In vitro evaluation of oral contraceptives on long-tailed macaque (Macaca fascicularis) primary ovarian cells.

Hormonal contraception has been advocated as an alternative population control method for the long-tailed macaque population, which has increased exponentially due to anthropogenic changes and incidental food subsidies from human food waste. Risks of increased zoonosis and conflict are imminent if the population growth of long-tailed macaques is unchecked. However, there's a gap in the literature about the effect of hormonal contraceptives on long-tailed macaque reproductive tissues cell line. The present study aims to investigate the effect of oral contraceptives (Nordette, Noriday, and Ella) on long-tailed macaque ovarian cells. We determine the cell viability and cytotoxicity as well as the morphological changes of the drugs on long-tailed macaque ovarian cells using the MTT assay, Acridine orange/propidium iodide double staining method, morphological examination, and the 4, 6-diamidino-2-phenylindole (DAPI) staining method. For the MTT assay, The drugs were dissolved in culture media before use to have a concentration ranging from 0.5 µg/mL, 2.5 µg/mL, 0.125 µg/mL, 0.0625 µg/mL, and 0.0315 µg/mL to have three replicates for each treatment. In contrast, the concentration of 0.0315 µg/mL was used for the morphological and histopathological analysis. The result of the study indicates that human oral contraceptives (Nordette, Noriday, and Ella) inhibit the growth of long-tailed macaque ovarian cells and induce apoptosis in a concentration- and time-dependent manner (at a concentration of 0.0315 µg/mL and an IC50 lower than 10 µg/mL), With a statistically significant value of ****P < 0.001 for each drug compared to the negative control. The result of the present study contributes toward addressing the gap in the literature on the effect of oral contraceptives in long-tailed macaque ovarian cells. Hence, we conclude that human oral contraceptives (Nordette, Noriday, and Ella) are safe and effective in long-tailed macaque ovarian cells as such could be used to develop non-invasive oral contraceptives for controlling the population of long-tailed macaques as an alternative population control method.

A GC-MS/MS Method for Trace Level Quantification of Six Nitrosamine Impurities (NDMA, NDEA, NEIPA, NDIPA, NDPA, and NDBA) in Commercially Used Organic Solvents: Dichloromethane, Ethyl Acetate, Toluene, and O-xylene.

Nitrosamines, the probable carcinogens have been reported with Angiotensin II Receptor Blocker (ARB) drugs, Ranitidine, and other medicines. Solvents play a vital role in the pharmaceutical industry in the separation, purification, and cleaning process for manufacturing APIs and drug products. According to the FDA and EMA, solvents used in the drug manufacturing process are potential root causes of Nitrosamine impurities. Hence, monitoring nitrosamines in solvents is an essential step for manufacturers. A sensitive direct injection GC-MS/MS, an essential analytical tool for low-level nitrosamine quantification in solvents, was developed by utilizing multiple reactions monitoring mode (MRM) for the simultaneous determination of six nitrosamines, namely, NDMA, NDEA, NEIPA, NDIPA, NDPA and NDBA in common solvents such as dichloromethane, ethyl acetate, toluene, and o-xylene. NDMA-d6 was used as an internal standard. The FDA reported a combined direct injection method for nitrosamine impurity assay by GC-MS/MS, which had several challenges for commercial-grade solvents in terms of interferences and resolution of unknown impurities and nitrosamine peaks. A novel method was developed to optimize the critical parameters of GC-MS/MS according to the solvent samples. The method validation was performed through the following parameters, sensitivity, linearity, accuracy, precision, specificity, and stability. The quantification of nitrosamines in commercial-grade solvents ranged from 100 ppb to 8000 ppb with respect to the sample concentration of 25 mg/mL with good sensitivity in LOQ level. The quantification ranged from 5 ppb (for NDMA, NDEA, NEIPA, NDIPA, NDPA) and 13 ppb (NDBA) to 2000 ppb with respect to the sample concentration of 100 mg/mL for analytical grade solvents with good sensitivity in the proposed method. Hence, it will be useful to quantify the low-level nitrosamines in commercial-grade solvents as well as analytical-grade solvents.

First study on phenotypic and morphological characteristics of Malaysian Kedah-Kelantan cattle (Bos indicus) and method of estimating their body weight.

Background and Aim: Indigenous Kedah-Kelantan (KK) cattle are well adapted with distinguished reproductive capabilities; they account for more than 70% of the domestic beef production in Malaysia. The published literature on the phenotypic and morphometric characteristics of KK cattle are sparse and require further improvement. Therefore, this study was aimed to determine the phenotypic and morphometric characteristics of Malaysian KK cattle and method of estimating live body weight (BW). Materials and Methods: Morphometric and phenotypic measurements were taken from 184 KK cattle (102 males and 82 females) sourced from three regions. Each animal's color pattern was recorded for their coat, muzzle, face, eyelashes, horns, tail switch, hoof, and legs through visual observation. Length measurements were taken of the body, face, ear, horn, tail, and rump. Several morphological features such as length, width, and girth were measured using a measuring tape, while wither height and hip height were assessed with a measuring scale. Results: Brown is the predominant coat color in KK cattle (>82%). The overall means of head length, face width (FW), ear length, horn length, wither height, heart girth (HG), body length (BL), and rump length were 42.5±4.5, 17.3±2.9, 19.8±3.1, 9.9±4.4, 104.3±7.1, 127.4±13.2, 98.3±12.3, and 32.4±4.1 cm, respectively. Different morphometric parameters of length, width, and circumference were significantly ( p<0.01) larger in males than females, except for tail length and TG. Correlation coefficient and multiple regression analysis clearly revealed that BL is the best parameter for estimating live BW in KK cattle. Conclusion: Phenotypic and morphometric measurements in this study showed that Malaysian KK cattle generally possess a brown coat pattern with smaller body size, while BL revealed to be the best parameter to predict BW. The data generated from this study would be useful as baseline data for the identification and selection of KK cattle based on their phenotypical- and morphological-features for further improvement of this breed.

Opportunities and challenges associated with fecal progesterone metabolite analysis.

Conventionally, plasma or milk progesterone evaluations are used to determine the reproductive status of female animals. Collection of such samples is often associated with difficulties of animal handling and restraint. Measurable quantities of progesterone metabolites are found in feces of animals. Their concentrations are known to be well correlated to plasma progesterone levels and are, therefore, used as non-invasive samples for assessing reproductive function in a wide range of animal species. Although the analysis of fecal progesterone metabolites has been widely accepted in many laboratories, several factors are known to affect the results from this valuable analytical technique. Some of these factors include storage/transportation media for fecal samples, type of solvent that is used for extraction of progesterone metabolites from feces, and the type and sensitivity of an assaying technique employed. Although fecal progesterone metabolites analysis is associated with some difficulties, it can effectively be used to monitor reproductive function in a wide range of animal species. This review aims to highlight the usefulness of fecal progesterone metabolite analysis as a non-invasive technique in monitoring reproductive function in animals. The article mainly focuses on the many opportunities and challenges associated with this analytical technique.

A Method for Mapping Glycosylation Sites in Proteins.

The analysis of protein glycosylation by mass spectrometry (MS) has been a challenging technical problem. Quantification by HPLC of N-linked glycans can be executed by the use of peptide-N-glycosidase F to release them from the protein, followed by attachment of a fluorescent label and subsequent fluorescence detection. Similar quantification of O-linked glycans is not possible, as a result of the lack of a universal deglycosylation enzyme. Site-specific analyses by MS, such as the use of proteases to digest the glycoprotein, are difficult to use for quantification of glycans, as a result of the presence of miscleavages. Here, we present a new application of a digestion method for native proteins using resin-bound, thermally stabilized proteases. The use of this enzymatic treatment eliminates miscleavages around the site of glycosylation, thereby allowing site-specific relative quantification of glycans on glycoproteins. A native, intact human mAb was digested using a thermally stable, resin-bound trypsin to produce glycopeptides from the Fc region using a single-step protocol. A 1 mg sample was treated with 60 µg trypsin for 3 h at 70°C. After digestion, acetonitrile was added, and the mixture was centrifuged to remove the resin before analysis. Liquid chromatography (LC)/MS with hydrophilic interaction chromatography was used to analyze the glycopeptides produced. All of the glycopeptides found resulted from a single peptide (EEQYNSTYR). The LC/MS analysis of the glycopeptides is compared with that of fluorescently labeled glycans. Quantitative analysis produced a correlation coefficient of 0.87 for the linear fit between the glycopeptide and released glycan methods.

Outcome prediction values of soluble human epidermal growth factor receptor-2 extracellular domain in metastatic breast cancer.

BACKGROUND: HER-2 overexpression is an independent predictor for poor prognosis of breast cancer patients. Recently, extracellular domain of HER-2 (ECD) was found detectable in the serum of breast cancer patients. In this prospective study, we wonder whether ECD levels predict the clinical outcome of metastatic breast cancer patients. METHODS: ECD were measured in 190 women with metastatic breast cancer. Chi-square test was performed to determine the relationship between ECD status and clinical outcomes. Kaplan-Meier curves were applied for survival analysis. RESULTS: Elevated ECD levels were significantly associated with short-term response to Herceptin treatment. The median PFS was significantly longer in ECD-Low patients. The patients who remained low ECD levels or achieved low ECD levels after treatments have significantly longer PFS than those whose levels remained high or converted from low to high. CONCLUSIONS: Overall, our results support the clinical utility of measuring serum HER2 ECD levels in patients with advanced breast cancer. Baseline and serial measurements of serum ECD levels are reliably predictive of clinical outcome of breast cancer patients.

Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

Novel blood-based, five-gene biomarker set for the detection of colorectal cancer.

PURPOSE: We applied a unique method to identify genes expressed in whole blood that can serve as biomarkers to detect colorectal cancer (CRC). EXPERIMENTAL DESIGN: Total RNA was isolated from 211 blood samples (110 non-CRC, 101 CRC). Microarray and quantitative real-time PCR were used for biomarker screening and validation, respectively. RESULTS: From a set of 31 RNA samples (16 CRC, 15 controls), we selected 37 genes from analyzed microarray data that differed significantly between CRC samples and controls (P < 0.05). We tested these genes with a second set of 115 samples (58 CRC, 57 controls) using quantitative real-time PCR, validating 17 genes as differentially expressed. Five of these genes were selected for logistic regression analysis, of which two were the most up-regulated (CDA and MGC20553) and three were the most down-regulated (BANK1, BCNP1, and MS4A1) in CRC patients. Logit (P) of the five-gene panel had an area under the curve of 0.88 (95% confidence interval, 0.81-0.94). At a cutoff of logit (P) >+0.5 as disease (high risk), <-0.5 as control (low risk), and in between as an intermediate zone, the five-gene biomarker combination yielded a sensitivity of 94% (47 of 50) and a specificity of 77% (33 of 43). The intermediate zone contained 22 samples. We validated the predictive power of these five genes with a novel third set of 92 samples, correctly identifying 88% (30 of 34) of CRC samples and 64% (27 of 42) of non-CRC samples. The intermediate zone contained 16 samples. CONCLUSION: Our results indicate that the five-gene biomarker panel can be used as a novel blood-based test for CRC.

Novel blood biomarkers of human urinary bladder cancer.

PURPOSE: Recent data indicate that cDNA microarray gene expression profile of blood cells can reflect disease states and thus have diagnostic value. We tested the hypothesis that blood cell gene expression can differentiate between bladder cancer and other genitourinary cancers as well as between bladder cancer and healthy controls. EXPERIMENTAL DESIGN: We used Affymetrix U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) to profile circulating blood total RNA from 35 patients diagnosed with one of three types of genitourinary cancer [bladder cancer (n = 16), testicular cancer (n = 10), and renal cell carcinoma (n = 9)] and compared their cDNA profiles with those of 10 healthy subjects. We then verified the expression levels of selected genes from the Affymetrix results in a larger number of bladder cancer patients (n = 40) and healthy controls (n = 27). RESULTS: Blood gene expression profiles distinguished bladder cancer patients from healthy controls and from testicular and renal cancer patients. Differential expression of a combined set of seven gene transcripts (insulin-like growth factor-binding protein 7, sorting nexin 16, chondroitin sulfate proteoglycan 6, and cathepsin D, chromodomain helicase DNA-binding protein 2, nell-like 2, and tumor necrosis factor receptor superfamily member 7) was able to discriminate bladder cancer from control samples with a sensitivity of 83% (95% confidence interval, 67-93%) and a specificity of 93% (95% confidence interval, 76-99%). CONCLUSION: We have shown that the gene expression profile of circulating blood cells can distinguish bladder cancer from other types of genitourinary cancer and healthy controls and can be used to identify novel blood markers for bladder cancer.

Comparative gene expression analysis of blood and brain provides concurrent validation of SELENBP1 up-regulation in schizophrenia.

Microarray techniques hold great promise for identifying risk factors for schizophrenia (SZ) but have not yet generated widely reproducible results due to methodological differences between studies and the high risk of type I inferential errors. Here we established a protocol for conservative analysis and interpretation of gene expression data from the dorsolateral prefrontal cortex of SZ patients using statistical and bioinformatic methods that limit false positives. We also compared brain gene expression profiles with those from peripheral blood cells of a separate sample of SZ patients to identify disease-associated genes that generalize across tissues and populations and further substantiate the use of gene expression profiling of blood for detecting valid SZ biomarkers. Implementing this systematic approach, we: (i) discovered 177 putative SZ risk genes in brain, 28 of which map to linked chromosomal loci; (ii) delineated six biological processes and 12 molecular functions that may be particularly disrupted in the illness; (iii) identified 123 putative SZ biomarkers in blood, 6 of which (BTG1, GSK3A, HLA-DRB1, HNRPA3, SELENBP1, and SFRS1) had corresponding differential expression in brain; (iv) verified the differential expression of the strongest candidate SZ biomarker (SELENBP1) in blood; and (v) demonstrated neuronal and glial expression of SELENBP1 protein in brain. The continued application of this approach in other brain regions and populations should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for SZ. The identification of valid peripheral biomarkers for SZ may ultimately facilitate early identification, intervention, and prevention efforts as well.