Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay

The first step of enzyme linked immunosorbent assay (ELISA), namely, adsorption of antigen or antibody to the plastic microtiter well plate, was studied as a function of insolubility of IgG in water. Immobilization efficiency was assessed in terms of number of wells coated per milliliter of primary antiserum. We have compared different coating/immobilization protocols, i.e., direct and indirect immobilization of primary antibody to the plastic microtiter well plate using carbonate buffer and phosphate buffer with glutaraldehyde. We have observed efficient coating when the immobilization of primary antibody through an immunobridge technique was performed, where water was used as a coating medium. It gave a higher number of wells coated per milliliter of anti-serum (primary or secondary) than other compared coating protocols and it allowed the use of serum (non-immune) and anti-serum (primary and secondary antibody) dilutions, avoiding the need for gamma-globulin purification from normal and immunized serum.     

N-myristoyltransferase: A potential novel diagnostic marker for colon cancer

Background  

Colon cancer is the second leading cause of cancer deaths in the western world. If detected early, colorectal cancer is one of the most treatable forms of cancer. Unfortunately, very few people are screened. N-myristoyltransferase (NMT) catalyzes myristoylation of various proteins including oncoproteins. We have demonstrated earlier the alteration of NMT activity during the progression of colorectal cancer and established NMT as a putative therapeutic target for cancer.     

Anatomical basis of antropyloric transposition for fecal incontinence in humans: the infrapyloric approach

Purpose

Antropylorus transposition in the perineum for end-stage anal incontinence has shown to be feasible in humans. Vascular anatomy of the antro-pyloro-duodenal area is critical in preventing complications and increasing pyloric graft survival. This study was undertaken to examine the vascular anatomy of antro-pyloro-duodenal area in an attempt to safeguard the graft blood supply and improve its survival.

Methods

After obtaining preoperative CT angiography to delineate the infrapyloric artery (IP a.), bench dissection of resected pancreaticoduodenectomy specimens was performed in 12 patients. Ex vivo angiography of these specimens were also performed. Subsequent to the information obtained from these dissections, the method of antropylorus mobilization during transposition was modified in terms of the site of division of the right gastroepiploic a. (Rt GEA). Perioperative outcomes (graft related complications, fecal incontinence scores, Doppler flow studies, and manometry studies of the graft) were compared between the two groups.

Results

IP a. originated only from the Rt GEA in 8 cases (66 %) and from both the gastroduodenal a. and the Rt GEA in the rest. However, its origin solely from the gastroduodenal a. was not observed. The pyloric graft survival, pyloric valve pressures and Doppler flow velocities were significantly (p < 0.05) better when the infrapyloric a. was preserved following this refinement. However, no immediate significant difference in incontinence scores was observed.

Conclusions

Careful preservation of the pyloric valve vascularity by preserving the IP a. by dividing the Rt GEA at its origin increases vascularity, survival and contractility of the pyloric graft in perineum.     

Challenges in optimizing sample preparation and LC‐MS/MS conditions for the analysis of carglumic acid,an N‐acetyl glutamate derivative in human plasma

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed‐phase solid‐phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic‐lipophilic balance, and mixed‐mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed‐mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid‐13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) ? 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00‐6000 ng/mL (r² ≥ 0.9987). Matrix effect was evaluated in terms of IS‐normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra‐ and inter‐batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject. Copyright © 2015 John Wiley & Sons, Ltd.     

Financial impact of ivabradine on reducing heart failure penalties under the Hospital Readmission Reduction Program

Objective: The introduction of the Hospital Readmission Reduction Program (HRRP) has led to renewed interest in developing strategies to reduce 30?day readmissions among patients with heart failure (HF). In this study, a model was developed to investigate whether the addition of ivabradine to a standard-of-care (SoC) treatment regimen for patients with HF would reduce HRRP penalties incurred by a hypothetical hospital with excess 30?day readmissions.

Research design: A model using a Monte Carlo simulation framework was developed. Model inputs included national hospital characteristics, hospital-specific characteristics, and the ivabradine treatment effect as quantified by a post hoc analysis of the Systolic Heart failure treatment with the I_f inhibitor ivabradine Trial (SHIFT).

Results: The model computed an 83% reduction in HF readmission penalty payments in a hypothetical hospital with a readmission rate of 22.95% (excess readmission ratio = 1.056 over the national average readmission rate of 21.73%), translating into net savings of $44,016. A sensitivity analysis indicated that the readmission penalty is affected by the specific characteristics of the hospital, including the readmission rate, size of the ivabradine-eligible population, and ivabradine utilization.

Conclusions: The results of this study indicate that the addition of ivabradine to an SoC treatment regimen for patients with HF may lead to a reduction in the penalties incurred by hospitals under the HRRP. This highlights the role ivabradine can play as part of a wider effort to optimize the care of patients with HF.     

Overexpression of Akt/PKB modulates N‐myristoyltransferase activity in cancer cells

N‐myristoyltransferase (NMT) catalyses the myristoylation reaction. Since NMT activity is elevated in various cancers and activated Akt/PKB leads to cell survival, we were interested in studying if activation of Akt/PKB has any effect on NMT. Overexpression of constitutively active Akt/PKB in HepG2 cells (HepG2‐CA‐Akt/PKB) led to an approximately 50% reduction of NMT compared with parental HepG2 cells. Reduced NMT activity in HepG2‐CA‐Akt/PKB was found to be due to the NMT1 phosphorylation. We determined NMT activity in various human breast cancer cell lines with differing metastatic potentials and pseudo‐normal breast cells (HBL‐100). Tumourigenic or metastatic breast cancer cell lines such as MDA‐MB‐231, MDA‐MB‐435, and Hs 578T displayed reduced NMT activity. Western blot analysis revealed that NMT1 is phosphorylated in these breast cancer cells. Furthermore, patients' breast cancer tissue array revealed strong positivity and high intensity for NMT in malignant breast tissues compared with normal breast cells. A gradation in the NMT staining was observed for grade I, II, and III infiltrating ductal carcinoma breast tissues. These studies demonstrate that overexpression of Akt/PKB results in NMT1 phosphorylation and that NMT1 is phosphorylated in breast cancer cells. Immunohistochemical analysis suggests that NMT may prove to be an added diagnostic biomarker for breast cancer. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Suppression of Puerperal Lactation Using Jasmine Flowers (Jasminum Sambac)

The efficacy of jasmine flowers (Jasminum Sambac) applied to the breasts to suppress puerperal lactation was compared that of Bromocriptine. Effectiveness of both regimens was monitored by serum prolactin levels, clinical evaluation of the degree of breast engorgement and milk production and the analgesic intake. While both bromocriptine and jasmine flowers brought about a significant reduction in serum prolactin, the decrease was significantly greater with bromocriptine. However, clinical parameters such as breast engorgement, milk production and analgesic intake showed the 2 modes of therapy to be equally effective. The failure rates of the 2 regimens to suppress lactation were similar; however, rebound lactation occurred in a small proportion of women treated with bromocriptine. Jasmine flowers seem to be an effective and inexpensive method of suppressing puerperal lactation and can be used as an alternative in situations where cost and nonavailability restrict the use of bromocriptine.