Inhibition of HSP90AA1 induces abnormalities in bovine oocyte maturation and embryonic development.

In brief: HSP90AA1 is a ubiquitous molecular chaperone that can resist cellular stress, such as oxidative stress and apoptosis, and mediate the efficacy and protein folding of normal cells during heat stress, as well as many other functions. This study further reveals the role of HSP90AA1 in bovine oocyte maturation and early embryonic development. Abstract: HSP90AA1, a highly abundant and ubiquitous molecular chaperone, plays important roles in various cellular processes including cell cycle control, cell survival, and hormone signaling pathways. In this study, we investigated the functions of HSP90AA1 in bovine oocyte and early embryo development. We found that HSP90AA1 was expressed at all stages of development, but was mainly located in the cytoplasm, with a small amount distributed in the nucleus. We then evaluated the effect of HSP90AA1 on the in vitro maturation of bovine oocytes using tanespimycin (17-AAG), a highly selective inhibitor of HSP90AA1. The results showed that inhibition of HSP90AA1 decreased nuclear and cytoplasmic maturation of oocytes, disrupted spindle assembly and chromosome distribution, significantly increased acetylation levels of α-tubulin in oocytes and affected epigenetic modifications (H3K27me3 and H3K27ac). In addition, H3K9me3 was increased at various stages during early embryo development. Finally, the impact of HSP90AA1 on early embryo development was explored. The results showed that inhibition of HSP90AA1 reduced the cleavage and blastocyst formation rates, while increasing the fragmentation rate and decreasing blastocyst quality. In conclusion, HSP90AA1 plays a crucial role in bovine oocyte maturation as well as early embryo development.

Social interaction anxiety and sleep quality in youth: Individual difference in childhood adversity and cardiac vagal control.

BACKGROUND: Social interaction anxiety and sleep problems are prevalent during adolescence. Social interaction anxiety undermines sleep quality, however, little is known whether the association between social interaction anxiety and sleep quality is moderated by environmental factors such as childhood adversity and individual factors such as cardiac vagal control. This study sought to investigate the moderating effects of childhood adversity and cardiac vagal control on the link between social interaction anxiety and sleep quality. METHOD: The Social Interaction Anxiety Scale, the Pittsburgh Sleep Quality Index and the Childhood Trauma Questionnaire were administered to 274 adolescents, who received 3-min resting ECG recording to assess respiratory sinus arrhythmia (RSA) as an index of cardiac vagal control. RESULTS: Social interaction anxiety was negatively associated with sleep quality, and this association was moderated by childhood adversity and cardiac vagal control. In specific, social interaction anxiety was negatively associated with sleep quality among adolescents with low childhood adversity regardless of cardiac vagal control. Sleep quality was generally disrupted when adolescents exposed to high childhood adversity, but the negative association between social interaction anxiety and sleep quality among adolescents with high childhood adversity could be amortized by high cardiac vagal control. LIMITATIONS: Cross-sectional design precluded establishing causality among variables. CONCLUSION: These findings suggest that high cardiac vagal control reflecting better self-regulation might buffer the negative effect of social interaction anxiety on sleep quality particularly among adolescents exposed to early life stress.

Triboelectric nanogenerator based on Teflon/vitamin B1 powder for self-powered humidity sensing.

Recently, there has been growing interest in triboelectric nanogenerators (TENGs) that can effectively convert various forms of mechanical energy input into electrical energy. In the present study, a novel Teflon/vitamin B1 powder based triboelectric nanogenerator (TVB-TENG) is proposed. Paper is utilized as a supporting platform for triboelectrification between a commercial Teflon tape and vitamin B1 powder. The measured open-circuit voltage was approximately 340 V. The TVB-TENG can be applied as a humidity sensor and exhibits a linear and reversible response to the relative humidity of the environment. Moreover, the change in relative humidity is also indicated by the change in luminosity of a set of light-emitting diodes (LEDs) integrated in the TVB-TENG system. The TVB-TENG proposed in this study illustrates a cost-effective method for portable power supply and sensing devices.

Balancing the Affinity and Pharmacokinetics of Antibodies by Modulating the Size of Charge Patches on Complementarity-Determining Regions.

A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.

Use of chelating agents to improve the resolution and consistency of cation-exchange chromatography of monoclonal antibodies.

Analytical cation-exchange chromatography (CEX) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). However, the consistency of CEX profiles of a mAb can be significantly reduced by metal ion impurities from sample, mobile phase or leachates from the stainless steel components of the pumping system. In this work, we have developed a new CEX method that dynamically removes metal ions during sample analysis by incorporating the use of chelating agents (1-5mM) in HPLC mobile phases. Among four different chelating agents that were evaluated, EDTA and oxalic acid showed excellent capability of removing metal ions and provided consistent CEX chromatograms for mAb1. Furthermore, the use of oxalic acid in mobile phases not only improved the reproducibility of CEX chromatograms, but also increased the resolution of charge isoforms. Oxalic acid appears capable of binding to mAbs and reducing the positive surface charge density, resulting in a modulation of chromatographic separation. Due to this modulation effect, the CEX resolution was dependent on the concentration of the chelating agent. Optimal resolution for mAb1 was obtained with 2mM of oxalic acid. The oxalic acid modulated CEX method was shown to be capable of monitoring the degradation of mAb1. We further qualified this method according to International Committee on Harmonization (ICH) guidelines and demonstrated that the oxalic acid modulated CEX method is precise and robust at different chromatographic conditions and is suitable for use in a development and/or GMP setting.

Improving pH gradient cation-exchange chromatography of monoclonal antibodies by controlling ionic strength.

Analytical ion exchange chromatography (IEC) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). Since conventional salt gradient IEC methods are product-specific and time-consuming to develop, a previously reported alternative pH gradient IEC (pH-IEC) method using a cation-exchange column has been shown to be a multiproduct charge sensitive separation method for mAbs with isoelectric points between 7.3 and 9.0. In the work presented here, we have extended the application of that pH-IEC method to also profile the charge heterogeneity of mAbs with extreme pI values (e.g. acidic with pI<7 or basic with pI>9). A key observation of our work is that for the buffer systems used by Farnan and Moreno, the ionic strength of the mobile phase containing multiple polyamine buffers is pH and concentration dependent, and the ionic strength decreases when the pH increases. For the mobile phase with high buffer concentration the ionic strength is high at low pH values, leading to the flow through of acidic mAbs on the cation-exchange column. The basic mAbs may not have an optimal elution profile as the relatively low ionic strength of the mobile phase reduces the resolution of pH-IEC. To modulate the ionic strength, we introduced a salt gradient in addition to the pH gradient. Studies were performed to optimize the buffer and salt concentrations simultaneously to improve the retention of low pI mAbs and the resolution of high pI mAbs. The optimized salt-mediated pH-IEC method was not only applicable to mAbs over a broader pI range from 6.2 to 9.4, but also offered better resolution for mAbs with pI values between 7.3 and 9.0 than the previously reported pH-IEC method. This salt-mediated pH-IEC method was demonstrated to be robust at various chromatography conditions and capable of assessing manufacturing consistency and monitoring degradation of mAbs.

Revealing a positive charge patch on a recombinant monoclonal antibody by chemical labeling and mass spectrometry.

During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometry-based footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.

De novo sequencing of tryptic peptides derived from Deinococcus radiodurans ribosomal proteins using 157 nm photodissociation MALDI TOF/TOF mass spectrometry.

Vacuum ultraviolet photodissociation of peptide ions in a matrix assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer is used to characterize peptide mixtures derived from Deinococcus radiodurans ribosomal proteins. Tryptic peptides from 52 proteins were separated by reverse-phase liquid chromatography and spotted onto a MALDI plate. From 192 sample spots, 492 peptide ions were isolated, fragmented by both photodissociation and postsource decay (PSD), and then de novo sequenced. Three-hundred seventy-two peptides yielded sequences with 5 or more amino acids. Homology searches of these sequences against the whole bacterial proteome identified 49 ribosomal proteins, 45 of which matched with two or more peptides. Peptide de novo sequencing identified slightly more proteins than conventional database searches using Mascot and was particularly advantageous in identifying unexpected peptide modifications. In the present analysis, 52 peptide modifications were identified by de novo sequencing, most of which were not recognized by database searches.

Peptide de novo sequencing using 157 nm photodissociation in a tandem time-of-flight mass spectrometer.

It has previously been shown that photodissociation of tryptic peptide ions with 157 nm light in a matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer generates an abundance of x-type ions. A peptide de novo sequencing algorithm has now been developed to interpret these data. By combination of photodissociation and postsource decay (PSD) spectra, the algorithm identifies x-type ions and derives peptide sequences. The confidence of amino acid assignments is evaluated by observing complementary y-, v-, and w-type ions that provide additional constraints to sequence identification. In the analysis of 31 tryptic peptides from 4 model proteins, the algorithm identified 322 (or 90.7%) of the 355 amino acids and made only 3 incorrect assignments. The other 30 amino acids were not identified because specific needed x-type ions were not detected. Based on the observation of v- and w-type ions, 45 of 50 detected leucine and isoleucine residues were successfully distinguished and there was only one mistake. The remaining four residues were not distinguished because the corresponding v- and w-type ions were not detected. These de novo sequencing results translated into successful identification of proteins through homology searches. To evaluate the robustness of the present sequencing approach, a collection of 266 tryptic peptides from 23 model proteins were analyzed and then sequenced. A total of 167 peptides yielded sequence tags of 5 or more residues. In 5 peptides, 1 or 2 residues were incorrectly assigned.

Peptide photodissociation with 157 nm light in a commercial tandem time-of-flight mass spectrometer.

Photodissociation with 157 nm light was implemented in an ABI model 4700 matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer for peptide analysis. With a homemade computer program to control the light timing based on the m/z of each precursor ion, the photodissociation setup was seamlessly automated with the mass spectrometer. Peptide photodissociation in this apparatus yielded fragments similar to those observed in previous experiments with a home-built tandem-TOF mass spectrometer. Peptides having arginine at their C-termini yielded high-energy x-, v-, and w- type fragments, while peptides with N-terminal arginine produced many a- and d- type ions. Abundant immonium ions were also generated. High-quality photodissociation spectra were obtained with as little as 5 fmol of peptides. In the analysis of various tryptic peptides, photodissociation provided much more sequence information than the conventional TOF-TOF collision induced dissociation (CID). Because of the high fragmentation efficiency, sensitivity was not sacrificed to achieve this.

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation.

Odd-electron a + 1 radical ions generated in the 157 nm photodissociation of peptide ions were investigated in an ion trap mass spectrometer. To localize the radical, peptide backbone amide hydrogens were replaced with deuterium. When the resulting radical ions underwent hydrogen elimination, no H/D scrambling was obvious, suggesting that without collisional activation, the radical resides on the terminal alpha-carbon. Upon collisional excitation, odd-electron radical ions dissociate through two favored pathways: the production of a-type ions at aromatic amino acids via homolytic cleavage of backbone C(alpha)-C(O) bonds and side-chain losses at nonaromatic amino acids. When aromatic residues are not present, nonaromatic residues can also lead to a-type ions. In addition to a-type ions, serine and threonine yield c(n-1) and a(n-1) + 1 ions where n denotes the position of the serine or threonine. All of these fragments appear to be directed by the radical and they strongly depend on the amino acid side-chain structure. In addition, thermal fragments are also occasionally observed following cleavage of labile Xxx-Pro bonds and their formation appears to be kinetically competitive with radical migration.

Extracting both peptide sequence and glycan structural information by 157 nm photodissociation of N-linked glycopeptides.

The 157 nm photodissociation of N-linked glycopeptides was investigated in MALDI tandem time-of-flight (TOF) and linear ion trap mass spectrometers. Singly charged glycopeptides yielded abundant peptide and glycan fragments. The peptide fragments included a series of x-, y-, v-, and w- ions with the glycan remaining intact. These provide information about the peptide sequence and the glycosylation site. In addition to glycosidic fragments, abundant cross-ring glycan fragments that are not observed in low-energy CID were detected. These fragments provide insight into the glycan sequence and linkages. Doubly charged glycopeptides generated by nanospray in the linear ion trap mass spectrometer also yielded peptide and glycan fragments. However, the former were dominated by low-energy fragments such as b- and y- type ions while glycan was primarily cleaved at glycosidic bonds.

Use of 157-nm photodissociation to probe structures of y- and b-type ions produced in collision-induced dissociation of peptide ions.

y- and b-type fragment ions produced in the collisional dissociation of arginine-terminated peptide ions are photodissociated with 157-nm light in a linear trap. y-type ions are shown to have the same structure as that of intact peptides of the same sequence with the ionizing proton located at the most basic residue(s). For generic b-type ions, the ionizing proton is shown to be sequestered at the N-terminal arginine, which is consistent with the proposed oxazolone structure.

Structures of alpha-type ions formed in the 157 nm photodissociation of singly-charged peptide ions.

One hundred fifty-seven nm photodissociation of singly-charged peptide ions induces the cleavage of alpha-carbon to carbonyl-carbon bonds along the backbone. a(n) + 1 radical ions are observed as the primary photolysis products of peptides with N-terminal arginines in a linear ion trap mass spectrometer. The radical elimination pathways undertaken by the a(n) + 1 radical ions to form more stable even-electron species are studied in hydrogen-deuterium (H/D) exchange experiments. Two types of a(n) ions along with d-type ions are observed as secondary elimination products. The relative abundance of each depends on the C-terminal residue of the radical fragment ion.

Quantitative determination of Freon gas using an electronic nose.

This paper reports a quantitative electronic nose (enose) for the quantitative determination of Freon gas within the concentration range 0-1000 ppm in the presence of interfering gases such as water, lubricant and petrol vapours. This quantitative enose is a new type of Freon detection system, composed of an array of four sensors. The artificial neural network (ANN) and fuzzy logic type of ANN (FNN), in combination with the relative error concept in analytical chemistry, are integrated for both quantification and discrimination. The predicted results are satisfied with a pass rate of > 80% within the permitted relative errors. The results show that the Freon enose developed in this study is reliable for both the qualitative and quantitative determination of Freon gas and exhibits the merits of high sensitivity, anti-interference and accuracy.