Acidotic and hypoxic tumor microenvironment induces changes to histone acetylation and methylation in oral squamous cell carcinoma.

Hypoxia and acidosis are ubiquitous hallmarks of the tumor microenvironment (TME), and in most solid cancers they have been linked to rewired cancer cell metabolism. These TME stresses are linked to changes in histone post-translational modifications (PTMs) such as methylation and acetylation, which lead to tumorigenesis and drug resistance. Hypoxic and acidotic TME cause changes in histone PTMs by impacting the activities of histone-modifying enzymes. These alterations are yet to be extensively explored in oral squamous cell carcinoma (OSCC), one of the most prevalent cancers in developing countries. Hypoxic, acidotic, and hypoxia with acidotic TME affecting histone acetylation and methylation in the CAL27 OSCC cell line was studied using LC-MS-based proteomics. The study identified several well-known histone marks, in the context of their functionality in gene regulation, such as H2AK9Ac, H3K36me3, and H4K16Ac. The results provide insights into the histone acetylation and methylation associated with hypoxic and acidotic TME, causing changes in their level in a position-dependent manner in the OSCC cell line. Hypoxia and acidosis, separately and in combination, cause differential impacts on histone methylation and acetylation in OSCC. The work will help uncover tumor cell adaptation to these stress stimuli in connection with histone crosstalk events.

PX-12 synergistically enhances the therapeutic efficacy of vorinostat under hypoxic tumor microenvironment in oral squamous cell carcinoma in vitro.

Hypoxia is a characteristic feature of solid tumors, including oral squamous cell carcinoma (OSCC), which causes therapeutic resistance. The hypoxia-inducible factor 1-alpha (HIF-1α) is a key regulator of hypoxic tumor microenvironment (TME) and a promising therapeutic target against solid tumors. Among other HIF-1α inhibitors, vorinostat (suberoylanilide hydroxamic acid, SAHA) is a histone deacetylase inhibitor (HDACi) targeting the stability of HIF-1α, and PX-12 (1-methylpropyl 2-imidazolyl disulfide) is a thioredoxin-1 (Trx-1) inhibitor preventing accumulation of HIF-1α. HDACis are effective against cancers; however, they are accompanied by several side effects along with an emerging resistance against it. This can be overcome by using HDACi in a combination regimen with Trx-1 inhibitor, as their inhibitory mechanisms are interconnected. HDACis inhibit Trx-1, leading to an increase in the production of reactive oxygen species (ROS) and inducing apoptosis in cancer cells; thus, the efficacy of HDACi can be elevated by using a Trx-1 inhibitor. In this study, we have tested the EC50 (half maximal effective concentration) doses of vorinostat and PX-12 on CAL-27 (an OSCC cell line) under both normoxic and hypoxic conditions. The combined EC50 dose of vorinostat and PX-12 is significantly reduced under hypoxia, and the interaction of PX-12 with vorinostat was evaluated by combination index (CI). An additive interaction between vorinostat and PX-12 was observed in normoxia, while a synergistic interaction was observed under hypoxia. This study provides the first evidence for vorinostat and PX-12 synergism under hypoxic TME, at the same time highlighting the therapeutically effective combination of vorinostat and PX-12 against OSCC in vitro.

Echinocystic Acid Bidesmoside Saponins from Microglossa afzelii O. Hoffm and Their Cytotoxic Activity against the CAL-27 Oral Squamous Carcinoma Cell Line.

This paper describes eight new triterpenoid saponins, including afzeliioside A (1), four acetylated afzeliiosides as pairs of inseparable regioisomers, called afzeliiosides B/C (2/3) and D/E (4/5), afzeliiosides F-H (6-8), and a known impatiprin C (9), which were isolated from the n-BuOH fraction of the liana of Microglossa afzelii. Their structures were established mainly by extensive spectroscopic analysis, including 1D and 2D NMR, HRFAB-MS, tandem ESI-MS/MS, and chemical methods, as well as a comparison of their spectral data with those of related compounds. All the isolates were screened for their cytotoxic activity against the CAL-27 oral squamous carcinoma cell line. Only compounds 4/5 (EC50 = 36.0 µg/mL (32.7 µM)) exhibited moderate cytotoxic activity. This work presents the first chemical and biological investigation of Microglossa afzelii and reports, for the first time, on the isolation of saponins in the genus Microglossa.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Genomic features of a high-risk mcr-1.1-positive Escherichia coli ST10 isolated from cattle farm environment.

The environment plays an important role in the dissemination of clinically relevant antimicrobial-resistant bacteria and genes. In this study, we described genomic features of a plasmid-mediated colistin-resistant mcr-1-positive Escherichia coli strains (PK-3225) isolated from a dairy farm wastewater sample. After initial isolation and PCR detection of mcr-1-positive E. coli, whole-genome sequencing was performed using Illumina Hiseq 2500 followed by in silico analysis. Genetic context surrounding the mcr-1 gene was determined and SNP-based phylogenomic analysis was performed. Furthermore, plasmid analysis and conjugation assays were performed to determine transferability of mcr-1. E. coli PK-3225 belonged to ST10 and carried a broad resistome that included colistin (mcr-1), beta-lactam (blaTEM-IB), tetracycline (tetB), phenicol (catA1), macrolide (mdfA), trimethoprim (dfrA17), aminoglycosides (aadA5, aph(3")-Ib, aph(6)-Id), and sulphonamide (sul2) resistance genes. The draft genome of E. coli calculated as 4.9 Mbp. Conjugation experiment showed successful transfer of the mcr-1 gene to E. coli recipient strain J53. In silico analysis showed that mcr-1 was located on IncI2 plasmid of > 59 kb in length, with the nikB-mcr-1-pap2 gene array, and lack ISApl1. The phylogenomic analysis revealed that the PK-3225 was closely related to human ST10 E. coli from Brazil and USA. To our knowledge, this is the first draft genome sequence of mcr-1 carrying E. coli isolated from the farm environment in Pakistan. Considering the high burden of colistin resistance in Pakistan, presence of pandemic high-risk E. coli clones in the environment requires strict surveillance.

Evaluation of cytotoxicity of areca nut and its commercial products on normal human gingival fibroblast and oral squamous cell carcinoma cell lines.

Consumption of areca nut products is the most common cause of oral cancers, particularly in South Asian countries. This study evaluates the cytotoxic and necrotizing effects of areca nut and its formulations on normal human gingival fibroblasts (HGF-1) and oral squamous cell carcinoma (OSCC, CAL-27) cell lines. Identification of various carcinogens and adulterants using LC-HR-ESI-MS/MS analysis was performed in the extracts of areca nut and its products. Apart from alkaloids and flavonoids, a major adulterant, saccharin was found in all the samples of chalia (one of the most common chewing products of areca nut) in the ranges between 1.697-7.170 mg/g of the sample. Cytotoxic studies showed that most of the areca nut products were found cytotoxic to HGF-1 cells while being relatively non-cytotoxic against CAL-27 cells, rather they promote the growth of cancer cells. Our findings revealed that the components of areca nut and its products were injurious to HGF-1 cells and caused necrosis, which may attenuate HGF-1 protection toward oral epithelial cells. Moreover, the non-cytotoxic effect of these products on cancer cell lines suggests further predisposal of the habitual chewers for developing oral carcinomas. This study will give a better understanding of the hazardous effects of areca nut products.

Phosphoproteomic strategies in cancer research: a minireview.

Understanding the cellular processes is central to comprehend disease conditions and is also true for cancer research. Proteomic studies provide significant insight into cancer mechanisms and aid in the diagnosis and prognosis of the disease. Phosphoproteome is one of the most studied complements of the whole proteome given its importance in the understanding of cellular processes such as signaling and regulations. Over the last decade, several new methods have been developed for phosphoproteome analysis. A significant amount of these efforts pertains to cancer research. The current use of powerful analytical instruments in phosphoproteomic approaches has paved the way for deeper and sensitive investigations. However, these methods and techniques need further improvements to deal with challenges posed by the complexity of samples and scarcity of phosphoproteins in the whole proteome, throughput and reproducibility. This review aims to provide a comprehensive summary of the variety of steps used in phosphoproteomic methods applied in cancer research including the enrichment and fractionation strategies. This will allow researchers to evaluate and choose a better combination of steps for their phosphoproteome studies.

Metabolite Profiling and Quantitation of Cucurbitacins in Cucurbitaceae Plants by Liquid Chromatography coupled to Tandem Mass Spectrometry.

Cucurbitaceae is an important plant family because many of its species are consumed as food, and used in herbal medicines, cosmetics, etc. It comprises annual vines and is rich in various bioactive principles which include the cucurbitacins. These steroidal natural products, derived from the triterpene cucurbitane, are mainly the bitter principles of the family Cucurbitaceae. Their biological activities include anti-inflammatory, hepatoprotective, and anti-cancer activities. A total of 10 species belonging to 6 genera of the Cucurbitaceae family along with Cissampelos pareira (Menispermaceae) were included in this study. A comprehensive profiling of certain natural products was developed using HPLC-QTOF-MS/MS analysis and a distribution profile of several major natural products in this family was obtained. A total of 51 natural products were detected in both positive and negative ionization modes, based on accurate masses and fragmentation patterns. Along with this, quantitation of four bioactive cucurbitacins, found in various important plants of the Cucurbitaceae family, was carried out using multiple reaction monitoring (MRM) approach on an ion trap mass spectrometer. Cucurbitacin Q was found to be the most abundant in C. pareira, while Citrullus colocynthis contained all four cucurbitacins in abundant quantities. The developed quantitation method is simple, rapid, and reproducible.

MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption ionization (LDI) matrix.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization technique, has become a method of choice for high-throughput analysis of proteins and peptides. In this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were employed as target analytes to check performance of olanzapine and compared with conventional MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA. Computational studies were performed to determine the thermochemical properties of olanzapine in order to further evaluate its similarity to MALDI matrices which were found in good agreement with the data of existing MALDI matrices.

Probing of metabolites in finely powdered plant material by direct laser desorption ionization mass spectrometry.

Natural products continue to serve as an important source of novel drugs since the beginning of human history. High-throughput techniques, such as MALDI-MS, can be techniques of choice for the rapid screening of natural products in plant materials. We present here a fast and reproducible matrix-free approach for the direct detection of UV active metabolites in plant materials without any prior sample preparation. The plant material is mechanically ground to a fine powder and then sieved through different mesh sizes. The collected plant material is dispersed using 1 µL solvent on a target plate is directly exposed to Nd:YAG 335 nm laser. The strategy was optimized for the analysis of plant metabolites after study of the different factors affecting the reproducibility and effectiveness of the analysis, including particle sizes effects, types of solvents used to disperse the sample, and the part of the plant analyzed. Moreover, several plant species, known for different classes of metabolites, were screened to establish the generality of the approach. The developed approach was validated by the characterization of withaferin A and nicotine in the leaves of Withania somnifera and Nicotiana tabacum, respectively, through comparison of its MS/MS data with the standard compound. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques were used for the tissue imaging purposes. This approach can be used to directly probe small molecules in plant materials as well as in herbal and pharmaceutical formulations for fingerprinting development.

The effects of magnesium particles in posterolateral spinal fusion: an experimental in vivo study in a sheep model.

OBJECT: Magnesium has recently become a material of interest as a biocompatible and biodegradable implant metal. Authors of several reports have noted the potential bone-cell activating or bone-healing effect of high Mg ion concentrations. The classic method for achieving intertransverse process fusion involves using an autologous iliac crest bone graft. Several studies have been performed to investigate enhancement of this type of autograft fusion. To the authors' knowledge, no research has been conducted in which the efficacy of pure Mg particles in posterolateral spinal fusion has been investigated. The objective of this study was to determine whether Mg particles enhance the effectiveness of intertransverse process lumbar fusion in a sheep model. METHODS: Sixteen skeletally mature female sheep were subjected to intertransverse process spinal fusions with pedicle screw fixation at L2-3 and L5-6. Each animal was given a 5-cm3 bone autograft at one fusion level, and a combined 5-cm3 bone autograft with the addition of 1 cm3 Mg at the other level. Six months after surgery, bone formation was evaluated by gross inspection and palpation, and by radiological, histological, scanning electron microscopic, and x-ray diffraction analyses. Radiological results were graded from 0 to 4 according to the status of the bridging bone, which was determined by evaluating both x-ray films and computed tomography scans. The quality of the spinal fusion was assigned a histological score of 0 to 7, in which a score of 0 represented an empty cleft and a score of 7 represented complete bridging of bone between the transverse processes. The trabecular bone formation at each fusion level and the Ca hydroxyapatite crystalline structure in core biopsy specimens were evaluated using scanning electron microscopy and x-ray diffraction analyses, respectively. The rate of rigid bone fusion, according to both palpation and radiological assessment, in the combined Mg and autologous bone treatment group was higher (81.25%) than in the autograft bone treatment group (62.5%), but this difference was not statistically significant. The quality of bone fusion, according to the histological grading system and scanning electron microscopy inspection, was higher in the bone fusion segments of the Mg and autologous graft combined group than in the group with autograft-only arthrodesis, and this difference was statistically significant. The x-ray diffraction analyses further confirmed the effect of Mg in promoting the formation of the crystalline portion of the bone (hydroxyapatite). CONCLUSIONS: Based on the results of this study, adding Mg particles to autologous corticocancellous bone in a posterolateral intertransverse process fusion enhances the quality of bone formation. However, radiological findings did not reveal a statistically significant effect of Mg on the rate of solid bone fusion formation between the two transverse processes.