miR-2940-1 is involved in the circadian regulation of oviposition in Aedes albopictus.

The vast majority of all global species have circadian rhythm cycles that allow them to adapt to natural environments. These regular rhythms are regulated by core clock genes and recent studies have also implicated roles for microRNAs in this regulation. Oviposition is an important circadian behavior in the reproductive cycle of insect vectors of diseases, and little is known about the rhythm or its regulation in mosquitoes. Aedes albopictus is a diurnal mosquito that transmits arboviruses and is the major cause of outbreaks of dengue fever in China. We analyzed the oviposition rhythm patterns of A. albopictus under different light/dark conditions and show that the mosquitoes have an oviposition peak between zeitgeber time 9 (ZT 9) and ZT 12. Furthermore, the antagomir-mediated knockdown of expression of the microRNA miR-2940-1 affected the oviposition rhythm of A. albopictus. These data support the conclusion that miR-2940-1 is involved in the regulation of oviposition rhythm in A. albopictus and provide a foundation for using oviposition rhythms as a new target for vector mosquito control.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity.

BACKGROUND: Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. RESULTS: A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. CONCLUSIONS: This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

A DBHS family member regulates male determination in the filariasis vector Armigeres subalbatus.

The initial signals governing sex determination vary widely among insects. Here we show that Armigeres subalbatus M factor (AsuMf), a male-specific duplication of an autosomal gene of the Drosophila behaviour/human splicing (DBHS) gene family, is the potential primary signal for sex determination in the human filariasis vector mosquito, Ar. subalbatus. Our results show that AsuMf satisfies two fundamental requirements of an M factor: male-specific expression and early embryonic expression. Ablations of AsuMf result in a shift from male- to female-specific splicing of doublesex and fruitless, leading to feminization of males both in morphology and general transcription profile. These data support the conclusion that AsuMf is essential for male development in Ar. subalbatus and reveal a male-determining factor that is derived from duplication and subsequent neofunctionalization of a member of the conserved DBHS family.

Differential proteomic patterns of plasma extracellular vesicles show potential to discriminate ß-thalassemia subtypes.

The observed specificity of ß-thalassemia-subtype phenotypes makes new diagnostic strategies that complement current screening methods necessary to determine each subtype and facilitate therapeutic regimens for different patients. Here, we performed quantitative proteomics of plasma-derived extracellular vesicles (EVs) of ß-thalassemia major (TM) patients, ß-thalassemia intermedia (TI) patients, and healthy controls to explore subgroup characteristics and potential biomarkers. Plasma quantitative proteomics among the same cohorts were analyzed in parallel to compare the biomarker potential of both specimens. EV proteomics showed significantly more abnormalities in immunity and lipid metabolism in TI and TM, respectively. The differential proteomic patterns of EVs were consistent with but more striking than those of plasma. Notably, we also found EV proteins to have a superior performance for discriminating ß-thalassemia subtypes. These findings allowed us to propose a diagnostic model consisting of five proteins in EVs with subtyping potential, demonstrating the ability of plasma-derived EVs for the diagnosis of ß-thalassemia patients.

Clock genes regulate mating activity rhythms in the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: Endogenous circadian rhythms result from genetically-encoded molecular clocks, whose components and downstream output factors cooperate to generate cyclic changes in activity. Mating is an important activity of mosquitoes, however, the key aspects of mating rhythm patterns and their regulatory mechanisms in two vector mosquito species, Aedes albopictus and Culex quinquefasciatus, remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: We determined and compared the diel mating activity rhythms of these two mosquito species and discovered that Ae. albopictus had mating peaks in the light/dark transition periods (ZT0-3 and ZT9-12), while Cx. quinquefasciatus only had a mating peak at ZT12-15. Knockouts of the clock (clk) orthologous genes (Aalclk and Cxqclk) resulted in phase delay or phase reversal of the mating peaks in Ae. albopictus and Cx. quinquefasciatus, respectively. In addition, the temporal expression pattern of the desaturase orthologous genes, desat1, in both mosquito species was also different in respective wild-type strains and showed phase changes similar to the mating rhythms in clk mutant strains. Inhibition of desat1 expression resulted in decreased mating activity in male mosquitoes of both species but not females. In addition, desat1 regulated cuticular hydrocarbons' synthesis in both species. Silencing desat1 in male Ae. albopictus resulted in decreases of nonadecane and tricosane, which promoted mating, with concomitant increases of heptacosane, which inhibited mating. Silencing desat1 in male Cx. quinquefasciatus also resulted in decreases of tricosane, which promoted mating. CONCLUSIONS/SIGNIFICANCE: These results suggest that Aalclk and Cxqclk have significant roles in the mating activity rhythms in both Ae. albopictus and Cx. quinquefasciatus by regulating the temporal expression of the desat1 gene under LD cycles, which affects sex pheromone synthesis and mating. This work provides insights into the molecular regulatory mechanism of distinct mating rhythm of Ae. albopictus and Cx. quinquefasciatus and may provide a basis for the control of these two important vector mosquitoes.

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae.

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.

Armigeres subalbatus is a potential vector for Zika virus but not dengue virus.

BACKGROUND: Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses primarily transmitted by Aedes mosquitoes. Armigeres subalbatus is an emerging and widely distributed mosquito, and ZIKV has been detected and isolated from it. However, it is not clear whether Ar. subalbatus could be a vector for ZIKV and DENV or not. In this study, we investigated the infection and transmission of Ar. subalbatus to ZIKV and DENV. METHODS: A line of Ar. subalbatus was isolated from Guangdong, China, and further identified by the mitochondrial cytochrome oxidase subunit 1 (COI) gene. The adults of Ar. subalbatus were fed with blood meal containing ZIKV or DENV-2. At 4, 7, 10, 14, and 21 days post-inoculation (dpi), the infections of ZIKV or DENV-2 in the midguts, ovaries and salivary glands were detected and quantified by RT-PCR and RT-qPCR. To assess the transmissibility, suckling mice were exposed to bites of ZIKV-infected mosquitoes, and ZIKV was detected in brain tissue by RT-qPCR and plaque assays. Furthermore, the larvae of Ar. subalbatus were reared in artificial urine containing ZIKV or DENV-2. The infection rates and viral titers of larvae and adults were analyzed by RT-PCR and RT-qPCR, and the viral distribution in larval tissues was observed by immunohistochemistry. Chi-square test and one-way ANOVA analysis were used for assessing the infection rate and viral titer in varied tissues and different time points, respectively. RESULTS: Following oral inoculation, ZIKV but not DENV-2 could be detected in Ar. subalbatus midguts at 4 dpi, ovaries at 7 dpi and salivary glands at 10 dpi. The highest infection rate (IR) of ZIKV was 27.8% in midgut at 7 dpi, 9.7% in ovary and 5.6% in salivary gland at 21 dpi. Eight days after being bitten by ZIKV-positive mosquitoes, ZIKV was detected in three brain tissues out of four suckling mice exposed to bites. ZIKV could be detected in the larvae reared in artificial urine contained ZIKV at a high concentration of 105 pfu/ml and various tissues of adults with a low infection rate (0.70-1.35%). ZIKV could be observed in anal papillae and midgut of larvae at 4 dpi under laboratory conditions. CONCLUSIONS: ZIKV but not DENV-2 can infect Ar. subalbatus by blood meal and artificial urine, and the infected mosquitoes can transmit ZIKV to suckling mice by bite. From these findings, we can conclude that the Ar. subalbatus isolated from Guangdong province, China, is a potential vector for ZIKV and should therefore be considered in vector control programs to prevent and control of Zika virus disease.

The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males.

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae. albopictus Nix (AalNix) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4, is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4-♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae. albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion.

Interspecific mating bias may drive Aedes albopictus displacement of Aedes aegypti during its range expansion.

Aedes albopictus is the most invasive mosquito in the world and often displaces Ae. aegypti in regions where their populations overlap. Interspecific mating has been proposed as a possible cause for this displacement, but whether this applies across the range of their sympatry remains unclear. Aedes albopictus and Ae. aegypti collected from allopatric and sympatric areas in China were allowed to interact in cage experiments with different crosses and sex-choices. The results confirm that asymmetric interspecific mating occurs in these populations with matings between allopatric Ae. albopictus males and Ae. aegypti females being significantly higher (55.2%) than those between Ae. aegypti males and Ae. albopictus females (27.0%), and sympatric mosquitoes showed a similar but lower frequency bias, 25.7% versus 6.2%, respectively. The cross-mated females can mate second time (remate) with the respective conspecific males and the 66.7% remating success of female Ae. albopictus was significantly higher than the 9.3% of Ae. aegypti females. Furthermore, 17.8% of the matings of Ae. albopictus males exposed to mixed pools of Ae. albopictus and Ae. aegypti females and 9.3% of the matings of Ae. aegypti males with mixed Ae. aegypti and Ae. albopictus females were interspecific. The difference in the length of clasper between male Ae. albopictus (0.524 mm) and Ae. aegypti (0.409 mm) may be correlated with corresponding mates. We conclude that stronger Ae. albopictus male interspecific mating and more avid female intraspecific remating result in a satyr effect and contribute to competitive displacement of Ae. aegypti as allopatric Ae. albopictus invade during range expansion.

Plasma proteome profiling combined with clinical and genetic features reveals the pathophysiological characteristics of ß-thalassemia.

The phenotype of ß-thalassemia underlies multigene interactions, making clinical stratification complicated. An increasing number of genetic modifiers affecting the disease severity have been identified, but are still unable to meet the demand of precision diagnosis. Here, we systematically conducted a comparative plasma proteomic profiling on patients with ß-thalassemia and healthy controls. Among 246 dysregulated proteins, 13 core protein signatures with excellent biomarker potential are proposed. The combination of proteome and patients' clinical data revealed patients with codons 41/42 -TTCT mutations have an elevated risk of higher iron burden, dysplasia, and osteoporosis than patients with other genotypes. Notably, 85 proteins correlating to fetal hemoglobin (Hb F) were identified, among which the abundance of 27 proteins may affect the transfusion burden in patients with ß-thalassemia. The current study thus provides protein signatures as potential diagnostic biomarkers or therapeutic clues for ß-thalassemia.

Mutations in surface-sensing receptor WspA lock the Wsp signal transduction system into a constitutively active state.

Pseudomonas aeruginosa rugose small-colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous in-frame deletion, wspAΔ280-307 , which led to hyper-biofilm and RSCV phenotypes. WspA is a homologue of methyl-accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic-di-guanosine monophosphate (c-di-GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspAΔ280-307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c-di-GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280-313 (aa280-313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude-oil environment.

LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles.

Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma.

Proteomics reveals the function reverse of MPSSS-treated prostate cancer-associated fibroblasts to suppress PC-3 cell viability via the FoxO pathway.

Prostate cancer-associated fibroblasts (prostate CAFs) are essential components of the tumor microenvironment and can promote tumor progression through their immunosuppressive functions. MPSSS, a novel polysaccharide purified from Lentinus edodes, has been reported to have anti-tumor activity. MPSSS could also inhibit the immunosuppressive function of prostate CAFs, which has been demonstrated through that the secretome of MPSSS-treated prostate CAFs could inhibit the proliferation of T cells. However, how the secretome of MPSSS-treated prostate CAFs influence prostate cancer progression is still unclear. Interestingly, we found that the low molecular weight (3-100kD) secretome of prostate CAFs (lmwCAFS) could promote the growth of PC-3 cells, while that of MPSSS-treated prostate CAFs (MT-lmwCAFS) could inhibit their growth. We carried out comparative secretomic analysis of lmwCAFS and MT-lmwCAFS to identify functional molecules that inhibit the growth of PC-3 cells, and proteomic analysis of lmwCAFS-treated PC-3 cells and MT-lmwCAFS-treated PC-3 cells to investigate the underlying molecular mechanism. These analyses suggest that TGF-ß3 from MT-lmwCAFS may inhibit the growth of PC-3 cells. The validated experiments revealed that TGF-ß3 from MT-lmwCAFS activated p21 expression in PC-3 cells by regulating the FoxO pathway thereby inducing G0/G1 cell cycle arrest of PC-3 cells. Overall, our data demonstrated that MPSSS reversed the ability of prostate CAFs to suppress the cell viability of PC-3 cells, which might provide a potential therapeutic strategy to prevent prostate cancer progression.

Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species/JUN Pathway.

Oleic acid (OA), a monounsaturated fatty acid (MUFA), has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, its underlying molecular mechanism is unclear. In addition, previous studies have shown that eicosapentaenoic acid (EPA), a ω-3 polyunsaturated fatty acid (PUFA), reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after PA+OA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PA+EPA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA and OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca2+) content. Importantly, H2O2-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.

Separation and characterization of extracellular vesicles from human plasma by asymmetrical flow field-flow fractionation.

It is a big challenge to isolate extracellular vesicles (EVs) from human plasma because of the contamination from high abundant lipoproteins, such as high density lipoprotein (HDL) and low density lipoprotein particles (LDL). In this study, the parameters of asymmetrical flow field-flow fractionation (AF4) technology and sample preparation, including cross flow gradient, focusing time, ultrafiltration condition, sample amount and injection volume have been optimized and successfully utilized for the separation and characterization of EVs from human plasma. This study demonstrated that the great potential of AF4 in the separation of EVs from HDL and LDL in human plasma with high reproducibility and purity. This study indicated excessive focusing time in the AF4 separation and 100-300 kDa MWCO membrane based ultrafiltration in the pre-preparation will cause loss of EVs. A total of 1038 proteins have been identified in seven replicates of purified EVs from pooled human plasma sample. They are mainly enriched in extracellular exosomes, involved in extracellular matrix structural constituent, and associated with extracellular matrix-receptor interaction pathway. This study also indicated that human plasma contains more EVs than the paired serum at the same volume, and showed age- and gender-independent individual variability of the amount of EVs in human plasma. This study displayed that AF4 technique can serve as a powerful platform for the separation of EVs from human plasma, serum or human body fluids and this technology will promote the studies on EVs, such as proteomics, biomarker discovery and functions.

Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma.

Low-molecular weight proteins and peptides (LMWPs, <30 kDa) in human plasma serve as potential biomarkers or drug targets and are endowed with desirable traits for biological and clinical studies. However, the identification of LMWPs from plasma is retarded by high-abundance proteins, high-molecular weight proteins, and lipids. Here, we present a sequential precipitation and delipidation (SPD) method for the efficient enrichment of LMWPs based on methyl-tert-butyl ether/methanol/water systems. The enriched LMWP sample was analyzed by single-shot liquid chromatography-tandem mass spectrometry employing both HCD and EThcD without tryptic digestion, and 725 peptides were identified on average. The LMWP sample was also digested and analyzed using a bottom-up proteomics pipeline, and 289 proteins were identified, of which 129 (44.6%) proteins were less than 30 kDa and lipoprotein-associated proteins were significantly enriched. Additionally, 25 neuropeptides and 19 long noncoding RNA-encoded polypeptides were identified. Taken together, the SPD method shows good sensitivity and reproducibility when compared with other enrichment methods and has great potential for clinical biomarker discovery and application.

Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and crrB Mutation-Mediated Colistin Resistance.

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually over the past few years. Although studies on polymyxin mechanisms are expanding, systemwide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapts to colistin (polymyxin E) pressure, we carried out proteomic analysis of a K. pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in K. pneumoniae involve several pathways, including (i) gluconeogenesis and the tricarboxylic acid (TCA) cycle, (ii) arginine biosynthesis, (iii) porphyrin and chlorophyll metabolism, and (iv) enterobactin biosynthesis. Interestingly, decreased abundances of class A ß-lactamases, including TEM, SHV-11, and SHV-4, were observed in cells treated with colistin. Moreover, we present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant K. pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of K. pneumoniae ATCC BAA 2146, showed a missense mutation in crrB This crrB mutant, which displayed lipid A modification with 4-amino-4-deoxy-l-arabinose (l-Ara4N) and palmitoylation, showed striking increases in the expression of CrrAB, PmrAB, PhoPQ, ArnBCADT, and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, the multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediated colistin resistance, which may offer valuable information on the management of polymyxin resistance.

Comprehensive characterization and proteoform analysis of the hydrophobic surfactant proteins B and C in calf pulmonary surfactant.

Calf pulmonary surfactant (CPS), which contains about 98% lipids and 2% hydrophobic surfactant proteins B (SP-B) and C (SP-C), has been used as a surfactant preparation for the clinical replacement therapy of respiratory distress syndrome (RDS). Characterization of SP-B and SP-C in CPS is informative for quality control and the evaluation of their biological activities. However, analysis of SP-B and SP-C is impeded by the high content of lipids in CPS. Here, we describe an integrated method by combining size exclusion chromatography (SEC)-based delipidation, SDS-PAGE separation, in-gel digestion and mass spectrometric analysis for comprehensive characterization and proteoform analysis of the extremely hydrophobic SP-B and SP-C in CPS. This study has shown that 30 proteoforms of SP-C with different truncations and modifications were identified and SP-B was found to be existed as a dimer form in the CPS.

Simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis.

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.

Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.