IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ～8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc.     

Predictors of Frequent Emergency Room Visits among a Homeless Population

Background

Homelessness, HIV, and substance use are interwoven problems. Furthermore, homeless individuals are frequent users of emergency services. The main purpose of this study was to identify risk factors for frequent emergency room (ER) visits and to examine the effects of housing status and HIV serostatus on ER utilization. The second purpose was to identify risk factors for frequent ER visits in patients with a history of illicit drug use.

Methods

A retrospective analysis was performed on 412 patients enrolled in a Boston-based health care for the homeless program (HCH). This study population was selected as a 2:1 HIV seronegative versus HIV seropositive match based on age, sex, and housing status. A subgroup analysis was performed on 287 patients with history of illicit drug use. Chart data were analyzed to compare demographics, health characteristics, and health service utilization. Results were stratified by housing status. Logistic models using generalized estimating equations were used to predict frequent ER visits.

Results

In homeless patients, hepatitis C was the only predictor of frequent ER visits (OR 4.49, p<0.01). HIV seropositivity was not predictive of frequent ER visits. In patients with history of illicit drug use, mental health (OR 2.53, 95% CI 1.07–5.95) and hepatitis C (OR 2.85, 95% CI 1.37–5.93) were predictors of frequent ER use. HIV seropositivity did not predict ER use (OR 0.45, 95% CI 0.21 – 0.97).

Conclusions

In a HCH population, hepatitis C predicted frequent ER visits in homeless patients. HIV seropositivity did not predict frequent ER visits, likely because HIV seropositive HCH patients are engaged in care. In patients with history of illicit drug use, hepatitis C and mental health disorders predicted frequent ER visits. Supportive housing for patients with mental health disorders and hepatitis C may help prevent unnecessary ER visits in this population.     

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass, notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tal1 and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tal1, lmo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development.