bluuebunny/crossref_metadata_2025_split huggingface dataset:
IP3R is localized to ER‐PM contact sites at unique endocytic structures involved in intercellular junction turnover events critical to spermatogenesis.
Authors: Lyon Kevin , Asghari Parisa , Scriven David RL , Moore Edwin DW , Vogl A Wayne 
Published: November 2023

Unique endocytic structures termed tubulobulbar complexes (TBCs) are critical for mammalian spermatogenesis. They function to internalize intercellular junctions located at the blood‐testis barrier basally in the seminiferous epithelium to allow spermatocytes to move from the basal compartment into the adluminal compartment. They also function to internalize apical attachments between spermatids and Sertoli cells prior to their release into the seminiferous tubule lumen. TBCs have three distinct regions: a proximal tubule, a bulb, and a short distal tubule. The tubular regions are cuffed by dendritic actin networks and the distal tubule is capped with a clathrin‐coated pit. The bulb region lacks an actin cuff, but is closely associated with cisternae of endoplasmic reticulum (ER). The function of this ER‐plasma membrane (PM) contact is unknown but we suspect it may be involved in calcium signaling. We previously used immunofluorescence to determine whether IP3R, a calcium channel in the ER that releases calcium, is present at these ER‐PM contacts. We previously probed cryo‐sections and epithelial fragments of perfusion‐fixed rat testes with antibodies against IP3R and an appropriate fluorescent secondary. Our previous data showed that IP3R labels the distal region of TBCs which is consistent with where we would expect the bulb region to be. To gather more convincing evidence, we used thicker cryo‐sections of 10 μm compared to our standard 5 μm to ensure that entire clusters of TBCs would be present in the section. We performed 3D reconstruction on deconvolved Z‐stacks to evaluate the localization of the IP3R labeling. The labeling is seen located between the distal and proximal tubules of TBCs indicating that it is at bulb region containing the ER‐PM contacts. To determine whether or not calcium release from IP3R channels occurs at these ER contacts, Adenophostin A, an IP3R agonist, was used to induce calcium release in isolated intact spermatids with associated Sertoli cell regions containing clusters of TBCs. Calcium release was monitored using Fluo4, a fluorescent calcium indicator. Our preliminary data suggests that Adenophostin A induces calcium release from IP3R channels in regions of isolated spermatids that is consistent with the bulb regions of TBCs. The data are consistent with our hypothesis that these ER‐PM contacts are involved in calcium signaling.Support or Funding InformationSupported by a NSERC Discovery Grant (#155397‐2013) to AWV.
Nerval danger zones for surgical procedures of the arm and the forearm
Authors: Hirtler Lena , Wlodek Vincent 
Published: November 2023

IntroductionThe course of cutaneous nerves at the distal forearm, the wrist joint and the hand is highly variable. This variability can cause complications in the different standardized surgical approaches for surgical interventions in these regions.The aim of this study was therefor (1) to document the courses of the lateral, medial and posterior antebrachial cutaneous nerves, the superficial branch of the radial nerve, the dorsal branch of the ulnar nerve and the palmar branches of the median and ulnar nerve and (2) to determine their relationship to osseous and tendinous landmarks. Afterwards danger zones for surgical approaches will be defined.Materials and methodsThe cutaneous nerves were dissected on 20 formalin/phenol – embalmed forearms and hands of anatomical specimens. The position and distribution of the nerves as well as landmarks were documented and distances were measured at pre‐determined positions.ResultsPalmar danger zone in a proximal to distal course: 10 cm proximal to the wrist joint: between 0 – 20%, 40 – 66,67 %, 80 – 86,67% and 93,34 – 100% of the width of the forearm; 5 cm prox. to the wrist: 0 – 26,67 %, 53,34 – 66,67 % and 86,7 – 100 % of the width; on the line between the styloid processes: 0 – 13,33 %, 20 – 26,67 % and 93,34 – 100% of the width; on the line between the distal border of the pisiform bone and the base of the first metacarpal bone: 0 – 20 %, 46,67 – 60 % and 66,67 – 73,33% of the width. Dorsal danger zones in a proximal to distal course: 10 cm prox. to the wrist: 0 – 6,67 %, 13,34 – 33,33 %, 40 – 66,67 % and 93,33 – 100 % of the width; 5 cm prox. to the wrist: 0 – 6,67 %, 20 – 60 % and 66,67 – 73,33% of the width; on the line between the styloid processes: 0 – 20 %, 73,33 – 80 % and 93,33 – 100 % of the width; proximal metacarpus: 0 – 40 %, 73,33 – 86,67 % and 93,33 – 100 % of the width; on 50% of the metacarpal height: 0 – 6,67%, 13,33 – 33,33 %, 46,67 – 53,33 %, 66,67 – 86,67 % and 93,33 – 100 % of the width; distal metacarpus: 0 – 5 %, 25 – 35 %, 50 – 65 %, 75 – 85 % and 95 – 100 % of the width. The anatomic snuffbox and the ulnar aspect of the wrist were considered to be not safe, because of the wide distribution of nerves.ConclusionAberrant and normal courses of the cutaneous nerves were documented and described in detail. Based on the results of this study, specific danger zones were defined to further help refining surgical procedures in future.Support or Funding Informationno funding was provided.
Acetylpyrazine Thiosemicarbazone Inhibiting Topoisomerase II
Authors: Ngo Lana C , Stults Garrett 
Published: November 2023

We have previously showed that Cu(II) acetylpyrazine methylthiosemicarbazone chloride ([Cu(APZ‐MTSC)Cl]) inhibits the ATP hydrolysis of human topoisomerase (Topo II) as catalytic inhibitor and share some common features as covalent poisons such as increasing TopoII‐mediate DNA cleavage which can be abolished by the presence of DTT. To further test if acteylpyrazine thiosemicarbazone metal complexes all have the similar characteristics, we synthesized a series of acetylpyrazine thiosemicarbazone metal (copper and palladium) complexes with different side chain substitutions including methyl, ethyl, turt‐butyl, benzyl, phenyl and dimethyl functional groups on the terminal nitrogen. We examined their effect on TopoII‐mediated DNA relaxation and DNA cleavage assays. Our data suggest that all metal acetylpyrazine thiosemicarbazones display similar characteristics as [Cu(APZ‐MTSC)Cl], but metal ions play a very important role in inhibiting TopoII.Support or Funding InformationThis project is funded by Faculty Development Research Grant from Tennessee Board of Regents and Faculty Research Grant from Tennessee Technological University.
Assessing DNA Cross‐Linking and Repair in Human Leukemia Cells
Authors: Le Phuong M. , Millard Julie T. 
Published: November 2023

The anti‐tumor effects of bifunctional alkylating agents have been recognized for more than 50 years, notably in the frontline chemotherapeutic drug, nitrogen mustard and its derivatives. These compounds are double‐edged swords: killing both cancerous and non‐cancerous cells by forming cytotoxic DNA adducts via interstrand and intrastrand cross‐links. Intrastrand crosslinks can be readily removed by nucleotide excision repair mechanisms while interstrand crosslinks (ICLs) pose a challenging block to DNA replication, transcription, and repair and are hence a potentially lethal event. Using human leukemia (HL‐60) cells as an in vitro model, we are investigating the cytotoxicity and DNA repair of ICLs induced by nitrogen mustard (HN2), epichlorohydrin (ECH), and 1,2,3,4‐diepoxybutane (DEB)‐the active form of the anti‐ovarian cancer drug Ovastat. The median lethal dose (LD50) of each compound was determined via the AlamarBlue assay over a wide a range of drug concentrations. DNA cross‐linking activity and repair were quantified via single cell gel electrophoresis (the comet assay). Drug‐induced ICL formation retarded the mobility of hydrogen peroxide‐treated DNA in an electric field, creating a comet‐like migration pattern at the individual cell level. The shorter and brighter the comet tail was, the more crosslinks were present. Preliminary data showed a consistent cytotoxicity trend after a 12‐hour or 24‐hour drug treatment with HN2 > ECH ≥ DEB. Comet assay analysis from treating cells with equitoxic doses revealed that the percentage decrease in tail moment, a combined parameter that includes the comet tail length and fluorescence intensity, varied between different drugs, corresponding to their different cross‐linking potentials. Interestingly, cross‐linking activity was maintained in some, but not all, compounds at 24 hours compared to 12 hours, suggesting differential rates of DNA repair. With these quantitative investigations, we hope to correlate the toxicity of these bifunctional alkylating agents with their cross‐linking specificity. Our ultimate goal is to elucidate the underlying mechanism by which ICLs induced by one drug are more susceptible to removal and repair than another. These findings can improve our understanding of dose‐dependent drug cocktail administration and the long‐term effects of these chemotherapeutic drugs.Support or Funding InformationThis research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.
Heme induces HAP4 transcription and mitochondrial respiration
Authors: Zhang Tiantian , Vancura Ales 
Published: November 2023

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors, as well as communication between the nuclear and mitochondrial genomes. We have shown previously that decreased expression of histones or a defect in nucleosome assembly in Saccharomyces cerevisiae result in increased mitochondrial DNA (mtDNA) copy number, oxygen consumption, ATP synthesis, and expression of genes encoding enzymes of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Transcription of genes involved in TCA cycle and OXPHOS induced by altered chromatin structure requires the activities of Hap1p and Hap2/3/4/5p complex as well as transport and metabolism of pyruvate in mitochondria. Here we show that this pyruvate‐derived metabolite is heme. Heme biosynthesis starts in mitochondria; it requires glycine and the TCA cycle intermediate succinyl‐CoA for synthesis of 5‐aminolevulinic acid (ALA), a key precursor for heme biosynthesis. Heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, in a Hap1p‐ and Hap2/3/4/5p‐dependent manner. Since the transcription of genes involved in TCA cycle and OXPHOS is repressed by glucose in yeast, heme levels are limiting for HAP4 transcription and the activity of the Hap2/3/4/5p complex. Increasing heme levels induces HAP4 transcription and mitochondrial respiration. Together, our data indicate that carbon flux into the TCA cycle regulates heme synthesis, which, in turn, regulates HAP4 transcription and mitochondrial respiration.Support or Funding InformationNIH/National Institute of Gerneral Medical Science provided funding to Ales Vancura under grant number 1R15GM120710‐01
A Novel Long Non‐coding RNA Modulates Macrophage Phenotype During Diet‐Induced Obesity
Authors: Stapleton Kenneth , Chen Zhou , Reddy Marpadga , Lanting Linda , Leung Amy , Dieuliis Jeffrey , Natarajan Rama 
Published: November 2023

Systemic low‐grade inflammation associated with obesity is a strong risk factor for the development of metabolic syndrome, which in turn raises the risk of diabetes and cardiovascular complications. This inflammatory state is primarily caused by the release of proinflammatory cytokines by macrophages in response to excess lipid levels. Macrophage phenotypic changes are associated with a shift in global gene expression profiles, which includes long non‐coding RNAs (lncRNAs). Although the misregulation of lncRNAs have been implicated in the development of many human diseases, including diabetic complications, their role in directing macrophage phenotype during diet‐induced obesity and pre‐diabetes has been largely unexplored. We assembled transcriptomes de novo from macrophages of high fat diet (HFD)‐ and control diet‐fed mice and identified novel lncRNAs that were differentially expressed. One of the top lncRNA candidates was termed lnc‐Fabp5, due to its proximity to Fabp5, a fatty acid transporter with known roles in insulin resistance and diabetes. HFD caused decreased expression of both lnc‐Fabp5 and Fabp5. We hypothesized that lnc‐Fabp5 affects macrophage dysfunction during obesity and diabetes. Gain‐ and loss‐of‐function studies were performed to evaluate the effect of lnc‐Fabp5 on macrophage gene expression and phenotype. A reduction in lnc‐Fabp5 transcripts with gapmers decreased Fabp5 levels, increased expression of immune response and inflammatory genes, and increased acetylated LDL uptake. Many of those changes were not replicated by Fabp5 knockdown, indicating that lnc‐Fabp5 knockdown‐induced changes are not simply a result of altered Fabp5 expression. Lnc‐Fabp5 overexpression caused inverse expression changes and partially blunted the inflammatory response to endotoxin. lnc‐Fabp5 levels were measured in human adipose tissue macrophages from obese and healthy donors, and expression was inversely correlated with patient BMI and fasting insulin levels. This previously uncharacterized lncRNA appears to have a functional role in macrophage response to environmental changes caused by high‐fat diet.Support or Funding InformationNIH: DK065073
CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival
Authors: Sheshadri Namratha , Sharan Shikha , Sterneck Esta 
Published: November 2023

CEBPD (C/EBP delta) is a member of the CCAAT‐enhancer binding protein (C/EBP) family of transcription factors characterized by a b‐Zip domain that mediates dimerization and DNA binding. CEBPD is induced in response to acute stressors such as cytokine stimulation, bacterial lipopolysaccharide (LPS), corticosteroids, radiation and hypoxia. We have previously reported that CEBPD has dual functions in breast cancer by both attenuating or enhancing oncogenic pathways depending on context (Balamurugan and Sterneck, 2013, Mendoza‐Villanueva et al., 2016).Recent studies reveal that elevated Endoplasmic Reticulum (ER) stress is associated with the pathology of several diseases including cancer. Limiting supply of nutrients and oxygen in growing tumor cells disrupts the protein folding homeostasis resulting in activation of the unfolded protein response (UPR). The UPR includes pathways that support adaptation to stress, and that are also implicated in promoting malignant features and therapy resistance in breast cancer. Hence, we investigated a possible role for CEBPD in this stress response pathway.Using breast cancer cell lines as model systems, we found that CEBPD is dynamically regulated in response to chemical and physiological inducers of ER stress. Kinetic analyses indicate that CEBPD induction is concurrent with the activation of the UPR effectors XBP1s and ATF4 suggesting a potential role in stress adaptation. Examination of the global transcriptional prolife of the Thapsigargin‐induced stress response in MDA MB‐231 cells indicated that CEBPD activates genes involved in cytokine and chemokine production/signaling, immune cell recruitment as well as angiogenesis pathways. While XBP1s has been previously ascribed to activating these pathways, the levels of activated XBP1 were not affected by CEBPD silencing. This is the first report of a similar role for CEBPD in the ER stress response and warrants the investigation of possible co‐operation of CEBPD and XBP1 in the UPR pathway. Further, we show that knock down of CEBPD in cancer cells increased PARP cleavage and susceptibility to ER stress induced cell death. CEBPD deficient cells also displayed reduced LC3II accumulation, suggesting impaired autophagy induction in response to ER stress.Taken together, our data suggests that CEBPD is a novel proximal effector of the ER stress response in breast cancer cells. Activation of this transcription factor regulates tumor promoting inflammatory and angiogenic signaling pathways. Additionally, CEBPD expression confers a survival advantage to cancer cells encountering ER stress. Therefore, we propose that targeting CEBPD may sensitize cancer cells to ER stress inducing therapeutics.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute and in part with Federal Funds under contract no. HHSN261200800001E
Translational Regulation of Gene Expression in Mycobacterium: A Means for Coordinating the Expression of Functionally Related Proteins
Authors: Saks Margaret E. , Oh John , Deets Austin C. , Mastorakos George M. , Martinis Susan Anne 
Published: November 2023

Mycobacterium tuberculosis (Mtb) is one of the world's deadliest infectious agents. Consequently, a top priority has been to identify the mechanisms that Mtb uses to regulate the expression of genes involved in its virulence/persistence. Our over arching focus is on the translational regulation of gene expression. Our studies indicate that Mtb leverages differential codon usage and tRNA expression during both initiation and elongation to regulate protein synthesis.Translation initiation plays a key role in determining protein expression levels through its influence on the density of ribosomes bound on an mRNA. In bacteria, the initiation complex typically assembles with tRNAfMet bound to an AUG codon. However, sometimes the complex must assemble on a non‐canonical GUG or UUG initiation codon. In Mtb, and in the model organism M. smegmatis (Msm) which we use for our experiments, approximately forty percent of the initiation codons are non‐canonical. By contrast, non‐canonical initiation codons are used in only approximately 10 percent of the Escherichia coli coding sequences (CDS). We engineered an mCherry reporter system for use in Msm and used it to determine whether protein synthesis is differentially affected by initiation codon usage. Our experiments demonstrate that, throughout the growth curve, protein synthesis is substantially reduced when translation is initiated on GUG and UUG, rather than AUG codons. We are currently investigating whether canonical vs non‐canonical initiation codons are distributed across Mycobacterium coding sequences as a means for regulating protein synthesis. A conceptually related series of experiments focuses on the idea that, during elongation, the synthesis of functionally related proteins can be regulated though a “supply‐demand” mechanism. Due to the high GC content of the Mtb genome, the genome‐wide fraction of UUA (leucine) codons is only approximately1.6%. Interestingly the UUA codon is not randomly distributed across Mtb genes. Indeed, it is over‐represented in certain groups of genes involved in virulence and it is not used in the majority of Mtb coding sequences. These UUA‐containing genes can only be translated when the cognate tRNA, tRNALeuUAA is expressed. Therefore, the expression of functionally related proteins can be coordinated through the expression of tRNALeuUAA.Support or Funding InformationNational Institutes of Health GM63789
Novel Bryostatin‐1 Targets: Mammalian Unc13‐1 and Unc13‐2 Isoforms
Authors: Blanco Francisco A , Pany Satyabrata , Ghosh Anamitra Alejandro , You Younki , Das Joydip 
Published: November 2023

Neuroprotective Bryostatin‐1 (Bryo) is currently under phase II clinical trials for Alzheimer's disease (AD). Bryo has been postulated to enact its neuroprotective properties through the modulation of protein kinase C (PKC) by interacting with the diacylglycerol/phorbol ester‐binding pocket of its C1 domain. Mammalian un13‐1 (Munc13‐1) and brain specific unc13‐2 (bMunc13‐2) contain C1 domain with a high degree of homology to that of PKC. Additionally, Munc13‐1 and bMunc13‐2 are expressed in the hippocampus and function in synaptic vesicular priming. In this study we explored the existence of an interaction between Bryo and these Munc13 isoforms. The long‐term goals of this project are to expand the understanding of Munc13‐C1‐domain and identify novel effectors of Bryo. We hypothesized that Bryo would interact with Munc13‐1 and bMunc13‐2, resulting in membrane translocation. To test this hypothesis, proliferating hippocampal mouse (HT22) cells were transiently transfected with plasmids containing Munc13 DNA tagged with green fluorescent protein (GFP) and a membrane translocation assay was implemented. Activation of the protein was identified by accumulation of GFP fluorescence around the peripheral of the cell. Effects of Bryo were quantified and compared to those of a phorbol ester previously established to activate Munc13s, 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA). In this study, we demonstrate Bryo activation of over‐expressed GFP‐tagged Munc13‐1 and bMunc13‐2 in HT22 cells. Furthermore, by employing immunocytochemistry and immunoblotting of differentiated HT22 cells and primary hippocampal neurons, we observed increased expression of Munc13‐1. To conclude this project, we plan to continue studying the effects of Bryo on bMunc13‐2 activation and exploring the effects of Bryo treatments on the expression levels of Munc13 isoforms. Findings of this study suggest that Bryo's neuroprotective properties should be further studied in terms of Munc13 isoforms modulation along with those of PKC. The interaction between Munc13 isoforms and Bryo provides a novel molecular target that could be utilized to modulate neuronal synapses under dysfunctional synaptic conditions, like those present during the onset of AD.Support or Funding InformationNIH R01 Grant (1R01AA022414‐01A1)
Expression, Purification and Initial Characterization of the Coiled‐Coil 2‐3 Region of Atg11, a Central Organizer of Selective Autophagy Initiation
Authors: Zois Carson , Kennedy Bridget , Backues Steven K 
Published: November 2023

Autophagy is physiological process for the destruction of internal cell components via the vacuole/lysosomes in order to promote cell survival. This process allows the cell to maintain homeostasis during times of starvation and recycle damaged cellular components. In humans, this helps to protect against neurogenerative diseases, infectious diseases and cancers. Of the ~30 proteins involved in the initiation and execution of autophagy in yeast, one of the most understudied is Atg11, which shows similarity to human Huntingtin protein. Atg11 has been shown to interact with a number of other autophagy proteins and is therefore thought to be a central organizer of the autophagy initation complex during selective autophagy. Many of these interactions, as well as Atg11's self‐interaction, have all been mapped to Atg11's coiled‐coil domains 2 and 3. However, it is not known how Atg11 organizes its binding partners spatially and temporally – for example, do some partners exhibit competitive binding? We have heterologously expressed and purified the Atg11CC2‐3 region and begun to investigate its behavior via in vitro binding assays. In parallel, we are performing targeted yeast‐2‐hybrid assays with Atg11CC2‐3 to allow us to directly compare the binding behavior of Atg11's CC2‐3 region in vitro and in vivo.Support or Funding InformationNSF RUI #1613653 “Scaffold or Assembly Line: How Does Atg11 Organize its Binding Partners for the Initiation of Selective Autophagy?” to S. K. Backues