A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis

Bostonia: The Boston University Alumni Magazine. Volume 9

Founded in 1900, Bostonia magazine is Boston University's main alumni publication, which covers alumni and student life, as well as university activities, events, and programs

Physician Accuracy in Interpreting Potential ST‐Segment Elevation Myocardial Infarction Electrocardiograms

Background: With adoption of telemedicine, physicians are increasingly asked to diagnose ST‐segment elevation myocardial infarctions (STEMIs) based on electrocardiograms (ECGs) with minimal associated clinical information. We sought to determine physicians' diagnostic agreement and accuracy when interpreting potential STEMI ECGs. Methods and Results: A cross‐sectional survey was performed consisting of 36 deidentified ECGs that had previously resulted in putative STEMI diagnoses. Emergency physicians, cardiologists, and interventional cardiologists participated in the survey. For each ECG, physicians were asked, “based on the ECG above, is there a blocked coronary artery present causing a STEMI?” The reference standard for ascertaining the STEMI diagnosis was subsequent emergent coronary arteriography. Responses were analyzed with generalized estimating equations to account for nested and repeated measures. One hundred twenty‐four physicians interpreted a total of 4392 ECGs. Among all physicians, interreader agreement (kappa) for ECG interpretation was 0.33, reflecting poor agreement. The sensitivity to identify “true” STEMIs was 65% (95% CI: 63 to 67) and the specificity was 79% (95% CI: 77 to 81). There was a 6% increase in the odds of accurate ECG interpretation for every 5 years of experience since medical school graduation (OR 1.06, 95% CI: 1.02 to 1.10, P=0.01). After adjusting for experience, there was no significant difference in the odds of accurate interpretation by specialty—Emergency Medicine (reference), General Cardiology (AOR 0.97, 95% CI: 0.79 to 1.2, P=0.80), or Interventional Cardiology physicians (AOR 1.24, 95% CI: 0.93 to 1.7, P=0.15). Conclusions: There is significant physician disagreement in interpreting ECGs with features concerning for STEMI. Such ECGs lack the necessary sensitivity and specificity to act as a suitable “stand‐alone” diagnostic test

Urinary Epidermal Growth Factor as a Marker of Disease Progression in Children With Nephrotic Syndrome

© 2019 International Society of Nephrology Introduction: Childhood-onset nephrotic syndrome has a variable clinical course. Improved predictive markers of long-term outcomes in children with nephrotic syndrome are needed. This study tests the association between baseline urinary epidermal growth factor (uEGF) excretion and longitudinal kidney function in children with nephrotic syndrome. Methods: The study evaluated 191 participants younger than 18 years enrolled in the Nephrotic Syndrome Study Network, including 118 with their first clinically indicated kidney biopsy (68 minimal change disease; 50 focal seNorthwell Healthntal glomerulosclerosis) and 73 with incident nephrotic syndrome without a biopsy. uEGF was measured at baseline for all participants and normalized by the urine creatinine (Cr) concentration. Renal epidermal growth factor (EGF) mRNA was measured in the tubular compartment microdissected from kidney biopsy cores from a subset of patients. Linear mixed models were used to test if baseline uEGF/Cr and EGF mRNA expression were associated with change in estimated glomerular filtration rate (eGFR) over time. Results: Higher uEGF/Cr at baseline was associated with slower eGFR decline during follow-up (median follow-up = 30 months). Halving of uEGF/Cr was associated with a decrease in eGFR slope of 2.0 ml/min per 1.73 m2 per year (P \u3c 0.001) adjusted for age, race, diagnosis, baseline eGFR and proteinuria, and APOL1 genotype. In the biopsied subgroup, uEGF/Cr was correlated with EGF mRNA expression (r = 0.74; P \u3c 0.001), but uEGF/Cr was retained over mRNA expression as the stronger predictor of eGFR slope after multivariable adjustment (decrease in eGFR slope of 1.7 ml/min per 1.73 m2 per year per log2 decrease in uEGF/Cr; P \u3c 0.001). Conclusion: uEGF/Cr may be a useful noninvasive biomarker that can assist in predicting the long-term course of kidney function in children with incident nephrotic syndrome

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites

URBAN TERRAIN CLIMATOLOGY AND REMOTE SENSING *

. Urban areas have been conceived of as monolithic heat islands because traditional ground observation techniques do not lend themselves to more specific analyses. Observations of urban energy-exchange obtained from calibrated electro-optical scanners combined with energy budget simulation techniques provide tools to relate the urban land use mosaic to the heat island phenomenon. Maps of surface energy-related phenomena were made from airborne scanner outputs for selected flightpaths across the city of Baltimore, Maryland. Conditions for the flight time were simulated according to the various types of land use using an energy budget simulation model which lends itself to extrapolation of simulated grid-point conditions into a map form. Maps made by simulation compare sufficiently well with those made by aerial observation to encourage further refinement of the simulation approach.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72392/1/j.1467-8306.1976.tb01110.x.pd

3-D struktura serumske paraoksonaze 1 objašnjava njezinu aktivnost, stabilnost, topljivost i kristalizaciju

Serum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the “good cholesterol”), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1’s active site and suggests possible mechanisms for its catalytic activity on certain substrates.Serumske paraoksonaze (PONs) imaju široki raspon fiziološki važnih hidrolitičkih aktivnosti uključujući metabolizam lijekova i detoksikaciju nervnih plinova. PON1 i PON3 smještene su na lipoproteinima visoke gustoće (engl. high-density lipoprotein; HDL - “dobri kolesterol”) i uključene su u ublažavanje ateroskleroze. Članovi skupine PON identificirani su ne samo u sisavaca i drugih kralježnjaka već i kod beskralješnjaka. Prije smo opisali prvu kristalnu strukturu člana PON skupine, direktno razrađenu varijantu PON1 pri rezoluciji 2,2 Å. PON1 je beta-propeler sa šest lopatica s jedinstvenim poklopcem aktivnog mjesta, koji je tako|er uključen u vezanje na HDL. 3-D struktura, gledana zajedno s direktnim razvojnim istraživanjima, omogućila je analizu mutacija koje povećavaju stabilnost, topljivost i kristalizaciju te PON1 varijante. Struktura dopušta detaljan opis aktivnog mjesta PON1 i sugerira moguće mehanizme za njezinu katalitičku aktivnost prema odre|enim supstratima

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening