bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mass Spectrometry Based Proteomic Analysis of Saliva from Children with Autism Spectrum Disorder (ASD) and Matched Controls
Authors: Wormwood Kelly L , Charette Laci , Ryan Jeanne P , Woods Alisa G , Darie Costel C 
Published: November 2023

Approximately 1/68 children are believed to be affected by Autism Spectrum Disorder (ASD). A survey study suggests numbers as high as 1/50. With the increased diagnosis of ASD, treatment and understanding of the disorder is a pressing health concern. Protein biomarkers found for ASD may be used for ASD diagnosis, subtyping, monitoring, treatment, identifying therapeutic targets and may also provide clues about the causes of ASD. Here, saliva samples from 14 children with ADOS‐2 diagnosed ASD and 14 age and gender matched controls were analyzed using a combination of gel electrophoresis, in gel digestion or in solution digestion and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate differences between the proteomes of ASD subjects and matched controls. These results will hopefully shed light on possible causes of ASD and confirm our previous findings. In a preliminary 6 vs. 6 analysis using saliva from males, in solution digestion and analysis identified numerous proteins involved in immune responses to be dysregulated. Two of these proteins were lactotransferrin, involved in gastrointestinal antimicrobial activity, and prolactin inducible protein, involved in immune responses, which were upregulated in ASD compared to controls. Additional dysregulated proteins were also detected in blood serum, suggesting an enhanced response to environmental toxic factors. This investigation is ongoing.Support or Funding InformationASPIRE Fellowship, Shire, T. Urling and Mabel Walker Research Fellowship Program, SUNY Plattsburgh Presidential Research Award
The Potential Urinary Aging Markers from 20‐month‐old Rats
Authors: Li Xundou , Gao Youhe 
Published: November 2023

Different organs may have different aging processes. Is it possible to find markers of aging from different organs?Biomarker is measurable changes associated with physiological and/or pathophysiological conditions. Urine can be better source for biomarker discovery because it accumulates the changes of body while body removes them from blood by homeostatic mechanisms.The urinary proteome is influenced by various factors, which is a major challenge in urinary biomarker discovery. To circumvent these problems, simpler systems, such as animal models, should be used to establish direct associations between physiological or pathological conditions and changes in the urinary proteome.In this study, the urinary proteome of young (2‐month‐old) and old rats (20‐month‐old; 9 in each group) were analyzed using LC‐MS/MS (liquid chromatography‐mass spectrometry) and quantified using the Progenesis LC‐MS software. A total of 371 proteins were identified, 194 of which were shared between young and old rats. Based on the criteria of a fold change ≥ 2, P < 0.05 and being identified in each rat in the high abundance group, 33 proteins were changed (15 up‐regulated and 18 down‐regulated in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), 8 proteins were changed consistently in all rats of between the groups, 2 of which are also altered in the urinary proteome of aging humans. There are no shared proteins between our results and the previous aging plasma proteome. Twenty of the 33 (60 %) changed proteins have been reported to be disease biomarkers, which implies that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs, which are strongly expressed in the kidney, intestine, cerebellum and lung, according to the human protein ATLAS. Therefore, the urinary proteome may reflect aging conditions in these organs. More age groups should be included in the study if more mass spectrometry time could be available for this study.Support or Funding InformationThis work was supported by the National Basic Research Program of China (2012CB517606, 2013CB530805, 2014CBA02005 and 2013FY114100).
New serum biomarkers of blood coagulation function for pulmonary tuberculosis using iTRAQ‐coupled 2D LC‐MS/MS
Authors: Jiang Tingting , Wei Li‐Liang , Shi Li‐Ying , Wang Chong , Xu Dan‐Dan , Chen Zhong‐Liang , Ping Ze‐Peng , Liu Chang‐Ming , Li Ji‐Cheng 
Published: November 2023

This study aimed to discover the novel protein biomarkers of blood coagulation function for pulmonary tuberculosis (TB) patients. Differentially expressed proteins of TB patients were screened and identified with the application of iTRAQ‐coupled 2D LC‐MS/MS technique. Clinical data of 204 TB cases, and their correlation with serum abnormal proteins were analyzed by statistical methods. A diagnostic model was constructed, and the accuracy was analyzed by receiver operation characteristic (ROC) curves. A total of 79 differentially expressed proteins were identified in the serum of TB patients, including 50 up‐regulated proteins and 29 down‐regulated proteins. Proteins closely related to clotting such as serum amyloid A (SAA) (P<0.0001), vitamin K‐dependent protein Z (PROZ) (P<0.0001), and C4b‐binding protein β chain (C4BPB) (P<0.0001) were significantly higher in TB patients than the healthy controls. Clinical analysis showed that coagulation indexes of TB patients, including PT, PT%, INR, FIB, APTT, and TT were significantly different with healthy controls. Correlation analysis revealed that PROZ was significantly correlated with PT% and INR, and C4BPB was significantly correlated with FIB. ROC curve analysis showed that the area under curve (AUC) value of the diagnostic model combined by SAA, PROZ, and C4BPB was 0.977 with 93.0% sensitivity and 93.1% specificity to discriminate TB patients from healthy controls. The results suggest that SAA, PROZ, and C4BPB may be potential biomarkers of blood coagulation function for TB.Support or Funding InformationThis work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Special Sci‐Tech Projects (No. 2012ZX10005001‐006), and National Natural Science Foundation of China (No.81273882, NO. 81573709).
Analysis of Fluorescent Proteins for the Development of an In Vivo System for Protease Evolution
Authors: Selby Thomas Lee 
Published: November 2023

The objective of this study is to develop a robust in vivo system using E. coli cells for screening protease activity based on intracellular Förster (fluorescence) resonance energy transfer (FRET). Toward this end, we expressed and purified the fluorescent proteins GFP, Orange‐FP (OFP), Clover, Ruby2, and td‐Tomato as single polypeptides, and as fusion partners linked by protease target sequences, to evaluate their stability, fluorescence intensity, chromophore maturation, kinetics, and FRET properties under various expression and reaction conditions. GFP and Clover showed similar spectroscopic properties when produced by constant induction, or mid‐log phase induction. However, the spectroscopic properties of OFP, Ruby2, and td‐Tomato varied significantly based on their induction conditions, cell density, and growth media. These differences appear to be related to the degree of maturation of the chromophore, which required optimization to achieve maximum FRET, but resulted in aggregation for some of the proteins under certain conditions. The rate of chromophore maturation in vitro varied depending on conditions and/or the presence of a fusion partner. In fusion constructs, the fully matured, fluorescent proteins demonstrated a constant decrease in FRET during proteolysis of the target sequence. However, interpretation of the kinetic profiles in vitro was complicated by samples with incomplete maturation. This complication is believed to be due a simultaneous decrease in FRET signal upon proteolysis, as well as an increase in FRET occurring during chromophore maturation. While the Clover‐Ruby2 fusion protein demonstrated a FRET efficiency that was superior to the GFP‐OFP protein, the hydrophobic Ruby2 protein was prone to aggregation under certain reaction conditions, resulting in a loss of fluorescent signal, challenges during purification, and difficulty interpreting kinetic results. Td‐Tomato, a less hydrophobic protein, gave lower yields during purification, and matured in vivo at a much slower rate than Ruby2 and OFP. In conclusion, these results revealed variable fluorescent properties for different fusion partners investigated in vitro, while providing data for comparison when optimizing fluorescent protein expression, and kinetics, during in vivo screening, with the ultimate goal of developing a system that will utilize fluorescent activated cell sorting (FACS) to evolve proteases with novel specificities.Support or Funding InformationThis work was supported by the National Institutes of Health under grant GM080691 and Mercer University School of Medicine, Division of Basic Medical Sciences.
Peptide‐induced inhibition of ATP Synthase as an alternative approach to combat microbial infections
Authors: Ahmad Zulfiqar 
Published: November 2023

BackgroundATP synthase is the fundamental means of cellular energy production. Inhibition of ATP synthase can deprive cell of required energy resulting in cell death. A wide variety of cationic α‐helical peptides are known to bind and inhibit ATP synthase. Our lab has studied the inhibitory effects of a variety of peptides with anti‐microbial and anti‐tumor properties on Escherichia coli ATP synthase. Peptide‐induced inhibition of E. coli ATP synthase establishes that the anti‐microbial and anti‐tumor effects of peptides are at least in part linked to the blocking of ATP synthesis in tumor and bacterial cells.MethodWild‐type E. coli strain, pBWU13.4 growth properties were checked on fermentable glucose and non‐fermentable succinate carbon sources before harvesting cells in minimal media to isolate ATP synthase. Peptide‐induced inhibitory studies were performed on membrane bound F1Fo ATP synthase. An additional positive charge in the form of a c‐terminal NH2 group was added to augment the extent of inhibition. Abrogation of cell growth was tested in presence and absence of peptides along with null control pUC118 strain.ResultsPeptides caused variable degree of inhibition. Potency of inhibition was found to be related to the complexity and number of positively charged residues. It was observed that the presence of a c‐terminal NH2 group on the peptides enhances the inhibitory potency on molar scale. Our studies also demonstrated that peptides follow a differential pattern of inhibition. These results confirm that incremental addition of positively charged residues can augment the inhibitory effects of peptides by about 100‐fold. Moreover, growth of wild‐type E. coli was fully abrogated while no effect was observed on the growth of null cells in presence of peptides. Thus, making ATP synthase a potent molecular drug target for anti‐microbial and anti‐tumor peptides. Currently, we are studying the inhibitory effects of venom peptides from different sources along with synergetic inhibitory effects of peptides on ATP synthase.ConclusionsWe conclude that cationic α‐helical peptides are potent inhibitors of E. coli ATP synthase. Our data suggests that the inhibitory potency of peptides can be enhanced by inserting additional positive charges including c‐terminal amide group. Finally, the selective inhibition of bacterial ATP synthase is an alternative novel approach to comate microbial infections.Support or Funding InformationNational Institutes of Health Grant GM085771
N‐terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme and Neprilysin
Authors: Kuruppu Sanjaya , Rajapakse Niwanthi , Kleifeld Oded , Small David H , Whisstock James , Smith Alexander Ian 
Published: November 2023

Neprilysin (NEP) and endothelin converting enzyme (ECE) are two enzymes that clear amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49‐P1‐20, from the venom of Bothrops asper which directly enhances the activity of both ECE and NEP. This is evidenced by a 1.8‐ and 5.6‐fold increase in the Vmax of ECE and NEP respectively. The K49‐P1‐20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE and NEP was 4.2 ± 0.2 and 2.9 ± 0.3 ng/μL respectively. Increase in activity is the result of a conformational change in the enzyme that occurs upon K49‐P1‐20 binding. Therefore, K49‐P1‐20 is both an excellent research tool to study enzyme activation, and a potential novel drug lead in the fight against Alzheimer's disease.Support or Funding InformationThis work was supported by funding made available through 1) the strategic grant scheme administered by the Monash University's Faculty of Medicine Nursing & Health Sciences; 2) Monash University's Interdisciplinary Research Funding Scheme; and 3) ANZ National Medical Foundation
Effect of the heavy metals cobalt and cadmium on LINE1 endonuclease activity
Authors: Ahmad Idrees , Smith Michaela , Meyers Jade , Russell Brittany , Dorrah Kimberly , DeFreece Cecily 
Published: November 2023

Long interspersed element ‐1 (LINE‐1) is a mobile element, or jumping gene, which employs a “copy and paste” method called retrotransposition to insert itself into the human genome. This retrotransposition mechanism utilizes an enzyme called the L1 endonuclease to initiate the double strand breaks necessary for LINE‐1 insertion. Studies have found that a significant portion of the human genome is composed of mobile elements, which have likely played a crucial evolutionary role in the current genetic variability observed in humans. However, in some instances the mutations that have arisen from increased LINE‐1 retrotransposition, have been linked to genetic diseases such as colon cancer, breast cancer, and have been linked to genome instability due to increased double strand breaks. Previous research suggests that heavy metals such as Ni2+, Co2+, and Cd2+, which occur in our industrialized environment as pollutants, potentially affect levels of LINE‐1 retrotransposition. These toxic metals have been observed to compete with Mg2+ as cofactors for proteins, and have been observed to inhibit essential reactions in DNA repair. Competition with Mg as a cofactor suggests these heavy metals likely have an effect on levels of LINE‐1 endonuclease activity in vitro since it also utilizes Mg2+ as a cofactor for enzymatic activity. We focused on the effects that the heavy metals cobalt and cadmium have on the LINE‐1 endonuclease activity to determine whether or not these metals also influence levels of double strand breaks in vitro. Preliminary results indicate that the metal cobalt does not prevent LINE‐1 endonuclease activity at doses up to 100uM.Support or Funding InformationThis work was funded by an Institutional Development Award (IDeA) from the NIGMS under grant number P20GM103424 and from the Louisiana Cancer Research Consortium, the NIH‐RCMI grant #8G12MD007595‐05.
Structural and inhibitor screening studies of a fungal form of aspartate semialdehyde dehydrogenase from Cryptococcus neoformans
Authors: Dahal Gopal P , Viola Ronald E 
Published: November 2023

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH‐dependent reductive dephosphorylation of β‐aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow selective targeting of pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. The structure of a new fungal form of ASADH from Cryptococcus neoformans has been determined to 2.6 Å resolution. The overall structure of CnASADH is similar to that of its bacterial orthologs, but with some critical differences both in biological assembly and in secondary structural features that can potentially be exploited for the development of species selective drugs with selective toxicity against only certain infectious organisms. A customized fragment library has been screened against several fungal ASADHs and initial hits have been identified with inhibition constants (Ki) in the low millimolar range. The ASADH obtained from this fungal species have shown different inhibition selectivity when compared to this enzyme from bacterial species and also differences between fungal species. Docking studies with the CnASADH structure are being used to guide the design and development of potent inhibitors. The long term goal of this project is to develop drugs that selectively target this enzyme and block the aspartate biosynthetic pathway.Support or Funding InformationThis work is supported by funding from the NIH (AI077720).
Functional Studies of Thiosemicarbozone Compounds Against Topoisomerase IIα
Authors: Brown Anthony Ray , Ngo Lana , Morris William , Medawala Wathsala , Khoo Vivien , Lisic Edward , Jiang Xiaohua 
Published: November 2023

Topoisomerase IIα is known to play a vital role in cell proliferation of all cells, especially cancer cells. It has been a popular target for the production of new drugs to combat cancer. This study looks at the inhibition of Topoisomerase IIα with a variety of thiosemicarbazone, such as ATZ ligands with different metals complexes. We studied three groups of ATZ compounds including ATZ‐ligand without metal, ATZ‐Copper, and ATZ‐Palladium complexes. In each of those groups, six different modifications on the side chain are examined. The compounds were tested using cleavage assays for stimulation of the relative DNA cleavage by Topoisomerase, which is a very important indicator for the potentiality to be an anticancer drug. Compounds that showed promising stimulation were selected and tested further in a dose dependent cleavage assay. Our results show that ATZ‐Copper complexes exhibited greater stimulation on Topoisomerase II. And Cu(ATZ‐ETSC)Cl is twice as effective as Etoposide‐ a leading anticancer drug in targeting Topoisomearse II.Support or Funding InformationThis project is funded by URECA! Undergraduate research program at Tennessee Tech University
Investigation of Solvent Hydron Exchange in the Reaction Catalyzed by the Antibiotic Resistance Protein, Cfr
Authors: Bauerle Matthew Richard , Grove Tyler L. , Booker Squire J. 
Published: November 2023

Cfr is a radical SAM methylase that catalyzes the radical‐mediated appendage of a methyl group at C8 of adenosine 2503 in 23S ribosomal RNA (rRNA). Methylation of C8 by Cfr confers resistance to several classes of antibiotics that bind to the peptidyl transferase center of the bacterial ribosome. RlmN, a homologous RS methyltransferase, exclusively methylates C2 of A2503, which tunes the ribosome's interaction with nascent peptide chains. Cfr can also catalyze methylation at C2 in vitro, but it is much slower than methylation at C8. The Cfr and RlmN reactions take place via a cross‐linked intermediate between the terminal carbon of a methylcysteine residue on Cfr or RlmN and the carbon atom undergoing methylation on the RNA. This intermediate is resolved by abstraction of the C8 proton during C8 methylation or the C2 proton during C2 methylation. However, the identity of the active site base that mediates this abstraction at C8 has not been determined. EPR and crystallographic studies on the C118A variant of RlmN provided evidence that C118 is the operative base in the RlmN reaction. Previous studies using deuterium‐labeled substrates showed that the C2 proton is transferred to the nascent methyl group without isotope dilution during C2 methylation, while partial solvent hydron exchange takes place during C8 methylation. These observations led to the proposal that a polyprotic base is responsible for abstracting the C8 proton to initiate resolution of the cross‐linked intermediate. Herein, using deuterated substrates in conjunction with mass spectrometry and EPR we provide evidence that C105 of Cfr, the cognate residue to C118 of RlmN, deprotonates C8 of A2503.Support or Funding InformationThis work was supported by NIH Grant GM 101957 (S.J.B.)
Search for New Pro‐apoptotic agents in Indigenous Antitumor Plants in Nigeria
Authors: James Ayorinde Babatunde , Saibu Gbemisola Morounke , Magbagbeola Olubunmi Abiola , Omilabu Sunday Aremu , Meyer Mervin 
Published: November 2023

Due to severe cytotoxicities exacerbated by cytotoxic drugs, newer therapies from natural products are desirable. This work aimed to partially purify pro‐apoptotic agents from indigenous natural products and to investigate the mechanism of action of the selected plant fractions.Sixteen indigenous medicinal plants were collected and fractionated into hexane (HF), chloroform (CF), ethylacetate (EAF), methanol (MF) and water (WF) fractions using solvent‐solvent partitioning and polyamide adsorption chromatography. The resultant ninety‐six test fractions were screened for lethality potential using brine shrimp lethality (BSL) assay. Fractions with LC50 less than 10 ug/ml were subjected to Water Soluble Tetrazolium (WST‐1) cytotoxicity assay and apoptosis evaluations using: morphological assessment, flow cytometric analysis of phosphatidylserine externalization (Annexin V) and propidium iodide (PI) staining, caspase 3 enzyme assay, immunoblot assessment of Poly ADP‐Ribose Polymerase‐1 (PARP‐1) Cleavage, carboxymethyl‐2′,7′‐dichlorodihydrofluorescein‐diacetate (CM‐H2DCFDA) fluorescent microscopic/microplate analysis of reactive oxygen species generation, and Terminal deoxynucleotidyl transferase (Tdt) mediated dUTP‐biotin Nick End Labelling (TUNEL) assessment of DNA fragmentation.Following BSL assay, C. afer (EAF), P. guineense (HF), P. guineense (CF), A. virides (EAF), C. roseus (CF), Z. zanthoxyloides (HF) and Z. zanthoxyloides (EAF) had LC50 values < 10 μg/ml. WST‐1 Cytotoxicity testing of these hit fractions on HeLa (Tumourigenic) and KMST‐6 (Non tumourigenic) cell Lines confirms their cytotoxic effects with the exception of Z. zanthoxyloides (HF). Significant (P<0.05) selective cytotoxic preference for HeLa to KMST‐6 Cell lines was exhibited by P. guineense (CF) and C. roseus (CF). Membrane blebs were observed in HeLa cells treated with the hit fractions in comparison to 17.42 μg/ml(50μM) Camptothecin. Annexin V‐PI analysis reveals that Z. zanthoxyloides (EAF) and P. guineense (HF) showed similar apoptotic effect in comparison to 21.8% apoptotic population in Camptothecin. A significant (P<0.05) increase in caspase‐3 enzyme activity was observed in C. roseus (CF) and Z. zanthoxyloides (EAF) in comparison with the negative control. PARP‐1 cleavage was confirmed in all the test groups except for A. virides (EAF) treated cells.Findings in this study suggest that all the hit test fractions elicited apoptotic effects but at varying degrees with Z. zanthoxyloides (EAF) showing the most prominent effect. While A. virides (EAF) elicits its cytotoxic effect via necrosis.