Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings

S. Gupta, K. L. Chachra, P. Bhadola and P. Sodhani
Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings Objective: To devise a simple, cost‐effective protocol for Papanicolaou (Pap) staining of cervicovaginal smears. Methods: Five hundred coded paired cervical smears were collected from women as part of routine cervical cancer screening. One set of smears was stained by conventional Pap staining protocol (CP) and the other by a modified protocol (MP) in which alcohol was replaced by 1% acetic acid in all the steps except during fixation and prior to mounting; in addition, one alcohol‐based counterstain, OG, was omitted. The smears were examined blindly by the pathologists and then decoded. Each pair of smears was compared and the two protocols were analysed for staining quality and diagnoses by McNemar and chi‐square tests. Results: The staining quality in the MP was satisfactory. The nuclear and cytoplasmic features were comparable to the CP. Cytoplasmic transparency was maintained in the MP and the differential staining of blue/green and pink was acceptable to the pathologists and technicians. The diagnoses agreed in all cases and there was no compromise in interpreting the smears. With MP it took only 3–4 minutes to stain a batch of 50 slides. in contrast to the 20 minutes taken by CP. The MP used almost one‐seventh of the amount of alcohol compared with CP, which translated into a significant cost reduction per smear. Conclusions: The improvised Pap staining protocol with minimum alcohol use is a simple, cost‐effective and technician‐friendly procedure that can be easily adopted in high‐volume, resource‐limited laboratories for mass cervical cancer screening.     

Efficacy of Ki-67 antigen staining in Papanicolaou (Pap) smears in post-menopausal women with atypia—an audit

In cervical smears from post-menopausal women with mucosal atrophy it can be difficult to distinguish atrophic epithelial cell groups from neoplastic cell groups on cytomorphological criteria only. The consequence of post-menopausal atypia is that the woman is referred for a repeat smear after local oestrogen treatment or for colposcopy. We investigated whether immunocytochemical expression of Ki-67 (MIB-1) on the primary Papanicolaou-stained smear could be of any diagnostic help. Our data showed that negative Ki-67 expression is a very reliable indicator of a normal atrophic cell pattern, and by using this method on the original smear we were able to reduce the false-positive cytologic diagnoses by 86%.     

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.     

Tor‐Sch9 deficiency activates catabolism of the ketone body‐like acetic acid to promote trehalose accumulation and longevity

In mammals, extended periods of fasting leads to the accumulation of blood ketone bodies including acetoacetate. Here we show that similar to the conversion of leucine to acetoacetate in fasting mammals, starvation conditions induced ketone body‐like acetic acid generation from leucine in S. cerevisiae. Whereas wild‐type and ras2Δ cells accumulated acetic acid, long‐lived tor1Δ and sch9Δ mutants rapidly depleted it through a mitochondrial acetate CoA transferase‐dependent mechanism, which was essential for lifespan extension. The sch9Δ‐dependent utilization of acetic acid also required coenzyme Q biosynthetic genes and promoted the accumulation of intracellular trehalose. These results indicate that Tor‐Sch9 deficiency extends longevity by switching cells to an alternative metabolic mode, in which acetic acid can be utilized for the storage of stress resistance carbon sources. These effects are reminiscent of those described for ketone bodies in fasting mammals and raise the possibility that the lifespan extension caused by Tor‐S6K inhibition may also involve analogous metabolic changes in higher eukaryotes.     

A decade with nucleic acid‐based microbiological methods in safety control of foods

In the last decade, nucleic acid‐based methods gradually started to replace or complement the culture‐based methods and immunochemical assays in routine laboratories involved in food control. In particular, real‐time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry‐over contamination. Basic advantages provided by nucleic acid‐based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid‐based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.     

Sulfanilic acid‐modified chitosan mini‐spheres and their application for lysozyme purification from egg white

A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico‐chemically characterized by ss‐NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g^?1 while the dissociation constant was 0.074 ± 0.012 mg mL^?1. The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP‐HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387–396, 2018     

Mass spectrometry‐compatible silver staining of histones resolved on acetic acid‐urea‐Triton PAGE

Acetic acid‐Urea‐Triton (AUT) PAGE is commonly used method to separate histone variants and their post‐translationally modified forms. Coomassie staining is the preferred method for protein visualization; however, its sensitivity is less than that of silver staining. Though silver staining of histones in AUT‐PAGE has been reported, the method is time‐consuming, dependent on prior staining by Amido black and has not been reported suitable for mass spectrometry. Here, we propose ‘SDS‐Silver’ method for rapid, sensitive and mass spectrometry‐compatible staining of histones resolved on AUT‐PAGE.     

Reduced blood‐brain barrier expression of fatty acid‐binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega‐3 fatty acid diets

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood‐brain barrier (BBB) transport of plasma‐derived DHA, a process facilitated by fatty acid‐binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of ¹⁴C‐DHA in 8‐month‐old AD transgenic mice (APPswe,PSEN1?E9) relative to wild‐type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short‐term spatial and recognition memory deficits were observed in AD mice on a 6‐month n‐3 fatty acid‐depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n‐3 fatty acid‐depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.

     

A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.     

Ultrasound‐mediated synthesis of camphoric acid‐based chiral salens for the enantioselective trimethylsilylcyanation of aldehydes

New chiral salen ligands were prepared by the ultrasound‐irradiated condensation of optically active (1R, 3S)‐1,2,2‐trimethyl‐1,3‐diaminocyclopentane with aromatic 1‐hydroxyaldehydes. The ultrasound‐mediated process is more convenient due to shorter reaction times, energy economy, and easier isolation of the products. The in situ formed Ti(IV)(salen) complexes, evaluated as catalysts in the enantioselective trimethylsilylcyanation of benzaldehyde, were found to be efficient for this process, originating the corresponding product in high yields (72–99%) and selectivities of up to 79%. The lowest energy transition states were determined by computational studies. These results were in qualitative agreement with the experimentally observed ones. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane

In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Ca²⁺ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors.     

On‐plate‐selective enrichment of glycopeptides using boronic acid‐modified gold nanoparticles for direct MALDI‐QIT‐TOF MS analysis

In this study, an on‐plate‐selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless‐steel plate, then modified with 4‐mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI‐MS simply by deposition of 2,5‐dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on‐plate strategy promising for online enrichment of glycopeptides, which could be applied in high‐throughput proteome research.