Miracle-dz/UniFAR-FTUs huggingface dataset:
Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet-B-induced CPDs in rice chloroplast and mitochondrial DNA.

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation.
Surviving meningococcal septic shock: Health consequences and quality of life in children and their parents up to 2 years after pediatric intensive care unit discharge

Objective: To assess health consequences and health-related quality of life (HR-QoL) in children with meningococcal septic shock up to 2 yrs after discharge from the pediatric intensive care unit and to assess their parents. To determine major predictors of that outcome. Patients and Methods: A prospective cohort study. Follow-up of all consecutive children with septic shock and purpura requiring intensive care treatment between 2001 and 2005, and their parents. HR-QoL was assessed with the Child Health Questionnaire (children) and Medical Outcomes Study 36-item Short-Form Health Survey (parents). Results: Of 53 eligible families, 47 participated (28 boys/19 girls; median age at the time of pediatric intensive care unit admission, 3.7 yrs; median Pediatric Risk of Mortality score, 21). A total of 26 children (55%) had scars as a result of skin necrosis; two (4%) had amputation of a digit or digits. In 21 children (45%), chronic complaints were reported. Children with and without chronic complaints did not differ significantly with regard to severity of illness and age at the time of pediatric intensive care unit admission. Significantly lower scores were found on HR-QoL scales concerning mainly physical functioning and health perception in comparison with normative data. There was a significant negative association between severity of illness and the HR-QoL scale concerning physical functioning. Children with chronic complaints had significantly lower scores on the HR-QoL scale concerning pain. Eight of 47 mothers (17%) at the time of the study had anxiety or depression requiring professional help. Mothers with and without these problems differed significantly with regard to age of their child at the time of pediatric intensive care unit admission. Parents showed significantly higher scores on HR-QoL scales concerning physical functioning and bodily pain in comparison with normative data. There was a significantly negative association between the presence of emotional problems and HR-QoL scores in mothers. Conclusions: Up to 2 yrs after discharge from the pediatric intensive care unit, there is still a considerable effect on health and HR-QoL in children, especially on the physical scales. Severity of illness and chronic complaints negatively affected HR-QoL scales in children. Quite a few mothers suffered from emotional problems.
The quality of tuberculosis diagnosis in districts of Tigray region of northern Ethiopia

Background : Low detection of smear-positive and over-diagnosis of smear-negative Pulmonary Tuberculosis (PTB) are major problems in Ethiopia. Non-adherence to diagnostic guidelines could be contributing to the poor detection of tuberculosis. Objective: To assess the quality of diagnosis based on the national tuberculosis guidelines. Methods : A retrospective diagnostic audit was made in eight districts among patients aged 815 years that were on TB treatment between 12/10/2001 and 15/05/2002. A team of three physicians reviewed patient charts, sputum microscopy and radiograph registers to assess diagnostic criteria used to each patient. Results : A total of 237 patients were reviewed: 42 were smear-positive, 101 were smear-negative PTB and 94 were extra-pulmonary tuberculosis. The diagnosis was considered correct in 33 of the 42 smear-positive PTB patients and incorrect in 9 patients. Of 101 smear-negative PTB patients, 31 (31%) were diagnosed as per the national diagnostic guideline. In more than half of patients treated for lymph node tuberculosis their diagnosis was inconsistent with the national diagnostic guideline. Conclusion : Non-adherence to the national guidelines is a major problem in district hospitals. This calls for action to promote clinicians' adherence to the national diagnostic guidelines. The Ethiopian Journal of Health Development Vol. 19 2005: 13-20
In-cell Selectivity Profiling of Serine Protease Inhibitors by Activity-based Proteomics*S

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction (“click chemistry”). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular “side effects” of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.
MR imaging of displaced meniscal tears of the knee: Importance of a “disproportional posterior horn sign”

Purpose: Meniscal tears associated with displaced fragments are clinically significant. We propose the "disproportional posterior horn sign" as a supportive criterion to identify a posterocentrally displaced meniscal fragment on MR imaging studies. If the meniscal posterior horn in the central portion appears larger than that in the peripheral section, it is considered positive for "disproportional posterior horn sign". Material and Methods: MR images obtained in 42 patients with 43 lesions, confirmed to have displaced meniscal tears, were included in this study. The MR images were retrospectively evaluated for the presence of the "disproportional posterior horn sign", as well as the other known signs. Results: The "disproportional posterior horn sign" was seen in 9 (20.9%) of 43 lesions, including 1 lateral discoid meniscal tear, 5 lateral meniscal tears and 3 medial meniscal tears. Five of them also had other signs of a displaced meniscal fragment. However, the remaining 4 cases only exhibited the "disproportional posterior horn sign". For the other MR signs, the "absent bow tie sign" was detected in 40 (93%) of 43 lesions, the "flipped meniscus sign" in 27 (62.8%) of 43 lesions, the "double posterior cruciate ligament sign" in 17 (39.5%) of 43 lesions and the "notch fragment sign" in 22 (51.2%) of 43 lesions. Conclusion: The "disproportional posterior horn sign" is helpful in demonstrating a posterocentrally displaced meniscal fragment, especially when other characteristic signs are unremarkable or absent.
Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method*S

Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli's salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli's salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets.
Identification and Characterization of Neuronal Mitogen-activated Protein Kinase Substrates Using a Specific Phosphomotif Antibody*S⃞

Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed “5557”) recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in δ-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that δ-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with δ-catenin degradation, and a δ-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.
Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides*

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.
Excretory/Secretory Proteome of the Adult Developmental Stage of Human Blood Fluke, Schistosoma japonicum*

Schistosomes are the causative agents of schistosomiasis, one of the most prevalent and serious of the parasitic diseases that currently infects ∼200 million people worldwide. Schistosome excretory/secretory (ES) proteins have been shown to play important roles in modulating mammalian host immune systems. In our current study, we performed a global proteomics identification of the ES proteins from adult worms of Schistosoma japonicum, one of the three major schistosome species. Our results unambiguously identified 101 proteins, including 53 putatively secreted proteins. By quantitative analysis, we revealed fatty acid-binding protein as a major constituent of the in vitro ES proteome. Strikingly the heat shock proteins HSP70s, HSP90, and HSP97 constituted the largest protein family in the ES proteome, implying a central role for these proteins in immunomodulation in the host-parasite relationship. Other important S. japonicum ES proteins included actins, 14-3-3, aminopeptidase, enolase, and glyceraldehyde-3-phosphate dehydrogenase, some of which have been considered as viable vaccine candidates and therapeutic targets. A comparison with previous studies suggests that 48.5% of S. japonicum ES proteins are common to other parasite ES products, indicating that the molecular mechanisms involved in evading the host immune response may be conserved across different parasites. Interestingly seven host proteins, including antimicrobial protein CAP18, immunoglobulins, and a complement component, were identified among in vitro S. japonicum ES products likely originating from the schistosome tegument or gut, indicating that host innate and acquired immune systems could defend against schistosome invasion. Our present study represents the first attempt at profiling S. japonicum ES proteins, provides an insight into host-parasite interactions, and establishes a resource for the development of diagnostic agents and vaccines for the control of schistosomiasis.
“ChopNSpice,” a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides

Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9.
Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature*

Exosomes are 40–100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.
Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants*

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.
A Solanum lycopersicum catechol-O-methyltransferase involved in synthesis of the flavor molecule guaiacol.

O-methyltransferases (OMT) are important enzymes that are responsible for the synthesis of many small molecules, which include lignin monomers, flavonoids, alkaloids, and aroma compounds. One such compound is guaiacol, a small volatile molecule with a smoky aroma that contributes to tomato flavor. Little information is known about the pathway and regulation of synthesis of guaiacol. One possible route for synthesis is via catechol methylation. We identified a tomato O-methyltransferase (CTOMT1) with homology to a Nicotiana tabacum catechol OMT. CTOMT1 was cloned from Solanum lycopersicum cv. M82 and expressed in Escherichia coli. Recombinant CTOMT1 enzyme preferentially methylated catechol, producing guaiacol. To validate the in vivo function of CTOMT1, gene expression was either decreased or increased in transgenic S. lycopersicum plants. Knockdown of CTOMT1 resulted in significantly reduced fruit guaiacol emissions. CTOMT1 overexpression resulted in slightly increased fruit guaiacol emission, which suggested that catechol availability might limit guaiacol production. To test this hypothesis, wild type (WT) and CTOMT1 that overexpress tomato pericarp discs were supplied with exogenously applied catechol. Guaiacol production increased in both WT and transgenic fruit discs, although to a much greater extent in CTOMT1 overexpressing discs. Finally, we identified S. pennellii introgression lines with increased guaiacol content and higher expression of CTOMT1. These lines also showed a trend toward lower catechol levels. Taken together, we concluded that CTOMT1 is a catechol-O-methyltransferase that produces guaiacol in tomato fruit.
Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe*

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.