Endocrine monitoring of the ovarian cycle and pregnancy in the saddle-back tamarin (Saguinus fuscicollis) by measurement of steroid conjugates in urine

Direct measurements of urinary immunoreactive estrone conjugates (E1C) and pregnanediol glucuronide (PdG), were applied to monitoring the ovarian cycle (n = 9) and pregnancy (3 full term pregnancies, 2 mid-term abortions) in Saguinus fuscicollis. During the ovarian cycle, urinary E1C concentrations revealed a high degree of day-to-day variability and appeared to be uninformative in reflecting cyclic ovarian function. In contrast, PdG was a reliable indicator of ovarian cyclicity with excretion patterns corresponding well with plasma progesterone profiles. Luteal phase PdG concentrations were on average 4–7–fold higher than corresponding follicular phase values. On the basis of changes in circulating progesterone, a mean cycle length of 25.7 ±1.0 days with an average follicular phase of 7.1 ± 0.6 days and a mean luteal phase of 18.6 ± 0.7 days, was found (n = 14 cycles). Following conception, both urinary steroid conjugate concentrations increased and elevated levels were maintained beyond the normal luteal phase length, allowing pregnancy to be determined at around day 25–30. During mid- to late pregnancy, PdG levels declined while E1C concentrations continued to be elevated until approximately 6 weeks before parturition when a decrease occurred. Both hormones showed a clear and rapid fall to follicular phase values following termination of pregnancy at either parturition or mid-term abortion. Post partum ovulations (n = 5) occurred on average 17–18 days following birth with four ovulations leading to conceptions. The results demonstrate the potential of urinary steroid conjugate analysis as a practical and reliable method for non-invasive monitoring of reproductive status in the female saddle-back tamarin. © 1995 Wiley-Liss, Inc.     

Mass Spectrometry–Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Tumor profiling using protein biomarker panels in malignant melanoma: application of tissue microarrays and beyond

In the past decade, analysis of the urinary proteome (urinary proteomics) has intensified in response to the need for novel biomarkers that support early diagnosis of kidney diseases. In particular, this also applies to acute kidney injury, which is a heterogeneous complex syndrome with a still-increasing incidence at the intensive care unit. Unfortunately, this major need remains largely unmet to date. The current report aims to explain why attempts to implement urinary proteomic-discovered acute kidney injury diagnostic candidates in the intensive care unit setting have not yet led to success. Subsequently, some key notes are provided that should enhance the chance of translating selected urinary proteomic candidates to valuable tools for the nephrologist and intensivist in the near future.     

Comparison of urinary creatine with other biomarkers for the detection of 2-methoxyethanol-induced testicular damage

Abstract

This study has compared different biomarkers of testicular damage, in particular evaluating urinary creatine as a non-invasive marker. Male rats were exposed to various doses of 2-methoxyethanol, a known testicular toxicant. Pathological damage, testis weight, urinary creatine and creatinine, serum lactate dehydrogenase, isozyme C4 (LDH-C4), and serum testosterone were determined. 2-Methoxyethanol caused dose-dependent pathological damage to the testes which was detectable at the lowest dose (100 mg kg^?1). Urinary creatine excretion was significantly raised at all doses but testis weight was only significantly decreased at the highest two doses (500, 750 mg kg^?1). Serum testosterone was only significantly decreased at 500 mg kg^?1 and LDH-C4 was not significantly increased at any dose. Therefore urinary creatine was the most sensitive marker of 2-methoxethanol-induced testicular damage and dysfunction.     

An in-depth Comparison of the Pediatric and Adult Urinary N-glycomes

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Highlights

• •N-glycan patterns are distinct in pediatric and adult urine.
• •Sex differences of N-glycans are much larger in adults.
• •Pediatric urine has almost no sex differences in N-glycan levels.
• •In adults, the majority of N-glycans were more abundant in males.

     

Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

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Highlights

• •Urinary proteomes of patients with recurrent UTI, renal scarring, and VUR.
• •80 proteins differentially expressed, compared to healthy controls.
• •62 proteins may be indicative of susceptibility for UTI.
• •Altered acute phase response, extracellular matrix and carbohydrate metabolism.

     

Optimal selection of 2D reversed-phase–reversed-phase HPLC separation techniques in bottom-up proteomics

Evaluation of: Stephanowitz H, Lange S, Lang D et al. Improved two-dimensional reversed phase–reversed phase LC-MS/MS approach for identification of peptide–protein interactions. J. Proteome Res. 11(2), 1175–1183 (2011).

Recent developments in bottom-up proteomics have supplanted the use of gel-based approaches in favor of multidimensional chromatographic separations of peptide mixtures followed by mass spectrometry analysis. This trend is driven by the desire to eliminate labor-intensive in-gel digestion procedures and increase proteome coverage through better recovery of proteolytic fragments. Introduction of reversed-phase–reversed-phase 2D separation techniques is one of the major improvements that have made this possible. In this article, we review recent developments in 2D HPLC and highlight variations in reversed-phase HPLC separation selectivity that allow for superior peak capacity in peptide fractionation.