HNE levels in ALDH2*2 mice, without interfering with IBA-1 and GFAP expressions. However, the microglia morphology analysis revealed that Alda-1 might induce a shift from inflammatory to anti-inflammatory profile. The co-localization study showed that 4-HNE accumulates in afferent axons, Schwann cells, as well as spinal cord neurons and oligodendrocytes. Finally, we also showed that CCI induced sciatic nerve degeneration in both genotypes, which was prevented by the treatment with Alda-1. Conclusion: The data suggest that 4-HNE plays an important role in neuropathic hypernociception by controlling aldehyde levels and central neuroinflammation, as well as regulates axonal degeneration and peripheral inflammation in this model. Interestingly, the genetic impairment in ALDH2 activity changes the dynamics of glial activation upon nerve injury. Finally, Alda-1 is a promising molecule for neuropathic pain treatment.

A aldeído desidrogenase-2 (ALDH2) é uma enzima mitocondrial responsável pela detoxicação de aldeídos gerados a partir da peroxidação lipídica. Mostramos anteriormente que a Alda-1, uma pequena molécula ativadora a ALDH2, inibe a hipernocicepção em um modelo de dor neuropática induzida pela constrição crônica do nervo isquiático (CCI) em camundongos, por levar a diminuição de aldeídos reativos, como 4-hidroxinonenal (4-HNE) no local lesionado. Sabe-se que um dos principais mecanismos envolvidos na instalação e na cronificação da dor neuropática é a sensibilização central e periférica, que ocorre por ativação de células imunes e gliais. Considerando que os aldeídos reativos são pró-inflamatórios e pró- nociceptivos, levantamos a hipótese que o 4-HNE participa dos processos de sensibilização central e periférica no modelo de neuropatia induzida por CCI. Métodos: O limiar nociceptivo foi determinado pelo método up and down por meio de filamentos de von Frey, que foi aplicado antes (0), 7 e 14 dias após a cirurgia. A função da ALDH2 foi modulada pelo tratamento com a Alda-1 (16 mg/Kg/dia, s.c. por bomba osmótica), ou por meio de camundongos transgênicos contendo uma mutação encontrada em populações asiáticas que reduz (~ 95%) a atividade de ALDH2 (ALDH2*2). A análise dos níveis de adutos de 4-HNE, ativação de células imunes e gliais e liberação de citocinas foram avaliados por meio de western blot, imunofluorescência e ensaio de multiplex, respectivamente. Ainda, a degeneração axonal foi determinada pela infiltração celular e fragmentação de mielina no preparo histológico do nervo isquiático. Resultados: a CCI diminui o limiar nociceptivo em 7 e 14 dias após a cirurgia, em animais selvagens (WT) e ALDH2*2. A administração de Alda-1 previne a hipernocicepção induzida pela CCI em ambos os períodos e genótipos. Adicionalmente, demonstramos que a CCI aumenta a imunomarcação para adutos de 4-HNE, GFAP (astrócitos), Iba-1 (microglia), juntamente com uma mudança na morfologia da micróglia para um perfil pró-inflamatório, no corno dorsal da medula espinal de animais selvagens. A Alda-1 previne o aumento nos níveis de 4-HNE e na ativação de células gliais. Curiosamente, o prejuízo na atividade da ALDH2 contribui para mudanças na dinâmica de ativação glial, já que a Alda-1 reduz os níveis de 4-HNE em camundongos ALDH2*2, sem interferir nas expressões de IBA-1 e GFAP. No entanto, a análise da morfologia da micróglia revelou que Alda-1 pode induzir uma mudança para um perfil anti-inflamatório. O estudo de co- localização mostrou que o 4-HNE se acumula em axônios aferentes, células de Schwann e macrófagos na periferia, bem como neurônios e oligodendrócitos da medula espinal. Por fim, também mostramos que a CCI induziu degeneração do nervo isquiático em ambos os genótipos, que foi prevenida pelo tratamento com Alda-1. Conclusão: Os dados sugerem que o 4-HNE desempenha um papel importante na hipernocicepção neuropática controlando os níveis de aldeídos e a neuroinflamação central, bem como regula a degeneração axonal e a inflamação periférica neste modelo. Curiosamente, o comprometimento genético na atividade da ALDH2 altera a dinâmica da ativação glial frente à lesão do nervo. Finalmente, a Alda-1 é uma molécula promissora para o tratamento da dor neuropática.