Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Jamun Seed-Derived Nitrogen-Doped Carbon Dots: A Novel Microwave-Assisted Synthesis for Ultra-Bright Fluorescence and Mn7+ Detection.

For the synthesis of heteroatom-doped carbon nanostructures, biomass is considered as a promising option. Utilizing the microwave-assisted method, we have demonstrated an easy and straightforward one-pot synthesis of nitrogen-doped luminous carbon dots (NCDs) from jamun seed powder and guanidine hydrochloride. Structural and morphological analyses were performed using various analytical techniques. Under ultraviolet light of 315 nm, NCDs emit a bright blue fluorescence, possess a high quantum yield of 26.90%, exhibit strong water dispersion, and demonstrated excellent stability. The average particle size of the NCDs was found to be 7.5±1.2 nm, with a spherical shape. NCDs exhibit high selectivity and sensitivity in fluorescence quenching when exposed to Mn7+ ions. Over a concentration range of 2-30 µM, the fluorescence response (F0/F) shows a linear relationship with Mn7+ concentration, with a detection limit of 0.81 µM. The probe exhibited negligible interference and proved to be effective in accurately quantifying Mn7+ in spiked real-water samples.

Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants.

The aim was to develop a reliable rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine the main bovine milk protein fractions, including their genetic variants. Compared to the previous studies, our method is able to separate the main protein fractions within 20 min of total run time. The method validation consisted of testing repeatability, reproducibility linearity, repeatability, and accuracy. The procedure was developed using raw individual, bulk, and commercially available heat-treated cow milk samples. The RSD of peak areas ranged from 1.43 to 3.16% within analytical day and from 3.29 to 6.70% across analytical days. The method can be applied to investigate both raw and heat-treated milk samples.

Pharmacokinetic investigation of branched oligohexamethyleneguanidine hydrochloride in a liquid composition for the treatment of stomatitis and gingivitis.

Branched oligohexamethyleneguanidine hydrochloride (branched OHMG-HC) possesses high biocidal activity. This study aimed at evaluating the pharmacokinetics of branched OHMG-HC and is mandatory for obtaining permission to conduct clinical studies. The thermal activation method was used to obtain radioactive-labeled drugs for the investigation of substance distribution in various tissues of experimental animals (rats and rabbits). Substance administration was carried out both orally (a dose was split into two equal volumes that were applied to buccal zones of the oral cavities of the animals) and intravenously to get a clear pharmacokinetic profile. In this research, the drug was applied in the concentration of 0.77 mg/kg with the addition of 0.037 mg/kg 3H-OHMG for rats, and 0.42 mg/kg with the addition of 0.015 mg/kg 3H-OHMG for Chinchilla rabbits. The selected samples of blood, organs, and urine underwent alkaline mineralization. A quantitative determination of 3H-OHMG was carried out using a liquid ß-, γ-counter according to the level of scintillation in the sample. Branched OHMG-HC displayed uniform distribution within all main organs and tissues upon oral administration. The highest concentrations were found in liver and kidney tissues, whereas the lowest in blood, cardiac muscle tissue, and brain. The closeness of the fabs values obtained from different animals (24.5% for rats and 29.0% for rabbits) demonstrated the absence of the species specificity in response to the pharmaceutical substance. The main parameters of excretion were established, and the half-life time was estimated to be 15 h.

Toxicity of humidifier disinfectant polyhexamethylene guanidine hydrochloride by two-week whole body-inhalation exposure in rats.

The use of polyhexamethylene guanidine hydrochloride (PHMG·HCl) as a humidifier disinfectant caused an outbreak of pulmonary disease, leading to the deaths of pregnant women and children in South Korea. However, limited information is available on the inhalation toxicity of PHMG·HCl. Therefore, this study aimed to characterize the subacute inhalation toxicity of PHMG·HCl by whole-body exposure in rats. F344 rats were exposed to 0 mg/m3, 1 mg/m3, 5 mg/m3, or 25 mg/m3 of PHMG·HCl for 6 h/day, 5 days/week for two weeks via whole-body inhalation. Emaciation and rale were observed in rats in the 25 mg/m3 PHMG·HCl group. Significant changes in body weight, hematology, serum chemistry and organ weight were observed in all PHMG·HCl-exposed groups. Gross lesions showed ballooning or red focus in the lungs of rats in the PHMG·HCl-exposed groups. In histopathological examination, most of histological lesions (including degeneration, atrophy, ulcer, inflammatory cell infiltration, inflammation, and fibrosis in nasal cavity, larynx, trachea, and lungs) indicated tissue damage by PHMG·HCl in all PHMG·HCl-exposed groups. Additionally, atrophy of the spleen, thymus, and reproductive organs; immaturity of the testes; and cell debris in the epididymides were affected by the reduction in body weight in PHMG·HCl-exposed groups. In conclusion, two-week repeated whole-body inhalation exposure of rats to PHMG·HCl reveled toxic effects on the respiratory system and secondary effects on other organs. The results of this study indicate that the no observable adverse effect level (NOAEL) for PHMG·HCl is below 1 mg/m3.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride.

BACKGROUND: Long-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line. METHODS: A number of common solutes were tested in lieu of sorbitol for synchronization of P. knowlesi A1-H.1 ring stage. RESULTS: Guanidine hydrochloride was found to selectively lyse trophozoite- and schizont-infected red blood cells, yielding highly synchronous and viable rings. CONCLUSIONS: A method for synchronization of P. knowlesi in human red blood cells was developed. Requiring only common laboratory reagents, this method is simple and should be applicable to most laboratory settings.

Effective and economical column-based method for RNA isolation from animal cells.

OBJECTIVES: To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. RESULTS: Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. CONCLUSIONS: The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Immunohistochemical characterization of oxidative stress in the lungs of rats exposed to the humidifier disinfectant polyhexamethylene guanidine hydrochloride.

Polyhexamethylene guanidine hydrochloride (PHMG-HCl), an antimicrobial additive in humidifier disinfectants, was associated with the pulmonary disease outbreak in South Korea. However, PHMG-mediated oxidative stress has only been studied in vitro. Here, we evaluated PHMG-induced oxidative stress in the lungs of rats exposed to PHMG-HCl. Male F344 rats were exposed to different concentrations of PHMG-HCl for 13-weeks via whole-body inhalation. Histopathological examination of the exposed rats showed the presence of lung lesions, including alveolar/interstitial fibrosis with inflammatory cell infiltration, bronchioalveolar hyperplasia, bronchiolar/alveolar squamous metaplasia, bronchial/bronchiolar epithelial detachment, and alveolar hemorrhage. Immunohistochemical analysis showed that 4-hydroxynonenal (4-HNE) was expressed in the bronchiolar epithelium, mainly in Clara cells and macrophages of the fibrotic tissue. The number of 4-HNE-positive cells increased significantly in a dose-dependent manner. This is the first in vivo study to report PHMG-induced oxidative stress. Our study provides clues to elucidate the mechanisms underlying PHMG-induced damage in patients affected by humidifier disinfectants.

Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

Osmolyte-Like Stabilizing Effects of Low GdnHCl Concentrations on d-Glucose/d-Galactose-Binding Protein.

The ability of d-glucose/d-galactose-binding protein (GGBP) to reversibly interact with its ligands, glucose and galactose, makes this protein an attractive candidate for sensing elements of glucose biosensors. This potential is largely responsible for attracting researchers to study the conformational properties of this protein. Previously, we showed that an increase in the fluorescence intensity of the fluorescent dye 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN) is linked to the holo-form of the GGBP/H152C mutant in solutions containing sub-denaturing concentrations of guanidine hydrochloride (GdnHCl). It was hypothesized that low GdnHCl concentrations might lead to compaction of the protein, thereby facilitating ligand binding. In this work, we utilize BADAN fluorescence spectroscopy, intrinsic protein UV fluorescence spectroscopy, and isothermal titration calorimetry (ITC) to show that the sub-denaturing GdnHCl concentrations possess osmolyte-like stabilizing effects on the structural dynamics, conformational stability, and functional activity of GGBP/H152C and the wild type of this protein (wtGGBP). Our data are consistent with the model where low GdnHCl concentrations promote a shift in the dynamic distribution of the protein molecules toward a conformational ensemble enriched in molecules with a tighter structure and a more closed conformation. This promotes the increase in the configurational complementarity between the protein and glucose molecules that leads to the increase in glucose affinity in both GGBP/H152C and wtGGBP.

Dissociated and Reconstituted Cartilage Microparticles in Densified Collagen Induce Local hMSC Differentiation.

Decellularized cartilage microparticles, and all associated native signals, are delivered to hMSC populations in a dense, type I collagen matrix. Hybrid usage of native tissue signals and the engineering control of collagen matrices show the ability to induce local infiltration and differentiation of hMSCs. Additionally, the solid cartilage microparticles inhibit bulk cell-mediated contraction of the composite.

Denaturation induced aggregation in α-crystallin: differential action of chaotropes.

α-Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α-crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α-crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α-crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α-crystallin at 216 nm, suggesting gain in ß-sheet structure in the presence of GdnHCl (0.5-1 M) followed by unfolding at higher concentration (2-6 M). However, only minor changes in the secondary structure of α-crystallin were observed in the presence of urea. Moreover, 8-anilinonaphthalene-1-sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α-crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α-crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α-crystallin in the presence of GdnHCl, whereas only aggregate-like structures were observed in α-crystallin treated with urea. Our results suggest that α-crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis, Characterization, and Bactericidal Evaluation of Chitosan/Guanidine Functionalized Graphene Oxide Composites.

In response to the wide spread of microbial contamination induced by bacterial pathogens, the development of novel materials with excellent antibacterial activity is of great interest. In this study, novel antibacterial chitosan (CS) and polyhexamethylene guanidine hydrochloride (PHGC) dual-polymer-functionalized graphene oxide (GO) (GO-CS-PHGC) composites were designed and easily fabricated. The as-prepared materials were characterized by Fourier transform infrared (FTIR), X-ray photoelectron spectrometer (XPS), field emission scanning electron microscopy (FE-SEM), transmission electron microscope (TEM), thermogravimetric analysis (TGA) and Raman spectroscopy. Their antibacterial capability towards bacterial strains was also studied by incubating both Gram-negative bacteria and Gram-positive bacteria in their presence. More significantly, the synergistic antibacterial action of the three components was assayed, and the findings implied that the as-prepared GO-CS-PHGC shows enhanced antibacterial activity when compared to its single components (GO, CS, PHGC or CS-PHGC) and the mixture of individual components. Not only Gram-negative bacteria but also Gram-positive bacteria are greatly inhibited by GO-CS-PHGC composites. The minimum inhibitory concentration (MIC) value of GO-CS-PHGC against E. coli was 32 µg/mL. With the powerful antibacterial activity as well as its low cost and facile preparation, GO-CS-PHGC has potential applications as a novel antibacterial agent in a wide range of biomedical uses.

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells.

We evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells. Although detergents were found to be highly suited for global proteome analysis, higher intensities were obtained for high-abundant nucleic acid-associated protein complexes, like the ribosome and histone proteins, using guanidine hydrochloride. Importantly, we show for the first time that the observable total proteome mass of a sample strongly depends on the sample preparation protocol, with some protocols resulting in a significant underestimation of protein mass due to incomplete protein extraction of biased protein groups. Furthermore, we demonstrate that some of the observed abundance biases can be overcome by incorporating a nuclease treatment step or, alternatively, a correction factor for complementary sample preparation approaches.

The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles.

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.

The importance of the last strand at the C-terminus in ßB2-crystallin stability and assembly.

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on ßB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of ßB2-crystallin Greek-key motif 4. All of the three mutations promoted ßB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected ßB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of ß-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between ß-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the ß-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of ß-crystallins are achieved by the precisely regulated oligomeric equilibrium.

Apolipoprotein A-I configuration and cell cholesterol efflux activity of discoidal lipoproteins depend on the reconstitution process.

Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.

Polyhexamethylene guanidine hydrochloride shows bactericidal advantages over chlorhexidine digluconate against ESKAPE bacteria.

More information regarding the bactericidal properties of polyhexamethylene guanidine hydrochloride (PHMG) against clinically important antibiotic-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens needs to be provided for its uses in infection control. The bactericidal properties of PHMG and chlorhexidine digluconate (CHG) were compared based on their minimum inhibitory concentrations (MICs), minimum bactericidal concentrations, and time-course-killing curves against clinically important antibiotic-susceptible and antibiotic-resistant ESKAPE pathogens. Results showed that PHMG exhibited significantly higher bactericidal activities against methicillin-resistant Staphylococcus aureus, carbapenem-resistant Klebsiella pneumoniae, and ceftazidime-resistant Enterobacter spp. than CHG. A slight bactericidal advantage over CHG was obtained against vancomycin-resistant Enterococcus faecium, ciprofloxacin- and levofloxacin-resistant Acinetobacter spp., and multidrug-resistant Pseudomonas aeruginosa. In previous reports, PHMG had higher antimicrobial activity against almost all tested Gram-negative bacteria and several Gram-positive bacteria than CHG using MIC test. These studies support the further development of covalently bound PHMG in sterile-surface materials and the incorporation of PHMG in novel disinfectant formulas.

An improved in-gel digestion method for efficient identification of protein and glycosylation analysis of glycoproteins using guanidine hydrochloride.

In-gel digestion followed by LC/MS/MS is widely used for the identification of trace amounts of proteins and for the site-specific glycosylation analysis of glycoproteins in cells and tissues. A major limitation of this technique is the difficulty in acquiring reliable mass spectra for peptides present in minute quantities and glycopeptides with high heterogeneity and poor hydrophobicity. It is considered that the SDS used in electrophoresis can interact with proteins noncovalently and impede the ionization of peptides/glycopeptides. In this study, we report an improved in-gel digestion method to acquire reliable mass spectra of a trace amount of peptides/glycopeptides. A key innovation of our improved method is the use of guanidine hydrochloride, which forms complexes with the residual SDS molecules in the sample. The precipitation and removal of SDS by addition of the guanidine hydrochloride was successful in improving the S/N of peptides/glycopeptides in mass spectra and acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide.