Mild Detergent Research Articles

Biochemical characterization of paralyzed flagellum proteins A (PflA) and B (PflB) from Helicobacter pylori flagellar motor.

Motility by means of flagella plays an important role in the persistent colonization of Helicobacter pylori in the human stomach. The H. pylori flagellar motor has a complex structure that includes a periplasmic scaffold, the components of which are still being identified. Here, we report the isolation and characterization of the soluble forms of two putative essential H. pylori motor scaffold components, proteins PflA and PflB. We developed an on-column refolding procedure, overcoming the challenge of inclusion body formation in Escherichia coli. We employed mild detergent sarkosyl to enhance protein recovery and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO)-containing buffers to achieve optimal solubility and monodispersity. In addition, we showed that PflA lacking the β-rich N-terminal domain is expressed in a soluble form, and behaves as a monodisperse monomer in solution. The methods for producing the soluble, folded forms of H. pylori PflA and PflB established in this work will facilitate future biophysical and structural studies aimed at deciphering their location and their function within the flagellar motor.

Adaptive immunity to retroelements promotes barrier integrity.

Maintenance of tissue integrity is a requirement of host survival. This mandate is of prime importance at barrier sites that are constitutively exposed to the environment. Here, we show that exposure of the skin to non-inflammatory xenobiotics promotes tissue repair; more specifically, mild detergent exposure promotes the reactivation of defined retroelements leading to the induction of retroelement-specific CD8+ T cells. These T cell responses are Langerhans cell dependent and establish tissue residency within the skin. Upon injury, retroelement-specific CD8+ T cells significantly accelerate wound repair via IL-17A. Collectively, this work demonstrates that tonic environmental exposures and associated adaptive responses to retroelements can be coopted to preemptively set the tissue for maximal resilience to injury.

Flavivirus Zika NS4A protein forms large oligomers in liposomes and in mild detergent

In flaviviruses such as Dengue or Zika, non-structural (NS) NS4A protein forms homo-oligomers, participates in membrane remodelling and is critical for virulence. In both viruses, mature NS4A has the same length and three predicted hydrophobic domains. The oligomers formed by Dengue NS4A are reported to be small (n = 2, 3), based on denaturing SDS gels, but no high-resolution structure of a flavivirus NS4A protein is available, and the size of the oligomer in lipid membranes is not known. Herein we show that crosslinking Zika NS4A protein in lipid membranes results in oligomers at least up to hexamers. Further, sedimentation velocity shows that NS4A in mild detergent C14-betaine appears to be in fast equilibrium between at least two species, where one is smaller, and the other larger, than a trimer or a tetramer. Consistently, sedimentation equilibrium data was best fitted to a model involving an equilibrium between dimers (n = 2) and hexamers (n = 6). Overall, the large, at least hexameric, oligomers obtained herein in liposomes and in mild detergent are more likely to represent the forms of NS4A present in cell membranes.

Abstract 3070: Investigating CD36 Oligomerization In Macrophages

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Atherosclerosis, the main cause of CVD, results from the buildup of cholesterol-filled plaques in artery walls. Accumulation of cholesterol from oxidized low-density lipoprotein (oxLDL) leads to the formation of macrophage foam cells, a process that is mediated by CD36, a scavenger receptor that serves as the primary receptor for oxLDL. The structure of full-length CD36 has not been resolved, nor have the details or role of quaternary structure been fully elucidated. Studies have shown that CD36 has the ability to form homodimers and multimers in some cell types. However, the role of CD36 oligomerization in oxLDL uptake remains unknown. The goal of this study is to investigate CD36 oligomerization in macrophages, a cell type important in the initiation of atherosclerosis. We hypothesize that CD36 oligomerizes in macrophages to facilitate oxLDL uptake. To begin testing this hypothesis, we harvested elicited peritoneal macrophages from wild-type (WT) or CD36 knockout (CD36-KO) mice. We performed crosslinking experiments in which macrophages were incubated with BS3, a membrane-impermeable crosslinker, to stabilize CD36 oligomers. When crosslinked samples were separated by SDS-PAGE electrophoresis, we observed a decrease in monomeric species with increasing crosslinker concentration, however, BS3 also induced aggregation of CD36 rather than oligomer formation. We next turned to Native-PAGE electrophoresis, which lacks detergent entirely, to identify oligomeric species within macrophages, as well as PFO-PAGE electrophoresis, which contains a milder detergent than SDS. Both methods provided evidence of higher-order complexes in WT, but not in CD36-KO macrophages. To further validate our studies, we purified human full-length CD36 using an Sf9 insect cell system and demonstrated that pure CD36 forms homo-oligomers by PFO-PAGE. Together, these experiments provide strong evidence for CD36 oligomerization. Future studies will focus on correlating CD36 oligomerization with its functions in oxLDL binding and uptake. Understanding the higher-order structure of CD36 and how it impacts function will allow us to target CD36 to prevent foam cell formation and atherosclerosis.

Expression and functional analysis of a recombinant aquaporin Z from Antarctic Pseudomonas sp. AMS3.

Aquaporin (AQP) is a water channel protein from the family of transmembrane proteins which facilitates the movement of water across the cell membrane. It is ubiquitous in nature, however the understanding of the water transport mechanism, especially for AQPs in microbes adapted to low temperatures, remains limited. AQP also has been recognized for its ability to be used for water filtration, but knowledge of the biochemical features necessary for its potential applications in industrial processes has been lacking. Therefore, this research was conducted to express, extract, solubilize, purify, and study the functional adaptations of the aquaporin Z family from Pseudomonas sp. AMS3 via molecular approaches. In this study, AqpZ1 AMS3 was successfully subcloned and expressed in E. coli BL21 (DE3) as a recombinant protein. The AqpZ1 AMS3 gene was expressed under optimized conditions and the best optimized condition for the AQP was in 0.5 mM IPTG incubated at 25°C for 20 h induction time. A zwitterionic mild detergent [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was the suitable surfactant for the protein solubilization. The protein was then purified via affinity chromatography. Liposome and proteoliposome was reconstituted to determine the particle size using dynamic light scattering. This information obtained from this psychrophilic AQP identified provides new insights into the structural adaptation of this protein at low temperatures and could be useful for low temperature application and molecular engineering purposes in the future.

Small extracellular vesicles in plasma carry luminal cytokines that remain undetectable by antibody-based assays in cancer patients and healthy donors

BackgroundSmall (30–150 nm) extracellular vesicles (sEV), also known as exosomes, play a key role in cell-to-cell signaling. They are produced by all cells, circulate freely and are present in all body fluids. Evidence indicates that cytokines are present on the surface and/or in the lumen of sEV. The contribution of intravesicular cytokines to cytokine levels in plasma are unknown.MethodssEV were isolated by ultrafiltration/size exclusion chromatography from pre-cleared plasma obtained from patients with head and neck squamous cell carcinoma (HNSCC) and healthy donors (HDs). Multiplex immunoassays were used to measure cytokine levels in paired untreated and detergent-treated (0.5% Triton X-100) plasma and plasma-derived detergent-treated sEV. Non-parametric tests were used to assess differences in cytokine levels.ResultsThe presence of cytokines in sEV isolated from patients’ and HDs’ plasma was confirmed by immunoblots and on-bead flow cytometry. sEV-associated cytokines were functional in various in vitro assays. Levels of cytokines in sEV varied among the HNSCC patients and were generally significantly higher than the levels observed in sEV from HDs. Compared to untreated plasma, levels for the majority (40/51) of the evaluated proteins were significantly higher in detergent-treated plasma (P < 0.0001–0.03). In addition, levels of 24/51 proteins in sEV, including IL6, TNFRII, IL-17a, IFNa and IFNg, were significantly positively correlated with the difference between levels detected in detergent-treated plasma and untreated plasma.DiscussionThe data indicate that sEV-associated cytokines account for the differences in cytokine levels measured in detergent-treated versus untreated plasma. Ab-based assays using untreated plasma detect only soluble cytokines and miss cytokines carried in the lumen of sEV. Permeabilization of sEV with a mild detergent allows for Ab-based detection of sEV-associated and soluble cytokines in plasma. The failure to detect cytokines carried in the sEV lumen leads to inaccurate estimates of cytokine levels in body fluids.A schema illustrating the presence of cytokines on the surface or in the lumen of small extracellular vesicles (sEV).

Advancing surgical instrument safety: A screen of oxidative and alkaline prion decontaminants using real-time quaking-induced conversion with prion-coated steel beads as surgical instrument mimetic.

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the establishment of TESSA (sTainlESs steel-bead Seed Amplification assay), a modified real-time quaking induced cyclic amplification (RT-QuIC) assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

Water absorption behavior and physico‐chemical and mechanical performance of PLA‐based biopolymers filled with degradable glass fibers

AbstractPoly(lactic acid) (PLA)‐based biopolymers filled with degradable glass fibers were submitted to real life conditions, selected based on their potential applications as beverage containers, to evaluate the feasibility of their usage at the required operational conditions. To do so, several media (i.e., buffer solutions at pH = 3, 5, 7.5, 12 and alcoholic solution) and temperatures (25, 37, 55 and 70°C) were applied to mimic the storage of food, beverage, and detergents inside or in direct contact with the bio‐based prototypes. Their mechanical, spectroscopic (FTIR) and morphological (SEM) features were evaluated and compared with samples after immersion. Water absorption increased with the increasing temperature, with maximum values achieved in alcoholic solutions indicating a more severe interaction of media with the functional groups of the samples. The diffusivity, D, increased with temperature, indicating faster water diffusion rate, especially in strong alkaline media. Treatments at pH 12 led to the most relevant degradation. This behavior was confirmed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) analysis that evidenced a strong decrease of the ester band at 1755 cm−1 and the dissolution of the external layer of the polymeric matrix, which became more evident at higher temperatures, where several glass fibers emerged from the bulk. Tensile and flexural tests indicated a softening of the material and an increase in the plasticity of the samples with the increasing temperature. Evident embrittlement of samples was observed at higher temperatures, suggesting an increase in the crystallinity degree of the biopolymer. The obtained results showed the suitability of the PLA‐based biocomposites for applications that do not require high temperatures for long periods of time. Also, the tested materials were proved to resist washing with mild detergents. These results will help in the designing of the new improved PLA‐based biocomposites to meet with the target objectives on their applications.

Augmented Placental Protein 13 in Placental-Associated Extracellular Vesicles in Term and Preterm Preeclampsia Is Further Elevated by Corticosteroids.

Placental protein 13 (PP13) is a regulatory protein involved in remodeling the vascular system of the pregnancy and extending the immune tolerance of the mother to the growing fetus. PP13 is localized on the surface of the syncytiotrophoblast. An ex vivo placental model shows that the PP13 is released via placental-associated extracellular vesicles (PEVs) to the maternal uterine vein. This exploratory study aimed to determine PEV-associated PP13 in the maternal circulation as compared to the known soluble fraction since each has a specific communication pathway. Patients admitted to Bnai Zion Medical Center for delivery were recruited, and included 19 preeclampsia (PE) patients (7 preterm PE gestational age < 37 weeks' gestation), 16 preterm delivery (PTD, delivery at GA < 37 weeks' gestation), and 15 matched term delivery controls. Treatment by corticosteroids (Celestone), which is often given to patients with suspected preterm PE and PTD, was recorded. The PEV proteome was purified from the patients' plasma by size exclusion chromatography (SEC) to separate the soluble and PEV-associated PP13. The total level of PP13 (soluble and PEV-associated) was determined using mild detergent that depleted the PEV proteome. PP13 fractions were determined by ELISA with PP13 specific antibodies. ELISA with alkaline phosphatase (PLAP)- and cluster differentiation 63 (CD63)-specific antibodies served to verify the placental origin of the PEVs. SPSS was used for statistical analysis. The patients' medical, pregnancy, and delivery records in all groups were similar except, as expected, that a larger number of PE and PTD patients had smaller babies who were delivered earlier, and the PE patients had hypertension and proteinuria. The SEC analysis detected the presence of PP13 in the cargo of the PEVs and on their surface, in addition to the known soluble fraction. The median soluble PP13 was not significantly different across the PE, PTD, and term delivery control groups. However, after depleting the PEV of their proteome, the total PP13 (soluble and PEV-associated) was augmented in the cases of preterm PE, reaching 2153 pg/mL [IQR 1866-2838] but not in cases of PTD reaching 1576 pg/mL [1011-2014] or term delivery groups reaching 964 pg/mL [875-1636]), p < 0.01. On the surface of the circulating PEV from PTD patients, there was a decrease in PP13. Corticosteroid treatment was accompanied by a massive depletion of PP13 from the PEV, especially in preterm PE patients. This exploratory study is, thus, the first to determine PEV-associated PP13 in maternal circulation, providing a quantitative determination of the soluble and the PEV-associated fractions, and it shows that the latter is the larger. We found an increase in the amount of PP13 carried via the PEV-associated pathway in PE and PTD patients compared to term delivery cases, which was further augmented when the patients were treated with corticosteroids, especially in preterm PE. The signal conveyed by this novel communication pathway warrants further research to investigate these two differential pathways for the liberation of PP13.

Adaptive immunity to ancient retroelements controls the skin tissue threshold of activation

Abstract A symbiotic dialogue between the microbiota and host is essential to promote protective immune responses in the skin. Integrated retroviral elements, or retrotransposons (RTNs), comprise up to 45% of the human genome. We previously found that immune responses to the skin microbiota are dependent on the reactivation and keratinocyte-intrinsic sensing of RTNs. Here, we explored the possibility that RTNs may also represent a source of antigens recognized by the skin adaptive immune system. We first established a model of sterile injury, where topical application of a mild detergent reactivated RTNs, and recruited CD8 T cells to the skin in an RTN-dependent manner. RNAseq analysis of RTN expression revealed Langerhans cells (LCs) have the most abundant RTN expression amongst skin dendritic cells. Our work demonstrated that CD8 T cell responses to sterile injury were entirely dependent on LCs, supporting the idea that LCs could present RTN antigens to CD8 T cells. To identify RTN-specific T cells, we selected peptides that were uniquely expressed in LCs, but not in thymic epithelial cells which mediate negative selection. Several peptides promoted cytokine production from injury-elicited CD8 T cells, confirming the presence of RTN-reactive CD8 T cells in the skin. RNA-seq analysis suggests RTN-specific CD8 T cells may contribute to wound repair and immunoregulation. Thus, mild stress, such as that induced by mild detergent, is sufficient to promote adaptive immunity to defined antigens derived from RTNs. Our work also proposes that homeostatic immunity to ancient retroviruses promotes a defined class of T cells responses aimed at maintaining barrier integrity in response to daily stressors encountered by the skin. Cancer Research Institute, Irvington Postdoctoral Fellowship

Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane-associated proteostasis in Arabidopsis.

Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.

Irritant potential of different washing procedures used for heavy-duty soiling: Short and intense or longer and mild?

To prevent irritant contact eczema even in occupational fields with heavy-duty soiling, it is generally recommended to use 'mild' hand cleansers (mild detergent without grits, MC). On the other hand, since grit-containing cleansers (GC) show a higher washing power that minimizes washing time, their usage is generally preferred in specific occupational fields. To compare whether a shorter, intense washing period might cause less skin damage than a longer washing period with an MC. Differences in cleaning time were first verified in a pilot study using standardized model dirt. In the main study, the forearms of 35 healthy volunteers were washed with three standardized procedures over a period of 3 days, either using 2 min of MC with/without hand brush or 1-min GC. Clinical scoring, transepidermal water loss (TEWL), corneometry, colourimetry and scaliness/roughness (Visioscan) were used to evaluate the epidermal barrier, topography and irritation. The pre-study showed that washing time doubled when using MC vs. GC. Using GC resulted in stronger barrier disruption, even after a shorter washing period - median ΔT4-T1 TEWL 0.96 g/m2 /h vs. 4.91 g/m2 /h respectively, p < 0.0001. The most harmful procedure for the skin was the additional application of a hand brush (18.86 g/m2 /h). Short-time washing with GC damages the skin barrier more significantly in comparison to a longer application of an MC. When washing with MC, the strongest irritant reaction occurred when accompanied with hand brushing.

Conformational and functional interrelation between mitochondrial carrier proteins: UCP4, ANT, PiT.

Mitochondrial uncoupling proteins (UCPs) attenuate proton motive force across the inner membrane of mitochondria (IMM) by transporting protons from the intermembrane space into matrix, thus reducing the ATP synthesis rate. UCPs together with adenine nucleotide translocase (ANT) and inorganic phosphate translocase (PiT) create a subgroup of carriers directly linked to mitochondrial energetics. While its potential role in neuroprotection of neurogenerative diseases has been speculated, the brain specific neuronal UCP4 is among the least investigated UCPs. This integrative study will focus on the structural features and functional interrelation of UCP4, ANT and PiT. We designed recombinant versions of these proteins that, using a modified autoinduction method, were overexpressed in the membrane of Escherichia coli, extracted with mild detergents, and purified as monomers. Purified proteins, embedded in a shell of bacterial membrane lipids, were then reconstituted in IMM-mimicking phospholipid vesicles for biophysical studies using circular dichroism (CD) and fluorescence spectroscopies. Native-like helical conformations of proteins in the mild detergent octyl glycoside (OG) and phospholipid membranes were confirmed by CD spectroscopy. Interestingly, in agreement with our previous studies, monomeric UCP4 spontaneously self-associated to form tetramers in lipid vesicles. Conversely, ANT and PiT maintained their molecular forms in both detergent and liposome milieus. Reconstituted proteins in lipid membranes exhibited different levels of proton transport activity in the presence of fatty acids and decreased with the addition of ATP. UCP4 showed significantly higher proton transport rates compared to ANT and PiT. Research on the functional interrelation between these three carrier proteins, as well as the effect of mitochondrial lipid cardiolipin on their conformational stability and proton transport activity, is in progress in our laboratory.

Structural basis for excited light transfer within bacterial photosynthetic supercomplex.

Single-particle cryogenic electron microscopy (cryo-EM) enables the visualization of membrane protein supercomplexes in their native-like conditions. In green sulfur bacteria (GSB), the photochemical reaction center (RC) features a homodimeric architecture for charge separation across the membrane. We extracted the photosynthetic supercomplex from Chlorobaculum tepidum cells with a mild detergent and determined high-resolution structures of the supercomplex using single-particle cryo-EM. In the cryo-EM density, the supercomplex is assembled based on a symmetric RC core with two Fenna-Matthews-Olson (FMO) trimers, two cytochrome c subunits (PscC), and small membrane subunits. The two FMO trimers bind to the dimeric RC cytoplasmic surface with different interfaces, resulting in an asymmetric feature of the supercomplex. The cryo-EM density also reveals two accessory protein subunits, PscE and PscF, which have not been identified before. We also discovered additional pigments within the complex, especially a linker bacteriochlorophyll (BChl) located in one of the two FMO-PscA interfaces, possibly mediating the exciton transfer from the FMO trimer to the RC core. We further computed the fluorescence resonance energy transfer (FRET) rate between determined pigments and found that the excited light energy transfer is biased on one of the two FMO-PscA branches. Our structure of the GSB photosynthetic supercomplex provides mechanistic insights into the routes for light excitation energy transfer and a possible evolutionary transition intermediate of the bacterial photosynthetic supercomplex from the primitive homodimeric RC.

Purification and characterization of eukaryotic ATP-dependent transporters homologously expressed in Pichia pastoris for structural studies by cryo-electron microscopy

Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins.

Alzheimer brains: increased soluble amyloid peptides, but decreased lipid rafts

AbstractBackgroundPET imaging studies of AD patients show progressive increases of fibrillar Aβ‐ amyloid. Because current PET ligands underestimate non‐fibrillar forms, we assayed soluble Aβ in AD and controls. To identify mechanisms responsible for soluble Aβ in AD brains, we examined lipid rafts (LR), where APP is enzymatically processed. Frontal cortex was compared with cerebellum, which has minimal AD pathology.MethodPostmortem samples (N=48) were age‐and sex matched for Braak scores. Soluble Aβ was extracted from homogenates of full thickness frontal cortex by isotonic buffer and mild detergent (RIPA; 1% NP40) and centrifuged (10,000g/1h). Supernates were assayed by dot blots with antibodies for Aβ40 and Aβ42 (Biolegend, San Diego, CA). Aβ was assayed as relative fluorescent units per gm brain. LR fractions were isolated from these same brains by kit (Invent Biotechnologies; LR‐039), validated by ultracentrifugal fractionation.ResultCompared with cognitively normal controls (Braak 0‐1), elevations of soluble Aβ40 and Aβ42 were similar for intermediate and later stage AD (Braak 2‐3 and 4‐6). Clinical grade AD had greater increase of soluble Aβ40 (+400%) than Aβ42 (+50%) (p&lt;0.05). LR raft yield per gram AD frontal cortex was 20% below controls (p&lt;0.05), while cerebellar LR did not differ by Braak score. The composition of LR had a similar pattern, with 35% AD decreases of total protein and cholesterol (p&lt;0.05); cerebellar LR again did not differ by Braak score. However, LR 4‐HNE, a marker of oxidative damage, was similarly increased in AD cortex and AD cerebellum.ConclusionThe extensive overlap of soluble Aβ levels in controls with AD contrasts with the PET findings on fibrillar Aβ. These findings further support fibrillar Aβ as a biomarker for AD treatments and show the need for more detailed postmortem analysis of diverse soluble and insoluble Aβ aggregates in relation to PET. The decreased LR in AD frontal cortex is consistent with its extensive neuronal loss, in contrast to the cerebellum. Further studies will examine LR enzymes of APP processing as a source of the increased soluble Aβ in AD, and subcellular clearance processes, which may be impaired in association with the oxidative damage indicated by increased 4‐HNE.

Methods to study adult hippocampal neurogenesis in humans and across the phylogeny.

The hippocampus hosts the continuous addition of new neurons throughout life-a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field.

Effects of Detergents on the Spectral Features of B820 Bacteriochlorophyll a in Light-Harvesting Complex 3.

Excitonic coupling of bacteriochlorophyll (BChl) a in light-harvesting (LH) proteins of purple photosynthetic bacteria is key for efficient photon capture and energy transfer. Environmental factors can affect the spectral features of these BChl a pigments and investigating these effects can provide insight into the molecular mechanisms underlying the photosynthetic spectral tuning. The present study analyzes the spectral alterations of the Qy band of B820 BChl a within the LH3 protein in relation to the type and concentration of detergents in the buffer. Changing the detergent from lauryl dimethylamine N-oxide (LDAO) to n-dodecyl-β-d-maltoside (DDM) caused a red shift in the B820 Qy band accompanied by hyperchromism; these spectral alterations were completely reversed by exchanging back from DDM to LDAO. These results reflect the different effects of harsh vs mild detergents on the perturbation of LH3. The B820 Qy band did not change when LDAO or NaCl concentration was altered, suggesting that electrostatic effects by external components have little influence on the spectral features of B820 BChl a in LH3.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection.

We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA). High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results. In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA. The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.