Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.

Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

The gradient-like separation and reduced running time with Tris-Tricine-HEPES buffer for SDS-PAGE.

Tris-Glycine-SDS is the most commonly used running buffer for SDS-PAGE. Relatively long running times, poor resolution of small molecular weight proteins and excessive heat at higher voltages impede its utility for high throughput downstream applications such as western blot. Here we describe a protocol for gradient-like simultaneous separation of small (<10 kDa) and large (>400 kDa) proteins in a single percentage polyacrylamide Tris-Acetate gel using a novel running buffer composed of Tris, Tricine and HEPES.

The role of essential fatty acids in cystic fibrosis and normalizing effect of fenretinide.

Cystic fibrosis (CF) is the most common autosomal-recessive disease in Caucasians caused by mutations in the CF transmembrane regulator (CFTR) gene. Patients are usually diagnosed in infancy and are burdened with extensive medical treatments throughout their lives. One of the first documented biochemical defects in CF, which predates the cloning of CFTR gene for almost three decades, is an imbalance in the levels of polyunsaturated fatty acids (PUFAs). The principal hallmarks of this imbalance are increased levels of arachidonic acid and decreased levels of docosahexaenoic acids (DHA) in CF. This pro-inflammatory profile of PUFAs is an important component of sterile inflammation in CF, which is known to be detrimental, rather than protective for the patients. Despite decades of intensive research, the mechanistic basis of this phenomenon remains unclear. In this review we summarized the current knowledge on the biochemistry of PUFAs, with a focus on the metabolism of AA and DHA in CF. Finally, a synthetic retinoid called fenretinide (N-(4-hydroxy-phenyl) retinamide) was shown to be able to correct the pro-inflammatory imbalance of PUFAs in CF. Therefore, its pharmacological actions and clinical potential are briefly discussed as well.

Fenretinide favorably affects mucins (MUC5AC/MUC5B) and fatty acid imbalance in a manner mimicking CFTR-induced correction.

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome. Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.

Biochemistry of very-long-chain and long-chain ceramides in cystic fibrosis and other diseases: The importance of side chain.

Ceramides, the principal building blocks of all sphingolipids, have attracted the attention of many scientists around the world interested in developing treatments for cystic fibrosis, the most common genetic disease of Caucasians. Many years of fruitful research in this field have produced some fundamentally important, yet controversial results. Here, we aimed to summarize the current knowledge on the role of long- and very-long- chain ceramides, the most abundant species of ceramides in animal cells, in cystic fibrosis and other diseases. We also aim to explain the importance of the length of their side chain in the context of stability of transmembrane proteins through a concise synthesis of their biophysical chemistry, cell biology, and physiology. This review also addresses several remaining riddles in this field. Finally, we discuss the technical challenges associated with the analysis and quantification of ceramides. We provide the evaluation of the antibodies used for ceramide quantification and we demonstrate their lack of specificity. Results and discussion presented here will be of interest to anyone studying these enigmatic lipids.

Biochemistry of very-long-chain and long-chain ceramides in cystic fibrosis and other diseases: The importance of side chain.

Ceramides, the principal building blocks of all sphingolipids, have attracted the attention of many scientists around the world interested in developing treatments for cystic fibrosis, the most common genetic disease of Caucasians. Many years of fruitful research in this field have produced some fundamentally important, yet controversial results. Here, we aimed to summarize the current knowledge on the role of long- and very-long- chain ceramides, the most abundant species of ceramides in animal cells, in cystic fibrosis and other diseases. We also aim to explain the importance of the length of their side chain in the context of stability of transmembrane proteins through a concise synthesis of their biophysical chemistry, cell biology, and physiology. This review also addresses several remaining riddles in this field. Finally, we discuss the technical challenges associated with the analysis and quantification of ceramides. We provide the evaluation of the antibodies used for ceramide quantification and we demonstrate their lack of specificity. Results and discussion presented here will be of interest to anyone studying these enigmatic lipids.

Depletion of BAFF cytokine exacerbates infection in Pseudomonas aeruginosa infected mice.

BACKGROUND: Cystic fibrosis (CF) is a genetic disease characterized by chronic inflammation of the lungs that is ineffective at clearing pathogens. B-cell activating factor (BAFF), a cytokine involved in the development of B-cells, is known to be elevated in CF patients with subclinical infections. We postulate that the elevated BAFF levels in CF patients might be triggered by Pseudomonas aeruginosa infection and it might play a protective role in the regulation of lung responses to infection. METHODS: To address this hypothesis, we used a well characterized model of CFTR.KO mice infected with a clinical strain of P. aeruginosa (PA508). We quantified cell types with flow cytometry, concentration of cytokines by ELISA tests, bacterial load by colony counting and lung physiology by metacholine-induced lung resistance. RESULTS: Our data demonstrates that BAFF is not elevated in uninfected CF mice, and infection with Pseudomonas leads to significant induction of this regulatory cytokine. We also demonstrate that the maintenance of BAFF levels and its induction during the infection is important for clearance of Pseudomonas infection as its depletion during the course of infection leads to decrease in the resolution of infection both in WT and CFTR-KO mice. Interestingly, the depletion of BAFF not only results in a depletion of B cells numbers but also to a significant decrease in the number of regulatory T cells in the non-infected lungs. CONCLUSIONS: Overall, our data demonstrate for the first time that BAFF is an important regulatory molecule helping to maintain the immunological response to infection and clearance of lung infection.

Fenretinide differentially modulates the levels of long- and very long-chain ceramides by downregulating Cers5 enzyme: evidence from bench to bedside.

Cystic fibrosis is the most common genetic disease, in which symptoms may be alleviated but not fully eliminated. Ceramides have long been implicated in the inflammatory etiology of cystic fibrosis, with contradicting reports with regards to their role. Recently, significant biological and biophysical differences have been observed between long- and very long-chain ceramides. This work reveals that long-chain ceramides are upregulated whereas very long-chain ceramides are downregulated in cell lines, mouse animal model, and patients with cystic fibrosis, compared with their controls. Treatment with fenretinide decreases the levels of long-chain ceramides and increases the levels of very long-chain ceramides. Our results show that restoration of cystic fibrosis conductance regulator (CFTR) expression is associated with normalization of aberrant levels of specific ceramides. This demonstrates for the first time a correlation between CFTR protein expression and regulation of specific ceramide levels. Furthermore, using cystic fibrosis lung epithelial cell lines, we demonstrate that this effect can be attributed to the transcriptional downregulation of ceramide synthase 5 (Cers5) enzyme. We also discovered a partial synergism between fenretinide and zinc (Zn2+), which deficiency has been reported in patients with cystic fibrosis. Overall, in addition to having direct translational application, we believe that our findings contribute to the understanding of ceramide metabolism in cystic fibrosis, as well as other inflammatory diseases where imbalances of ceramides have also been observed. KEY MESSAGES: Long- and very long-chain ceramides (LCCs and VLCCs) are biochemically distinct. LCCs are upregulated whereas VLCCs are downregulated in cystic fibrosis. Fenretinide downregulates the levels of LCCs and upregulates the levels of VLCCs. Fenretinide changes the balance of LCCs and VLCCs by downregulating Cers5 enzyme. Fenretinide and zinc ions cooperate in the modulation of ceramide levels.

Development of buffers for fast semidry transfer of proteins.

Western blot is an extensively used method for protein detection in cell biology. To optimize this procedure, here we examined a panel of buffers for their ability to efficiently transfer proteins from SDS-polyacrylamide gels onto nitrocellulose membranes in a short 12-min period, designated here as fast semidry transfer. Our results show for the first time that HEPES- and HEPPS/EPPS-based buffers represent the most efficient buffers for fast semidry transfer.