Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.     

Use of hydrogen cyanamide for apple and plum thinning

Effects of various concentrations of Dormex (a.i. 49% hydrogen cyanamide) on fruit thinning of Rome Beauty apple (Malus domestica Borkh.), Friar and Simka plums (Prunus salicina Lindley) were studied. A full bloom application of Dormex at all tested concentrations decreased Rome Beauty apple fruit set and yield, and increased fruit weight. Dormex at 0.25% (v/v) resulted in adequate apple thinning, indicated by production of an optimum fruit weight (320 g). Prebloom and full bloom applications of Dormex at greater than 0.75% reduced plum fruit set and yield in Friar. Full bloom application of Dormex at 0.50% showed a satisfactory fruit set, yield, and fruit weight in Friar plum. Prebloom Dormex application had no significant effect on `Simka' plum fruit set or yield, but full bloom application decreased fruit set and yield.     

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.     

Presence and dynamics of leptin,GLP‐1, and PYY in human breast milk at early postpartum

Objective: The presence of appetite hormones, namely glucagon‐like peptide‐1 (GLP‐1), peptide YY (PYY), and leptin in breast milk may be important in infant feeding regulation and infant growth. This study evaluated whether concentrations of GLP‐1, PYY, and leptin change across a single feeding (from fore‐ to hindmilk), and are associated with maternal and infant anthropometrics. Design and Methods: Thirteen postpartum women (mean ± SD: 25.6 ± 4.5 years, 72.0 ± 11.9 kg) provided fore‐ and hindmilk samples 4‐5 weeks after delivery and underwent measurements of body weight and composition by Dual X‐ray Absorptiometry. GLP‐1, PYY, and leptin concentrations were measured using radioimmunoassay, and milk fat content was determined by creamatocrit. Results: Concentration of GLP‐1 and content of milk fat was higher in hindmilk than foremilk (P ≤ 0.05). PYY and leptin concentrations did not change between fore‐ and hindmilk. Both leptin concentration and milk fat content were correlated with indices of maternal adiposity, including body mass index (r = 0.65‐0.85, P < 0.02), and fat mass (r = 0.65‐0.84, P < 0.02). Hindmilk GLP‐1 was correlated with infant weight gain from birth to 6 months (r = ?0.67, P = 0.034). Conclusion: The presence of appetite hormones in breast milk may be important in infant appetite and growth regulation.     

Ssq1, a mitochondrial Hsp70 involved in iron-sulfur (Fe/S) center biogenesis. Similarities to and differences from its bacterial counterpart

The results of in vivo and in organellar experiments indicate that the Hsp70 Ssq1 and the J-protein Jac1 function together to assist in the biogenesis of iron-sulfur (Fe/S) centers in the mitochondrial matrix. Here we present biochemical evidence supporting this idea. Isu, the proposed scaffold on which Fe/S centers are assembled, is a substrate for both Jac1 and Ssq1. Jac1 and Isu1 cooperatively stimulate the ATPase activity of Ssq1. In addition, Jac1 facilitates the interaction of Ssq1 with Isu1 in the presence of ATP. These findings are consistent with the role in Fe/S biogenesis previously proposed for the bacterial Hsp70 Hsc66 and J-protein Hsc20 that interact with the bacterial Isu homologue IscU. However, unlike the bacterial Hsp70, we found that Ssq1 has a high affinity for nucleotide, and shares a nucleotide exchange factor, Mge1, with a second mitochondrial Hsp70, Ssc1. Thus, whereas the bacterial and mitochondrial chaperone systems share critical features, they possess significant biochemical differences as well.     

A method for benchmarking genetic screens reveals a predominant mitochondrial bias

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome‐wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene‐pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria‐associated signal within co‐essentiality networks derived from these data and explore the basis of this signal. Our analysis and time‐resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.     

Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n = 5) or 150% (Fat; n = 6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P < 0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P < 0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P = 0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) β tended (P = 0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-β was greater in female fetuses than male fetuses from Control ewes (P = 0.05). Although amount of ER-β mRNA did not differ among male fetuses (P = 0.7), amounts tended to be less (P = 0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P ≥ 0.8) from male fetuses. Hypothalamic ER-α mRNA tended (P = 0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P = 0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P = 0.06) to be greater in male fetuses than female fetuses and tended to be less (P = 0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P = 0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P ≥ 0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-α, and decrease amount of ER-β in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.     

Oxygen partial pressure effects on metabolic rate and behavior of tethered flying locusts

Resting insects are extremely tolerant of hypoxia. However, oxygen requirements increase dramatically during flight. Does the critical atmospheric P (O)(2) (P(c)) increase strongly during flight, or does increased tracheal conductance allow even flying insects to possess large safety margins for oxygen delivery? We tested the effect of P(O)(2) on resting and flying CO(2) emission, as well as on flight behavior and vertical force production in flying locusts, Schistocerca americana. The P(c) for CO(2) emission of resting animals was less than 1 kPa, similar to prior studies. The P(c) for flight bout duration was between 10 and 21 kPa, the P(c) for vertical force production was between 3 and 5 kPa, and the P(c) for CO(2) emission was between 10 and 21 kPa. Our study suggests that the P(c) for steady-state oxygen consumption is between 10 and 21 kPa (much higher than for resting animals), and that tracheal oxygen stores allowed brief flights in 5 and 10 kPa P(O)(2) atmospheres to occur. Thus, P(c) values strongly increased during flight, consistent with the hypothesis that the excess oxygen delivery capacity observed in resting insects is substantially reduced during flight.     

Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme

U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m(7)G and m(227)G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.