Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.     

Mechanism of oxygen availability from hydrogen peroxide to aerobic cultures of Xanthomonas campestris

Fundamental studies on the availability of oxygen from the decomposition of H(2)O(2), in vivo, by Xanthomonas campestris, when H(2)O(2) is used as an oxygen source are presented. It was found that the H(2)O(2) added extracellularly (0.1-6 mM) was decomposed intracellularly. Further, when H(2)O(2) was added, the flux of H(2)O(2) into the cell, is regulated by the cell. The steady-state H(2)O(2) flux into the cell was estimated to be 9.7 x 10(-8) mol m(-2) s(-1). In addition, it was proved that the regulation of H(2)O(2) flux was coupled to the protonmotive force (PMF) using experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which disrupts PMF. The coupling constant between the rate of free energy availability from PMF and the rate of reduction of H(2)O(2) flux, was found to be 46.4 mol m(-2) s(-1) J(-1) from simulations using a developed model. Also, the estimated periplasmic catalase concentration was 1.4 x 10(-9) M.     

Brugia filariasis differentially modulates persistent Helicobacter pylori gastritis in the gerbil model

In select Helicobacter pylori-infected populations with low gastric cancer, nematode coinfections are common and both helicobacter gastritis and filariasis are modeled in gerbils. We evaluated gastritis, worm counts, tissue cytokine gene expression levels and Th1/Th2-associated antibody responses in H. pylori and Brugia pahangi mono- and coinfected gerbils. H. pylori-associated gastritis indices were significantly lower 21 weeks post-infection in coinfected gerbils (p ≤ 0.05) and were inversely proportional to worm counts (r² = ?0.62, p < 0.003). Additionally, IFN-γ, IL-1β, CXCL1, IL-4 and IL-10 mRNA levels in the gastric antrum reflected a significant host response to gastric H. pylori and as well as systemic filariasis (p ≤ 0.05). Despite increasing worm burden (p < 0.05), gastritis progressed in coinfected gerbils (p < 0.03) becoming equivalent to H. pylori-infected gerbils at 42 weeks (p = 0.7). Pro- and anti-inflammatory mediator mRNA levels were notably downregulated in B. pahangi infected gerbils below uninfected control values, suggesting hyporesponsiveness to B. pahangi. Consistent with an increasing Th1 response to H. pylori, IgG2a (p < 0.01), IL-1β (p = 0.04) and CXCL1 (p = 0.006) responses significantly increased and IL-4 (p = 0.05) and IL-10 (p = 0.04) were decreased in coinfected gerbils at 42 weeks. Initial systemic responses to B. pahangi resulted in attenuated gastritis in coinfected gerbils, but subsequent filarid-associated hyporesponsiveness appears to have promoted H. pylori gastritis.     

Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.     

Hepatotoxicity of diazepam

Studies of effects of diazepam on liver parenchyma are very scantly. In this study, adult albino rats were treated with diazepam in two different doses (0.25 mg and 0.30 mg/kg body wt) daily for 30 and 60 d. Through light microscopy and electron microscopy, prenecrotic and necrotic changes were noted in the high-dose group. Trace metal analysis indicated that zinc (Zn) was reduced by 30 and 60 day under both the doses, whereas iron (Fe) and copper (Cu) were reduced significantly in these groups only after 60 d of treatment. This reduction in metal contents may have some correlations with necrotic changes in liver parenchyma.     

Identifying Novel Drug Indications through Automated Reasoning

Background

With the large amount of pharmacological and biological knowledge available in literature, finding novel drug indications for existing drugs using in silico approaches has become increasingly feasible. Typical literature-based approaches generate new hypotheses in the form of protein-protein interactions networks by means of linking concepts based on their cooccurrences within abstracts. However, this kind of approaches tends to generate too many hypotheses, and identifying new drug indications from large networks can be a time-consuming process.

Methodology

In this work, we developed a method that acquires the necessary facts from literature and knowledge bases, and identifies new drug indications through automated reasoning. This is achieved by encoding the molecular effects caused by drug-target interactions and links to various diseases and drug mechanism as domain knowledge in AnsProlog, a declarative language that is useful for automated reasoning, including reasoning with incomplete information. Unlike other literature-based approaches, our approach is more fine-grained, especially in identifying indirect relationships for drug indications.

Conclusion/Significance

To evaluate the capability of our approach in inferring novel drug indications, we applied our method to 943 drugs from DrugBank and asked if any of these drugs have potential anti-cancer activities based on information on their targets and molecular interaction types alone. A total of 507 drugs were found to have the potential to be used for cancer treatments. Among the potential anti-cancer drugs, 67 out of 81 drugs (a recall of 82.7%) are indeed known cancer drugs. In addition, 144 out of 289 drugs (a recall of 49.8%) are non-cancer drugs that are currently tested in clinical trials for cancer treatments. These results suggest that our method is able to infer drug indications (original or alternative) based on their molecular targets and interactions alone and has the potential to discover novel drug indications for existing drugs.     

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic,Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring,SWATH, and MSE

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes.Diabetes is a complex metabolic disorder characterized by prolonged hyperglycemia resulting from defects in insulin secretion, insulin action, or both, leading to abnormalities in carbohydrate, fat, and protein metabolism (1). According to the projection by the International Diabetes Foundation, around 592 million people will be affected by diabetes by the year 2040 (2). Diabetes and its associated complications are becoming global public health problems and posing a serious challenge in disease management. Many studies have implicated advanced glycation end products (AGEs)¹ in the development of insulin resistance, as well as in pathogenesis of diabetic complications (3). The levels of AGEs increase substantially in diabetic plasma due to the hyperglycemic condition. Factors such as oxidative stress, overnutrition, and foods rich in glycating agents promote the formation of AGEs even in nondiabetic condition (4). Oral AGEs foster insulin resistance and diabetes by down-regulation of anti-AGE receptor-1(AGER1), sirtuin 1, and up-regulation of receptor for AGEs (RAGE) (5). AGEs affect glucose uptake, transport and promote insulin resistance in adipocytes (6). While in skeletal muscle cells AGEs inhibit insulin action, mediated through RAGE (7). The AGE-RAGE axis induces oxidative stress, activates proinflammatory pathways and has been considered as a principal pathway in the pathogenesis of diabetes and its complications (8). AGE interacts with RAGE in different cells and tissues, contributing to pathogenesis in diabetes (9). By and large, AGEs contribute to development of insulin resistance leading to diabetes, as well as in the pathogenesis of diabetic complications. Therefore, analysis of plasma AGEs can possibly provide information about the severity of diabetes.Human serum albumin (HSA), one of the most abundant plasma proteins, is highly glycated and contributes predominantly to the plasma AGEs. Apart from its role in pathogenesis, AGE-modified HSA (AGE-HSA) has been suggested as an alternative diagnostic marker to glycated hemoglobin (HbA1c) for monitoring glycemic status in diabetes (10). Although HbA1c is considered the “gold standard” marker, reflecting the glycemic status over the period of 8–10 weeks (1, 10), factors like anemia, blood loss, splenomegaly, and iron deficiency affect HbA1c levels (11). AGE-HSA reflects glycemic status over the preceding 3–4 weeks and has been recommended in gestational diabetes (12). In diabetes, the levels of AGE-HSA increase and were found to be positively correlated with hyperglycemia (13, 14). In addition, several recent studies have suggested that the levels of AGE-HSA are associated with prediabetic condition (15) and microalbuminuria (16). Therefore, quantification of AGE-HSA is of utmost clinical significance. Thus, understanding the site-specific modification and their dynamic transformation to heterogeneous AGEs is quite critical for mass spectrometric quantification.AGEs can be quantified by various approaches, including colorimetric assay, ketoamine oxidase assay, enzyme-linked boronate immunoassay, fluorescence spectroscopy, boronic acid affinity chromatography assay, and mass spectrometry (MS) (17). Among these approaches, MS offers precise characterization of protein glycation, including the amino acid involved in the modification. Most of the AGEs reported in vitro and in vivo were discovered by MS-based techniques (18). AML modification has been extensively studied by different MS approaches. The fragmentation pattern and diagnostic ions for AML rearrangement product has been well established (19, 20). Further specific neutral loss ions of 162 Da, 120 Da, and 84 Da and water loss of 36 Da arising from hexose moiety of glycated peptide were also considered as signature ions to validate the glycation of peptides in HSA (21, 22). Similar characteristic patterns of water loss (18, 36, and 54 Da) ions and immonium ions derived from lysine arising from AML-modified peptide were also used to identify glycated peptides (23, 24). Diagnostic ions serve as the most reliable way of identifying glycated peptide by tandem mass spectrometry. Thus, having a good MS/MS fragment ion is key for precise characterization of glycation. However, the ratio of in vivo AGE-modified to unmodified protein is significantly low, which limits better MS/MS. Therefore, to achieve efficient identification, enrichment of glycated peptides using boronate affinity chromatography (BAC) was adopted prior to MS analysis (25). Further, by using a combination of immunodepletion, enrichment and fractionation strategies, a total of 7,749 unique glycated peptides corresponding to 1,095 native human plasma proteins, 1,592 in vitro glycated human plasma proteins, and 1,664 erythrocyte proteins were identified (26). In these lines, we have previously reported a database search approach for the identification of glycated peptide in a crude or nonenriched sample by untargeted MS/MS or data-independent workflow (27). Glycation is chronic process; a given protein can undergo dynamic heterogeneous transformations as these proteins have varying biological lifespans, influencing the function of a protein. Thus, to assess the degree of glycation at a given pathophysiological condition, precise identification of glycation becomes critical. In this regard, a stable-isotope-dilution tandem mass spectrometry method was employed for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins (28), and ¹³C6-glucose was utilized to quantify glycated proteins in the plasma and erythrocytes (29, 30). In a recent study, the glycation-sensitive peptides of HSA that could serve as markers for early diagnosis of type 2 diabetes were quantified by using an MS-based ¹⁸O-labeling technique (31). However, most of the previous studies have focused on AML modification, rather than other AGE modification. In fact, CML and CEL are the predominant AGEs, constituting up to 80% of total AGEs (32, 33). Diagnostic reporter ions for CML and CEL were reported recently by Prof. Ralf Hoffmann''s group (34). Here, for the first time, we report comprehensive development of an MS/MS fragment ion library for AML, CML, and CEL modifications of albumin. Further, fragment ion library was used as reference for quantification of AML-, CML-, and CEL-modified peptides of albumin in clinical plasma of healthy, prediabetic, diabetic, and microalbuminuria. Targeted SWATH analysis has led to quantification of 13 glycated peptides representing nine lysine sites. These peptides could serve as novel markers in diabetes.