bluuebunny/crossref_metadata_2025_split huggingface dataset:
Are Enzymes Required for the Extraction and Isolation of DNA in Conjunction with Mechanical Homogenization?
Authors: Easparro Brandon A. , Garrett Shari , Atwood James 
Published: November 2023

Enzymes are biological catalysts that play a role in the isolation of nucleic acids from tissue and microbial sample types. In 1973, Maria Gross‐Bellard was one of the first researchers to use Proteinase K in the isolation of DNA from mammalian cells1. Today, enzymes are commonly provided in DNA isolation kits to enable cellular and organelle disruption and for the removal of contaminating proteins. DNA kit enzymes vary based on the target sample. While Proteinase K is commonly used in the isolation of DNA from mammalian cells and tissues, lyticase and lysozyme are enzymes used to degrade the cell walls of yeast and bacteria and are frequently included in microbial DNA isolation kits. While effective, enzymes represent a significant percentage of DNA kit cost and require special storage and handling conditions. While standard lysis buffers coupled with enzymes can be used to disrupt soft tissues and cells, mechanical homogenizers are routinely employed for disruption of tougher tissues and cells with robust cell walls. In these cases, are enzymes necessary to obtain high concentrations of DNA?In this study, DNA extractions were performed using commercially available DNA extraction kits from three sample types: bacteria, yeast and tissues. DNA was extracted per the kit manufacturer's instructions in conjunction with mechanical homogenization via bead milling. The analysis was then repeated without the use of enzymes. DNA concentration and quality was evaluated by spectrophotometry and electrophoresis respectively for each sample type and processing method. High quality DNA was recovered with and without the use of enzymes. Although samples with enzyme digestion brought higher yields of DNA, the integrity of DNA was equivalent in samples without the enzyme digestion. Enzyme requirement in DNA isolation is based on down stream analysis.
Dissection of DNA Synthesis in crystallo Reveals a Third Mg2+ in the Transition State
Authors: Gao Yang , Yang Wei 
Published: November 2023

A conserved two‐metal‐ion mechanism has been proposed for all DNA and RNA polymerases. However, a third divalent cation coordinated by the reaction products was recently observed during in crystallo DNA synthesis by Pol η and Pol β (ref. 1, 2). Using time‐lapse X‐ray crystallography, we show that the well established binding of two Mg2+ or Mn2+ ions by Pol η leads to alignment of the DNA substrate and the incoming nucleotide for catalysis, but acquisition of the third metal ion is necessary for the phosphoryl‐transfer reaction. The third metal ion binding site is absent in the enzyme‐substrate complexes and relies on thermal energy for its formation. Pol η appears to acquire the third metal ion as a cofactor in the transition state to overcome the energy barrier for product formation. Identification of the non‐enzyme bound but essential third metal ion in the Pol η catalysis leads us to anticipate that acquisition of additional catalytic cofactors in transition states may be a general feature of enzyme catalysis.Support or Funding InformationThis work is funded by NIH intramural program (DK036146‐08, W.Y.).
IN VITRO EVALUATION OF CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL AND TERPENES OF Satureja macrostema
Authors: Salgado‐Garciglia Rafael , Torres‐Martínez Rafael , Hernández‐García Alejandra , Saavedra‐Molina Alfredo , López‐Meza Joel Edmundo , Ochoa‐Zarzosa Alejandra 
Published: November 2023

Satureja macrostema is a Mexican medicinal plant known as nurhitini té or nurite, whose antioxidant properties are mainly attributed to the volatile compounds from aerial part. Therefore, the goal of the present study was to investigate the in vitro cytotoxic and antioxidant potential of essential oil and some related terpenes of S. macrostema. Essential oil from fresh aerial parts of S. macrostema plants were prepared by hydrodistillation and characterized by GC‐MS, finding that the major terpenes were caryophyllene, limonene, linalool, menthone, pulegone and thymol. The effects of essential oil and terpenes from S. macrostema on bovine umbilical vein endothelial cells were determined. Different concentrations of essential oil and terpenes (1, 10, 100, 1000 μM) were added to cultured cells and incubated for 24 h. Cell survival was evaluated using the MTT‐based cytotoxicity assay. The essential oil and linalool, menthone and pulegone did not affect the BUVEC viability (> 86%), while limonene, caryophyllene and thymol showed toxicity at 1000 μM, causing less than 60% viability. Nitric oxide (NO) was determined using Griess reaction method in BUVEC induced with Staphylococcus aureus and treated with essential oil and terpenes from S. macrostema. The essential oil and terpenes at 1, 10 and 100 μM showed a diminution of NO production, indicative of the antioxidant activity. In addition, antioxidant effectiveness of essential oil and terpenes was examined by two different radical scavenging methods, DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl) and ABTS (2,2′‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulfonic acid), testing 1, 10, 100 and 1000 μM. The essential oil showed the highest antioxidant capacity, reaching 53.10% capture of radicals in the DPPH and 92.13% in the ABTS method. Thymol was the terpene with higher antioxidant activity (94%) at 1 μM determined with both methods. The results obtained show that essential oil and the major terpenes of S. macrostema have antioxidant activity, at concentrations that do not compromise BUVEC viability.Support or Funding InformationFinancial support grant from CONACYT (RTM) and CIC/UMSNH (Project 2.10 to RSG and 14.1 to AOZ).
Small Molecule Binding Sites Explored in Variable Heavy Chain Antibodies
Authors: White Brandy R , Gee Shirley J , Hammock Bruce J , Brooks Cory R 
Published: November 2023

Polybrominated diphenyl ethers (PBDEs) are chemical flame retardants that have been used extensively over the last 40 years in furniture, plastics, wiring, and electronics. Some PBDEs have been classified as possible human carcinogens. PBDEs have been widely found in human blood, breast milk, and body fat. While PBDEs have recently been placed on the California Biomonitoring Program and have been banned by the EPA, these PBDEs persist in the environment. 2,2′,4,4′‐tetrabrominated diphenyl ether (BDE‐47) and Tetrabromobisphenol A (TBBPA) are two of these chemical flame retardants that are persistent in the environment and detecting them with an VHH‐based method has been developed. However, it is not well understood how these detection methods work and it is difficult to improve on the methods without this understanding. The variable heavy chain domain antibodies (VHH) from camelids have many advantages compared to conventional monoclonal antibodies including increased stability and ease of recombinant production. Although it is well established how VHH interact with proteins, it is less well understood how these antibodies interact with small molecules and haptens. There are currently four known binding modes available for VHH to interact with small molecules including: combining VHH's three CDR loops, domain swapping utilizing a 2:1 binding stoichiometry, and tunneling under the CDR1 loop. Using VHH specific for BDE‐47 and TBBPA as a model for PBDEs we aim to examine VHH small molecule interactions. We have successfully completed expression tests of seven VHH and purified 2 VHH. Crystallization trails have yielded preliminary crystals. Future work will focus on crystal optimization and structure determination.Support or Funding Information‐BW was supported with funds from the Undergraduate rGrant from The Associated Students, Inc. at California State University, Fresno.
Targeting Peptide Bound to Major Histocompatibility Complex Allows Visualization of Insulin Secreting Beta Cells in vivo
Authors: Miller Keith Russell , Taft Joseph , Koide Akiko , Johnson Mark , Tisch Roland , Koide Shohei , Collins Edward 
Published: November 2023

The breaking of T cell tolerance in autoimmune disease results in selective killing of healthy cells. In Type 1 Diabetes (T1D), autoreactive T cells use their T cell receptor (TCR) to recognize β cells by binding to the β cells’ antigen‐peptide bound to Class I Major Histocompatibility (pMHC) molecules. The interaction of the TCR with the pMHC is required for T1D to develop; however, precise mechanisms and timing of β cell destruction have not been defined. A significant problem in developing T1D therapies and studying its disease initiation and progression is the absence of a noninvasive in vivo β cell imaging agent for early diagnosis and evaluation of potential treatments. To address this problem, we used phage display technology to isolate recombinant antibody fragments (Fabs) that mimic the TCR. Surface plasmon resonance showed that two Fabs, fIGRP and fHA, that target an islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP) peptide and influenza hemagglutinin (HA) peptide respectively, restricted to the histocompatibility‐2 Kd (H2‐Kd) have nanomolar binding affinity. Using immunofluorescence microscopy, both Fabs were shown to successfully localize to β cells on cryopreserved mouse pancreas sections. The Fabs also blocked cytokine production of autoreactive T cells when presented with cognate pMHC in vitro. The ability to generate affinity reagents with high affinity and specificity for pMHC has vast potential for understanding where and when specific autoantigens are presented and by which cell types during the progression of T1D and developing targeted therapies for β cell recovery.Support or Funding InformationThis research was supported by the National Institutes of Health AI088081 (EJC), NIH R01‐GM072688 (SK), the University of Chicago Comprehensive Cancer Center supported by P30‐CA014599, the DANA Foundation (http://www.dana.org), the University of North Carolina at Chapel Hill Center for AIDS Research (CFAR), an NIH funded program P30 AI50410 and a University of North Carolina University Cancer Research Fund pilot project award.
Novel small molecule, WZB117, competitively inhibit GLUT1‐mediated glucose transport to halt cancer growth
Authors: Ojelabi Ogooluwa , DeZutter Julie , Lloyd Kenneth , Carruthers Anthony 
Published: November 2023

GLUT1‐mediated cellular glucose transport is required to fuel anaerobic metabolism in proliferating cancer cells. Accordingly, GLUT1 inhibition by small molecules is a viable strategy to halt the proliferation of cancers. However, GLUT1 is presently not targeted therapeutically in cancers because of lack of potent and specific inhibitors. Recently, a novel small molecule, WZB117, was shown to kill lung and breast cancer cells by inhibiting GLUT1‐mediated glucose transport, without affecting non‐tumorigenic cells. Here, we studied the detailed kinetics of WZB117 inhibition of GLUT1, and found that WZB117 inhibits uptake of the non‐metabolizable sugar, 3‐O‐methylglucose, by red blood cells with an inhibition constant of 2.83 μM. Our data show that WZB117 is a competitive inhibitor of GLUT1‐mediated zero‐trans sugar uptake and exchange transport but is a non‐competitive inhibitor of zero‐trans exit. These results, together with our docking studies, suggest that WZB117 binds at or near the exofacial sugar‐binding site of GLUT1. In addition, our preliminary data on the specificity of WZB117 for GLUT1 suggests that, while WZB117 inhibits GLUT1 with a high affinity, it is also a potent inhibitor of the skeletal muscle and adipose specific transporter GLUT4 and the neuronal glucose transporter GLUT3. We conclude that the ability of WZB117 to serve as a tumor‐specific inhibitor derives, not from specificity of transport inhibition but rather, from the absolute dependence of tumor cells on anaerobic metabolism and glucose‐dependent anabolism.Support or Funding InformationThis work is supported by NIH grant DK 44888.
Mitochondrial Transfer by Photothermal Nanoblade Restores Respiration in Mammalian Cells with Dysfunctional Mitochondria
Authors: Patananan Alexander Nikolich , Wu Ting‐Hsiang , Sagullo Enrico , Case Dana , Zheng Xin , Li Yanjing , Hong Jason , TeSlaa Tara , McCaffery J. Michael , Niazi Kayvan , Braas Daniel , Koehler Carla , Graeber Thomas , Chiou Pei‐Yu , Teitell Michael 
Published: November 2023

The reliable delivery of large cargo into mammalian cells could have profound impacts on cell engineering and medicine. Current techniques, such as electroporation, sonoporation, and microcapillary injection, can deliver DNAs, RNAs, and proteins into cells, but they are limited by cargo size, endosomal cargo degradation, and poor efficiency. To circumvent these problems, we invented a photothermal nanoblade. The nanoblade is a titanium‐coated glass pipet that is placed near a cell membrane and heated by a laser pulse. A resulting vapor bubble generates a transient membrane incision that allows the pressure driven delivery of cargo up to 3 μm in size into cells with high efficiency and cell viability. Here, we evaluated whether the nanoblade could transfer isolated mitochondria into cells to rescue mitochondrial dysfunction. The transfer of MDA‐MB‐453 mitochondria into 143BTK‐ρ0 cells, which lack mtDNA, do not respire, and require uridine supplementation to proliferate, resulted in the emergence of three rescued clones in uridine‐free medium 2 weeks after delivery. Interestingly, clones 1 and 3 had ATP levels, oxygen consumption rates, and ETC complex activities similar to the donor and parent cells, while clone 2 continued to have abnormal levels. qRT‐PCR of genes encoding anaplerotic and catapleurotic enzymes showed that rescue lines 1 and 3 were similar to the 143 BTK‐ parent, whereas clone 2 remained similar to the 143BTK‐ ρ0 cells. This distinction between the three clones was maintained in the metabolic profiling of TCA cycle and cytosolic metabolites by mass spectrometry. In conclusion, the ability of transferring functional mitochondria to cells by the photothermal nanoblade is not only important for basic research studies, but also has potential for therapies targeted at correcting mitochondrial diseases.Support or Funding InformationUCLA Tumor Immunology Training Grant (Ruth L. #Kirschstein Institutional National Research Service Award # T32 CA 009120)
Crosstalk Between MAP Kinase And Hedgehog Pathways by Direct Phosphorylation of Gli Transcription Factors
Authors: Bardwell Lee , Bardwell A Jane , Wu Beibei , Waterman Marian 
Published: November 2023

MAP kinase pathways are hyperactivated in many of the same tumor types in which Hedgehog signaling is deregulated. Indeed, crosstalk between the Hedgehog/Gli pathway and Ras/mitogen‐activated protein kinase (MAPK) signaling pathways occurs in many types of cancer, including pancreatic cancer, melanoma, colorectal cancer, and medulloblastoma, and may contribute to the emergence of clinical resistance to Hedgehog pathway inhibitors (reviewed in [1, 2]). However, the molecular mechanism(s) of this crosstalk are unclear. We have shown that the transcription factors Gli1 and Gli3, which are downstream targets of the Hedgehog pathway, are also efficient substrates of ERK and JNK‐family MAPKs [3]. Here we show that MAPKs phosphorylate Gli1, Gli2 and Gli3 on residues located near the binding site for Sufu, a key negative regulator of Gli function. Furthermore, we show that MAPK‐mediated phosphorylation weakens the binding of Sufu to Gli1‐3. We have mapped the MAPK phosphorylation sites in Gli1, and show that three sites are collectively necessary and sufficient for Sufu release. Cells expressing Gli1 alleles mutated in these residues exhibit perturbed Hedgehog signaling. Hence, we identify a functional mechanism of MAPK‐Hedgehog crosstalk that could contribute to developmental signaling, tumorigenesis and clinical resistance.Support or Funding InformationThis work was supported by NIH‐NIGMS grant P50‐GM76516 and by a grant from the University of California Cancer Research Coordinating Committee
Specific Inhibition of Smad3 Nuclear Translocation with TAT Fusion Peptides Prevents Profibrotic TGFβ Activity
Authors: Kang Jeong‐Han , Jung Mi‐Yeon , Yin Xueqian , Andrianifahanana Mahefatiana , Hernandez Danielle , Leof Edward B. 
Published: November 2023

Transforming growth factor‐β (TGFβ) is the prototype of a family of secreted polypeptide growth factors with key roles in cellular growth and differentiation. The primary mediators of TGFβ action are the Smad proteins, specifically Smad2 and Smad3. Upon receptor activation these 55–60 kDa proteins are phosphorylated (p) by the type I TGFβ receptor and undergo nuclear translocation to function as co‐modulators of gene expression. While the specific roles of Smad2 and Smad3 are being actively investigated, recent findings have shown that while Smad3 is clearly pro‐fibrotic/pro‐carcinogenic, Smad2 can induce anti‐fibrotic/anti‐carcinogenic responses. Thus, approaches designed to target more downstream TGFβ receptor signaling components may provide equivalent efficacy and greater specificity. To that end, as we previously determined that sorting nexin 9 (SNX9) plays an integral role in the nuclear import of pSmad3 (but not pSmad2), we have extended those findings and identified the functional element in SNX9 critical for pSmad3 binding. Furthermore, we show that a TAT‐fusion encompassing this region can act in a dominant manner to block SNX9/pSmad3 binding, pSmad3 nuclear translocation, and subsequent pro‐fibrotic responses dependent upon pSmad3 both in vitro and in vivo.Support or Funding InformationThis work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.
Structured RNAs that Manipulate the Translation Machinery
Authors: Kieft Jeff S 
Published: November 2023

Translation is a complex process that is subject to precise regulation throughout all of its phases, but the repertoire of mechanisms by which this regulation is achieved remains unknown. We are interested in understanding one particular class of regulation mechanism: how structured RNA elements embedded within mRNAs can alter or regulate translation through direct interaction with the ribosome. Such interactions can drive non‐canonical modes of translation initiation (e.g. cap‐independent or IRES‐driven translation), programmed ribosomal frameshifting, enhancement of the rate of translation, etc. Many of the most useful model systems for understanding how diverse structured RNAs can manipulate the ribosome come from viral RNAs, therefore these have been the focus of much of our research. We employ a combination of structural biology, biophysics, biochemistry, and virology. In this talk, I will present some of our latest findings and efforts to link the structure of a given RNA with its ability to manipulate the translation machinery.Support or Funding InformationHoward Hughes Medical Institute early Career Scientist AwardNational Institutes of Health R01GM081346 & R01GM097333Cancer League of Colorado
Dissecting Functions of the Turnip yellows virus Suppressor of RNA Silencing P0 in Evasion and Elicitation of Plant Defenses
Authors: Dughbaj Mansour , Valdez Kevin , Wang Ken‐Der , Nguyen Tan Tri , Oza Shyamal Vishnu , Sacco Melanie Ann 
Published: November 2023

The Turnip yellows virus (TuYV) protein P0 (P0Tu) is a viral suppressor of RNA silencing (VSR) that targets Argonaute (AGO) proteins for degradation. P0Tu is also recognized in Nicotiana glutinosa accession TW59, in which it elicits hypersensitive response (HR) and resistance to TuYV. We are interested in understanding how P0Tu interacts with the host plant cell as a VSR versus an elicitor of HR. To identify amino acids that are critical for these activities, P0Tu has been subjected to serial deletion analysis and site‐directed mutagenesis at either several conserved sites or by systematic substitutions with the 6 amino acid sequence NAAIRS along the entire length of the coding sequence. We predicted that the modifications could potentially cause loss of VSR activity in Nicotiana benthamiana and/or loss of HR activation in N. glutinosa to dissect these two functions. Mutant clones were co‐infiltrated with GFP into the leaves of N. glutinosa accession TW59 and N. benthamiana to observe for suppression of RNA silencing and induction of cell death. Substitution of two conserved central arginine residues with lysine caused P0Tu to activate more rapid and robust HRs on N. glutinosa. Deletion or mutation of the amino terminus inactivated P0Tu, while modifications within or deletion of the last 22 carboxy‐terminal amino acid residues (227–249) resulted in mutants that maintained VSR activity, but had impaired ability to elicit HR on N. glutinosa, suggesting that P0 activities in suppression of silencing and elicitation of HR may be distinct.Support or Funding InformationNational Science Foundation (NSF) Grant #1122256 to M. A. Sacco.
Exploring the Relationship between Corruption and Food Security Status on a Global Scale
Authors: Helal Gabrielle , Ahmadigheidari Davod , Kosoy Nicolas , Melgar‐Quiñonez Hugo 
Published: November 2023

Food insecurity is a global problem that has yet to be properly addressed. Its determinants are part of a greater scheme of food security governance and overall governance. Presence of corruption occurs when there are failures in governance. There are currently no studies exploring corruption and food security, on a global scale, with internationally validated tools. This study aimed to fill this gap in the literature. The main objective was to explore the relation between corruption and food security status on a global scale.Data from 2014 Gallup World Poll (GWP) were analyzed. The sample included 185,341 individuals. Food security status, the dependant variable, was assessed using the Food and Agriculture Organization's Food Insecurity Experience Scale. Corruption, the independent variable, was measured using the GWP Corruption Index. Several statistical analyses formed the basis of the current work. Cross‐tabs and logistic regression were conducted to evaluate the relationship between socio‐demographic characteristics and corruption on food security, using IBM ® SPSS ® version 23, using the complex samples module.Descriptive statistics shows that an absence of perceived corruption was significantly higher in food secure population, when compared to food insecure. Women had higher rates of food insecurity than men. Lastly, higher level of education, higher income and full‐time employment were found among food secure population. All of these results were significant.Findings using a logistic regression model show that food insecurity was significantly higher in a population that perceives corruption (OR 1.192), after adjusting for other variables. Food insecurity was significantly higher among women (OR 1.123). Also, individuals who were unemployed (OR 1.077), or had part‐time employment (OR 1.492) had significantly higher odds of being food insecure. When compared to the high‐income group, low‐income individuals had significantly higher odds of being food insecure (OR 19.498). Finally, in terms of age, food insecurity was higher in the younger population (p=0.03).The findings of this study help fuel a new approach in the global fight against food insecurity. These findings suggest that amongst diverse population demographics, an absence of corruption has a positive impact on food security. The results of this novel study will promote governmental accountability in regards to corruption, and will contribute to emerging research in the field of food security governance.