Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 µL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

Postoperative fatigue negatively impacts the daily lives of patients recovering from hysterectomy.

OBJECTIVE: To assess, from the patient's perspective, the prevalence and impact of postoperative fatigue after hysterectomy and to increase understanding of physician-patient communications before and after surgery regarding recovery and diminished postoperative energy level. METHODS: We completed a telephone survey of 300 women aged 25-50 who had undergone a hysterectomy or myomectomy within the past 2 years. The patients were recruited randomly from two national, random samples of households: 1) women aged 25-50, and 2) women taking hormone replacement therapy in the target age range. Patients were asked a series of questions about their postoperative fatigue after surgery. RESULTS: Overall, 74% of patients experienced moderate-to-severe fatigue within the first few weeks after surgery. Fatigue occurred more frequently and persisted twice as long as pain, the next most frequent symptom, which was experienced by 63% of patients overall. Fatigue was the symptom that most interfered with daily activities (37%) and also contributed to feelings of frustration (52%), to depression (37%), and to difficulty in concentrating (42%). Patients employed at the time of surgery missed an average of 5.8 weeks of work; 69% of those surveyed required 2 or more weeks of caregiver assistance. Postoperative fatigue was discussed by 68% of patients' physicians before surgery, and 57% of patients discussed postoperative fatigue with their physicians after surgery. Oral iron therapy and dietary supplements were the most frequently recommended treatments; 52% of patients were not offered any treatments or recommendations to alleviate their fatigue. CONCLUSIONS: Fatigue is a highly prevalent posthysterectomy and myomectomy symptom and has substantial negative physical, psychosocial, and economic effects on patients during recovery.