bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structural Recognition of G‐quadruplex DNA by PC4: How Does a ssDNA Binding Protein Decide?
Authors: Griffin Wezley C. , Gao Jun , Byrd Alicia K. , Chib Shubeena , Raney Kevin D.
Published: November 2023

Over the past two decades, guanine‐rich nucleic acids have received considerable attention due to their potential to fold into a secondary DNA structure that has been termed a G‐quadruplex. G‐quadruplex sequences are distributed throughout the genome in distinct, non‐random locations such as the promoters of proto‐oncogenes and telomeric regions of chromosomes. Consequently, because of their non‐random location within the genome G‐quadruplex structures have been implicated in the regulation of genomic processes and their mis‐regulation can result in detrimental disease phenotypes such as cancer or neurodegenerative disorders. G‐quadruplexes are primarily formed by appropriately spaced di‐ or tri‐ guanine nucleotide repeats that self‐associate through Hoogsteen hydrogen bonding to form a planar G‐quartet. G‐quartets then stack onto one another, aided by the stabilization of monovalent cations, such as K+ or Na+ to form a complete G‐quadruplex. Utilizing the G‐quadruplex forming sequence from the c‐MYC gene promoter, we identified positive coactivator of transcription (PC4), a single‐stranded DNA (ssDNA) binding protein, as a novel G‐quadruplex interactor that binds with a low nano‐molar Kd value of ~2 nM, similar to that observed with ssDNA. Based on the structural differences between G‐quadruplex and ssDNA, we surmised that substantial differences might exist between the binding mode for each DNA form. Thus, we set out to investigate what differences exist between these two complexes. Equilibrium binding experiments with alternative G‐quadruplex structures did not influence the Kd value for the binding interaction. Fluorescence resonance energy transfer (FRET) stopped‐flow unwinding experiments revealed that formation of the PC4‐G‐quadruplex complex does not result in complete destabilization of the G‐quadruplex structure. Intrinsic protein fluorescence quenching experiments revealed the stoichiometry of the PC4‐G‐quadruplex complex to be a 1:1 ratio. Mutation of key amino acid residues involved in DNA binding revealed a difference in the requirement for the base‐stacking residues W89 and F77 in ssDNA and G‐quadruplex DNA binding. Taken together, these results provide a novel mode of binding by PC4 through the recognition of DNA secondary structure. The binding data indicates that PC4 can interact with G‐quadruplex DNA in a manner distinct from its binding to ssDNA, consistent with a potential role for this activity in vivo.

Support or Funding Information

This work was supported by NIH Grant R01 GM098922 and the Department of Biochemistry and Molecular Biology, UAMS.
Integrative Teaching of Breast Histology in Clinical Case‐Based Learning
Authors: Leo Joyce Mo‐Ting , Naus Gregory , Ionescu Diana , Hayes Malcolm , Kuo Kuo‐Hsing
Published: November 2023

The objective of integrative teaching of breast histology aims to improve learning efficiency for medical students in both content knowledge and clinical application.

The traditional content structure of Histology is designed to assist students in acquiring in‐depth knowledge of cell and tissue in a systemic order. However, current trends in medical curriculum are headed towards clinical presentation‐based formats to better assist students’ in developing critical thinking abilities under clinical settings. The system‐based content structure of Histology has therefore required revision to focus on presenting content relevant to clinical applications. The topic of breast pathology was identified for a pilot project to develop an integrative model of histology teaching. In the traditional system‐based structure, the topic of breast histology was either delivered within the systems of reproduction or endocrine, which lacked correlation with clinical applications and negatively impacted the effect of learning. With the integration of breast pathology, the learning experience of breast histology is expected to be more meaningful.

A team of histologists and breast pathologists developed an integrative module of breast histology and pathology, incorporating content knowledge from histology and pathology for a clinical approach.

The delivery of the integrative module contains two stages: (1) on‐line self‐learning and (2) clinical‐case discussion, which are designed to fulfill the purpose of developing content knowledge, while further fostering critical thinking. The on‐line component feature the designs of outcome‐based learning with escalating levels of knowledge. The outcome‐based design aims to prepare students with competent levels of knowledge for case discussion and to further enhance learning experience during the clinical application. The design of escalating knowledge levels allows students to learn based on individuals’ learning pace as well as allowing for real‐time review of fundamental material. The integrative teaching model is currently undergoing the piloting phase.

Support or Funding Information

The Teaching and Learning Enhancement Fund/University of British Columbia
SP101, a novel synthetic compound reduces the drug resistance of EGFR (T790M) and cancerous stemness in non‐small cell lung carcinoma
Authors: Chao Jui‐I
Published: November 2023

Epidermal growth factor receptor (EGFR) mutation at T790M and cancerous stemness have shown the drug resistance of non‐small‐cell lung cancer (NSCLC) in lung cancer therapy. Development of novel drugs in the overcoming drug resistances in the NSCLC therapy is highly desired. SP101 is a novel gefitinib derivative, which can bind the ATP‐binding pocket of EGFR to inhibit its EGFR kinase activity. Interestingly, SP101 has the ability to reduce the drug resistances of EGFR (T790M) and cancerous stemness in NSCLC cells. SP101 displays the anticancer ability in the gefitinib‐resistant EGFR (T790M) of H1975 cells. SP101 inhibited the survivin protein expression and induced the caspase 3 activation and poly ADP‐ribose polymerase (PARP) protein cleavage for apoptosis induction. Moreover, SP101 was effective on the reduction of cell viability in the A549‐ON cells, which expressed Oct4 and Nanog proteins in lung cancer cells displaying cancerous stemness and drug resistance. SP101 inhibited the protein expression of Nanog and Oct4. Taken together, this study demonstrates that SP101 is a novel potential compound in the overcoming drug resistance of NSCLC therapy.
Effect of Smallanthus sonchifolius (Ss) leaf extract on glucose metabolism in mice fed a high fructose diet (HF)
Authors: de Melo Nunes Núbia Alexandre , Simoni Eduardo Bassi , Oliveira Michael Eder , Chagas Viviany Santos , Oliveira Letícia Fernandes , Santos Maria Emilia Soares Martins
Published: November 2023

A high fructose diet (HF) might play a role in the onset of metabolic disorders and excess weight gain. Usually, the consumption of this diet can induce insulin resistance and increase intrahepatocellular lipids. Smallanthus sonchifolius (Ss) leaf have been studied for their hypoglycemic activity in diabetes type I models. It seems that effect could be related with the high content of phenolic compounds and their in vitro antioxidant activity. For most of the studies using Ss, the leaf extract was obtained using classic techniques of extraction such as maceration followed by organic extraction and evaporation. During this manipulation process, several active substances can be lost. In this study, our Ss extract was obtained using a spray drying encapsulation technique with the intuit to prevent as much as possible the bioactive loss. The aim of this work was evaluate the Ss extract capacity to prevent disorders in glucose metabolism (in a diabetes type II model). Male mice (n=6/group) were fed with a control (C) or high fructose (HF‐ 30%) diets for 8 weeks (C,HF). They also received regular water or a Ss enriched water for the same period (CS, HFS). Food (g) and water or tea intake (ml) were measured daily. Body weight (g) was evaluated weekly. After 8 weeks, non fasting blood glucose levels (GLU) (mg/dl) were measured using a commercial kit. Glucose tolerance test (GTT) was also performed. A basal blood sample was obtained after a 6 hours fasting. A bolus of glucose (2g/kg) was then ip administered. Blood samples were acquired at 30, 60, and 120 min following the glucose bolus for measurement of blood glucose using a glucometer. Results were expressed as mean ± SEM. Data were analyzed using T‐test. P<0.05 was considered significant for all data.

No difference was observed in body weight, food and water or Ss water intake in any group. There was an increase of ~30% in HF(403.2) vs C (311.0) and a decrease ~13% in HFS (348.9) vs HF (403.2) at GLU. No difference in GTT was observed between HF and HFS at 0, 30 and 60 minutes. However, a decrease ~9 % HFS (285) vs HF (311) was observed at 120 minutes.

In conclusion, we observed that Smallanthus sonchifolius was able to prevent glucose metabolism alterations also in a model of diabetes type II. We believe that more pronounced results could be achieved if we increase the dose and also the period of Ss enriched water administration.

Support or Funding Information

CNPq, Labtest
Radiology: a Powerful Ally of Human Anatomy in Medical Education
Authors: Silva Vivian Alessandra , Vilela Daniel Mendonça , Ribeiro Fábio , Regacini Rodrigo , Curcio Daniella Franco
Published: November 2023

The undergraduate medical curriculum is constantly changing to absorb the exponentially growing knowledge generated by advances in science and technology, particularly in medicine and associated health care fields. This increasing demand for adaption has pushed many educational settings worldwide to experiment and develop innovative pedagogic strategies, in order to add clinical perspective to the traditional knowledge of gross anatomy, and thus enhance student's learning experience. In countries where new teaching methods are replacing human cadaver dissections, the use of imaging exams has become a valuable resource for students to understand human body structures and functions. This study reports the perception of first‐year medical students regarding the inclusion of radiology modules in the Gross Anatomy course of a Brazilian medical school. One hundred students volunteered to answer a survey comprised by 30 questions, using a 5‐point Likert scale. The majority of the students reported a positive outcome of exploring radiology concepts in the anatomy classes (84%). The experience was considered fun (67%), stimulating and challenging (73%). The participants agreed that the use of imaging modules has facilitated learning (96%) and engagement in active search for further and complementary knowledge (69%). Most participants agreed that the strategy has stimulated the study of clinical applications of anatomical concepts, which made them feel more prepared to interact with patients in the future (78%), and helped them to understand the significance of human anatomy in medical clinical practice (93%). After the radiology modules, 71% of the students felt more capable of identifying structures and 69% reported increased interest in imaging diagnosis as a future practice and specialization (69%). The use of radiology images was perceived as a useful resource to learn human anatomy (87%), because it has raised students’ interest in anatomy (77%) and made them more open and prone to acquire new vocabulary, memorize facts and comprehend concepts (74%). Overall, 84% of participants consider that the radiology modules has facilitated learning and helped them to retain anatomical knowledge. An early overview and introduction to diagnostic imaging was perceived as an exciting experience, stimulating students to engage in proactive learning actions, both individually and in teams. The clinical knowledge from radiology was perceived as an excellent complement to the often arid area of Gross Anatomy, especially in the absence of dissection practice of human bodies in lab.

Support or Funding Information

CNPq ‐ Brazilian National Counsel of Technological Development
FRET Cytometric Method for High Throughput Screening of Potential Metabolic Inhibitors in Trypanosoma brucei
Authors: Voyton Charles Michael , Morris Meredith T , Ackroyd P. Christine , Morris James C , Christensen Kenneth A
Published: November 2023

The infectious form of Trypanosoma brucei survives in the mammalian host by converting blood glucose into ATP in a specialized peroxisome‐like organelle called the glycosome. Compartmentalization of glycolysis and the requirement of glucose as the sole carbon source for parasite survival make glucose metabolism and hexose transport in T. brucei an important area of investigation and a promising pathway for targeted anti‐parasite therapy. Here we describe the application of a genetically encoded fluorescent biosensor (FLII12Pglu‐600u) which we endogenously expressed in the cytosol of the infectious bloodstream form (BSF) and in the glycosome of the insect intermediate procyclic form (PCF) allowing for quantification of cytosolic and glycosomal glucose concentration. This methods utilizes flow cytometry for high throughput analysis, as opposed lower throughput microscopy methods typically used with fluorescent protein sensors. Using this method we have measured glucose flux in live BSF and PCF cells showing that under normal growth conditions glycosomal glucose is nearly ten times that of the cytosol. Cellular glucose concentrations were monitored in the presence of candidate glycolytic and hexose transporter inhibitors allowing for inhibition efficacy to be scored. Using this methodology we have now developed high throughput screening (HTS) assays for inhibitors of glucose metabolism and hexose transport in PCF and BSF parasites. The screen allows scoring of 250 test compounds per hour and a robust Z’‐values of 0.56 and 0.74 for BSF and PCF assays respectively. We have demonstrated that flow cytometric analysis of FRET biosensors can serve as a potent platform for analysis of metabolite flux in live T. brucei as well as implementing this strategy in a high throughput screen for molecules that inhibit glucose metabolism and transport in live T. brucei.

Support or Funding Information

R21AI105656 from NIH and Brigham Young University
Thermogenic adipocytes regulate axonal growth, innervation, and thermogenic remodeling of white adipose tissue via retinoic acid‐ephrin A5‐dependent pathway
Authors: Ziouzenkova Ouliana , Shen Qiwen , Rumana Yasmeen , Popovich Phillip
Published: November 2023

Innervation regulates lipolysis, thermogenesis, and vascularization in white adipose tissue (WAT) and undergoes pathological changes in obesity. It is widely believed that mechanisms responsible for innervation during WAT remodeling depend on the central nervous system or neural precursor cells.

Objective

We test thermogenic adipocytes in WAT regulate neuronal growth.

Methods

We examined this hypothesis using gene expression analysis, neronal and adipose co‐cultures in vitro. We also used diet induced obesity models including a xenograft model inducing thermogenesis in WAT in obese mice.

Results

We showed that thermogenic adipocytes in WAT efficiently stimulate neurite outgrowth in vitro and innervation in vivo. We identified their axon guiding secretome that includes ephrin A5 and A4, semaphorins and adamtS9 protease. Diet‐induced and genetically obese mice have decreased expression of these axon guiding molecules in WAT. However, engraftment of encapsulated Aldh1a1−/− adipocytes in intra‐abdominal WAT rescues diminished innervation in obese WT mice. The gain of function studies identified ephrin A5 as an inducer of innervation. Ephrin A5 and A4 are under the control of aldehyde dehydrogenase 1a1 (Aldh1a1) and retinoic acid which represses expression of ephrins and axon growth.

Conclusions

Our data support a neuroregulatory role of thermogenic adipocytes and a mechanism for WAT innervation via bidirectional communication between thermogenic adipocytes and the nervous system.

Support or Funding Information

This research was supported by the American Egg Board (20020728), from Novo Nordisk Pharmaceuticals (10040042), and the Ray W. Poppleton Endowment (P.G.P., J.M.), and NSF Center funding (EEC‐0914790). The project was supported by NIH grants R21OD017244 and R01NS047175 (P.G.P., J.M.), and by the National Center for Research Resources UL1RR025755, UL1TR001070, and NCI P30CA16058 (OSUCCC), and supported by the NIH Roadmap for Medical Research.
Reactive Sulphydryl Groups present in Horse (Equus Ferus Caballus) Carbonmonoxyhaemoglobin by Titrating with 5, 5′‐Dithiobis‐2‐Nitrobenzoate (DTNB)
Authors: Iheagwam Franklyn Nonso , OMOTOSHO Omolola Elizabeth , Oloruntola Ayobami , Edobor‐Osor Abiola , Omotosho Temidayo Victor , Chinedu Shalom N
Published: November 2023

This research was carried out to determine the number of reactive sulphydryl groups in horse (Equus ferus caballus) haemoglobin. Haemolysate was prepared from the horse blood. The haemoglobin was separated into major and minor fractions using carboxymethyl cellulose (CMC‐52). Each haemoglobin fraction, in phosphate buffer pH 7.6, was accurately measured into several clean, dry test tubes. Increasing volumes of stock 5,5′‐dithiobis‐2‐nitrobenzoate (DTNB) were added to the different test tubes and left to equilibrate for 3 hours. Absorbance of each solution in each test tube was read at 412 nm. A graph of change in absorbance against the volume of DTNB was plotted; maximum change in absorbance was obtained at the point where the graph levels off. The ratios of 5‐thio‐2‐nitrobenzoate (TNB) concentration to the concentration of haemoglobin tetramer (Hb4) were calculated and plotted against the volume of DTNB. The number of sulphydryl groups reacting with DTNB was found to be two in both the major and minor haemoglobin.

Support or Funding Information

Covenant University, Ota.
Reinforcement of medical Spanish through bilingual presentation of anatomical terminology using dual language labeling of virtual anatomical images
Authors: Ontiveros Steven J , Rollag Mark D , Ketchum Robert J , Donohue Miriam L , Howell Sherri J , Vilchis Hugo , Minugh‐Purvis Nancy
Published: November 2023

Traditionally, one of the primary contributions of gross anatomy to medical school curricula has been the introduction and development of proficiency in medical terminology and parlance. Training at the Burrell College of Osteopathic Medicine, situated in the Borderland region of the US southwest, includes serving communities in which Spanish is the majority language. In preparing primarily English speaking students for clinical rotations in such communities, Burrell's DO curriculum is being designed to contain formal coursework as well as a variety of additional opportunities for exposure to medical Spanish, culminating in a required history and physical conducted entirely in Spanish by students during their second year of study. Burrell's gross anatomy virtual instructional presentation within an integrated, spiral systems‐based curriculum, affords a unique opportunity to imbed Spanish (and other languages) within gross anatomy teaching media. Using the Anatomage platform, separate labeling systems will enable users to toggle between languages, thus facilitating an association between a visual image of a structure and its name in either language. Images will include cross‐sectional and 3‐D male and female cadaver images using the Anatomage system, as well as diagnostic patient images imported into the system. Such additional exposure to Spanish will not only contribute to student competency in Spanish, but also complement other areas of the preclinical curriculum designed to provide students with cultural awareness enabling them to interact and communicate with their Spanish‐speaking patients with sensitivity, ultimately creating a more trusting physician‐patient relationship.
In Utero Electroporation(IUE) and mouse genetic model approach to assess PITP‐dependent inositol lipid signaling in NSC pool maintenance in embryonic brain
Authors: Hur Seong Kwon , Xie Zhigang , Bankaitis Vytas
Published: November 2023

Inositol (Ins) and phosphoinositide (PIP) signaling pathways are major intracellular regulatory systems of eukaryotic cells. Phosphatidylinositol transfer proteins (PITPs) execute novel modes of inositol lipid signal control by tightly channeling PIP production to specific biological outcomes. PITPα and PITPβ are highly expressed in mouse embryonic neocortex. We exploit an in utero electroporation approach to investigate the role of PITP‐dependent inositol lipid signaling in the embryonic neural stem cell (NSC) pool. We found that the combination of a PITPα null mouse line and PITPβ silencing evokes a dramatic depletion of NSC pools in embryonic brain. And, we finally have generated PITPαflox/flox and PITPβflox/flox single mutants, and PITPαflox/flox, PITPβflox/flox double mutant mouse line. In an experiment using Emx1‐Cre driver, which is forebrain specific driver, eviction of both PITPα and PITPβ in forebrain leads to a mouse that is born but has an amazing microcephaly that is due to virtual loss of the forebrain. Neither PITPα nor PITPβ eviction alone has any such effect. This experiment result is consistent with In utero electroporation data. Thus, we demonstrate that PITPα and PITPβ redundantly control NSC homeostasis in mouse brain development. Based on these data, we will observe carefully daughter cell fate during NSC division to investigate whether PITPs deficiency defeats NSC division program, asymmetric self‐renewing/differentiating cell divisions. Moreover, we will exploit our unique library of mutant PITPs with defined biochemical defects, to assess whether PtdCho‐ and PtdIns‐binding are required for PITP function in embryonic NSCs. Our research will generate completely new information regarding how lipid signaling contributes to NSC homeostasis in the developing mammalian brain.

Support or Funding Information

NIH 1R01GM112591‐01A1
Acute exercise rescues high fat diet induced reductions in PGC‐1alpha protein content in mouse cortex
Authors: MacPherson Rebecca EK , Wright David C
Published: November 2023

High fat diet induced obesity and insulin resistance have been directly implicated in the neuropathology of Alzheimer's disease. Specifically, previous work has demonstrated that a HFD suppresses PGC‐1alpha in male mice, leading to a down‐regulation of estrogen receptor alpha and increased inflammation. Exercise can improve neuroinflammation but the underlying mechanisms remain unknown. The purpose of this study was to determine the effects of a single bout of exercise on PGC‐1alpha, ER‐alpha, and markers of inflammation in brains from mice fed a high fat diet. Male C57BL/6 mice were fed a low (LFD, 10% kcals from lard) or a high fat diet (HFD, 60% kcals from lard) for 7wks. HFD mice underwent an acute bout of exercise (treadmill running: 15m/min, 5% incline, 120min) followed by a recovery period of 2h. The HFD increased body mass and glucose intolerance (both p<0.05). The HFD resulted in a decline in both PGC‐1alpha and ER‐alpha protein content (p<0.05). There were no diet‐induced alterations in either PGC‐1alpha or ER‐alpha mRNA expression. Similarly, the HFD did not alter the mRNA expression of inflammatory markers (IL‐6, TNF‐alpha, or IL‐1beta). Acute exercise rescued PGC‐1alpha protein content (p>0.05) and increased ER‐alpha protein content. Exercise did not affect gene expression of PGC‐1alpha, ER‐alpha, or inflammatory markers, 2 hours post‐exercise. This indicates post‐translational modification of PCG‐alpha and ER‐alpha proteins post‐exercise. Our findings demonstrate for the first time that an acute bout of exercise can increase PCG‐1alpha and ER‐alpha protein content in obese male mice.

Support or Funding Information

Rebecca EK MacPherson is supported by a Postdoctoral Fellowship from the Alzheimer's Society of Canada
Using Optical Structural Maps to Improve the Canine Genome
Authors: Silver Kristopher , Coleman Michelle , Biswell Rebecca , Lillich James , Brown Susan J
Published: November 2023

Genome models have almost unlimited potential for understanding the biology and origins of disease. Efforts on the human genome have resulted in significant advances in human medicine and understanding of human biology. Similarly, recent efforts have resulted in the completion of a high‐quality, sequence‐based assembly of the genome of the domestic dog (Canis lupus familiaris). Though comprehensive, the canine genome assembly, based solely on sequence data, may have misassembled contigs and scaffolds in addition to having multiple gaps in it (more than 3000 sequences are still unanchored to the assembly). We are validating the current genome assembly and incorporating orphaned sequences into the dog genome assembly by using next generation mapping technology to create a structural map based on detection of fluorescent tags in ultra‐long molecules of DNA. The original sequence of the canine genome was based on a purebred boxer named Tasha. Accordingly, we selected three closely related boxers (a dame and two sibling offspring) to produce optical maps as similar to each other and Tasha as possible for maximum resolution. Ultra‐high molecular weight DNA from these three individuals was labeled at BspQ1 sites and imaged in nanochannels designed to contain single DNA molecules. Whole genome restriction maps were produced by overlap layout consensus for comparison to the CanFam3.1 assembly. Our results lay the groundwork for improving the canine genome assembly and for developing this technology as a potential tool for identifying significant chromosomal alterations that are associated with disease in dogs.

Support or Funding Information

This work was supported by a grant from Morris Animal Foundation.
Optimization ratio of ethanolic extract of Quercus acutissima, Castanea crenata and Perilla frutescens var. acuta using response surface methodology
Authors: Lee Young Seok , Yang Sung‐Yong , Nam Mi‐Hyun , Lee Kwang‐Won
Published: November 2023

The Korean traditional food materials like Quercus acutissima, Castanea crenata and Perilla frutescens var. acuta were well known as antioxidant‐rich plants (i.e., Quercetin, Rosmarinic acid). We want to know whether the ratio of these materials was high antioxidant activities. In this study, the central composite design (CCD) were used to set up 17 ratios of antioxidant extract from Quercus acutissima (X1), Castanea crenata (X2) and Perilla frutescens var. acuta (X3). The dried food materials were extracted in 50% EtOH, followed by refluxing at 60°C for 4h and then cooled. The undissolved remains were removed by filtration through a Whatman 2 filter paper. The filtrate was concentrated rotary vacuum evaporator, and then immediately freezed in deep freezer, followed by drying in a freeze dryer. The ratio of extract was optimized by measured antioxidant activities from 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay, Ferric‐reducing power (FRAP) assay, Total polyphenol content assay and 2, 2‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical scavenging assay. The results were analyzed by using response surface methodology (RSM) to show that the optimal antioxidant extract ratio obtained as follows: X1 : X2 : X3 = 11 : 1: 7. The ratio of extract containing three plants set by RSM can be applied to standard process in industry.
Acylation of SOD1 Provides Tool to determine if Mitochondrial Aggregation of SOD1 is Main Driver of ALS
Authors: Banks Courtney , Rodriguez Nathan , Gashler Kyle , Bikman Benjamin , Andersen Joshua
Published: November 2023

Most cases of ALS are sporadic but about 10% are familial (referred to as fALS), caused by mutations in a few known genes, including the ROS‐scavenging enzyme superoxide dismutase Cu/Zn (SOD1). These fALS‐linked mutations in SOD1 have no consistent effect on SOD1 ROS‐scavenging but rather are thought to cause a poorly understood toxic gain‐of‐function, believed by some groups to be an aggregation of mutant SOD1 in mitochondria that leads to neuronal death. However, the link between SOD1 mitochondrial aggregation and ALS is based on correlative data and has yet to be proven experimentally. Our preliminary experiments identified a novel mode of post‐translational SOD1 regulation by acylation (both acetylation and succinylation) at lysine 123 (K123). We observed that while acyl‐mimicking SOD1 mutants, K123E and K123Q, do not alter SOD1's ability to dimerize or scavenge ROS, they do abolish SOD1 mitochondrial localization even when combined with the fALS‐causing G93A mutation that is known to aggregate in the mitochondria. We also found that the acyl‐mimicking mutants of SOD1 abolish SOD1's ability to suppress respiration. In yeast, SOD1 is thought to suppress respiration in the cytosol through stabilization of casein kinase‐γ1 and the WNT pathway. However, the fact that the acyl‐mimicking mutants of SOD1 do not import into the mitochondria and also cannot suppress respiration suggests that SOD1's anti‐respiratory effect may be linked to SOD1's accumulation in mitochondria. We are now using the acyl‐mimicking SOD1 mutants as tools to determine if mitochondrial aggregation of mutant SOD1 is the main driver of ALS and if SOD1 mitochondrial localization is necessary for its anti‐respiratory activity.

Support or Funding Information

Simmons Center for Cancer Research, BYU Internal Funding
Metal Specificity of Protein Kinases ‐ Implications in Pseudokinase Function
Authors: Herberg Friedrich W. , Knape Matthias J.
Published: November 2023

cAMP‐dependent protein kinase (PKA) is regulated primarily in response to extracellular physiological signals, while nucleotides and metal ions may provide fine‐tuning inside the cell.

PKA can use different metal ions for phosphoryl transfer, yet some, like Ca2+, do not support steady‐ state catalysis. Surface Plasmon Resonance (SPR) was used to study how metals can act differentially as a result of their inherent coordination properties. Ca2+ works in a single turnover mode to allow for phosphoryl‐ transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with substrate in the presence of Mg2+ATP. This allows us to depict the entire kinase reaction including complex formation, transition state as well as release of the phosphorylated substrate. In contrast to Mg2+, Ca2+ apparently slows down the enzymatic reaction. Ca2+ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg2+. We therefore hypothesize that the physiological metal ions Mg2+ and Ca2+ both play a role in kinase activity and regulation under different circumstances. In a more detailed approach, the role of the metal binding sites 1 and 2 were also investigated in the context of pseudokinase function. The invariant DFG‐Asp184, coordinating both the lynchpin and the low affinity metal site, and the secondary metal binding site Asn171, were modified using site directed mutagenesis to mimic the situation in pseudokinases.
Engineering Protein Matrices for Production of β‐like Cells for Treatment of Type 1 Diabetes
Authors: Chigumba Desnor Nicole , Tirrell David A , Kozlowski Mark T
Published: November 2023

Type 1 diabetes is a chronic condition in which patients cannot produce their own insulin. Previous studies have shown that it is possible to manipulate post‐natal progenitor cells to differentiate directly into β‐cells for transplantation by using laminin and methylcellulose to provide the appropriate biochemical and mechanical cues respectively. By using engineered protein hydrogels with an encoded laminin sequence and leucine zippers, we can make a material that provides both cues. As the cell culture is 3‐dimensional, this would enable us to suspend the cells in solution. Our primary objective was to produce a hydrogel which would be soluble at 37°C. Previous protein constructs were not soluble and we suspect this was because they had low LCSTs. Therefore, we altered the sequence to increase solubility. We cloned a PC10‐lam‐C10P protein where the C domain is hydrophilic and does not have an LCST. PE6‐lam‐E6P, PE6‐trunc lam‐E6P and PE6‐lam‐E6P C3A were also constructed as modifications of a previous elastin PEP construct. All constructs were soluble and MALDI‐TOF MS suggests we obtained our protein of interest. All constructs are sent to City of Hope Hospital for cell culture studies.

Support or Funding Information

California Institute of Technology SURF program
Mutation of the Omega‐Loop Impacts Stability and Function of Human Cytochrome C
Authors: Saxena Manoj , Ramirez‐Paz Josell , Ponce De León Guzmán Solimar L. , Sharma Rohit kumar , Griebenow Kai
Published: November 2023

Cytochrome c (cyt c) is an evolutionarily conserved globularprotein often used as a model for studying the protein stability and folding. Although its role as an electron carrier in the electron transport chain is well documented, findings about its role as a protein that could trigger apoptosis upon its release from mitochondria have reignited interest in cyt c structure and function. We have studied the role of two alanine‐to‐cys mutants that were designed in the omega loop of the cytochrome c. The importance of this loop is evident from the finding that the only reported natural variants associated with disease in human cyt c are found in this loop. Also, in vitro studies have shown that mutations in this loop impact the apoptotic function of cyt c. Our DSC data suggests that the A44C and A51C mutants are less stable than the wild type protein. A44C being least stable while the A51C's stability being intermediate between A44C and the wild type, as judged by the decline in their Tm. Additionally, the preliminary results from the in vitro cell free caspase 3 activity assay indicate altered apoptotic activities of these mutants. Results from the secondary structure comparison using CD suggests that the secondary structure of these mutants is not changed much while a change in the tertiary structure is clearly evident. We propose that on mutating Ala to Cys at these locations a possible conformational change occurs which could facilitate the change in caspase activity while decreasing the overall stability of the protein.

Support or Funding Information

This work was supported in part by the Institute for Functional Nanomaterials (NSF Cooperative Agreement 1002410)
Immunochemical and biochemical characterization of immunogenic proteins of Giardia lamblia
Authors: ROMERO GLORIA CAROLINA LOPEZ , SAMANIEGO RAUL IGNACIO RASCON , LOPEZ MARIA ALEJANDRA VALDEZ , LIZARRAGA THANIA MARIA GARZON , SOTO BRENDA GUADALUPE SAMANIEGO , DE JESUS QUINTERO VARGAS JAEL TERESA , CORONA IVAN ANDURO , GARCIA HUMBERTO ASTIAZARAN , CONTRERAS CARLOS ARTURO VELAZQUEZ
Published: November 2023

Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. It is known that to eliminate the parasite from host intestine is necessary the activation of B‐cell and T‐cell dependent mechanisms. However, little is known about which Giardia antigens stimulate effective immune responses. Our research group, using a model of G. lamblia infection in C3H‐heJ mice, previously described a Giardia protein of 70 kDa (5G8 protein) which was strongly recognized by serum from infected mice. This antigen is expressed both intracellular and on the surface of the trophozoite. Mass spectrometry analysis (ESI‐MS/MS) indicated that 5G8 could be the Binding Immunoglobulin Protein (BIP), a heat shock protein of 70 kDa (HSP 70). The main aim of this study is determine whether the Giardia 5G8 antigen is the BIP protein. We synthesize a recombinant BIP protein (BIPr) in E. coli system and it was purified by immobilized metal affinity chromatography (IMAC). To identify BIP protein, we generated polyclonal antibodies by immunizing C3H‐heN mice with BIPr protein. Finally, we analyze the expression profile of both proteins on Giardia trophozoites by immunochemical and flow cytometry assays. The immunochemical assays showed a differential profile of recognition in the protein extracts of two strains of G. lamblia: GS/M83‐H7 and GS/M83‐H7 5G8 (+). The monoclonal antibody 5G8.B5 recognized a 70 kDa band in the protein extract of Giardia GS/M83‐H7 5G8 (+), while BIP recombinant protein was not recognized. On the other hand, anti‐BIP polyclonal antibodies reacted with a 70 kDa protein band of both Giardia strains at a similar level. Additionally, the Flow Cytometry analysis determine that BIP is expressed in the intracellular compartments of Giardia trophozoites, while 5G8 is strongly expressed on the surface of Giardia trophozoites GS/M83‐H7 5G8 (+). Our data indicate that 5G8 and BIP proteins are different molecules of Giardia. Additional studies are required to determine the nature and function of 5G8 protein.
Regulation of Protein Kinase CK2 Catalytic Activity by Protein Kinase C and Phospholipase D2
Authors: Bae Young‐Seuk , Lee Young‐Hoon , Park Jeong‐Woo
Published: November 2023

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase that catalyzes the phosphorylation of a large number of cytoplasmic and nuclear proteins. The holoenzyme of CK2 is a heterotetramer, composed of two catalytic (α and/or α’) and two non‐catalytic (β) subunits. The β subunit enhances the catalytic activity of the α or α’ subunit and mediates tetramer formation and substrate recognition. We have previously demonstrated that phospholipase D2 (PLD2) overexpression antagonizes protein kinase CK2 (CK2) inhibition‐mediated cellular senescence. In the current paper, we show the molecular mechanism of CK2 activation by PLD2 and protein kinase C (PKC). Elevated expression and chemical activation of PLD2 increased the catalytic activity of CK2 and PKC in human colon cancer HCT116 and embryonic kidney HEK293 cells, whereas utilization of PLD2 chemical inhibitors and siRNA suppressed this activity. Inhibition of PKC in these cells suppressed PLD2‐induced CK2 activation, suggesting that PLD2 enhances CK2 activity through PKC. Overexpression of the PKC isoforms: PKCα, PKCβ, and PKCζ, but not PKCγ, stimulated CK2 activity in the cells. Importantly, purified conventional PKC (cPKC) and atypical PKC (aPKC) enzymes enhanced CK2 activity and phosphorylated serines 194 and 277 within CK2α, as well as serine 148 within CK2β. Furthermore, PKC‐mediated phosphorylation of these serines on CK2a was crucial for the stimulation of CK2 activity. Taken together, the present results suggest that PLD2 can stimulate CK2 activity through PKC‐mediated CK2α phosphorylation.

Support or Funding Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010‐0024102) and the Ministry of Science, ICT and future Planning (NRF‐2015R1A2A2A01004593).
TRAX mediates the protective role of the A2A adenosine receptor in detecting the DNA damage induced by oxidative stress
Authors: Chern Yijuang , Chien Ting , Weng Yu‐Ting , Chang Shu‐Yung
Published: November 2023

The translin‐associated protein X (TRAX) was originally identified as an interacting protein of Translin, a DNA/RNA binding protein that controls mRNA transport, translation, DNA repair, and DNA recombination. It interacts specifically with an activator (i.e., C1D) of DNA‐dependent protein kinase (DNA‐PK) upon γ‐irradiation and thus is implicated in repair of double‐strand breaks (DSBs). We recently reported that TRAX interacts with phosphorylated ATM (Ser1981) and contributes to the ATM‐mediated DNA repair in response to genotoxic stresses. The A2A adenosine receptor (A2AR) is a G‐protein‐coupled receptor that binds to adenosine and exists in many brain areas. Using a yeast two‐hybrid approach, we found that TRAX is an interacting protein of the cytoplasmic C terminus of A2AR, and mediates the neuritogenesis process via KIF2A. Given that stimulation of A2AR by two A2AR‐selective agonists markedly ameliorates the DSBs evoked by elevated oxidative stress in human iPSCs‐derived neurons, the role of TRAX in mediating the protective role of A2AR is of great interest. In the present study, we present evidence to suggest that TRAX is required for the A2AR‐mediated facilitation of DNA repair in a neuronal cell line (PC12) and primary neurons. Stimulation of A2AR appears to enhance the non‐homologous end joining (NHEJ) repair activity by increasing the activation and phosphorylation of DNA‐PK at Thr2609. Collectively, TRAX contributes to the activation of DNA‐PK and the detection of DSBs evoked by oxidative stress.
Zinc finger protein ZPR9 interacts with MPK38, an AMPK‐related serine/threonine kinase, and stimulates MPK38‐dependent ASK1‐, TGF‐β‐, and p53 function
Authors: Ha Hyunjung , Seong Hyun‐A
Published: November 2023

Murine protein serine‐threonine kinase 38 (MPK38) is a member of the AMP‐activated protein kinase (AMPK)‐related serine/threonine kinase family and plays a role in inducing ASK1‐, TGF‐β‐, and p53‐mediated activity. Here, zinc finger protein ZPR9 was found to be a positive regulator of MPK38. The redox‐dependent association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252, indicating ZPR9 as an endogenous substrate for MPK38. Expression of wild‐type ZPR9, but not the ZPR9 mutants C305S/C308S and T252A, enhanced MPK38‐induced ASK1, TGF‐β, and p53 function by stabilizing MPK38. Consistently, knockdown of endogenous ZPR9 using an inducible ZPR9 shRNA system showed an opposite trend and inhibited the MPK38‐induced ASK1, TGF‐β, and p53 function. These results suggest that ZPR9 functions as an activator in MPK38‐dependent ASK1‐, TGF‐β‐, and p53 signaling pathways.

Support or Funding Information

This work was supported by National Research Foundation of Korea Grant 2015R1A2A2A01006098
Effect of Targeting pathway inter‐connectors on gene expression pattern in Lipopolysaccharide (LPS) Induced Human Peripheral Blood Mononuclear Cells (PBMCs)
Authors: Mendis Chanaka
Published: November 2023

Lipopolysaccharide (LPS) is an endotoxin that induces the secretion of pro‐inflammatory cytokines in many cells and has been known to induce septic shock in humans. This project is aimed at developing a rapid diagnostic method to combat the effects of LPS and eventually other toxins by utilizing signal transduction pathways induced by of LPS in Peripheral Blood Mononuclear Cells (PBMC) as a prototype module. We have identified a set of LPS specific genes in human PBMCs through micro array analysis as well as a central signaling pathways consisting some of the LPS specific genes. Previously identified pathway inter‐connectors such as JNK and p38 were then blocked by specific inhibitors and the gene expression pattern of the same set of LPS specific markers were evaluated. The main objective was to investigate the effect of two inhibitors on the time dependent gene expression pattern and then, the impact on the components that are indirectly associated with the inhibited signaling pathways. In addition, the specific inhibitors were evaluated by correlating the observed gene expression pattern though reverse transcription polymerase chain reactions (RT‐PCR) to the protein expression pattern though enzyme‐linked immunosorbent assay (ELISA)

Support or Funding Information

N/A
BioCyc: Online Resource for Genome and Metabolic Pathway Analysis
Authors: Caspi Ron , Billington Richard , Foerster Hartmut , Fulcher Carol A. , Keseler Ingrid , Kothari Anamika , Krummenacker Markus , Latendresse Mario , Mueller Lukas A. , Ong Quang , Paley Suzanne , Subhraveti Pallavi , Weaver Dan S. , Karp Peter D.
Published: November 2023

BioCyc.org is a genomic resource that contains more than 7600 Pathway/Genome Databases (PGDBs) for organisms whose genomes have been completely sequenced. Although most PGDBs are bacterial, PGDBs exist for key experimental organisms, including humans (HumanCyc), yeast (YeastCyc), and Arabidopsis (AraCyc).

Each BioCyc database integrates the genome sequence of one organism with its predicted metabolic network, including metabolic pathways, reactions, and chemical compounds, all presented via a user‐friendly graphical interface. The BioCyc databases contain additional computed information such as predicted operons and candidate enzymes for catalyzing predicted enzymatic steps that have no assigned enzymes in the genome annotation.

The BioCyc.org website enables users to perform many types of searches and data analyses online. Analysis tools include comparisons of pathways and genomes among multiple organisms, alignment of orthologous genes in a multi‐genome browser, and searching for minimal‐cost metabolic network routes that connect two substrates. Tools are also provided for visualizing omics data – such as metabolomics and gene expression data – on individual pathway diagrams, on personalized groups of multiple pathway diagrams called pathway collages, and on diagrams depicting the full metabolic network of an organism.

A powerful tool in BioCyc is Smart Tables, which are user‐defined groups of metabolites or genes that can be transformed easily into other types of objects, analyzed, saved in a BioCyc account, and shared with other users.

All BioCyc databases save one were created computationally using MetaCyc as a reference. MetaCyc (MetaCyc.org) is a comprehensive manually‐curated metabolic database whose contents are derived from experimentally studies. It currently contains more than 2,400 experimentally determined metabolic pathways from more than 2,700 organisms, curated from more than 47,800 scientific publications. It also contains extensive information on metabolic enzymes, reactions, and substrates.

The BioCyc databases can be accessed through the BioCyc.org web site and can be downloaded. In addition, the Pathway Tools software can be downloaded and installed locally to create BioCyc‐like databases for more genomes, curate existing databases, or perform additional analyses that are not available online.

Support or Funding Information

National Institute of General Medical Sciences of the National Institutes of Health (NIH) [GM080746, GM077678, GM75742].