Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.

A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.     

Rapid Grading of Fundus Photographs for Diabetic Retinopathy Using Crowdsourcing

Background

Screening for diabetic retinopathy is both effective and cost-effective, but rates of screening compliance remain suboptimal. As screening improves, new methods to deal with screening data may help reduce the human resource needs. Crowdsourcing has been used in many contexts to harness distributed human intelligence for the completion of small tasks including image categorization.

Objective

Our goal was to develop and validate a novel method for fundus photograph grading.

Methods

An interface for fundus photo classification was developed for the Amazon Mechanical Turk crowdsourcing platform. We posted 19 expert-graded images for grading by Turkers, with 10 repetitions per photo for an initial proof-of-concept (Phase I). Turkers were paid US $0.10 per image. In Phase II, one prototypical image from each of the four grading categories received 500 unique Turker interpretations. Fifty draws of 1-50 Turkers were then used to estimate the variance in accuracy derived from randomly drawn samples of increasing crowd size to determine the minimum number of Turkers needed to produce valid results. In Phase III, the interface was modified to attempt to improve Turker grading.

Results

Across 230 grading instances in the normal versus abnormal arm of Phase I, 187 images (81.3%) were correctly classified by Turkers. Average time to grade each image was 25 seconds, including time to review training images. With the addition of grading categories, time to grade each image increased and percentage of images graded correctly decreased. In Phase II, area under the curve (AUC) of the receiver-operator characteristic (ROC) indicated that sensitivity and specificity were maximized after 7 graders for ratings of normal versus abnormal (AUC=0.98) but was significantly reduced (AUC=0.63) when Turkers were asked to specify the level of severity. With improvements to the interface in Phase III, correctly classified images by the mean Turker grade in four-category grading increased to a maximum of 52.6% (10/19 images) from 26.3% (5/19 images). Throughout all trials, 100% sensitivity for normal versus abnormal was maintained.

Conclusions

With minimal training, the Amazon Mechanical Turk workforce can rapidly and correctly categorize fundus photos of diabetic patients as normal or abnormal, though further refinement of the methodology is needed to improve Turker ratings of the degree of retinopathy. Images were interpreted for a total cost of US $1.10 per eye. Crowdsourcing may offer a novel and inexpensive means to reduce the skilled grader burden and increase screening for diabetic retinopathy.     

Postoperative complications associated with 25-gauge pars plana vitrectomy.

BACKGROUND AND OBJECTIVE: To report postoperative complications in eyes undergoing 25-gauge pars plana vitrectomy (PPV). PATIENTS AND METHODS: Seventy consecutive eyes that underwent 25-gauge PPV for various indications, including epiretinal membrane, non-clearing vitreous hemorrhage, and idiopathic macular hole, and had a minimum follow-up of 12 weeks were reviewed retrospectively. Main outcome measures included best-corrected Snellen visual acuity, intraocular pressure (IOP), intraoperative complications, and postoperative complications. RESULTS: The mean visual acuity improved from 20/368 preoperatively to 20/105 postoperatively (P < .00005). Intraoperative complications included retinal tears in 2 eyes (2.9%). Postoperative complications included cataract progression in 17 eyes (42.5%), cystoid macular edema exacerbation in 5 eyes (7.1%), and retinal detachment in 1 eye (1.4%). Postoperative day 1 IOP was statistically lower than preoperative IOP in fluid-filled eyes (P = .031) but not in eyes filled with intravitreal air (P = .30) or gas (P = .52). Sclerotomy sutures were required intraoperatively in 5 eyes (7.1%) and postoperative day 1 hypotony was noted in 4 eyes (5.7%). All of these complications were noted in fluid-filled eyes except for one case of postoperative day 1 hypotony with gas tamponade. CONCLUSIONS: Intraoperative and postoperative complications were rare in this series of 25-gauge PPV. Postoperative cataract progression and hypotony were the most common complications. Fluid-filled eyes appear to have a higher risk of wound leakage and postoperative hypotony after 25-gauge PPV than eyes with air or gas tamponade.     

Comparison of some biological effects of epidermal growth factor and commercial serum albumin on the induction of alpha-lactalbumin in rat and rabbit mammary explants

Commercial preparations of serum albumin from six species can markedly enhance the prolactin-independent induction of alpha-lactalbumin in mammary explants from pregnant rats, and evoke such induction in the tissue from virgin rats. These effects are similar to those of epidermal growth factor (EGF) reported previously. The stimulatory activity of bovine serum albumin (BSA) resides in a putative impurity in the albumin. Charcoal extraction and gel filtration of the BSA results in complete loss of activity. Of the five milk protein mRNAs studied, only alpha-lactalbumin mRNA is induced by insulin, glucocorticoid and serum albumin in the absence of prolactin. Despite these similarities, the biological effects of serum albumin and EGF on mammary tissue diverge in some respects. They appear to operate by different mechanisms since their effects on the rat system are additive. Furthermore, while both inhibit prolactin-mediated induction of alpha-lactalbumin in rabbit mammary explants, cortisol converts EGF into a stimulatory agent, but merely blocks the inhibitory effect of serum albumin. The results emphasize that commercial serum albumin is not to be regarded simply as an inert protein additive to culture media.     

Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.