Collision-Induced Unfolding, Tandem MS, Bottom-up Proteomics, and Interactomics for Identification of Protein Complexes in Native Surface Mass Spectrometry.

Endogenously occurring salts and nonvolatile matrix components in untreated biological surfaces can suppress protein ionization and promote adduct formation, challenging protein identification. Characterization of labile proteins within biological specimens is particularly demanding because additional purification or sample treatment steps can be time-intensive and can disrupt noncovalent interactions. It is demonstrated that the combined use of collision-induced unfolding, tandem mass spectrometry, and bottom-up proteomics improves protein characterization in native surface mass spectrometry (NSMS). This multiprong analysis is achieved by acquiring NSMS, MS/MS, ion mobility (IM), and bottom-up proteomics data from a single surface extracted sample. The validity of this multiprong approach was confirmed by the successful characterization of nine surface-deposited proteins, with molecular weights ranging from 8 to 147 kDa, in two separate mixtures. Bottom-up proteomics provided a list of proteins to match against observed proteins in NSMS and their detected subunits in tandem MS. The method was applied to characterize endogenous proteins from untreated chicken liver samples. The subcapsular liver sampling for NSMS analysis allowed for the detection of endogenous proteins with molecular weights of up to ∼220 kDa. Moreover, using IM-MS, collision cross sections and collision-induced unfolding pathways of enzymatic proteins and protein complexes of up to 145 kDa were obtained.

Tissue-resident memory T cell maintenance during antigen persistence requires both cognate antigen and interleukin-15.

Our understanding of tissue-resident memory T (TRM) cell biology has been largely developed from acute infection models in which antigen is cleared and sterilizing immunity is achieved. Less is known about TRM cells in the context of chronic antigen persistence and inflammation. We investigated factors that underlie TRM maintenance in a kidney transplantation model in which TRM cells drive rejection. In contrast to acute infection, we found that TRM cells declined markedly in the absence of cognate antigen, antigen presentation, or antigen sensing by the T cells. Depletion of graft-infiltrating dendritic cells or interruption of antigen presentation after TRM cells were established was sufficient to disrupt TRM maintenance and reduce allograft pathology. Likewise, removal of IL-15 transpresentation or of the IL-15 receptor on T cells during TRM maintenance led to a decline in TRM cells, and IL-15 receptor blockade prevented chronic rejection. Therefore, antigen and IL-15 presented by dendritic cells play nonredundant key roles in CD8 TRM cell maintenance in settings of antigen persistence and inflammation. These findings provide insights that could lead to improved treatment of chronic transplant rejection and autoimmunity.

Tuning CARs: recent advances in modulating chimeric antigen receptor (CAR) T cell activity for improved safety, efficacy, and flexibility.

Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility.

Native Mass Spectrometry and Collision-Induced Unfolding of Laser-Ablated Proteins.

Infrared laser ablation sample transfer (LAST) was used to collect samples from solid surfaces for mass spectrometry under native spray conditions. Native mass spectrometry was utilized to probe the charge states and collision-induced unfolding (CIU) characteristics of bovine serum albumin (BSA), bovine hemoglobin (BHb), and jack-bean concanavalin A (ConA) via direct injection electrospray, after liquid extraction surface sampling, and after LAST. Each protein was deposited from solution on solid surfaces and laser-ablated for off-line analysis or sampled for online analysis. It was found that the protein ion gas-phase charge-state distributions were comparable for direct infusion, liquid extraction, and laser ablation experiments. Moreover, calculated average collision cross section (CCS) values from direct injection, liquid extraction, and laser ablation experiments were consistent with previously reported literature values. Additionally, an equivalent number of mobility features and conformational turnovers were identified from unfolding pathways from all three methods for all charge states of each protein analyzed in this work. The presented work suggests that laser ablation yields intact proteins (BSA, BHb, and ConA), is compatible with native mass spectrometry, and could be suitable for spatially resolved interrogation of unfolding pathways of proteins.

CD8+ T cells specific for cryptic apoptosis-associated epitopes exacerbate experimental autoimmune encephalomyelitis.

The autoimmune immunopathology occurring in multiple sclerosis (MS) is sustained by myelin-specific and -nonspecific CD8+ T cells. We have previously shown that, in MS, activated T cells undergoing apoptosis induce a CD8+ T cell response directed against antigens that are unveiled during the apoptotic process, namely caspase-cleaved structural proteins such as non-muscle myosin and vimentin. Here, we have explored in vivo the development and the function of the immune responses to cryptic apoptosis-associated epitopes (AEs) in a well-established mouse model of MS, experimental autoimmune encephalomyelitis (EAE), through a combination of immunization approaches, multiparametric flow cytometry, and functional assays. First, we confirmed that this model recapitulated the main findings observed in MS patients, namely that apoptotic T cells and effector/memory AE-specific CD8+ T cells accumulate in the central nervous system of mice with EAE, positively correlating with disease severity. Interestingly, we found that AE-specific CD8+ T cells were present also in the lymphoid organs of unprimed mice, proliferated under peptide stimulation in vitro, but failed to respond to peptide immunization in vivo, suggesting a physiological control of this response. However, when mice were immunized with AEs along with EAE induction, AE-specific CD8+ T cells with an effector/memory phenotype accumulated in the central nervous system, and the disease severity was exacerbated. In conclusion, we demonstrate that AE-specific autoimmunity may contribute to immunopathology in neuroinflammation.

Chemometric techniques coupled with NMR for matabolic profiling of lettuce exposed to polycyclic aromatic hydrocarbones.

Treated waste water (TWW) quality varies due to the occurrence of polycyclic aromatic hydrocarbons (PAHs) up to low µg L-1. In this study, a non-targeted metabolomic analysis was performed on lettuce (Lactuca sativa L) exposed to 4 PAHs by irrigation. The plants were watered with different concentrations of contaminants (0-100 µg L-1) for 39 days under controlled conditions and then harvested, extracted and analyzed by nuclear magnetic resonance (NMR). Different chemometric tools based on principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) are proposed for the analysis of the complex data sets generated in the different exposure experiments. Furthermore, Analysis of Variance Simultaneous Component Analysis (ASCA) of changes on metabolite peaks showed significant PAHs concentration and exposure time-dependent changes, clearly differentiating between exposed and non-exposed samples.

Supramolecular solvent-based high-throughput sample treatment platform for the biomonitoring of PAH metabolites in urine by liquid chromatography-tandem mass spectrometry.

Monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), present in human urine at trace concentrations (viz. from ng L-1 to µg L-1), are considered the main biomarkers of human exposure to PAHs. In this work, we report a simple and high-throughput sample treatment platform to facilitate the biomonitoring of OH-PAHs by making it easier, greener and most cost-effective. This platform is based on the integration of analyte extraction and sample cleanup in a single step by the use of supramolecular solvents with restricted access properties (SUPRAS-RAM). The SUPRAS was spontaneously formed in situ in the urine by the addition of a colloidal suspension of decanoic acid in THF. Metabolites from naphthalene, fluorene, phenanthrene and pyrene were quantitatively extracted (absolute recoveries in the range 91-109%). Polysaccharides and proteins in the urine were excluded from extraction by physical and chemical mechanisms, which allowed the direct analysis of the SUPRAS extract by liquid chromatography tandem mass spectrometry. Absolute matrix effects for OH-PAHs were in the range 92-103%. Method quantification limits for OH-PAHs, without the need for evaporation of the SUPRAS extracts, were in the interval 1.0-6.7 ng L-1. The precision, evaluated in terms of repeatability and reproducibility, varied between 1.1 and 13.8%. The method was successfully applied to the analysis of urine from 16 smoking and non-smoking volunteers. Both analytical and operational features of this method make it suitable to evaluate human exposure to PAHs.

Harmonized Autophagy Versus Full-Fledged Hepatitis B Virus: Victorious or Defeated.

Autophagy is a finely tuned process in the regulation of innate immunity to avoid excessive inflammatory responses and inflammasome signaling. In contrast, the results of recent studies have shown that autophagy may disease-dependently contribute to the pathogenesis of liver diseases, such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) during hepatitis B virus (HBV) infection. HBV has learned to subvert the cell's autophagic machinery to promote its replication. Given the great impact of the autophagy mechanism on the HBV infection and HCC, recognizing these factors may be offered new hope for human intervention and treatment of chronic HBV. This review focuses on recent findings viewing the dual role of autophagy plays in the pathogenesis of HBV infected hepatocytes.

Extraction of antidepressant drugs in biological samples using alkanol-based nano structured supramolecular solvent microextraction followed by gas chromatography with mass spectrometric analysis.

In the present study, a supramolecular solvent was formed from reverse micelle aggregates of octanol. The proposed supramolecular solvent was used for rapid extraction of some antidepressants drugs including amitriptyline, imipramine, desipramine, maprotiline, sertraline, and doxepin from biological samples. Alkanol-based supramolecular solvents have a unique array of physicochemical properties, making them a very attractive alternative to replace organic solvents in analytical extractions. The parameters affecting the extraction of target analytes (i.e., the volume of tetrahydrofuran and octanol as the major components comprising the supramolecular solvent, chain length of alkanols, sample solution pH, salt addition, and ultrasonic time) were investigated and optimized by factor by factor optimization method. Under the optimum conditions, preconcentration factors of 470, 490, 460, 385, 370, and 430 were obtained for amitriptyline, doxepin, imipramine, desipramine, maprotiline, and sertraline, respectively. The linear ranges and coefficients of determination (R2 ) were obtained in the range of 0.01-100 µg/L and 0.9974-0.9991, respectively. Also the limits of detection (S/N = 3) of 0.003-0.03 µg/L, and precisions (n = 5) of 4.9-8.9% were calculated. Finally, the method was successfully applied for the extraction of antidepressant drugs in biological samples, and relative recoveries in the range of 91-102% were obtained.

Nanostructured gemini-based supramolecular solvent coupled with ultrasound-assisted back extraction as a preconcentration step before GC-MS.

Liquid-phase microextraction based on gemini-based supramolecular solvent was successfully applied as a preconcentration step before gas chromatography with mass spectrometry. To eliminate the interferences of gemini surfactant, the analytes were back-extracted into an immiscible organic solvent in the presence of ultrasonic sound waves. Three phthalate esters (di-n-butyl-, butylbenzyl-, bis(2-ethylhexyl)-, and di-n-octyl phthalatic esters) were used as target analytes. The effective parameters on extraction efficiency of the target analytes (i.e., the amount of surfactant and volume of propanol as major components making up the supramolecular solvent, ionic strength, hexane volume, and ultrasound time) were investigated and optimized by a one-variable-at-a-time method. Under the optimum conditions, the preconcentration factors of the analytes were in the range of 95-182. The linear dynamic range of 0.05-200.00 µg/L with a correlation of determination of (R2 ) ≥ 0.9935 was obtained. The proposed method had an excellent limit of detection (S/N = 3) of 0.01 for di-n-octyl and 0.02 µg/L for butylbenzyl- and di-n-butyl-phthalatic ester. Good relative recoveries in the range of 85.7-105.2% guaranteed the accuracy of the amount of phthalates distinguished in the nonspiked samples.

Autophagy induction regulates influenza virus replication in a time-dependent manner.

PURPOSE: Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research. METHODOLOGY: In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. CONCLUSION: This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production.

MicroRNA-Mediated Post-Transcriptional Regulation of Epithelial to Mesenchymal Transition in Cancer.

Epithelial to mesenchymal transition (EMT) program participates in tissue repair, embryogenesis and numerous pathological conditions, particularly cancer progression and tumor metastasis. A highly complex and strongly controlled post-transcriptionally regulated network of microRNAs (miRNAs) regulates the EMT process. miRNAs are critical parts of the post-transcriptional regulation of gene expression. A set of miRNAs target multiple components of major signaling pathways and downstream effectors of EMT. miRNAs associated with this process are involved in controlling tumor progression and invasiveness either as oncogenes or as tumor suppressors. Since several miRNAs directly affect EMT-related master regulators, they have been discovered to have the potential to be used as biomarkers or targets in EMT-based pathological conditions such as cancer. Therefore, comprehensive understanding of miRNA-EMT correlation with tumor metastatic spread may provide improvements to diagnostic tools or therapeutics for cancer. This review summarizes our current knowledge about some of these important miRNAs and focuses on their specific roles in regulation of the EMT process in cancer.

A new generation of nano-structured supramolecular solvents based on propanol/gemini surfactant for liquid phase microextraction.

A new supramolecular solvent (SUPRAS) made up of aggregates of gemini surfactant was introduced. A microextraction method, based on the SUPRAS followed with high performance liquid chromatography-ultraviolet detection, was applied for the determination of parabens in cosmetics, beverages and water samples. A SUPRAS is a nano-structured liquid made up of surfactant aggregates synthesized through a self-assembly process. In the present work, a new gemini-based SUPRAS was introduced. Methyl paraben (MP), ethyl paraben (EP), and propyl paraben (PP) were extracted on the basis of π-cation and Van der Waals interactions into the SUPRAS. The parameter affecting the extraction of target analytes (i.e., the amount of surfactant and volume of propanol as major components comprising the supramolecular solvent, sample solution pH, salt addition, ultrasonic and centrifugation time) were investigated and optimized by one-variable-at-a-time method. Under the optimum conditions, the preconcentration factors of 98, 143 and 156 were obtained for MP, EP and PP, respectively. The linearity ranged from 0.5 to 0.7-200 µg L-1 with the correlation of determination of (R2) ≥ 0.9938. The gemini-based SUPRAS followed by HPLC-UV has been found to have excellent detection sensitivity with a limit of detection (LOD, S/N = 3) of 0.5 µg L-1 for EP and PP, and 0.7 µg L-1 for MP. Good recoveries over the range of 92.0-108.3% assured the accuracy of the amount of parabens distinguished in the non-spiked samples.

Nano-structured gemini-based supramolecular solvent for the microextraction of cyhalothrin and fenvalerate.

A novel supramolecular solvent-based microextraction followed by high-performance liquid chromatography with ultraviolet detection method has been developed for the extraction and determination of two pyrethroid analytes, cyhalothrin and fenvalerate, in water and soil samples. The liquid-liquid-phase separation of surfactants has been used in analytical extraction. The surfactant-rich phase is a nano-structured liquid, recently named as a supramolecular solvent, generated from the amphiphiles. The alkyl carboxylic acid based supramolecular solvents were introduced before. Coacervates made up of gemini surfactant, consisting of two amphiphilic moieties, were first used as solvent. The effective parameters on extraction (i.e., type of organic solvent, the amount of surfactant and volume of tetrahydrofuran, sample solution pH, salt addition, ultrasonic and centrifugation time) were investigated and optimized. Under the optimum conditions, preconcentration factors of 110 and 145 were obtained for the analytes. The linearity was 0.5-200.0 µg/L with the correlation of determination of (R(2) ) ≥ 0.9984. The limit of detection of the method was (S/N = 3) 0.2 µg/L, and precisions in the range of 6.3-10.3% (RSDs, n = 5) were obtained. This method has been successfully applied to analyze real samples, and good recoveries in the range of 101.2-108.8% were obtained.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production.

OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Over-expression of NOTCH1 as a biomarker for invasive breast ductal carcinoma.

Breast cancer is the leading cause of cancer-related death in women worldwide. Invasive ductal carcinoma (IDC) is the most frequent invasive form of breast cancer followed by metastasis. There is no accepted marker for distinguishing this form from other less aggressive forms of breast cancer. Therefore, finding new markers especially molecularly detectable ones are noteworthy. It has been shown that NOTCH1 has been overexpressed in the patients with breast cancer, but no study has investigated the expression of NOTCH1 and its correlation with other molecular and hormonal markers of breast cancer so far. In the current study, 20 breast cancer tissues and 20 matched adjacent normal breast tissue from breast cancer patients were obtained and categorized in two groups: patients with IDC and patient with other types of breast cancer. Gene expression analysis using real-time PCR showed that the NOTCH1 gene was significantly overexpressed in patients with IDC. We also found a slight correlation between NOTCH1 overexpression and p53 accumulation in the cancerous cells confirmed by Immunohistochemistry (IHC). This results showed that it is possible to introduce NOTCH1 expression as a novel biomarker of IDC, alone or preferably accompanied by IHC of p53. We also can design new therapeutic agents targeting NOTCH1 expression for inhibition of metastasis in ductal breast carcinoma.