RESUMO
Microalgae biofilm emerged as a solid alternative to conventional suspended cultures which present high operative costs and complex harvesting processes. Among several designs, rotating biofilm-based systems stand out for their scalability, although their primary applications have been in wastewater treatment and aquaculture. In this work, a rotating system was utilized to produce a high-value compound (astaxanthin) using Haematococcus pluvialis biofilms. The effect of nitrogen regime, light intensity, and light history on biofilm traits was assessed to better understand how to efficiently operate the system. Our results show that H. pluvialis biofilms follow the classical growth stages described for bacterial biofilms (from adhesion to maturation) and that a two-stage (green and red stages) allowed to reach astaxanthin productivities of 204 mg m-2 d-1 . The higher light intensity applied during the red stage (400 and 800 µmol m-2 s-1 ) combined with nitrogen depletion stimulated similar astaxanthin productivities. However, by training the biofilms during the green stage, using mild-light intensity (200 µmol m-2 s-1 ), a process known as priming, the final astaxanthin productivity was enhanced by 40% with respect to biofilms pre-exposed to 50 µmol m-2 s-1 . Overall, this study shows the possibility of utilizing rotating microalgae biofilms to produce high-value compounds laying the foundation for further biotechnological applications of these emerging systems.
Assuntos
Clorofíceas , Clorófitas , Microalgas , Luz , Nitrogênio , XantofilasRESUMO
In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. KEY POINTS: â¢EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. â¢Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. â¢The anti-adhesive effect is strain-specific and affects both Gram - and Gram + bacteria.
Assuntos
Clorófitas , Closterium , Microalgas , Humanos , Aderência Bacteriana , Matriz Extracelular de Substâncias Poliméricas , Escherichia coli , Staphylococcus aureus , BiofilmesRESUMO
Mouse models of Alzheimer's disease (AD) show progression through stages reflective of human pathology. Proteomics identification of temporal and sex-linked factors driving AD-related pathways can be used to dissect initiating and propagating events of AD stages to develop biomarkers or design interventions. In the present study, we conducted label-free proteome measurements of mouse hippocampus tissue with variables of time (3, 6, and 9 months), genetic background (5XFAD versus WT), and sex (equal males and females). These time points are associated with well-defined phenotypes with respect to the following: Aß42 plaque deposition, memory deficits, and neuronal loss, allowing correlation of proteome-based molecular signatures with the mouse model stages. Our data show 5XFAD mice exhibit increases in known human AD biomarkers as amyloid-beta peptide, APOE, GFAP, and ITM2B are upregulated across all time points/stages. At the same time, 23 proteins are here newly associated with Alzheimer's pathology as they are also dysregulated in 5XFAD mice. At a pathways level, the 5XFAD-specific upregulated proteins are significantly enriched for DNA damage and stress-induced senescence at 3-month only, while at 6-month, the AD-specific proteome signature is altered and significantly enriched for membrane trafficking and vesicle-mediated transport protein annotations. By 9-month, AD-specific dysregulation is also characterized by significant neuroinflammation with innate immune system, platelet activation, and hyper-reactive astrocyte-related enrichments. Aside from these temporal changes, analysis of sex-linked differences in proteome signatures uncovered novel sex and AD-associated proteins. Pathway analysis revealed sex-linked differences in the 5XFAD model to be involved in the regulation of well-known human AD-related processes of amyloid fibril formation, wound healing, lysosome biogenesis, and DNA damage. Verification of the discovery results by Western blot and parallel reaction monitoring confirm the fundamental conclusions of the study and poise the 5XFAD model for further use as a molecular tool for understanding AD.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/metabolismo , Amiloide , Peptídeos beta-Amiloides/metabolismo , Animais , Apolipoproteínas E/metabolismo , Biomarcadores , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , ProteomaRESUMO
Methionine oxidation to the sulfoxide form (MSox) is a poorly understood post-translational modification of proteins associated with non-specific chemical oxidation from reactive oxygen species (ROS), whose chemistries are linked to various disease pathologies, including neurodegeneration. Emerging evidence shows MSox site occupancy is, in some cases, under enzymatic regulatory control, mediating cellular signaling, including phosphorylation and/or calcium signaling, and raising questions as to the speciation and functional nature of MSox across the proteome. The 5XFAD lineage of the C57BL/6 mouse has well-defined Alzheimer's and aging states. Using this model, we analyzed age-, sex-, and disease-dependent MSox speciation in the mouse hippocampus. In addition, we explored the chemical stability and statistical variance of oxidized peptide signals to understand the needed power for MSox-based proteome studies. Our results identify mitochondrial and glycolytic pathway targets with increases in MSox with age as well as neuroinflammatory targets accumulating MSox with AD in proteome studies of the mouse hippocampus. Further, this paper establishes a foundation for reproducible and rigorous experimental MSox-omics appropriate for novel target identification in biological discovery and for biomarker analysis in ROS and other oxidation-linked diseases.
Assuntos
Envelhecimento , Doença de Alzheimer , Glicólise , Hipocampo , Metionina , Camundongos Endogâmicos C57BL , Mitocôndrias , Proteômica , Animais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Hipocampo/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteômica/métodos , Metionina/metabolismo , Metionina/análogos & derivados , Envelhecimento/metabolismo , Masculino , Feminino , Oxirredução , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Modelos Animais de DoençasRESUMO
We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 µm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass.
Assuntos
Microalgas , Fotossíntese , Biofilmes , Carbono , Simulação por ComputadorRESUMO
Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.
Assuntos
Aberrações Cromossômicas , Proteínas de Ligação a DNA/genética , Doenças Fetais/genética , Mutação , Obstrução do Colo da Bexiga Urinária/congênito , Obstrução do Colo da Bexiga Urinária/genética , Adulto , Animais , Criança , Feminino , Doenças Fetais/patologia , Genes Dominantes , Idade Gestacional , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Gravidez , Obstrução do Colo da Bexiga Urinária/patologia , Peixe-ZebraRESUMO
Microalgae biofilms have great ecological importance and high biotechnological potential. Nevertheless, an in-depth and combined structural (i.e., the architecture of the biofilm) and physiological characterization of microalgae biofilms is still missing. An approach able to provide the same time physiological and structural information during biofilm growth would be of paramount importance to understand these complex biological systems and to optimize their productivity. In this study, monospecific biofilms of a diatom and a green alga were grown under dynamic conditions in custom flow cells represented by UV/Vis spectroscopic cuvettes. Such flow cells were conceived to characterize the biofilms by several techniques mostly in situ and in a nondestructive way. Physiological traits were obtained by measuring variable chlorophyll a fluorescence by pulse amplitude modulated fluorometry and by scanning the biofilms in a spectrometer to obtain in vivo pigments spectral signatures. The architectural features were obtained by imaging the biofilms with a confocal laser scanning microscopy and an optical coherence tomography. Overall, this experimental setup allowed us to follow the growth of two biofilm-forming microalgae showing that cell physiology is more affected in complex biofilms likely as a consequence of alterations in local environmental conditions.
Assuntos
Biofilmes , Tomografia de Coerência Óptica , Clorofila A , Microscopia Confocal/métodos , Tomografia de Coerência Óptica/métodosRESUMO
The maintenance of the stem cell niche in the shoot apical meristem, the structure that generates all of the aerial organs of the plant, relies on a canonical feedback loop between WUSCHEL (WUS) and CLAVATA3 (CLV3). WUS is a homeodomain transcription factor expressed in the organizing centre that moves to the central zone to promote stem cell fate. CLV3 is a peptide whose expression is induced by WUS in the central zone and that can move back to the organizing centre to inhibit WUS expression. Within the past 20 years since the initial formulation of the CLV-WUS feedback loop, the mechanisms of stem cell maintenance have been intensively studied and the function of WUS has been redefined. In this review, we highlight the most recent advances in our comprehension of the molecular mechanisms of WUS function, of its interaction with other transcription factors and hormonal signals, and of its connection to environmental signals. Through this, we will show how WUS can integrate both internal and external cues to adapt meristem function to the plant environment.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Meristema/metabolismo , Brotos de Planta/metabolismoRESUMO
Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Flavonoides/química , Glicogênio Fosforilase/metabolismo , Humanos , Hipoglicemiantes/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Transforming growth factor-ß (TGFß) has been reported to be dysregulated in malformed ureters. There exists, however, little information on whether altered TGFß levels actually perturb ureter development. We therefore hypothesised that TGFß has functional effects on ureter morphogenesis. Tgfb1, Tgfb2 and Tgfb3 transcripts coding for TGFß ligands, as well as Tgfbr1 and Tgfbr2 coding for TGFß receptors, were detected by quantitative polymerase chain reaction in embryonic mouse ureters collected over a wide range of stages. As assessed by in situ hybridisation and immunohistochemistry, the two receptors were detected in embryonic urothelia. Next, TGFß1 was added to serum-free cultures of embryonic day 15 mouse ureters. These organs contain immature smooth muscle and urothelial layers and their in vivo potential to grow and acquire peristaltic function can be replicated in serum-free organ culture. Such organs therefore constitute a suitable developmental stage with which to define roles of factors that affect ureter growth and functional differentiation. Exogenous TGFß1 inhibited growth of the ureter tube and generated cocoon-like dysmorphogenesis. RNA sequencing suggested that altered levels of transcripts encoding certain fibroblast growth factors (FGFs) followed exposure to TGFß. In serum-free organ culture exogenous FGF10 but not FGF18 abrogated certain dysmorphic effects mediated by exogenous TGFß1. To assess whether an endogenous TGFß axis functions in developing ureters, embryonic day 15 explants were exposed to TGFß receptor chemical blockade; growth of the ureter was enhanced, and aberrant bud-like structures arose from the urothelial tube. The muscle layer was attenuated around these buds, and peristalsis was compromised. To determine whether TGFß effects were limited to one stage, explants of mouse embryonic day 13 ureters, more primitive organs, were exposed to exogenous TGFß1, again generating cocoon-like structures, and to TGFß receptor blockade, again generating ectopic buds. As for the mouse studies, immunostaining of normal embryonic human ureters detected TGFßRI and TGFßRII in urothelia. Collectively, these observations reveal unsuspected regulatory roles for endogenous TGFß in embryonic ureters, fine-tuning morphogenesis and functional differentiation. Our results also support the hypothesis that the TGFß up-regulation reported in ureter malformations impacts on pathobiology. Further experiments are needed to unravel the intracellular signalling mechanisms involved in these dysmorphic responses. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Morfogênese , Fator de Crescimento Transformador beta/metabolismo , Ureter/anormalidades , Ureter/metabolismo , Anormalidades Urogenitais/metabolismo , Urotélio/anormalidades , Urotélio/metabolismo , Animais , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Humanos , Camundongos , Técnicas de Cultura de Órgãos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Ureter/efeitos dos fármacos , Anormalidades Urogenitais/genética , Urotélio/efeitos dos fármacosRESUMO
Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract.
Assuntos
Glucuronidase/genética , Glicoproteínas de Membrana/genética , Doenças do Sistema Nervoso Periférico/genética , Obstrução do Colo da Bexiga Urinária/genética , Bexiga Urinária/inervação , Doenças Urológicas/genética , Animais , Criança , Análise Mutacional de DNA , Fácies , Feminino , Perfilação da Expressão Gênica , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso Periférico/patologia , Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/patologia , Doenças Urológicas/patologiaRESUMO
Radiographs are an important diagnostic tool available in wildlife hospitals to evaluate the size of the avian heart. Despite the large variety wild birds in the Iberian peninsula, clinical studies addressing these species are lacking. To establish reference values for cardiac size in the Bonelli's eagle (Aquila fasciata), ventrodorsal radiographs of 20 healthy birds were obtained, and the width of the cardiac silhouette, sternum, thorax, coracoid, and hepatic silhouette were measured. The ratios between cardiac width and other mentioned indices were calculated. Results showed that cardiac silhouette width should occupy 81%-93% of sternal width, 48%-57% of thoracic width, and 506%-673% of coracoid width. The cardiac silhouette width was strongly correlated with sternal and thoracic widths. A moderate correlation was found between the width of the heart and the width of the coracoid. No significant correlation was found between width of the cardiac silhouette and the hepatic silhouette. These results support that sternal and thoracic widths should be used preferentially when evaluating the width of the cardiac silhouette in this species. The values obtained in this study can be used as a reference for normal cardiac size when evaluating radiographs of Bonelli's eagles.
Assuntos
Águias/anatomia & histologia , Coração/diagnóstico por imagem , Animais , Processo Coracoide/diagnóstico por imagem , Fígado/diagnóstico por imagem , Radiografia/veterinária , Valores de Referência , Estudos Retrospectivos , Esterno/diagnóstico por imagem , Cavidade Torácica/diagnóstico por imagemRESUMO
A kinetic model of the simultaneous saccharification, protein hydrolysis, and fermentation (SSPHF) process for lactic acid production from wheat flour has been developed. The model describes the bacterial growth, substrate consumption, lactic acid production, and maltose hydrolysis. The model was fitted and validated with data from SSPHF experiments obtained under different dilution rates. The results of the model are in good agreement with the experimental data. Steady state concentrations of biomass, lactic acid, glucose, and maltose as function of the dilution rate were predicted by the model. This steady state analysis is further useful to determine the operating conditions that maximize lactic acid productivity.
Assuntos
Farinha , Ácido Láctico/metabolismo , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Modelos Teóricos , Triticum/metabolismo , Fermentação , Hidrólise , Maltose/metabolismoRESUMO
γ-Herpesviruses express proteins that modulate B lymphocyte signaling to achieve persistent latent infections. One such protein is the M2 latency-associated protein encoded by the murid herpesvirus-4. M2 has two closely spaced tyrosine residues, Tyr(120) and Tyr(129), which are phosphorylated by Src family tyrosine kinases. Here we used mass spectrometry to identify the binding partners of tyrosine-phosphorylated M2. Each M2 phosphomotif is shown to bind directly and selectively to SH2-containing signaling molecules. Specifically, Src family kinases, NCK1 and Vav1, bound to the Tyr(P)(120) site, PLCγ2 and the SHP2 phosphatase bound to the Tyr(P)(129) motif, and the p85α subunit of PI3K associated with either motif. Consistent with these data, we show that M2 coordinates the formation of multiprotein complexes with these proteins. The effect of those interactions is functionally bivalent, because it can result in either the phosphorylation of a subset of binding proteins (Vav1 and PLCγ2) or in the inactivation of downstream targets (AKT). Finally, we show that translocation to the plasma membrane and subsequent M2 tyrosine phosphorylation relies on the integrity of a C-terminal proline-rich SH3 binding region of M2 and its interaction with Src family kinases. Unlike other γ-herpesviruses, that encode transmembrane proteins that mimic the activation of ITAMs, murid herpesvirus-4 perturbs B cell signaling using a cytoplasmic/membrane shuttling factor that nucleates the assembly of signaling complexes using a bilayered mechanism of phosphotyrosine and proline-rich anchoring motifs.
Assuntos
Linfócitos B/metabolismo , Infecções por Herpesviridae/metabolismo , Complexos Multiproteicos/metabolismo , Muromegalovirus/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Linfócitos B/virologia , Membrana Celular/genética , Membrana Celular/metabolismo , Infecções por Herpesviridae/genética , Camundongos , Complexos Multiproteicos/genética , Muromegalovirus/genética , Ligação Proteica , Transporte Proteico/genética , Proteínas Virais/genética , Domínios de Homologia de srcRESUMO
In the framework of environment preservation, microalgae biotechnology appears as a promising alternative for CO2 mitigation. Advanced control strategies can be further developed to maximize biomass productivity, by maintaining these microorganisms in bioreactors at optimal operating conditions. This article proposes the implementation of Nonlinear Predictive Control combined with an on-line estimation of the biomass concentration, using dissolved carbon dioxide concentration measurements. First, optimal culture conditions are determined so that biomass productivity is maximized. To cope with the lack of on-line biomass concentration measurements, an interval observer for biomass concentration estimation is built and described. This estimator provides a stable accurate interval for the state trajectory and is further included in a nonlinear model predictive control framework that regulates the biomass concentration at its optimal value. The proposed methodology is applied to cultures of the microalgae Chlorella vulgaris in a laboratory-scale continuous photobioreactor. Performance and robustness of the proposed control strategy are assessed through experimental results.
Assuntos
Dióxido de Carbono/química , Microalgas/metabolismo , Fotobiorreatores , Algoritmos , Biomassa , Biotecnologia , Técnicas de Cultura de Células , Chlorella vulgaris/metabolismo , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Modelos Teóricos , Fatores de TempoRESUMO
The interest by biofilm-based microalgae technologies has increased lately due to productivity improvement, energy consumption reduction and easy harvesting. However, the effect of light, one key factor for system's operation, received less attention than for planktonic cultures. This work assessed the impact of Photon Flux Density (PFD) on Chlorella vulgaris biofilm dynamics (structure, physiology, activity). Microalgae biofilms were cultivated in a flow-cell system with PFD from 100 to 500 [Formula: see text]. In the first stage of biofilm development, uniform cell distribution was observed on the substratum exposed to 100 [Formula: see text] while cell clusters were formed under 500 [Formula: see text]. Though similar specific growth rate in exponential phase (ca. 0.3 [Formula: see text]) was obtained under all light intensities, biofilm cells at 500 [Formula: see text] seem to be ultimately photoinhibited (lower final cell density). Data confirm that Chlorella vulgaris showed a remarkable capability to cope with high light. This was marked for sessile cells at 300 [Formula: see text], which reduce very rapidly (in 2 days) their chlorophyll-a content, most probably to reduce photodamage, while maintaining a high final cell density. Besides cellular physiological adjustments, our data demonstrate that cellular spatial organization is light-dependent.
Assuntos
Chlorella vulgaris , Microalgas , Iluminação , Luz , BiofilmesRESUMO
Biofilm-based cultivation systems are emerging as a promising technology for microalgae production. However, efficient and non-invasive monitoring routines are still lacking. Here, a protocol to monitor microalgae biofilms based on reflectance indices (RIs) is proposed. This framework was developed using a rotating biofilm system for astaxanthin production by cultivating Haematococcus pluvialis on cotton carriers. Biofilm traits such as biomass, astaxanthin, and chlorophyll were characterized under different light and nutrient regimes. Reflectance spectra were collected to identify the spectral bands and the RIs that correlated the most with those biofilm traits. Robust linear models built on more than 170 spectra were selected and validated on an independent dataset. Astaxanthin content could be precisely predicted over a dynamic range from 0 to 4% of dry weight, regardless of the cultivation conditions. This study demonstrates the strength of reflectance spectroscopy as a non-invasive tool to improve the operational efficiency of microalgae biofilm-based technology.
Assuntos
Clorofíceas , Microalgas , Xantofilas , Biomassa , BiofilmesRESUMO
Rare early-onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders, whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy.
Assuntos
Dependovirus , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Glucuronidase , Bexiga Urinária , Animais , Camundongos , Humanos , Bexiga Urinária/fisiopatologia , Glucuronidase/genética , Glucuronidase/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Pseudo-Obstrução Intestinal/genética , Pseudo-Obstrução Intestinal/terapia , Pseudo-Obstrução Intestinal/fisiopatologia , Doenças Urológicas , FáciesRESUMO
Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.
Assuntos
Fator 1-beta Nuclear de Hepatócito , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Fator 1-beta Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Heterozigoto , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Mutação , Rim/patologia , Rim/metabolismo , Rim/anormalidades , Sistemas CRISPR-Cas , Células-Tronco Pluripotentes/metabolismo , Edição de GenesRESUMO
CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.