Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

BACKGROUND: In the genus Streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression (CCR). This mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. CCR also affects the synthesis of secondary metabolites and morphological differentiation. Even when the outcome effect of CCR in different bacteria is the same, their essential mechanisms can be quite different. In several streptomycetes glucose kinase (Glk) represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in CCR. To evaluate the paradigmatic model proposed for CCR in Streptomyces, a high-density microarray approach was applied to Streptomyces coelicolor M145, under repressed and non-repressed conditions. The transcriptomic study was extended to assess the ScGlk role in this model by comparing the transcriptomic profile of S. coelicolor M145 with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from Zymomonas mobilis (Zmglk), insensitive to CCR but able to grow in glucose (ScoZm strain). RESULTS: Microarray experiments revealed that glucose influenced the expression of 651 genes. Interestingly, even when the ScGlk protein does not have DNA binding domains and the glycolytic flux was restored by a heterologous glucokinase, the ScGlk replacement modified the expression of 134 genes. From these, 91 were also affected by glucose while 43 appeared to be under the control of ScGlk. This work identified the expression of S. coelicolor genes involved in primary metabolism that were influenced by glucose and/or ScGlk. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. CONCLUSIONS: The transcriptome of a classical model of CCR was studied in S. coelicolor to differentiate between the effects due to glucose or ScGlk in this regulatory mechanism. Glucose elicited important metabolic and transcriptional changes in this microorganism. While its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. Glucose also triggered the CCR by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. Our results confirm and update the agar model of the CCR in Streptomyces and its dependence on the ScGlk per se. Surprisingly, the expected regulatory function of ScGlk was not found to be as global as thought before (only 43 out of 779 genes were affected), although may be accompanied or coordinated by other transcriptional regulators. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. These findings offer new opportunities to study and understand the CCR in S. coelicolor by increasing the number of known glucose and ScGlk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the CCR.

Evaluation of Poly-3-Hydroxybutyrate (P3HB) Scaffolds Used for Epidermal Cells Growth as Potential Biomatrix.

Advances in tissue engineering have made possible the construction of organs and tissues with the use of biomaterials and cells. Three important elements are considered: a specific cell culture, an adequate environment, and a scaffold. The present study aimed to develop P3HB scaffolds by 3D printing and evaluate their biocompatibility with HaCaT epidermal cells, as a potential model that allows the formation of functional tissue. By using a method of extraction and purification with ethanol and acetone, a biopolymer having suitable properties for use as a tissue support was obtained. This polymer exhibited a higher molecular weight (1500 kDa) and lower contact angle (less than 90°) compared to the material obtained using the conventional method. The biocompatibility analysis reveals that the scaffold obtained using the ethanol-acetone method and produced by 3D printing without pores was not cytotoxic, did not self-degrade, and allowed high homogenous cell proliferation of HaCaT cells. In summary, it is possible to conclude that the P3HB scaffold obtained by 3D printing and a simplified extraction method is a suitable support for the homogeneous development of HaCaT keratinocyte cell lineage, which would allow the evaluation of this material to be used as a biomatrix for tissue engineering.

Asociación de marcadores inflamatorios en COVID-19 severo al ingreso con mortalidad y disfunción orgánica / Association of severe COVID-19 inflammatory markers at hospitalization with mortality and organic dysfunction / Associação de marcadores inflamatórios em COVID-19 grave na admissão com mortalidade e disfunção orgânica

Resumen: Introducción: se ha demostrado que los niveles iniciales de marcadores inflamatorios involucrados en COVID-19 (ej. ferritina, proteína C reactiva, procalcitonina, dímero D e interleucina-6) se relacionan con la mortalidad, sin encontrar resultados similares en pacientes con COVID-19 severo o quienes se encuentran bajo ventilación mecánica invasiva. Objetivo: determinar el nivel sérico con mayor sensibilidad y especificidad en los marcadores inflamatorios con relación a la mortalidad y gravedad de la disfunción orgánica en pacientes con COVID-19 severo usuarios de ventilación mecánica invasiva en las primeras 48 horas tras el ingreso hospitalario. Material y métodos: se realizó un estudio descriptivo de tipo cohorte retrospectiva y longitudinal en pacientes con diagnóstico de COVID-19 severo que fueran intubados antes de 48 horas tras el ingreso hospitalario por falla respiratoria aguda de enero de 2021 a agosto de 2021. Se determinó la relación entre los niveles de estos marcadores con las escalas pronósticas (SOFA, APACHE-II y SAPS-II), días de estancia hospitalaria, días en la Unidad de Terapia Intensiva Respiratoria, días de ventilación mecánica invasiva y las características de la mecánica ventilatoria inicial. Se agruparon los marcadores en niveles elevados y bajos para determinar su papel individual y en conjunto con los desenlaces. Resultados: se estudió una N = 218, con predominio de género masculino (77.5%) con media de edad de 60.3 ± 12.8 años. La hipertensión arterial sistémica y la diabetes mellitus tipo 2 fueron las comorbilidades más prevalentes (50.5% y 26.1%, respectivamente). La mediana de la relación PaO2/FiO2 fue de 128 mmHg (83.3-204.2), con una mortalidad total de 24.8%. Los niveles de biomarcadores con mayor sensibilidad para mortalidad y disfunción orgánica fueron: proteína C reactiva: ≥ 16 mg/dL, procalcitonina: ≥ 0.83 ng/mL, dímero D: ≥ 1,290 ng/mL, ferritina: ≥ 1,450 ng/mL e interleucina-6: ≥ 195 pg/mL. La procalcitonina y la interleucina-6 de manera aislada demostraron mayor riesgo de mortalidad y peor disfunción orgánica. Los marcadores inflamatorios se relacionaron a peor desenlace con respecto a las características del sistema respiratorio y el grado de alteración en gases arteriales. De forma conjunta (≥ 3 altos), los marcadores inflamatorios se relacionaron a mayor número de días de estancia hospitalaria, días en la Unidad de Terapia Intensiva Respiratoria y de días de ventilación mecánica invasiva. La proteína C reactiva, procalcitonina e interleucina-6 se asociaron a mayor riesgo de peor grado de disfunción orgánica por SOFA y peor pronóstico por APACHE-II y SAPS-II. Conclusión: la medición individual y conjunta de marcadores inflamatorios al ingreso hospitalario puede identificar a pacientes con mayor riesgo de estancia hospitalaria prolongada, así como ventilación mecánica invasiva, con mayor riesgo de mortalidad en el caso de procalcitonina e interleucina-6.

Abstract: Introduction: it has being demonstrated that the initial levels of inflammatory markers involved in COVID-19 (eg. C-reactive protein, procalcitonin, D-dimer, ferritin and interleukine-6) have an association with mortality, in different degree on severe COVID-19 patients or in those on invasive mechanical ventilation secondary to COVID-19 related acute respiratory distress syndrome. Objective: to determine the serum levels of these markers with the greatest sensibility and specificity for mortality and worst organ dysfunction in patients under invasive mechanical ventilation within the first 48 hours of hospitalization. Material and methods: in a retrospective and longitudinal cohort of severe COVID-19 patients on invasive mechanical ventilation within first 48 hours of hospitalization due to respiratory failure through January 2021 to August 2021, we determined the relation of inflammatory markers with prognostic scores (SOFA, APACHE-II and SAPS-II), hospital length-of-stay (LOS), intensive care LOS, invasive ventilation's days and initial ventilatory mechanics. We divided markers in high and low levels to identify the relation between each one and by groups with the outcomes. Results: we studied a N = 218, with male predominance (77.5%) and mean age of 60.3 ± 12.8 years. Arterial hypertension and diabetes mellitus type 2 were the most prevalent co-comorbidities (50.5% y 26.1%, respectively). The median initial PaO2/FiO2 was 128 mmHg (83.3-204.2), with a total mortality rate of 24.8%. Inflammatory markers levels with the highest sensibility for mortality were: C-reactive protein: ≥ 16 mg/dL, procalcitonin: ≥ 0.83 ng/mL, D-dimer: ≥ 1,290 ng/mL, ferritin: ≥ 1,450 ng/mL and interleukin-6: ≥ 195 pg/mL. Procalcitonin and interleukin-6 were associated to higher risk of mortality and worst organ dysfunction. The inflammatory markers were related with worst outcome in relation to respiratory mechanics and the amount of arterial-blood gases' alteration. Having ≥ 3 inflammatory markers within high levels was associated with prolonged LOS, more intensive care LOS and more days under invasive mechanical ventilation. The c-reactive protein, procalcitonin and interleukin-6 had higher organic dysfunction defined by SOFA and worst outcome defined by APACHE-II and SAPS-II. Conclusion: individual and joint measurement of inflammatory markers at hospitalization can identify patients with greater risk of longer hospital LOS, intensive care LOS and longer mechanical ventilation's days, with greater risk of mortality with higher procalcitonin and interleukine-6 serum levels.

Resumo: Introdução: demonstrou-se que os níveis iniciais de marcadores inflamatórios envolvidos no COVID-19 (por exemplo, ferritina, proteína C reativa, procalcitonina, D-dímero e interleucina-6) estão relacionados à mortalidade, sem encontrar resultados semelhantes em pacientes com COVID-19 grave ou que estejam sob ventilação mecânica invasiva. Objetivos: nosso objetivo foi determinar o nível sérico com maior sensibilidade e especificidade em marcadores inflamatórios em relação à mortalidade e gravidade da disfunção orgânica em pacientes com COVID-19 grave que usaram ventilação mecânica invasiva nas primeiras 48 horas após a admissão hospitalar. Material e métodos: realizou-se um estudo descritivo do tipo coorte retrospectivo e longitudinal em pacientes diagnosticados com COVID-19 grave que foram intubados nas primeiras 48 horas após a internação hospitalar por insuficiência respiratória aguda no período de janeiro de 2021 a agosto de 2021. A relação entre os níveis desses marcadores com as escalas de prognóstico (SOFA, APACHE-II e SAPS-II), dias de internação, dias na unidade de terapia intensiva respiratória, dias de ventilação mecânica invasiva e as características da ventilação mecânica inicial. Agrupou-se marcadores em níveis altos e baixos para determinar seu papel individualmente e em conjunto com os resultados. Resultados: estudou-se uma N = 218, com predominância do sexo masculino (77.5%) com idade média de 60.3 ± 12.8 anos. A hipertensão arterial sistêmica e a diabetes mellitus tipo 2 foram as comorbidades mais prevalentes (50.5% e 26.1%, respectivamente). A mediana da relação PaO2/FiO2 foi de 128 mmHg (83.3-204.2), com mortalidade total de 24.8%. Os níveis de biomarcadores com maior sensibilidade para mortalidade e disfunção orgânica foram: proteína C reativa: ≥ 16 mg/dL, procalcitonina: ≥ 0.83 ng/mL, dímero: ≥ 1.290 ng/mL, ferritina: ≥ 1.450 ng/mL, e interleucina-6: ≥ 195 pg/mL. A procalcitonina e a interleucina-6 sozinhas demonstraram maior risco de mortalidade e pior disfunção orgânica. Os marcadores inflamatórios foram relacionados a pior evolução quanto às características do sistema respiratório e ao grau de alteração dos gases arteriais. Juntos (≥ 3 altos), os marcadores inflamatórios foram relacionados a um maior número de dias de internação, dias na unidade de terapia intensiva respiratória e dias de ventilação mecânica invasiva. A proteína C-reativa, procalcitonina e interleucina-6 foram associadas a maior risco de pior grau de disfunção orgânica pelo SOFA e pior prognóstico pelo APACHE-II e SAPS-II. Conclusão: a medida individual e conjunta de marcadores inflamatórios na admissão hospitalar pode identificar pacientes com maior risco de internação prolongada e ventilação mecânica invasiva, com maior risco de mortalidade no caso da procalcitonina e interleucina-6.