Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation

Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the <i>Camelidae</i> species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP). Guided by predictive modeling, we show that customizing the NanoDeg rate of synthesis, rate of degradation, and mode of degradation enables quantitative and predictable control over the target’s levels. Integrating the GFP-specific NanoDeg within a genetic circuit based on stimulus-dependent GFP output results in enhanced dynamic range and resolution of the output signal. By providing predictable control over cellular proteins’ levels, the NanoDeg system could be readily used for a variety of systems-level analyses of cellular protein function

Dual-Functional Lipid Coating for the Nanopillar-Based Capture of Circulating Tumor Cells with High Purity and Efficiency

Clinical studies of circulating tumor cells (CTC) have stringent demands for high capture purity and high capture efficiency. Nanostructured surfaces have been shown to significantly increase the capture efficiency yet suffer from low capture purity. Here we introduce a dual-functional lipid coating on nanostructured surfaces. The lipid coating serves both as an effective passivation layer that helps prevent nonspecific cell adhesion and as a functionalized layer for antibody-based specific cell capture. In addition, the fluidity of lipid bilayers enables antibody clustering that enhances the cell–surface interaction for efficient cell capture. As a result, the lipid-coating method helps promote both the capture efficiency and capture purity of nanostructure-based CTC capture

Monitoring the Prevalence of <i>Leucocytozoon sabrazesi</i> in Southern China and Testing Tricyclic Compounds against Gametocytes

<div><p><i>Leucocytozoon</i> parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss on the poultry industry. Two major species of <i>Leucocytozoon</i> parasites have been reported in China, <i>L</i>. <i>sabrazesi</i> and <i>L</i>. <i>caulleryi</i>, although <i>L</i>. <i>sabrazesi</i> appears to be more widespread than <i>L</i>. <i>caulleryi</i> in southern China. The traditional method for detecting <i>Leucocytozoon</i> infection is microscopic examination of blood smears for the presence of mature gametocytes in circulation, which may miss infections with low parasitemia (gametocytemia) or immature gametocytes. Here we developed a PCR-based method to monitor <i>L</i>. <i>sabrazesi</i> infections at seven sites in four provinces of China after testing two PCR primer pairs based on parasite mitochondrial cytochrome b (<i>cytb</i>) and cytochrome c oxidase III (<i>coxIII</i>) genes. We compared the results of PCR detection with those of microscopic observation. As expected, the PCR assays were more sensitive than microscope examination in detecting <i>L</i>. <i>sabrazesi</i> infection and were able to detect parasite DNA after gametocytes disappeared in the blood stream. Using these methods, we investigated monthly dynamics of <i>L</i>. <i>sabrazesi</i> in chickens from a free-range farm in Xiamen, Fujian province of China, over one year. Our results showed that chickens were infected with <i>L</i>. <i>sabrazesi</i> year-round in southern China. Finally, we tested several compounds for potential treatment of <i>Leucocytozoon</i> infections, including primaquine, ketotifen, clomipramine hydrochloride, desipramine hydrochloride, sulfaquinoxaline, and pyrimethamine. Only primaquine had activity against <i>L</i>. <i>sabrazesi</i> gametocytes. Our results provide important information for controlling parasite transmission in southern China and disease management.</p></div

Weekly or biweekly infections of three chickens from a free-range farm in Xiamen, southern China, detected by blood smear or parasite-specific PCR.

<p>Weekly or biweekly infections of three chickens from a free-range farm in Xiamen, southern China, detected by blood smear or parasite-specific PCR.</p

Primer design and detection of diluted DNA samples from an infected chicken.

<p><b>A</b> and <b>B,</b> Aligned primer sequences based on genes encoding mitochondrial cytochrome b (<i>cytb</i>, <b>A</b>) and cytochrome oxidase subunit III (<i>coxIII</i>, <b>B</b>) from <i>L</i>. <i>sabrazesi</i> (NCBI accession No. AB299369.1), <i>Leucocytozoon caulleryi</i> (Accession No. AB302215.1), <i>Haemoproteus columbae</i> (NCBI accession No. FJ168562.1), <i>Plasmodium gallinaceum</i> (Accession No. AB250690.1), and chicken (<i>Gallus gallus domesticus</i>, Accession No. KM096864.1). <b>C</b>, Amplifications of diluted DNA from an infected chicken with known gametocytemia. DNA sample from infected chicken (#2HC with parasitemia of 0.02%) blood obtained from Haicang, Fujian province, was diluted in water at ratios of 1:10; 1:50; 1:200; 1:1,000; 1:5,000; 1:20,000; 1:100,000; 1:500,000; 1:2×10⁶; and 1:1×10⁷ and was amplified using Ls-coxIIIF2/R2 and Ls-cytbF1/R1 primers, respectively. PCR products (4 μl each) were separated on a 2% agarose gel. A DNA band could be easily detected at 1:1,000 dilution using Ls-coxIIIF2/R2, and a band at 1:5,000 could be seen using the Ls-cytbF1/R1 primers. “+” indicates un-diluted DNA control. The figure is representative of two replicates with the same results.</p

PCR detection of parasite DNAs extracted from chicken blood samples with or without positive identification of gametocytes.

<p><b>A</b>, PCR products amplified using the Ls-coxIIIF2/R2 and Ls-cytbF1/R1 primers, respectively. The numbers on top of the gels are gametocytemia per 1,000 red blood cells (RBCs) after counting 10,000 RBCs. DNAs were extracted from 20 μl infected blood, amplified using the Ls-coxIIIF2/R2 and Ls-cytbF1/R1 primers, respectively, and separated on a 2% agarose gel (4 μl PCR products loaded). DNA ladders of one hundred bp (100–500 bp) are on the left side of the gels. “+” indicates PCR positive; “?” positive with uncertainty; UD, undetectable gametocyte; The figure is representative of two replications of the same results. <b>B</b>, PCR products from microscopic negative samples amplified using the Ls-coxIIIF2/R2 and Ls-cytbF1/R1primers, respectively. <b>C</b>, Electropherograms from four microscopic negative samples by Ls-coxIIIF2/R2 primers showing double peaks in two samples and two nucleotide substitutions in one sample (0315#2).</p

Detection of <i>Leucocytozoon</i> gametocytes or DNA after drug treatments.

<p><b>A-C</b>, Curves of gametocytemia from two chickens after treatment (daily doses of 2 mg/kg for 14 days) with primaquine (<b>A</b>), ketotifen (<b>B</b>), or no-treatment controls (<b>C</b>). Each line in the graphs of A-C represents gametocytemia from an infected chicken. <b>D</b>, Agarose gels of PCR products from the Ls-coxIIIF2/R2 primers. #1 and #5 were treated with a dose of 2 mg/kg primaquine daily for 14 days; #3 and #4 were treated with a dose of 2 mg/kg ketotifen daily for 14 days; #6 and #7 were controls without treatment. All the chickens were from the Haicang (HC) farm. DNA sample preparation and PCR amplifications were as described in Materials and Methods.</p

Comparison of microscopic examination of blood smear and the Ls-coxIIIF2/R2 PCR method in detecting <i>Leucocytozoon sabrazesi</i> infection.

<p>Comparison of microscopic examination of blood smear and the Ls-coxIIIF2/R2 PCR method in detecting <i>Leucocytozoon sabrazesi</i> infection.</p

Monthly surveys of <i>Leucocytozoon</i> infections using microscopy of blood smear and the Ls-coxIIIF2/R2 PCR assay.

<p>Monthly surveys of <i>Leucocytozoon</i> infections using microscopy of blood smear and the Ls-coxIIIF2/R2 PCR assay.</p

Static Electricity Powered Copper Oxide Nanowire Microbicidal Electroporation for Water Disinfection

Safe water scarcity occurs mostly in developing regions that also suffer from energy shortages and infrastructure deficiencies. Low-cost and energy-efficient water disinfection methods have the potential to make great impacts on people in these regions. At the present time, most water disinfection methods being promoted to households in developing countries are aqueous chemical-reaction-based or filtration-based. Incorporating nanomaterials into these existing disinfection methods could improve the performance; however, the high cost of material synthesis and recovery as well as fouling and slow treatment speed is still limiting their application. Here, we demonstrate a novel flow device that enables fast water disinfection using one-dimensional copper oxide nanowire (CuONW) assisted electroporation powered by static electricity. Electroporation relies on a strong electric field to break down microorganism membranes and only consumes a very small amount of energy. Static electricity as the power source can be generated by an individual person’s motion in a facile and low-cost manner, which ensures its application anywhere in the world. The CuONWs used were synthesized through a scalable one-step air oxidation of low-cost copper mesh. With a single filtration, we achieved complete disinfection of bacteria and viruses in both raw tap and lake water with a high flow rate of 3000 L/(h·m²), equivalent to only 1 s of contact time. Copper leaching from the nanowire mesh was minimal