[Detection and analysis of moving reaction boundary-based electrophoresis distance using smartphone images].

Electrophoresis titration (ET) based on the moving reaction boundary (MRB) theory can detect the analyte contents in different samples by converting content signals into distance signals. However, this technique is only suitable for on-site qualitative testing, and accurate quantification relies on complex optical equipment and computers. Hence, applying this method to real-time point-of-care testing (POCT) is challenging. In this study, we developed a smartphone-based ET system based on a visual technique to achieve real-time quantitative detection. First, we developed a portable quantitative ET device that can connect to a smartphone; this device consisted of five components, namely, an ET chip, a power module, a microcontroller, a liquid crystal display screen, and a Bluetooth module. The device measured 10 cm×15 cm×2.5 cm, weighed 300 g, and was easy to hold. Thus, it is suitable for on-site testing with a run time of only 2-4 min. An assistant mobile software program was also developed to control the device and perform ET. The colored electrophoresis boundary can be captured using the smartphone camera, and quantitative detection results can be obtained in real time. Second, we proposed a quantitative algorithm based on ET channels. The software was used to recognize the boundary migration distance of three channels, a standard curve based on two given contents of the standards was established using the two-point method, and the content of the test sample was calculated. Human serum albumin (HSA) and uric acid (UA) were used as a model protein and biosample, respectively, to test the performance of the detection system. For HSA detection, different HSA solutions were mixed with a polyacrylamide gel (PAG) stock solution, phenolphthalein was added as an indicator, and sodium persulfate and tetramethyl ethylenediamine (TEMED) were used to promote polymerization to form a gel. For UA detection, agarose gel was filled into the ET channel, the UA sample, urate oxidase, and leucomalachite green were added into the anode cell and incubated for 20 min. ET was then performed. The fitting goodness (R2) values of HSA and UA were 0.9959 and 0.9935, respectively, with a linear range of 0.5-35.0 g/L and a log-linear range of 100-4000 µmol/L. The limits of detection for HSA and UA were 0.05 g/L and 50 µmol/L, respectively, and the corresponding relative standard deviations (RSDs) were not greater than 2.87% and 3.21%, respectively. These results demonstrate that the detection system has good accuracy and sensitivity. Clinical samples collected from healthy volunteers were used as target blood samples, and the developed system was used to measure serum total protein and UA levels. Serum samples from five volunteers were selected, standard curves of total serum protein and UA were established, and the test results were compared with hospital standard testing results. The relative errors for serum total protein and UA were less than 6.03% and 6.21%, respectively, and the corresponding RSDs were less than 3.72% and 5.84%, respectively. These findings verify the accuracy and reliability of the proposed detection system. The smartphone-based ET detection system introduced in this paper presents several advantages. First, it enables the portable real-time detection of total serum protein and UA. Second, compared with traditional ET strategies based on colored boundaries, it does not rely on optical detection equipment or computers to obtain quantitative detection results; as such, it can reduce the complexity of the operation and provide portability and real-time metrics. Third, the detection of two biomarkers, serum total protein and UA, is achieved on the same device, thereby improving the multitarget detection potential of the ET method. These advantages render the developed method a promising detection platform for clinical applications and real-time POCT.

[Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection].

Serum total protein refers to the sum of all proteins in the serum, and its content determination is relevant to human health monitoring and disease diagnosis. However, existing detection techniques present a number of limitations; for example, the Kjeldahl method suffers from the negative effects of interfering substances such as non-protein nitrogen (NPN). Although the electrophoresis titration (ET) method has solved interference problems to some extent, the current ET technique relies on optical detection methods, which increases the tediousness of the operation. This study addresses the challenge of accurate serum total protein detection by combining the traditional ET technique with capacitively coupled contactless conductivity detection (C4D). The research contributions of this work are multifold. First, it presents the first development of an ET-C4D detection system, which consists of six components: an ET power module, an ET chip, a C4D sensing module, a detection module, a data acquisition card, and software. The developed system can capture the conductivity of substances in the channel using the software developed by our laboratory during ET. The detection system can be used to quantify the total protein content in human serum without the addition of specific labeling reagents or using optical detection equipment, and its running time is approximately 300 s. Second, this research proposes the corresponding principle of the system. Under an electric field, ion migration results in different pH levels before and after the boundary, leading to a protein surface charge difference. The maintenance of the electrical neutrality of the substances in the detection channel is related to the protein surface charge; therefore, the ion concentration distribution of the substances in the detection channel changes as the protein surface charge varies. A plot of conductivity as a function of running time showed an "inverted clock shape", first falling and then rising. Owing to the addition of different types and concentrations of proteins, the microenvironment of the entire system changes, resulting in different changes in conductivity. Third, the performance of the detection system was tested using human serum albumin (HSA) standard protein, which was mixed with polyacrylamide gel (PAG) mother liquor, riboflavin, etc., and irradiated under ultraviolet light for 10 min to form a gel. The ET experiments were then carried out. The shape of the conductivity curve was consistent with the proposed principle, and the higher the HSA concentration, the lower the conductivity curve trough, followed by a lagged time of the trough. Quantitative analysis of the conductivity signals showed that the linear range was 0.25-3.00 g/L, with a linearity of up to 0.98. The limit of detection (LOD) was 0.01 g/L, the relative standard deviation (RSD) was 1.90%, and the relative error of the test values was <7.20%, indicating the good detection stability and sensitivity of the system. Clinical samples collected from healthy volunteers were used as target blood samples for serum total protein content measurement using our detection system. Blood samples from a volunteer were used to obtain a standard curve, and the serum samples of other four volunteers were selected for ET-C4D and biuret detection. The results showed that the relative errors between the two methods were within 4.43%, indicating the accuracy and reliability of the detection system. The advantages of the ET-C4D detection system proposed in this paper are as follows: (i) ET-C4D realizes the rapid detection of total serum protein content based on the ET technique; (ii) compared with the traditional protein ET technique, the ET-C4D method does not rely on specific labeling components or optical detection equipment, thereby reducing the complexity of the operation; and (iii) the output signal of ET-C4D can be used for quantitative analysis with excellent analytical performance and high accuracy. These merits highlight the potential of the developed system for clinical application and biochemical analysis.

Roles of acyl-CoA-binding proteins in plant reproduction.

Acyl-CoA-binding proteins (ACBPs) constitute a well-conserved family of proteins in eukaryotes that are important in stress responses and development. Past studies have shown that ACBPs are involved in maintaining, transporting and protecting acyl-CoA esters during lipid biosynthesis in plants, mammals, and yeast. ACBPs show differential expression and various binding affinities for acyl-CoA esters. Hence, ACBPs can play a crucial part in maintaining lipid homeostasis. This review summarizes the functions of ACBPs during the stages of reproduction in plants and other organisms. A comprehensive understanding on the roles of ACBPs during plant reproduction may lead to opportunities in crop improvement in agriculture.

Interactions between plant lipid-binding proteins and their ligands.

Lipids participate in diverse biological functions including signal transduction, cellular membrane biogenesis and carbon storage. Following de novo biosynthesis in the plastids, fatty acids (FAs) are transported as acyl-CoA esters to the endoplasmic reticulum where glycerol-3-phosphate undergoes a series of acyl-CoA-dependent acylation via the Kennedy pathway to form triacylglycerols for subsequent assembly into oils. Alternatively, newly synthesized FAs are incorporated into phosphatidylcholine (PC) by a PC:acyl-CoA exchange process defined as "acyl editing". Acyl-CoA-binding proteins (ACBPs) at various subcellular locations can function in lipid transfer by binding and transporting acyl-CoA esters and maintaining intracellular acyl-CoA pools. Widely distributed in the plant kingdom, ACBPs are found in all eukaryotes and some eubacteria. In both rice and Arabidopsis, six forms of ACBPs co-exist and are classified into four groups based on their functional domains. Their conserved four-helix structure facilitates interaction with acyl-CoA esters. ACBPs also interact with phospholipids as well as protein partners and function in seed oil regulation, development, pathogen defense and stress responses. Besides the ACBPs, other proteins such as the lipid transfer proteins (LTPs), annexins and lipid droplet-associated proteins are also important lipid-binding proteins. While annexins bind Ca2+ and phospholipids, LTPs transport lipid molecules including FAs, acyl-CoA esters and phospholipids.

Oxylipin signaling in salt-stressed soybean is modulated by ligand-dependent interaction of Class II acyl-CoA-binding proteins with lipoxygenase.

Plant lipoxygenases (LOXs) oxygenate linoleic and linolenic acids, creating hydroperoxy derivatives, and from these, jasmonates and other oxylipins are derived. Despite the importance of oxylipin signaling, its activation mechanism remains largely unknown. Here, we show that soybean ACYL-COA-BINDING PROTEIN3 (ACBP3) and ACBP4, two Class II acyl-CoA-binding proteins, suppressed activity of the vegetative LOX homolog VLXB by sequestering it at the endoplasmic reticulum. The ACBP4-VLXB interaction was facilitated by linoleoyl-CoA and linolenoyl-CoA, which competed with phosphatidic acid (PA) for ACBP4 binding. In salt-stressed roots, alternative splicing produced ACBP variants incapable of VLXB interaction. Overexpression of the variants enhanced LOX activity and salt tolerance in Arabidopsis and soybean hairy roots, whereas overexpressors of the native forms exhibited reciprocal phenotypes. Consistently, the differential alternative splicing pattern in two soybean genotypes coincided with their difference in salt-induced lipid peroxidation. Salt-treated soybean roots were enriched in C32:0-PA species that showed high affinity to Class II ACBPs. We conclude that PA signaling and alternative splicing suppress ligand-dependent interaction of Class II ACBPs with VLXB, thereby triggering lipid peroxidation during salt stress. Hence, our findings unveil a dual mechanism that initiates the onset of oxylipin signaling in the salinity response.

In silico Analysis of Acyl-CoA-Binding Protein Expression in Soybean.

Plant acyl-CoA-binding proteins (ACBPs) form a highly conserved protein family that binds to acyl-CoA esters as well as other lipid and protein interactors to function in developmental and stress responses. This protein family had been extensively studied in non-leguminous species such as Arabidopsis thaliana (thale cress), Oryza sativa (rice), and Brassica napus (oilseed rape). However, the characterization of soybean (Glycine max) ACBPs, designated GmACBPs, has remained unreported although this legume is a globally important crop cultivated for its high oil and protein content, and plays a significant role in the food and chemical industries. In this study, 11 members of the GmACBP family from four classes, comprising Class I (small), Class II (ankyrin repeats), Class III (large), and Class IV (kelch motif), were identified. For each class, more than one copy occurred and their domain architecture including the acyl-CoA-binding domain was compared with Arabidopsis and rice. The expression profile, tertiary structure and subcellular localization of each GmACBP were predicted, and the similarities and differences between GmACBPs and other plant ACBPs were deduced. A potential role for some Class III GmACBPs in nodulation, not previously encountered in non-leguminous ACBPs, has emerged. Interestingly, the sole member of Class III ACBP in each of non-leguminous Arabidopsis and rice had been previously identified in plant-pathogen interactions. As plant ACBPs are known to play important roles in development and responses to abiotic and biotic stresses, the in silico expression profiles on GmACBPs, gathered from data mining of RNA-sequencing and microarray analyses, will lay the foundation for future studies in their applications in biotechnology.

Investigations of Lipid Binding to Acyl-CoA-Binding Proteins (ACBP) Using Isothermal Titration Calorimetry (ITC).

Isothermal titration calorimetry (ITC) is a quantitative, biophysical method to investigate intermolecular binding between biomolecules by directly measuring the heat exchange in the binding reaction. The assay is carried out in solution when the molecules interact in vitro. This allows to determine values for binding affinity (Kd), binding stoichiometry (n), as well as changes in Gibbs free energy (ΔG), entropy (ΔS), and enthalpy (ΔH). This method also addresses the kinetics of enzymatic reactions for a substrate during conversion to a product. ITC has been used to study the interactions between proteins and ligands such as those of acyl-CoA-binding proteins (ACBPs) and acyl-CoA thioesters or ACBPs with protein partners. ITC has also been used in investigating interactions between antiserum and antigen, as well as those involving RNA and DNA and other macromolecules. We describe the methods used to isolate and purify a recombinant rice ACBP (OsACBP) for ITC. To study OsACBP binding to long-chain acyl-CoA thioesters, a microcalorimeter was used at 30 °C, and the ligand (acyl-CoA thioesters or a protein partner in the first cell), was mixed with the ACBP protein solution in a second cell, for more than 40 min comprising 20 injections. Subsequently, the binding parameters including the heat-release data were analyzed and various thermodynamic parameters were calculated.

Crystal structure of the rice acyl-CoA-binding protein OsACBP2 in complex with C18:3-CoA reveals a novel pattern of binding to acyl-CoA esters.

Acyl-CoA-binding proteins (ACBPs) are a family of proteins that bind acyl-CoA esters at a conserved acyl-CoA-binding domain. ACBPs maintain intracellular acyl-CoA pools to regulate lipid metabolism. Here, we report on the structure of rice OsACBP2 in complex with C18:3-CoA ester. The residues Y33, K34 and K56 of OsACBP2 play a crucial role in binding the CoA group, while residues N23, L27, K52 and Y55 in one molecule of OsACBP2 cooperate with L27, L28, A59 and A62 from another anchoring the fatty acyl group. Multiangle light scattering assays indicate that OsACBP2 binds C18:3-CoA as a monomer. The first complex structure of a plant ACBP binding with C18:3-CoA is therefore presented, providing a novel model for the interaction between an acyl-CoA ester and the acyl-CoA-binding domain(s).

Arabidopsis cytosolic acyl-CoA-binding proteins function in determining seed oil composition.

As plant seed oils provide animals with essential fatty acids (FAs), genes that regulate plant lipid metabolism have been used in genetic manipulation to improve dietary seed oil composition and benefit human health. Herein, the Arabidopsis thaliana cytosolic acyl-CoA-binding proteins (AtACBPs), AtACBP4, AtACBP5, and AtACBP6 were shown to play a role in determining seed oil content by analysis of atacbp (atacbp4, atacbp5, atacbp6, atacbp4atacbp5, atacbp4atacbp6, atacbp5atacbp6, and atacbp4atacbp5atacbp6) seed oil content in comparison with the Col-0 wild type (WT). Triacylglycerol (TAG) composition in electrospray ionization-mass spectrometer (ESI-MS) analysis on atacbp6 seed oil showed a reduction (-50%) of C58-TAGs in comparison with the WT. Investigations on fatty acid composition of atacbp mutants indicated that 18:2-FA accumulated in atacbp6 and 18:3-FA in atacbp4, both at the expense of 20:1-FA. As TAG composition can be modified by acyl editing through phosphatidylcholines (PC) and lysophosphatidylcholines (LPC), total PC and LPC content in atacbp6 mature seeds was determined and ESI-MS analysis revealed that LPC had increased (+300%) at the expense of PC. Among all the 14 tested PC species, all (34:1-, 34:2-, 34:3-, 34:4-, 34:5-, 34:6-, 36:2-, 36:3-, 36:5-, 36:6-, 38:2-, 38:3-, and 38:4-PCs) but 36:4-PC were lower in atacbp6 than the WT. In contrast, all LPC species (16:0-, 18:1-, 18:2-, 18:3-, and 20:1-LPC) examined were elevated in atacbp6. LPC abundance also increased in atacbp4atacbp5, but not atacbp4 and atacbp5. Interestingly, when LPC composition in atacbp4atacbp5 was compared with atacbp4 and atacbp5, significant differences were observed between atacbp4atacbp5 and each single mutant, implying that AtACBP4 and AtACBP5 play combinatory roles by affecting LPC (but not PC) biosynthesis. Furthermore, PC-related genes such as those encoding acyl-CoA:lysophphosphatidylcholine acyltransferase (LPCAT1) and phospholipase A2 alpha (PLA2α) were upregulated in atacbp6 developing seeds. A model on the role of AtACBP6 in modulating TAG through regulating LPCAT1 and PLA2α expression is proposed. Taken together, cytosolic AtACBPs appear to affect unsaturated TAG content and are good candidates for engineering oil crops to enhance seed oil composition.

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while ß-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

The first plant acyl-CoA-binding protein structures: the close homologues OsACBP1 and OsACBP2 from rice.

Acyl-CoA-binding proteins (ACBPs) are a family of proteins that facilitate the binding of long-chain acyl-CoA esters at a conserved acyl-CoA-binding domain. ACBPs act to form intracellular acyl-CoA pools, transport acyl-CoA esters and regulate lipid metabolism. In the model plant Arabidopsis thaliana, a family of six ACBPs has been demonstrated to function in stress and development. Six ACBPs (OsACBPs) have also been identified in Oryza sativa (rice), but they are not as well characterized as those in Arabidopsis thaliana. To understand the need in rice for the two 10 kDa ACBPs, namely OsACBP1 and OsACBP2, which share 79% sequence identity, their crystal structures were elucidated and their affinities toward acyl-CoA esters were compared using isothermal titration calorimetry. OsACBP2 was found to display a higher binding affinity for unsaturated acyl-CoA esters than OsACBP1. A difference between the two proteins is observed at helix 3 and is predicted to lead to different ligand-binding modes in terms of the shape of the binding pocket and the residues that are involved. OsACBP1 thus resembles bovine ACBP, while OsACBP2 is similar to human liver ACBP, in both structure and binding affinity. This is the first time that ACBP structures have been reported from plants, and suggests that OsACBP1 and OsACBP2 are not redundant in function despite their high sequence identity and general structural similarity.

How Work Organization Affects the Prevalence of WMSDs: A Case-control Study.

OBJECTIVE: In this study, we aimed at exploring the association between work-related musculoskeletal disorders (WMSDs) and work organization based on a case-control study. METHODS: A total of 1938 workers who claimed to suffer from WMSDs were selected from Beijing, Henan, Hubei, and the Guangdong province. The control group consisted of 2009 workers employed in similar industries without severe disease or musculoskeletal discomforts. We used a modified version of the questionnaire developed by the NMQ and the DMQ to investigate individual and work-related factors. RESULTS: A total of 13 variables (P<0.1) were selected by the chi-square test and finally, 7 variables entered into the equation, with 6 variables reaching statistical significance (P<0.05). The odds ratios (OR) of 'work changing with season' and 'sufficient rest time' did not reach 1 (0.749 and 0.441, respectively). In addition, 'sufficient rest time' seemed to be the stronger protective factor according to its higher standardized coefficient. And 'repetitive work every minute', 'constantly repetitive work' (every day), 'shortage of site personnel', and 'often switching shifts with others' seemed to be the risk factors. CONCLUSION: Work organization may have comprehensive effects on the occurrence of WMSDs. This pattern of associations suggests that further investigation into the mechanism of how work organization affects the prevalence of WMSDs is required.