Fluorescence Intensity Of Protein Research Articles

Intestinal Alkaline Phosphatase Exerts Anti-Inflammatory Effects Against Lipopolysaccharide by Inducing Autophagy

Intestinal alkaline phosphatase (IAP) regulates bicarbonate secretion, detoxifies lipopolysaccharide (LPS), regulates gut microbes, and dephosphorylates proinflammatory nucleotides. IAP also exhibits anti-inflammatory effects in a Toll-like Receptor-4 (TLR-4) dependent manner. However, it is not known whether IAP induces autophagy. We tested the hypothesis that IAP may induce autophagy which may mediate the anti-inflammatory effects of IAP. We found that exogenous IAP induced autophagy in intestinal epithelial cells and in macrophages. TLR4INC34 (C34), a TLR4 signaling inhibitor, suppressed IAP-induced autophagy. IAP also inhibited LPS-induced IL-1β mRNA expression and activation of NF-κB. When autophagy was blocked by 3-methyladenine (3MA) or by Atg5 siRNA, IAP failed to block LPS-mediated effects. IAP also upregulated autophagy-related gene expression in small intestine in mice. We administered either vehicle or IAP (100 U/ml) in drinking water for 14 days in C57BL/6 mice. Mice were sacrificed and ileal tissues collected. Increased expression of Atg5, Atg16, Irgm1, Tlr4, and Lyz genes was observed in the IAP treated group compared to the vehicle treated group. Increase in Atg16 protein expression and fluorescence intensity of LC3 was also observed in IAP-treated tissues compared to the vehicle-treated tissues. Thus, our study lays the framework for investigating how IAP and autophagy may act together to control inflammatory conditions.

Glucose-modified carbosilane dendrimers: Interaction with model membranes and human serum albumin.

Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM2-3Glu, significantly affect the type and extent of interaction with biological structures.

Abstract TMP30: M2 Microglia Derived Exosomes Promote White Matter Repair and Long-Term Functional Recovery After Focal Cerebral Ischemia in Mice

Objectives: White matter injury aggravates neurological and cognitive impairment in experimental ischemic stroke. M2 microglia promote oligodendrocyte precursor cells survival and differentiation, and further enhance white matter repair. However, the molecular mechanism is unclear. Here, we explored the effect and mechanism of M2 microglia-derived exosomes on white matter repair after focal cerebral ischemia in mice. Methods: Microglia BV2 cells were polarized to M2 phenotype by IL-4 stimulation. Exosomes were isolated from M2 microglia (M2-Exo) and unstimulated microglia as a control (M0-Exo). M2-Exo and M0-Exo (100 μg) were intravenously injected after 90-minute middle cerebral artery occlusion in mice (n=72). Brain atrophy volume and neuro behavioral outcomes were examined in 28 days following focal cerebral ischemia. Oligodendrocyte precursor cells survival, differentiation and white matter integrity were evaluated. Exosomal miRNA and target gene were further examined to explore molecular mechanism. Results: M2-Exo treatment promoted sensorimotor and memory function recovery ( p <0.05), and further reduced brain atrophy compared to the M0-Exo control group ( p <0.001). Immunostaining showed that M2-Exo increased the number of BrdU + /Pdgfr-α + and BrdU + /adenomatous polyposis coli + cells, enhanced myelin basic protein fluorescence-intensity compared to the control ( p <0.05). M2-Exo increased oligodendrocyte precursor cell survival under OGD in vi tro , ( p <0.05) and differentiation ( p <0.05). Exosomal miRNA sequencing and PCR identified that miR-23a-5p was enriched in M2-Exo. Conclusion: Our results showed that M2-Exo treatment enhanced oligodendrocyte precursor cell survival and differentiation, further promoted white matter repair and long-term functional recovery, suggesting that M2-Exo is a novel therapeutic strategy for the white matter repair after ischemic brain injury.

MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water

Mercury ion (Hg2+ ) is a universal pollutant and its detection is crucial for public healthcare. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+ -induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40nM. The limit of detection was 0.5nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependent on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5nM concentration. However, the interference effect was more pronounced at 40nM concentration of metal ions. The measurement was completed in less than 1Min with no need for sample preparation or preincubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry.

Pretreatment-promoted sludge fermentation liquor improves biological nitrogen removal: Molecular insight into the role of dissolved organic matter

Waste activated sludge (WAS) can be used as carbon sources to support biological nutrient removal (BNR). In this study, thermal-alkaline (THALK), ozonation (OZN), electrolysis (EC) and NaClO-promoted electrolysis (EC-AOP) were investigated to facilitate WAS solubilization and production of volatile fatty acids (VFAs). EEMF-PARAFAC and FT-ICR-MS were employed to characterize the transformation of dissolved organic matter (DOM) in WAS fermentation liquors at molecular level. THALK achieved the highest fluorescence intensity of C1 protein after pretreatment. Proteins and lipids were the dominant DOM in the pretreated WAS, while the DOM shifted towards substances with higher H/C and lower O/C after fermentation. The BNR results showed that THALK (100%) and EC-AOP (96.9%) outperformed other groups (78.9–90.3%) in terms of NO3-N removal, indicating the significant impact of DOM compositions. Overall, these results demonstrated that THALK and EC-AOP effectively enhanced release of VFAs and DOM, which subsequently improved NO3-N removal efficiency.

MHC-mismatched Allotransplantation of Induced Pluripotent Stem Cell-derived Cardiomyocyte Sheets to Improve Cardiac Function in a Primate Ischemic Cardiomyopathy Model.

Although allogeneic-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) exhibit potential in cardiomyogenesis for heart failure, whether major histocompatibility complex (MHC)-matched allogenic iPSC implantation (MMAI) minimizes immune rejection for cell survival or functional recovery remains unknown. We herein explored whether MMAI with an iPSC-CM sheet is stable for a longer period and therapeutically more effective than MHC-mismatched AI in a primate ischemic cardiomyopathy model. Green fluorescent protein-transfected iPSC-CM sheets, derived from cynomolgus macaques with homozygous MHC haplotypes ''HT1,'' were transplanted on the left ventricle, generated by ligating the left anterior descending artery for 2 weeks in an ischemic model with or without heterozygous HT1 as MMAI and MHC-mismatched AI. Sham models were made by opening the chest at 14 days after left anterior descending ligation without any treatment. Stereomicroscopy revealed that at 4 months after transplantation, green fluorescent protein intensity was higher in the MMAI group than in the MHC-mismatched AI group and the sham group. Immunohistochemistry staining revealed that host immune reaction with CD3-positive cells was stronger in MHC-mismatched AI than in MMAI at 3 months. Cardiac function improved both in MMAI and MHC-mismatched AI at 1 month after transplantation and was preserved until 6 months, whereas in the sham group, functional deterioration progressed over time. Although MHC-homo-iPSCs are preferred to avoid immune rejection, MHC-mismatched iPSC-CMs can also induce comparable cardiac functional recovery at late follow-up, suggesting that MHC-mismatched iPSC-based cardiac regenerative therapy with immunosuppressants is a feasible option for treating heart failure in clinical settings.

Wood Microfluidics

As nonbiodegradable plastics continue to pollute our land and oceans, countries are starting to ban the use of single-use plastics. In this paper, we demonstrated the fabrication of wood-based microfluidic devices and their adaptability for single-use, point-of-care (POC) applications. These devices are made from easily sourced renewable materials for fabrication while exhibiting all the advantages of plastic devices without the problem of nonbiodegradable waste and cost. To build these wood devices, we utilized laser engraving and traditional mechanical methods and have adapted specific surface coatings to counter the wicking effect of wood. To demonstrate their versatility, wood microfluidic devices were adapted for (i) surface plasmon coupled enhancement (SPCE) of fluorescence for detection of proteins, (ii) T-/Y-geometry microfluidic channel mixers, and (iii) devices for rapid detection of microbial contamination. These provide proof of concept for the use of wooden platforms for POC applications. In this study, we measured the fluorescence intensities of recombinant green fluorescent protein (GFP) standards (ranging from 1.5-25 ng/μL) and 6XHis-G-CSF (ranging from 0.1-100 ng/μL) expressed in cell-free translation systems. All tested devices perform as well as or better than their plastic counterparts.

Blockade of spinal α5-GABAA receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats

The role of spinal α5 subunit-containing GABAA (α5-GABAA) receptors in chronic pain is controversial. The purpose of this study was to investigate the participation of spinal α5-GABAA receptors in the reserpine-induced pain model. Reserpine administration induced tactile allodynia and muscle hyperalgesia in female and male rats. Intrathecal injection of L-655,708 and TB 21007 (7 days after the last reserpine injection) decreased tactile allodynia and, at a lesser extent, muscle hyperalgesia in female rats. The effects of these drugs produced a lower antiallodynic and antihyperalgesic effect in male than in female rats. Contrariwise, these drugs produced tactile allodynia and muscle hyperalgesia in naïve rats and these effects were lower in naïve male than female rats. Intrathecal L-838,417 prevented or reversed L-655,708-induced antiallodynia in reserpine-treated female rats. Repeated treatment with α5-GABAA receptor small interfering RNA (siRNA), but not scramble siRNA, reduced reserpine-induced allodynia in female rats. Accordingly, α5-GABAA receptor siRNA induced nociceptive hypersensitivity in naïve female rats. Reserpine enhanced α5-GABAA receptors expression in spinal cord and dorsal root ganglia (DRG), while it increased CD11b (OX-42) and glial fibrillary acidic protein (GFAP) fluorescence intensity in the lumbar spinal cord. In contrast, reserpine diminished K+-Cl- co-transporter 2 (KCC2) protein in the lumbar spinal cord. Data suggest that spinal α5-GABAA receptors play a sex-dependent proallodynic effect in reserpine-treated rats. In contrast, these receptors have a sex-dependent antiallodynic role in naïve rats.

Contaminant removal and microorganism response of activated sludge in sulfamethazine wastewater treatment

The contaminant removal and microorganism response in activated sludge exposed to sulfamethazine (SMT) for 66 days was investigated. The average removal efficiency of chemical oxygen demand (COD), total nitrogen (TN), ammonia nitrogen (NH4+-N), total phosphorus (TP) and SMT were 93%, 71%, 98%, 69% and 84%, respectively. The content of extracellular polymeric substance (EPS) increased significantly from day 0 to day 66 (p < 0.05). Microorganisms secreted more PN to protect cells from SMT damage under the stimulation of SMT. Three-dimensional excitation-emission matrix (3D-EEM) results showed that fluorescence intensity of tyrosine like aromatic protein in EPS increased significantly from day 0 to day 66. The total reads of Pseudomonas (denitrifying bacteria, DNB) and Acinetobacter (phosphorus-accumulating bacteria, PAO) increased significantly from day 0 to day 66 (p < 0.05), which indicated that the above-cited bacteria could resist the SMT stress.

Long noncoding RNA H19 prevents neurogenesis in ischemic stroke through p53/Notch1 pathway

Background and purposeLong non-coding RNA H19 (H19) is one of the earliest discovered long non-coding RNAs. H19 induced the onset of ischemic stroke through regulating neuronal autophagy and microglial polarization. And we aimed to study whether H19 participated the neurogenesis process after ischemic stroke. MethodsCirculating H19 levels in ischemic stroke patients and the mRNA levels of p53 target genes were tested by real-time polymerase chain reaction. H19 small interference RNA and pifithrin-α were used to inhibit H19 and p53 expression in the mice suffered middle cerebral artery occlusion, respectively. The expression of neurogenesis related proteins was assessed by Immunofluorescence and Western blot. ResultsCirculating H19 levels were positively associated with the National Institute of Health Stroke Scale Scores of the patients in 7d, 30d and 90d after stroke attack., H19 small interference RNA significantly decreased the volume of brain tissue loss at 14d after middle cerebral artery occlusion and reperfusion in mice and promoted the neurological deficit recovery of the mice. It was confirmed by immunofluorescence that H19 knockdown could decrease the fluorescence intensity of neurogenesis related proteins. While inhibiting p53 on the basis of H19 knockdown reversed the pro-neurogenesis effect of H19 inhibition. Furthermore, H19 decreased the transcriptional activity of p53 and the expression of Notch1, and p53 inhibition abolished these effects of H19. ConclusionsOur findings demonstrate that H19 prevents the process of neurogenesis after ischemic stroke through p53/Notch1 pathway and strengthen the novel role of H19-based therapy for ischemic stroke.

DEVELOPING A SENSOR SYSTEM FOR IDENTIFICATION OF POLYCYCLIC AROMATIC HYDROCARBONS AS ENVIRONMENTAL TOXICANTS IN AQUEOUS AND PROTEIN MEDIA

Aim: to investigate the possibility of identifying toxic polycyclic aromatic hydrocarbons (PAHs) in aqueous and protein media using a fluorescent sensor system based on serum albumins. Methods: The studies were conducted using fluorescent methods, in particular, by quenching the intrinsic fluorescence of proteins, recording the fluorescence of PAHs and using the pyrene luminescent probe. Results: It was experimentally found that when PAHs were introduced into serum albumin solutions, a general decrease in the fluorescence intensity of proteins was observed, indicating PAHs' binding to protein macromolecules and, probably, the formation of a non-fluorescent serum protein-PAH complex. The analysis of the fluorescence spectra of PAHs in serum albumins was carried out. The dependences of changes in the intensity of the fluorescence spectra maxima of PAHs in albumins with an increase in the concentration of PAHs were linear, which makes it possible to use these systems for analytical purposes to determine toxic PAHs in protein and aqueous media. Conclusions: The results of the luminescent study on the interaction of PAHs with protein molecules can be used for monitoring of environmental toxicants in various media, as well as for the development of the methods for early diagnosis of diseases associated with the effect of various agents on the proteins.

Development of a purification tag to produce thermostable fused protein

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.

Profound weight loss induces reactive astrogliosis in the arcuate nucleus of obese mice

ObjectiveObesity has been linked to an inflammation like state in the hypothalamus, mainly characterized by reactive gliosis (RG) of astrocytes and microglia. Here, using two diet models or pharmacological treatment, we assessed the effects of mild and drastic weight loss on RG, in the context of high-fat diet (HFD) induced obesity. MethodsWe subjected HFD-induced obese (DIO) male C57BL/6J mice to a weight loss intervention with a switch to standard chow, calorie restriction (CR), or treatment with the Glp1 receptor agonist Exendin-4 (EX4). The severity of RG was estimated by an ordinal scoring system based on fluorescence intensities of glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1 positive (Iba1), cell numbers, and morphological characteristics. ResultsIn contrast to previous reports, DIO mice fed chronically with HFD showed no differences in microglial or astrocytic RG, compared to chow controls. Moreover, mild or profound weight loss had no impact on microglial RG. However, astrocyte RG was increased in CR and EX4 groups compared to chow fed animals and strongly correlated to body weight loss. Profound weight loss by either CR or EX4 was further linked to increased levels of circulating non-esterified free fatty acids. ConclusionsOverall, our data demonstrate that in a chronically obese state, astrocyte and microglial RG is indifferent from that observed in age-matched chow controls. Nonetheless, profound acute weight loss can induce astrocyte RG in the hypothalamic arcuate nucleus, possibly due to increased circulating NEFAs. This suggests that astrocytes may sense acute changes to both the dietary environment and body weight.

Pseudomonas aeruginosa alkaline protease exhibits a high renaturation capability.

Thermally induced unfolding and renaturation capability of alkaline proteases (AprA) of three Pseudomonas aeruginosa strains, i.e. ATCC 27853 and two clinical isolates, was examined. Sequence analyses demonstrated a high level of aprA genes identity (99.24-99.8%) in these bacterial strains. The proteases retained 45-60% and 15% of their activity after pre-treatment at 60oC and 80oC, respectively, whereas pre-incubation at 90-95oC resulted in a higher level of activity than at 80oC. Zymography analyses and immunoblotting with AprA antiserum suggested a high thermostability and renaturation capability of the studied enzymes in comparison to another P. aeruginosa protease, elastase B. An intrinsic capability of renaturation of P. aeruginosa AprA was confirmed by fluorescence spectra of the native, thermally denatured, and renatured enzyme. The value of the fluorescence intensity of the denatured and subsequently cooled enzyme recovered to about 80% of the value of the native protein fluorescence intensity. Moreover, pre-incubation of the enzyme at 60oC and 90oC exerted only a slight effect on the intensity of absorbance and the shape of the amide I band, as demonstrated by Fourier transform infrared (FTIR) spectroscopy performed after subsequent cooling of the pre-treated enzyme. The results indicated a high renaturation capability of the P. aeruginosa AprA proteins.

Metastasis‐inducing protein S100A4 interacts with p53 in the nuclei of living cells

AbstractObjectiveS100A4 can bind to p53 in vitro. However, it is unclear whether the proteins interact in the nuclei of living cells.MethodsInitially, using an optical biosensor, the ability of CFP‐S100A4 to bind with p53‐YFP was examined in vitro. The fluorescence resonance energy transfer (FRET) technique using laser confocal microscopy was applied to detect the interaction of S100A4 with p53 in living cells.ResultsImaging FRET in HeLa cells expressing S100A4 and p53 showed an increase in cyan fluorescent protein fluorescence intensity, after photobleaching yellow fluorescent protein in cell nuclei. In comparison, the imaging of cell nuclei expressing mutant S100A4‐C and p53 or S100A4 and yellow fluorescent protein as the negative control showed the occurrence of FRET, and demonstrated that the interaction between S100A4 and p53 occurred in the nuclei of living HeLa cells. However, FRET did not occur in the cytoplasm of cells, showing that S100A4 did not interact with p53 in the cytoplasm.ConclusionS100A4 can interact directly with p53 in the nuclei of living cells. This might be a molecular basis for metastasis‐inducing S100A4 in vivo. The results also suggest that the inhibition of the calcium‐binding site of S100A4 might be a possible anticancer therapy.

Effects of microwave and water bath heating on the interactions between myofibrillar protein from beef and ketone flavour compounds

AbstractThis paper presents the effects of microwave on the structure changes of myofibrillar protein compared with traditional water bath heating method. Under microwave or water bath heating conditions, α‐helix conformation of myofibrillar protein was converted into β‐turn, β‐sheet and randomly coiled structures gradually. T he fluorescence intensity of myofibrillar protein exhibited a marked red shift of the maximum absorption wavelength after microwave or water bath treatment. Total sulfhydryls groups and surface hydrophobicity contents were increased with progressing microwave or water bath treatment time. Results indicated that the thermal effects of microwave dominated the structure changes of myofibrillar protein, and microwaving also had an apparent influence on myofibrillar protein structure compared with water bath heating due to its nonthermal effects, and thus affecting the retention and release of ketone flavour compounds.

The Mechanism of Fluorescence Quenching of Protein Photosensitizers Based on miniSOG During Internalization of the HER2 Receptor

The protein photosensitizer miniSOG is a promising agent for photodynamic therapy. The genetically encoded phototoxins 4D5scFv-miniSOG and DARPin-miniSOG specifically bind to the HER2 receptor overexpressed on the surface of cancer cells and promote receptor-mediated internalization of HER2. We show that ingestion of proteins in a complex with the receptor reduces the fluorescent signal of the phototoxic module in endosomes. In order to clarify the mechanism of decrease in the fluorescence intensity of miniSOG-based proteins as they enter a cancer cell during internalization, we analyzed the influence of different factors, including low pH, proteolysis, cofactor reduction, and shielding, on changes in the fluorescence of photosensitizers. Shielding and absorption of miniSOG fluorescence by cell fluorophores, including cytochrome c, were found to contribute significantly to the changes in the fluorescent properties of miniSOG.

Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants

Glucaric acid (GA), a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants (K59V/R60A, R171S and D276A) screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21(DE3)/MU-R171S accumulated more GA, 136.5% of that of the parent strain.

Boosting Secretion of Extracellular Protein by Escherichia coli via Cell Wall Perturbation.

Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced in E. coli by deleting two d,d-carboxypeptidase genes (dacA and dacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion of dacA and dacB enhanced the accumulation of intracellular soluble peptidoglycan in E. coli and affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion of dacA and dacB appears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacA pETDuet-amyk and BL21 ΔdacB pETDuet-amyk was increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion of dacA and dacB increased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCE Cell surface structure stabilization is important for extracellular secretion of proteins in Escherichia coli As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deleting dacA and dacB genes encoding d,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity of E. coli as a microbial cell factory for extracellular secretion of proteins and chemicals.

Fluorimetric Studies of a Transmembrane Protein and Its Interactions with Differently Functionalized Silver Nanoparticles

Transmembrane proteins play important roles in intercellular signaling to regulate interactions among the adjacent cells and influence cell fate. The study of interactions between membrane proteins and nanomaterials is paramount for the design of nanomaterial-based therapies. In the present work, the fluorescence properties of the transmembrane receptor Notch2 have been investigated. In particular, the steady-state and time-resolved fluorescence methods have been used to characterize the emission of tryptophan residues of Notch2 and then this emission is used to monitor the effect of silver colloids on protein behavior. To this aim, silver colloids are prepared with two different methods to make sure that they bear hydrophilic (citrate ions, C-AgNPs) or hydrophobic (dodecanethiol molecules, D-AgNPs) capping agents. The preparation procedures are tightly controlled to obtain metal cores with similar size distributions (7.4 ± 2.5 and 5.0 ± 0.8 nm, respectively), thus, making the comparison of the results easier. The occurrence of strong interactions between Notch2 and D-AgNPs is suggested by the efficient and statistically relevant quenching of the stationary protein emission already at low nanoparticle (NP) concentrations (ca. 12% quenching with [D-AgNPs] = 0.6 nM). The quenching becomes even more pronounced (ca. 60%) when [D-AgNPs] is raised to 8.72 nM. On the other hand, the addition of increasing concentrations of C-AgNPs to Notch2 does not affect the protein fluorescence (intensity variations below 5%) indicating that negligible interactions are taking place. The fluorescence data, recorded in the presence of increasing concentrations of silver nanoparticles, are then analyzed through the Stern-Volmer equation and the sphere of action model to discuss the nature of interactions. The effect of D-AgNPs on the fluorescence decay times of Notch2 is also investigated and a decrease in the average decay time is observed (from 4.64 to 3.42 ns). The observed variations of the stationary and time-resolved fluorescence behavior of the protein are discussed in terms of static and collisional interactions. These results document that the capping shell is able to drive the protein-particle interactions, which likely have a hydrophobic nature.