bluuebunny/crossref_metadata_2025_split huggingface dataset:
Utilizing the CRISPR‐Cas System to Excise and Study the Insulin Enhancer
Authors: Yang Sophia , Rose Robert 
Published: November 2023

The discovery of the CRISPR‐Cas system as an adaptive immune system for bacteria and archaea has stimulated much excitement in the scientific press. In type two CRISPR‐Cas systems, Cas9 is the RNA‐mediated endonuclease. Using a FLAG tagged Cas9, we propose to utilize the CRISPR‐Cas9 system to excise and pull down the insulin promoter both in vitro and in vivo to study the proteins associated with the enhancer. Guide sequences needed to target the insulin enhancer have been generated and we are currently in the process of expressing and purifying Cas9. In order to pull down the excised enhancer, we must determine how Cas9 releases products. Cas9 is a unique enzyme that has single turnover and must undergo significant conformational changes during catalysis. In vitro studies indicate Cas9 does not release cleaved DNA after cutting. We will crystallize the product form of the Cas9/DNA complex to characterize the structural rearrangement. Knowing the structure of the product complex will improve our understanding of how Cas9 associates and releases DNA post‐cleavage during genome editing.Support or Funding InformationNCSU CALS Dean's Enrichment Grant – Wynne Innovation Category 07/01/14–06/30/16Enhancer Excision based on CRISPR technology: a tool for deciphering gene regulation.
Mode of Ubiquitination Determines Complete or Partial Degradation of GFP Substrates by the 26S Proteasome
Authors: Chirico Giavanna Gabriella , Kraut Daniel Adam 
Published: November 2023

The 26S proteasome is responsible for the bulk of intracellular protein degradation in eukaryotes. Most proteins are targeted to the proteasome by the addition of one or more polyubiquitin tags. K11‐and K48‐linked chains typically signal for degradation in the cell, although K63‐linked chains can also target proteins to the proteasome in vitro. After binding to the proteasome via the ubiquitin tag and engaging proteasomal ATPases via an unstructured region, substrates are deubiquitinated, processively unfolded, translocated into the core of the proteasome, and degraded into small peptides. The factors that allow the proteasome to successfully unfold a folded domain (or prevent it from doing so) are still poorly understood. Green Fluorescent Protein (GFP) is commonly used, both in vivo and in vitro, as a model substrate to follow protein degradation. However, there are also reports that GFP can be difficult to unfold and degrade, perhaps because of its stable β‐barrel structure. In particular, Saeki et. al. reported that GFP resists degradation when tagged with K63‐linked polyubiquitin chains, while other proteins were degraded efficiently. To investigate whether chain linkage or mode of ubiquitination affected the proteasome's ability to unfold and degrade GFP, we constructed a degron that could be ubiquitinated with either the E3 ligase Rsp5 (reported to give K63‐linked chains) or the E3 ligase complex Cul3/Rbx1/Keap1 (reported to give primarily K48‐linked chains) and attached it to a GFP domain. Although full‐length substrates ubiquitinated with either E3 were degraded by the proteasome, degradation of the Rsp5‐ubiquitinated substrate led to the production of a large protein fragment containing the GFP domain while degradation of the Keap1‐ubiquitinated substrate proceeded without fragment formation. Our results suggest that the ability of the proteasome to unfold its substrates is tuned by the specific polyubiquitin modifications used to target it to the proteasome, and give additional insight into why K63‐linked chains are not typically used for proteasomal degradation in vivo.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1515229
Conformational Switching in Proteasomal ATPases Allows for Proper Substrate Processing
Authors: Snoberger Aaron M. , Smith David M. 
Published: November 2023

Virtually every cellular process relies on properly regulated protein degradation, and thus, improper regulation of this degradation can lead to a wide range of human diseases (e.g. Neurodegeneration, Cancers, Cardiomyopathies). The ubiquitin‐proteasome system is responsible for the majority of targeted degradation within eukaryotes, so it is critical to understand how this system functions to degrade proteins. However, due in part to the size and complexity of the proteasome, we still do not understand how it functions at a molecular level. The 26S proteasome from eukaryotes is a 2.5 megadalton macromolecular complex composed of 2 subcomplexes: a hollow 20S core particle with protease sites sequestered on its interior, and a 19S regulatory particle that caps one or both ends of the 20S. Proteins destined for degradation (e.g. polyubiquitinated proteins) bind and are recognized by the 19S regulatory particle, and then in an ATP‐dependent manner are unfolded and injected into the 20S core particle, where proteins are then degraded. The 19S ATPase is a heterohexamer, and although each of the 6 subunits are highly similar to one another, recent cryo‐EM structures reveal that that these subunits form an asymmetric “lockwasher” conformation. However the structural elements that give rise to this asymmetric conformation are unknown, and may come from the hetero‐oligomeric subunit arrangement of the ATPase ring. Furthermore, cryo‐EM structures reveal different conformations of the 19S ATPases in the ADP (i.e. ATPh) or ATPgS‐bound states. In the present study, we employ an exhaustive crosslinking approach to show that Proteasome Activating Nucleotidase (PAN), a homolog of the 19S ATPases, adopts similar conformations as the 19S ATPases in the ATP‐ and ADP‐bound states, even though PAN is composed of 6 identical subunits (i.e. a homohexamer). Thus, the asymmetrical state of the proteasomal ATPases appears to be an inherent property of the ATPase subunit and its allosteries that arise from oligomerization rather than the heteroligomeric nature of the 19S ATPases. Most interestingly, in the conformationally restricted variants that we designed we observed functional defects when the subunits are prevented from switching out of the different nucleotide‐bound conformations, suggesting that the asymmetrical subunit switching observed in the cryo‐EM structures is part of the proteasomal ATPase's normal mechanochemical cycle that allows for proper processing of substrates.Support or Funding InformationNIH‐R01GM107129, NIH‐F31GM115171
Resveratrol Modulates Cytokine Expression in LPS‐induced Human Monocytes: Role of Proteasome Subunits
Authors: Silswal Neerupma , Reddy Nidhi , Qureshi Nilofer 
Published: November 2023

Ubiquitin proteasome system (UPS) maintains the basic cellular processes of the eukaryotic system by catalyzing the degradation of proteins. The proteolytic active sites of proteasomes reside on three important constitutively expressed subunits: X (β5), Y (β1) and Z (β2) which exhibit chymotrypsin‐like (CT), post‐acidic (PA) and trypsin‐like (T) activities respectively. The constitutive subunits of proteasomes (P) can be induced to those containing immunosubunits (immunoproteasomes, IP) LMP7 (β5i), LMP2 (β1i) and LMP10 (β2i) after priming with LPS or cytokines. We have reported earlier that trans‐resveratrol decreases LPS‐induced inflammatory cytokine levels and proteasome enzyme activities by inhibiting NF‐κB activation in mouse macrophages. Recently, there has been an increased interest to develop natural, less toxic and potent proteasome inhibitors to reduce inflammation. Our objective here was to compare the effect of resveratrol and a known LMP7 inhibitor (ONX‐0914, obtained from UBPBio) on proteasome's activities in human monocytes/macrophages. Using fluorescence‐based assays, resveratrol (0–100 μM) inhibited all three protease activities of purified human blood IP and P, although predominantly IP as compared to P (n=2). In contrast, ONX‐0914 was more selective and potent for CT activity of IP and P (n=2). Similarly, using cell‐based luminescence assay (THP1, monocytes), resveratrol inhibited all three protease activities. Moreover, resveratrol (Ki 80 μM) and ONX‐0914 (Ki 0.25 μM), both inhibited expression of LPS‐induced inflammatory cytokines TNFα and ICAM1, as well as immuno‐proteasome subunits LMP7 and LMP2 in THP1 and CD14+ blood monocytes (p < 0.05, n=3). We observed that 90% cells (THP1) survived even at 80 μM dose of resveratrol, while < 50% survival was observed with 10 μM dose of ONX‐0914 after trypan‐blue staining as well as MTT‐assay. Collectively, our data suggest that resveratrol is an inhibitor of all three; CT (predominantly), T and PA protease activities and is less toxic to human monocytes/macrophages than ONX‐0914. In addition, resveratrol decreases the LPS‐induced inflammatory cytokine expression by decreasing the expression and activities of IP. Therefore, resveratrol, as a proteasome inhibitor can be used to curb inflammation in diseased states.Support or Funding InformationThis work was supported, in whole, by the National Institutes of Health Grants 1 R01 GM102631‐01 to Dr. Nilofer Qureshi.
Finding the Elusive Pathway to Promotion as an Educator
Authors: Lambert H. Wayne , Zdilla Matthew J. , Klinkhachorn Penprapa S. 
Published: November 2023

Most faculty members are required to meet specific benchmarks in their yearly academic assignment in the areas of research/scholarship, teaching, and service. Teaching faculty are charged with providing the majority of the student instruction and support within an academic department, and these educators often struggle to find the time and a venue to produce the required scholarship to achieve promotion to the next academic rank. Therefore, this career development talk will highlight important steps an educator can take to achieve career success both personally and professionally.Teaching faculty can follow varied pathways while progressing through the Promotion and Tenure Process. Most often, designing impactful scholarly projects around the teacher's personal passion is the best place to start. Activities, such as publishing in scholarly peer‐reviewed journals, developing effective teaching and assessments tools to be published in peer‐reviewed educational repositories like MedEdPortal, becoming involved with textbook projects, taking on administrative positions pertinent to the success of your department or school, and/or becoming a leader in national and international professional organizations, are a great place to carve out your own niche to derive satisfaction and enjoyment from your career.General themes of this talk will include: 1) identifying mentors that can provides tools to ensure your career success; 2) attending professional meetings to share your scholarly endeavors; 3) building collaborative relationships with fellow faculty members; and 4) recognizing your career goals may change as you adapt to internal and external pressures that exist both within and outside your institution. The goal of this talk is to help teaching faculty advance their academic careers by unlocking their potential and seizing opportunities that will arise in teaching, scholarship, research, and service, while avoiding obstacles (or pitfalls) that are often present.
Downregulation of the G6PDH Pathway Can Promote Oxidative Stress‐Mediated Reversal of Drug Resistance in B‐Cell Lymphoma cell Lines
Authors: Mousavi‐fard Seyed Hossein 
Published: November 2023

The Nrf2/G6PDH pathway plays a critical role in antioxidant defenses and cell signaling. A low oxidative stress state due to upregulation of Nrf2/G6PDH Pathway in cancer cells is one mechanism underlying acquisition of resistance to CHOP‐based chemotherapy in DLBCL.Normally under oxidative stress, Nrf2 activates expression of the glucose‐6‐phosphate dehydrogenase (G6PDH) gene, which leads to increase production of reducing equivalents (NADPH) through the pentose phosphate shunt. NADPH is then utilized to increase production of reduced glutathione (GSH) to balance the redox state. The present study explored the role of the Nrf2 downstream pathway, G6PDH, in CHOP resistance in DLBCL cells.The G6PDH inhibitor, DHEA, increased oxidative stress as evident by increases in ROS in CHOP‐resistant DBLCL cells. DHEA not only repressed G6PDH activity, but also G6PDH protein expression after 16 hrs. In addition, DHEA treatment decreased levels of NADPH and glutathione (GSH). Moreover, DHEA markedly increased sensitivity of chemoresistant cells to CHOP.In conclusion, repression of the G6PDH pathway leads to decreases in both NADPH and GSH, and increases in ROS (oxidative state), thereby sensitizing chemoresistant cells to chemotherapeutics.Abbreviations used: Nrf2, Nuclear factor erythroid–related factor 2; G6PDH, glucose‐6‐phosphate dehydrogenase; CHOP, (C)yclophosphamide, (H)ydroxydaunorubicin, (O)ncovin, (P)rednisone; DLBCL, Diffuse large B‐cell lymphoma; NADPH, Nicotinamide adenine dinucleotide phosphate; GSH, Glutathione; DHEA, didehydroepiandrosterone; ROS, reactive oxygen species;Support or Funding InformationDepartment of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843‐1114, USA
Identification of small molecule inhibitors of the LINE1 endonuclease
Authors: Meyers Jade , DeFreece Cecily 
Published: November 2023

Long interspersed element‐1 (L1) is the only active autonomous retroelement in the human genome. Studies characterizing L1 function show that the L1 endonuclease activity is associated with the formation of DNA double strand breaks (DSBs). While L1 mutagenic insertion events have been associated with diseases such as breast and colon cancer as well as muscular dystrophy, the full effect of L1 endonuclease upon stability of the genome is undetermined. Data suggests that the damage from L1 endonuclease activity may be greater than previously considered seeing as though only a small fraction of L1‐induced DSBs results in a retrotransposition event. We have utilized an in vitro fluorescence based assay to identify novel small molecule inhibitors of the L1 endonuclease. A short term cellular viability assays was also used to determine the toxicity of the drugs to cells. A cellular retrotransposition assay was performed to determine if the drugs prevent LINE1 retrotransposition. We have identified three small molecules that inhibit L1 endonuclease activity in our fluorescence‐based assay. The drugs also prevent L1 retrotransposition but do not appear toxic under the conditions examined. This study shows that novel small molecule inhibitors have been identified that can be further modified to create potent and selective inhibitors of the L1 endonuclease.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.
Novel Cell‐Penetrating Peptides Overcome Endosomal Escape and Deliver Protein Cargos into the Cell
Authors: NGWA Verra , Salerno John , Nowak Scott , Chrestensen Carol , McMurry Jonathan 
Published: November 2023

Over the last decade a number of peptides that are rapidly internalized by mammalian cells have been discovered or designed. Cell‐penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargoes to the cell interior. We have developed a novel CPP‐adaptor protein technology that allows any user‐defined cargo delivery and release into the cytoplasm. Our hypothesis is that a CPP‐adaptor with a moiety allowing high‐affinity but reversible non‐covalent cargo binding would lead to more efficient penetration and release than current CPP delivery strategies. Delivery of proteins to the interiors of cells has many applications. In addition to detecting and mapping the location of the components of living cells with fluorescent tags in real time, the availability of our system will enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers.CPP‐adaptors and cargo proteins (horse radish peroxidase, myoglobin and Beta‐galactosidase) were expressed in and purified from E. coli BL21 (DE3)pLysS. Optical biosensing experiments demonstrated that affinity and kinetics between the novel CPP and cargo proteins did not significantly differ from wild‐type interactions; all had subnanomolar affinities. Cargo proteins were labelled with DyLight 550. CPP‐cargo complexes or cargo alone were incubated with subconfluent baby hamster kidney (BHK) cells. After washing, cells were imaged by fluorescence confocal microscopy. All user define cargos exhibited penetration and release to the cytoplasm whereas cargo‐only controls exhibited no measurable penetration (though some adherence to the outside of the cells was observed.) Time courses and dose‐dependency studies characterizing penetration and release kinetics will be presented as well as initial efforts to deliver cargo that will alter cell‐signaling pathways will be described.The present results demonstrate the feasibility of delivery of a wide variety of cargo proteins and open up an array of potential research, diagnostic and therapeutic applications.Support or Funding InformationNIH grant R25GM111565
Regulation of the Unfolded Protein Response by Fic‐mediated Adenylylation/AMPylation
Authors: Mattoo Seema , Sanyal Anwesha 
Published: November 2023

Maintenance of ER (endoplasmic reticulum) homeostasis is a critical aspect of determining cell fate, and requires a properly functioning unfolded protein response (UPR) to combat such stress. We have discovered a hitherto unknown role for adenylylation/AMPylation, a post‐translational modification that involves the covalent attachment of an adenosine monophosphate (AMP) to the target protein, in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this reaction. A single Fic protein, called HYPE (Huntingtin yeast interacting protein), exists in humans. We reported that the ER molecular chaperone, BiP, is a target of HYPE‐mediated adenylylation. BiP serves as a sentinel for activating UPR and maintaining ER homeostasis by monitoring protein folding in cells. Our data indicate that HYPE localizes to the ER lumen where it interacts with BiP and adenylylates it, to alter BiP's ATPase activity in vitro. Further, HYPE expression increases upon induction of UPR stress. Accordingly, we demonstrated that cells knocked down for HYPE are unable to mount an appropriate UPR and are more susceptible to stress induced cell death. As such, HYPE represents a new player in UPR signaling. Here, we assess the cellular consequences of BiP adenylylation during UPR progression and describe the structural and kinetic parameters that govern the HYPE‐BiP interaction.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Core Pilot Grant, 2) a Showalter Research Trust Award, 3) an American Cancer Society (ACS) Institutional Grant, and 4) the National Institute Of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.
Obesity Risks Associated with Low‐ and High Birth Weight and They Varied by Population Obesity Prevalence: A systematic Review and Meta‐analysis
Authors: Song Taixia , Wang Youfa , Huang Wenjie , Lu Lu , Zhao Yong , Sharma Manoj , Zhang Yong 
Published: November 2023

ObjectivesThis systematic review examined obesity susceptibility in populations with different obesity prevalence, assessed obesity risk associated with low‐ and high birth weight( ie, tested “fetal origins” of obesity). Nutrition affects birth weight and obesity, while during prenatal stage it may modify metabolisms in that fetus can adapt to maternal nutritional status. Therefore, individual with different birth weight might have different susceptibility to obesity and its susceptibility might change in different nutrition conditions.METHODSPubmed and Embase databases were searched for relevant studies. Relative risk (RR) of overweight/obesity (OW/OB) compared with normal birth weight was analyzed by description and meta‐analysis. During meta‐regression, the OW/OB prevalence of samples was introduced to analyzing of association of birth weight and risk of OW/OB.RESULTSForty‐one studies met inclusion criteria. The pooled RR of OW/OB for low birth weight (LBW) was 0.80 (95%CI, 0.75–0.84, I2=73.0%), and 1.52 (95%CI, 1.45–1.60, I2=87.2%) for high birth weight (HBW). Meta‐regression showed that RR of OW/OB for LBW was positively associated with OW/OB prevalence (β=0.496, SE=0.244, p=0.046), and, for HBW, it was negatively associated with OW/OB prevalence (β=−0.722, SE=0.268, p=0.009).CONCLUSIONSLBW was associated with lower risk of subsequent OW/OB; the relation was stronger in countries with high obesity prevalence. HBW incases risk of subsequent OW/OB, and the relations was weaker in countries with high obesity prevalence. The findings support “fetal origins” hypothesis in obesity development.Support or Funding InformationNational Natural Science Foundation of China (No. 30901189) and NIH U54 center grant, HD070725
Effect of Daily Iron Supplementation in Healthy Exclusively Breastfed Infants: A Systematic Review with Meta‐Analysis
Authors: Cai Chenxi , Friel James 
Published: November 2023

BackgroundMaintaining adequate iron is critical to infants’ physical and neurological development. The literature on iron requirements of exclusively breastfed infants contains conflicting data and contrary views. We summarized the evidence for benefits and risks of daily oral iron supplementation with regard to hematologic, growth, cognitive parameters and adverse effects in exclusively breastfed infants.MethodsStructured electronic searches were conducted until August 26, 2015. We searched PubMed, Cochrane Library databases and Google Scholar for randomized controlled trials (RCTs) involving daily iron supplementation in healthy term, exclusively breastfed infants. Random‐ and fixed‐effects models were used to calculate the pooled estimates. Heterogeneity was assessed by Q‐test.ResultsFour RCTs with 511 infants were included in the meta‐analysis. Iron interventions had no significant effect on iron deficiency and iron deficiency anemia (OR=0.56; 95% CI, 0.23–1.33), serum ferritin level, or hemoglobin level. Iron interventions did result in a significant increase in Bayley psychomotor developmental indexes in later life (MD= 7.00, 95% CI, 0.99 to 13.01) and mean corpuscular volume (MD=2.17 fL; 95% CI, 0.99–43.5 fL). Iron supplementation was associated with slower growth in the exclusive breastfeeding period, but its long‐term effect is unclear.ConclusionsLimited available evidence suggests that daily iron supplementation has beneficial effects on the hematologic parameter and later cognitive development, but delays the physical growth in healthy term, exclusively breastfed infants. There was no evidence to suggest iron supplementation could cause adverse effects.