Reversing the miRNA -5p/-3p stoichiometry reveals physiological roles and targets of miR-140 miRNAs

The chondrocyte specific miR-140 miRNAs are necessary for normal endochondral bone growth in mice. miR-140 deficiency causes dwarfism and craniofacial deformity. However, the physiologically important targets of miR-140 miRNAs are still unclear. The miR-140 gene (Mir140) encodes three chondrocyte-specific microRNAs, miR-140-5p, derived from the 5′ strand of primary miR-140, and miR140-3p.1 and -3p.2, derived from the 3′ strand of primary miR-140. miR-140-3p miRNAs are ten times more abundant than miR-140-5p likely due to the non-preferential loading of miR-140-5p to Argonaute proteins. To differentiate the role of miR-140-5p and -3p miRNAs in endochondral bone development, two distinct mouse models, miR140-C>T, in which the first nucleotide of miR-140-5p was altered from cytosine to uridine, and miR140-CG, where the first two nucleotides of miR-140-3p were changed to cytosine and guanine, were created. These changes are expected to alter Argonaute protein loading preference of -5p and -3p to increase -5p loading and decrease -3p loading without changing the function of miR140-5p. These models presented a mild delay in epiphyseal development with delayed chondrocyte maturation. Using RNA-sequencing analysis of the two models, direct targets of miR140-5p, including Wnt11, were identified. Disruption of the predicted miR140-5p binding site in the 3′ untranslated region of Wnt11 was shown to increase Wnt11 mRNA expression and caused a modest acceleration of epiphyseal development. These results show that the relative abundance of miRNA-5p and -3p can be altered by changing the first nucleotide of miRNAs in vivo, and this method can be useful to identify physiologically important miRNA targets.

Optimization of Claudin-5 and ICAM-1 protein detection by using capillary-based immunoassay method in human brain endothelial cells

Background: Human brain endothelial cells (HBECs) are part of the blood-brain barrier (BBB). BBB acts as a barrier to control the passage of molecules or materials from the blood into the brain. Identification of specific proteins changes in their expressions that are related to disease state is important in order to understand the disease mechanism involving brain vasculature. To achieve that, the techniques involve in identifying the proteins of interest must be optimized prior to further investigation. Methodology: In this study, identification of Claudin-5 in HBEC lysates was tested using different sample preparation techniques such as; 1) reducing with Dithiothreitol (DTT) and non-reducing conditions; 2) denaturing by heating at 95°C for 5 minutes or 70°C for 20 minutes and 3) protein loading at 3 and 4 µg. The samples were then subjected to an automated capillary-based immunoassay, Jess. Results and Discussion: The results showed that HBEC samples loaded at 4 µg and heated for 5 minutes at 95°C with DTT produced clearer and intense bands for Claudin-5 identification compared to the other set ups. As reducing condition and denaturing by heated at 95°C for 5 minutes conditions demonstrated good results, the conditions were used to identify ICAM-1 expression at different protein loading (3 and 4 µg). The result demonstrated that HBEC samples heated for 5 minutes at 95°C with DTT and loaded at 4 µg produced a good detection for ICAM-1. Conclusion: These optimized conditions could be served as a standard procedure for further identification of Claudin-5 and ICAM-1 proteins in HBEC using a capillary immunoassay instrument.

Optimization of Claudin-5 and ICAM-1 protein detection by using capillary-based immunoassay method in human brain endothelial cells

Background: Human brain endothelial cells (HBECs) are part of the blood-brain barrier (BBB). BBB acts as a barrier to control the passage of molecules or materials from the blood into the brain. Identification of specific proteins changes in their expressions that are related to disease state is important in order to understand the disease mechanism involving brain vasculature. To achieve that, the techniques involve in identifying the proteins of interest must be optimized prior to further investigation. Methodology: In this study, identification of Claudin-5 in HBEC lysates was tested using different sample preparation techniques such as; 1) reducing with Dithiothreitol (DTT) and non-reducing conditions; 2) denaturing by heating at 95°C for 5 minutes or 70°C for 20 minutes and 3) protein loading at 3 and 4 µg. The samples were then subjected to an automated capillary-based immunoassay, Jess. Results and Discussion: The results showed that HBEC samples loaded at 4 µg and heated for 5 minutes at 95°C with DTT produced clearer and intense bands for Claudin-5 identification compared to the other set ups. As reducing condition and denaturing by heated at 95°C for 5 minutes conditions demonstrated good results, the conditions were used to identify ICAM-1 expression at different protein loading (3 and 4 µg). The result demonstrated that HBEC samples heated for 5 minutes at 95°C with DTT and loaded at 4 µg produced a good detection for ICAM-1. Conclusion: These optimized conditions could be served as a standard procedure for further identification of Claudin-5 and ICAM-1 proteins in HBEC using a capillary immunoassay instrument.

Ultrasonographic Intraparenchymal Renal Resistive Index Variation for Assessing Renal Functional Reserve in Patients Scheduled for Cardiac Surgery: A Pilot Study

<b><i>Introduction:</i></b> Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common complication in patients undergoing cardiac surgery. Preoperative renal functional reserve (RFR) has been demonstrated to be highly predictive of CSA-AKI. We have previously demonstrated that intraparenchymal renal resistive index variation (IRRIV) measured by ultrasound (US) can identify the presence of RFR in healthy individuals. This study aimed (1) to examine the correlation between the US IRRIV test and RFR measured through the protein loading test in patients undergoing elective cardiac surgery and (2) to determine the value of the 2 methods for predicting occurrence of AKI or subclinical AKI after cardiac surgery. <b><i>Methods:</i></b> Consecutive patients scheduled for cardiac surgery were enrolled for this pilot study. The protein loading test and the IRRIV test were performed in all patients 2 days before cardiac surgery. Correlation between IRRIV and RFR was tested using Pearson correlation analysis. Association between presence of RFR and positive IRRIV test, presence of RFR and AKI and subclinical AKI, and positive IRRIV test and AKI and subclinical AKI was evaluated using logistic regression analysis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the values of IRRIV for predicting RFR, RFR for predicting AKI and subclinical AKI, and IRRIV for predicting AKI and subclinical AKI. <b><i>Results:</i></b> Among the 31 patients enrolled, significant association was found between IRRIV and RFR (<i>r</i> = 0.81; 95% CI: 0.63–0.90; <i>p</i> &#x3c; 0.01). The association between RFR and IRRIV was described in 27/31 (87.1%) patients. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the IRRIV test were 100, 84, 60, and 100%, respectively. In ROC curve analysis, the area under the curve (AUC) was 0.80 (95% CI: 0.64–0.96). After cardiac surgery, 1/31 (3.2%) patient had AKI and 12/31 (38.7%) had subclinical AKI. RFR predicted subclinical AKI (odds ratio [OR] = 0.93; 95% CI: 0.87–0.98; <i>p</i> = 0.02). The sensitivity, specificity, PPV, and NPV of the RFR were 61, 88.8, 80, and 76%, respectively; the AUC was 0.75 (95% CI: 0.59–0.91). IRRIV predicts subclinical AKI (OR = 0.79; 95% CI: 0.67–0.93; <i>p</i> = 0.005). The sensitivity, specificity, PPV, and NPV of the IRRIV test were 46.1, 100, 100, and 72%, respectively; the AUC was 0.73 (95% CI: 0.58–0.87). <b><i>Conclusion:</i></b> This pilot study suggests that a positive IRRIV test can significantly predict the presence of RFR in patients scheduled for cardiac surgery. RFR measured by the protein loading test or by the US IRRIV test can predict the occurrence of subclinical postoperative AKI. The findings of this study need to be confirmed in large patient cohorts.

Protein Loading into Spongelike PLGA Microspheres

A self-healing microencapsulation process involves mixing preformed porous microspheres in an aqueous solution containing the desired protein and converting them into closed-pore microspheres. Spongelike poly-d,l-lactide-co-glycolide (PLGA) microspheres are expected to be advantageous to protein loading through self-healing. This study aimed to identify and assess relevant critical parameters, using lysozyme as a model protein. Several parameters governed lysozyme loading. The pore characteristics (open-pore, closed-pore, and porosity) of the preformed microspheres substantially affected lysozyme loading efficiency. The type of surfactant present in the aqueous medium also influenced lysozyme loading efficiency. For instance, cetyltrimethylammonium bromide showing a superior wetting functionality increased the extent of lysozyme loading more than twice as compared to Tween 80. Dried preformed microspheres were commonly used before, but our study found that wet microspheres obtained at the end of the microsphere manufacturing process displayed significant advantages in lysozyme loading. Not only could an incubation time for hydrating the microspheres be shortened dramatically, but also a much more considerable amount of lysozyme was encapsulated. Interestingly, the degree of microsphere hydration determined the microstructure and morphology of closed-pore microspheres after self-healing. Understanding these critical process parameters would help tailor protein loading into spongelike PLGA microspheres in a bespoke manner.

ACTB and GAPDH appear at multiple SDS-PAGE positions, thus not suitable as reference genes for determining protein loading in techniques like Western blotting

We performed polyacrylamide gel electrophoresis of human proteins with sodium dodecyl sulfate, isolated proteins at multiple positions, and then used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 41.7 and 36 kDa proteins, respectively, LC-MS/MS identified their peptides at all the positions studied. The National Center for Biotechnology Information (USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one noncoding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatics analysis identified a 17.6 kDa isoform encoded by the noncoding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with significant similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to the ACTB. These data lead us to conclude that GAPDH and ACTB are poor reference genes for determining the protein loading in such techniques as Western blotting, a leading role these two genes have been playing for decades in biomedical research.

Synthesis of Zwitterionic Chimeric Polymersomes for Efficient Protein Loading and Intracellular Delivery

A novel zwitterionic triblock copolymer of poly(dimethylamino carbonate)-polycaprolactone-poly((2-(methacryloyloxy)ethyl)dimethyl-(3-sulfopropyl)ammonium) [PAC(DMA)-PCL-PMDMSA] was designed and synthesized via sequential ring-opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization successively. The 1H NMR and...

Synthesis of Mesoporous Silica Nanowires and Their Application in Enzyme Immobilization

Hydrophobic mesoporous silica nanowires were synthesis and then employed as support for immobilization of lipase from Candida antarctica via covalent bonding (CALB@MSW). The parameters were optimized and the optimum conditions were as follows: GA concentration 5.5 wt.%, activation time 60 min and CALB concentration 4 mg/mL. Under these conditions, the protein loading and specific activity of CALB@MSW were 138.3 mg/gsupport and 41.1 U/mgsupport, respectively. Compared with free CALB, CALB@MSW showed better thermal stability and pH stability. The maximum yield of biodiesel catalytic by CALB@MSW was 93.4 %. After reused 8 times, CALB@MSW still remained 95.75 % initial activity showing better stability than free CALB.

Centromeric Transcription: A Conserved Swiss-Army Knife

In most species, the centromere is comprised of repetitive DNA sequences, which rapidly evolve. Paradoxically, centromeres fulfill an essential function during mitosis, as they are the chromosomal sites wherein, through the kinetochore, the mitotic spindles bind. It is now generally accepted that centromeres are transcribed, and that such transcription is associated with a broad range of functions. More than a decade of work on this topic has shown that centromeric transcripts are found across the eukaryotic tree and associate with heterochromatin formation, chromatin structure, kinetochore structure, centromeric protein loading, and inner centromere signaling. In this review, we discuss the conservation of small and long non-coding centromeric RNAs, their associations with various centromeric functions, and their potential roles in disease.