bluuebunny/crossref_metadata_2025 huggingface dataset:
Self‐organization of zebrafish lateral line primordium morphogenesis and migration
Authors: Chitnis Ajay , Nogare Damian Dalle
Published: November 2023

The zebrafish Posterior Lateral Line Primordium (PLLp) pioneers the establishment of the Posterior Lateral Line system. Interactions between the Wnt, FGF, Notch, and chemokine pathways in the PLLp provide a framework for understanding how cell fate and morphogenesis are coordinated in this group of about a hundred cells, as they collectively migrate under the skin from the ear to the tip of the tail. We have used a combination of experiment and computational modeling to define the mechanisms that determine both the periodic formation of neuromasts and their deposition along with inter‐neuromast cells, as the PLLp migrates along a path defined by chemokine expression. Furthermore, our high resolution imaging of the morphology and dynamics of individual cells in the primordium reveal unexpected features of how cells with distinct morphologies in distinct parts of the PLLp contribute to collective migration and communication between leading and trailing cells in the primordium. Together, our studies reveal novel features of the self‐organization of this remarkable system.

Support or Funding Information

Eunice Kennedy Shriver National Institute of Child Health and Human Development intramural Program
Elucidation of Saw1 and Rad1–Rad10 Recruitment to Induced Single‐stand Annealing Sites as a Function of 3′ Flap Length
Authors: Felix Juan Camberos , Fregoso Fred , Odango Rowen , Iannolo Bailey , Fischhaber Paula
Published: November 2023

Unrepaired DNA damage leads to cancer and aging. A more detailed understanding of DNA damage repair mechanisms, specifically of double‐strand breaks (DSB's), can lead to new cancer prevention and treatment strategies. Two major DSB repair pathways include Synthesis‐Dependent Strand Annealing (SDSA), a conservative pathway resulting in perfect repair, and Single‐Strand Annealing (SSA), repair characterized by the loss of genetic material.

During SSA in S. cerevisiae, complementary strands deriving from DNA repeats flanking a DSB become annealed, creating non‐homologous 3′ overhanging DNA flaps that are cleaved by the Rad1–Rad10 protein complex endonuclease activity. Prior research shows Saw1 is required to recruit Rad1–Rad10 to the DSB. However, recent studies show Rad1–Rad10 recruitment is Saw1‐independent if the overhanging flaps to be cleaved are short leaving ill‐defined the precise flap length triggering Saw1‐dependent Rad1–Rad10 recruitment.

To elucidate the flap length at which Rad1–Rad10 requires Saw1 for recruitment during SSA, DSB's will be triggered by restriction digest in fluorescently labeled yeast strains containing a fluorescently‐labeled I‐SceI cut site flanked by a variable number of non‐homologous base pairs (bp) between two DNA repeats. Repair sites containing variable length overhanging flaps produced by SSA will be monitored for recruitment of Rad1–Rad10 via fluorescence microscopy in pairs of strains that are either wild‐type in SAW1 or SAW1 deletion mutants.

The specialized yeast strains needed are not available and the current goal of this project is their preparation. Strains are being prepared by integration of DNA cassettes bearing the I‐SceI cut site flanked on either side by 20, 30 or 50 bp of non‐homologous DNA and the HIS3 gene. The 663 bp HIS3 gene provides the DNA repeat sequence. DNA plasmids bearing the cassettes were prepared by standard cloning methods. DNA for the 50 bp flap yeast strain has been successfully integrated into the yeast genome with PCR screens of yeast transformants showing incorporation of the correct DNA cassettes at the target chromosomal locus. Our 50 bp flap strain screen results show that the cloning scheme envisioned for the preparation of these specialized yeast strains is viable and preparation of one strain is complete.

Support or Funding Information

NIH‐SC3GM093858 and the Dr. Lan K. Wong Undergraduate Research Award.
Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates
Authors: LaCava John , Molloy Kelly R. , Fenyö David , Taylor Martin S. , Chait Brian T. , Boeke Jef D. , Rout Michael P.
Published: November 2023

LINE‐1 (L1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. L1 ribonucleoprotein particles incorporate a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against L1's mutagenic activity. We previously characterized host proteins associated with human L1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics.

To build on these analyses, we have executed a suite of quantitative proteomic comparisons, yielding interactomic maps of affinity isolated L1s. These studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. We report the proteins partitioning within these distinct L1 populations and associated in vitro activities. Our observations provide a basis for the classification of L1 interactors into physical and functional modules and have enabled the development of in vitro systems to study L1 activity.

Support or Funding Information

This work was supported by NIH grants P41 GM109824, 1P50GM107632‐01, and P41 GM103314
Neuroprotective actions of hydrogen sulfide (H2S) and sulfhydration in the brain
Authors: Paul Bindu Diana , Snyder Solomon H.
Published: November 2023

Hydrogen sulfide (H2S) has emerged as an important signaling molecule joining the ranks of the gasotransmitters, nitric oxide (NO) and carbon monoxide (CO). Analogous to NO, H2S signals via a posttranslational modification termed sulfhydration, wherein H2S mediates the conversion of –SH groups of reactive cysteine residues on target proteins to –SSH or persulfide groups. Ever since its characterization, sulfhydration has been shown to participate in a myriad of physiological processes ranging from vasorelaxation to neuroprotection. Recently we have shown that aberrant H2S metabolism and sulfhydration play key roles in the progression of neurodegeneration in Parkinson's disease (PD) and Huntington's disease (HD). In HD, mutant huntingtin disrupts the transcriptional regulatory machinery of cystathionine gamma lyase (CSE), the biosynthetic enzyme for H2S as well as its precursor cysteine. mHtt affects transcription factors responsible for expression of CSE leading to its depletion in HD. H2S also modulates the disposition of second messengers such as cGMP which results in cognitive deficits, a feature shared by several neurodegenerative disorders including PD and HD. This presentation focuses on how H2S regulates redox homeostasis via gene regulatory mechanisms, the disruption of which is a contributor to disease progression in neuronal degeneration.

Support or Funding Information

Representative Recent Recognition/Honor

ASBMB/jbc Herbert Tabor Young Investigator Award (May 2015) at the third European Conference on the Biology of Hydrogen Sulfide at Athens, Greece.
BRD4 IS A HISTONE ACETYLTRANSFERASE THAT EVICTS NUCLEOSOMES FROM CHROMATIN
Authors: Ballachanda Devaiah Nanjappa , Case‐Borden Chanelle , Gegonne Anne , Hsu Chih Hao , Chen Qingrong , Meerzaman Daoud , Dey Anup , Ozato Keiko , Singer Dinah
Published: November 2023

Bromodomain protein 4 (BRD4) is a master transcriptional and epigenetic regulator which plays a pivotal role in cancer development and immune diseases. As a chromatin binding protein that remains bound during mitosis, BRD4 has been described as a mitotic bookmark. Inhibiting its interaction with chromatin has been a successful therapeutic strategy against many cancers including acute myeloid leukemia, Burkitt's lymphoma, breast, colon and lung cancer. While BRD4 association with chromatin is known to activate transcription of key proto‐oncogenes, the mechanism involved is currently unknown. Here, we report that BRD4 has novel and intrinsic histone acetyltransferase (HAT) activity through which it acetylates histones H3 and H4 in nucleosomes. BRD4 HAT activity maps to two consensus acetyl CoA binding sites and a 40 aa catalytic site. BRD4 HAT activity is distinct from all other known HAT's with a unique lysine acetylation ‘fingerprint’ that includes acetylation of H3K4, H3K9, H3K18 and H3K27 but not H3K14. Most importantly, BRD4 acetylates H3K122, a key lysine residue critical for nucleosome stability located on the dyad axis of the nucleosome. BRD4 HAT activity and H3K122 acetylation is responsible for nucleosome eviction and opening of the chromatin, as evidenced by the ability of BRD4, but not BRD4 HAT mutants, to evict nucleosomes both in vitro and in vivo. Nucleosome clearance by BRD4 is selective and localized to the gene loci it regulates such as c‐Myc, c‐Fos and Aurora B kinase. Consistent with its role in localized chromatin de‐compaction, BRD4 HAT activity regulates transcription at these gene loci as well. BRD4 HAT activity thus plays a key role in chromatin remodeling and transcription.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Expanding the Roles of tRNA Thiolation in Bacteria: Involvement of 2‐thiouridine tRNA in Signaling Bacterial Sulfur Metabolism
Authors: Black Katherine Ann , Kus Nicole Joanne , Dos Santos Patricia Coutinho
Published: November 2023

Post‐transcriptional modifications of tRNA are found in all organisms and are critical for their structure and function during translation. 2‐thiouridine (s2U) modification at the wobble position of tRNAGlu, Gln, Lys is required for accurate and efficient protein synthesis and its absence impacts cellular viability. In addition to its role in translation, in yeast, the s2U modification is used as a sensor to gauge nutrient availability and regulate translational capacity and amino acid homeostasis, in which s2U abundance reflects the availability of sulfur‐containing amino acids, cysteine and methionine. In previous work, we showed that B. subtilis s2U biosynthesis involves only two proteins: the cysteine desulfurase YrvO and the thiouridylase MnmA. Genes coding for both proteins are located in an operon directly downstream of the master regulator of cysteine metabolism, cymR, suggesting a link between s2U formation and sulfur amino acid metabolism in this bacterium.

In this study we investigated s2U's role in metabolic homeostasis and translational regulation as a sensor of sulfur availability in bacteria. B. subtilis wild‐type was cultured in minimal media containing varying concentrations of an individual sulfur source: sulfate, cysteine, or methionine. Using our established LC‐MS method, analysis of tRNA‐thionucleosides showed that the abundance of s2U correlates to the concentration, but not the source, of sulfur available in the growth media. This relationship appears to be specific to s2U, as the levels of an additional thionucleoside, s4U, remained constant across all conditions; supporting the notion that only s2U is responsive to sulfur availability. Western blot analysis of YrvO and MnmA in these cultures showed patterns consistent with those of CymR expression. That is, higher expression of YrvO and MnmA was observed in cultures using sulfate, cysteine or cystine as the sole sulfur source when compared to thiosulfate, methionine or homocystine. Surprisingly, expression levels of YrvO and MnmA did not correlate with the concentration of sulfur source, or accumulation of s2U thionucleoside, suggesting that the activity of these enzymes is likely controlled by the concentration of cysteine, which affects the catalytic turnover rate of YrvO, the first enzyme in the s2U pathway. In this model, the availability of sulfur controls the flux of sulfur transfer which impacts the overall levels of the signaling metabolite s2U tRNA. Current work is aimed towards evaluating whether s2U levels dictate expression of proteins involved in translation and growth specific processes. Furthermore, these analyses are being expanded to other bacterial species to potentially establish this regulatory role across a diverse group of bacteria.

Support or Funding Information

This work has been funded through the Wake Forest University Center for Molecular Communication and Signaling graduate fellowship.
The Effect of Ultraviolet Stress on Nonfunctional rRNA Decay
Authors: Gomez Karen Janina Villarroel , LaRiviere Frederick J
Published: November 2023

Nonfunctional Ribosomal RNA Decay (NRD) is a quality control pathway that degrades structurally intact but functionally defective rRNAs. To date, there are two recognized NRD pathways in S. cerevisiae: 18S NRD and 25S NRD. These pathways are responsible for degrading nonfunctional 18S rRNAs and the 25S rRNAs respectively. 18S NRD is translation dependent and involves the translation factor‐like protein Dom34 and its binding partner Hbs1. In contrast, 25S NRD is translation independent and involves the E3 ligase components Rtt101 and Mms1. These pathways were identified using specifically engineered mutant rRNAs, but NRD may normally function to remove accumulated nonfunctional rRNAs that can arise as a result of genotoxic stress such as UV radiation. Understanding rRNA damage and decay is of special importance because rRNA makes up to 90% of the total RNA in S. cerevisiae. Therefore, we are interested in determining the effect of ultraviolet (UV) radiation on NRD to better understand how the 18S and 25S NRD pathways affect cell survival. We hypothesize that by removing damaged rRNAs, NRD can increase cell survival under certain stress conditions. To investigate the effect of UV radiation on NRD function, we transformed BY4741 S. cerevisiae with various rRNA expression plasmids. These plasmids encode either functional or nonfunctional versions of the 18S and 25S rRNAs. The plasmid‐derived rRNAs also contain short, unique sequence tags that allow specific detection of these rRNAs in the presence of total cellular rRNA. The transformed cells were exposed to increasing amounts of UV radiation and cell growth after exposure was monitored in both liquid and solid media. Currently, we are determining relative expression levels of WT and nonfunctional rRNAs using an RT‐qPCR assay. Furthermore, we are continuing these same experiments in NRD‐deficient yeast strains. This work may ultimately help elucidate the role of NRD in cellular recovery from UV damage and contribute to a better understanding of the NRD pathway.

Support or Funding Information

This work is supported by the Howard Hughes Medical Institute.
Development of a Natural Protein Synthesis System from Pseudomonas aeruginosa
Authors: Hughes Casey , Escamilla Yaritza , Hu Yanmei , Bullard James M
Published: November 2023

We have developed a protein synthesis system from P. aeruginosa composed of the ribosome and 15 accessory proteins. We successfully over‐expressed, purified, and characterized all fifteen of these proteins. These proteins consisted of: PheRS, ArgRS, AspRS, GlnRS, GluRS, LeuRS, MetRS, ProRS, initiation factor‐1 (IF‐1), initiation factor‐2 (IF‐2), initiation factor‐3 (IF‐3), elongation factor Tu (EF‐Tu), elongation factor Ts (EF‐Ts), elongation factor G (EF‐G) and methionyl‐tRNA formyltransferase (MFT). To confirm that the proteins were isolated in active forms, each was subjected to individual activity assays. The aaRS proteins were analyzed using aminoacylation assays. We developed a ribosome dependent GTPase assay to monitor the activity of IF‐2 and EF‐G. EF‐Tu and EF‐Ts activity was monitored by GDP exchange assays. We also devised a ribosome dissociation assay to monitor the activity of IF‐1 and IF‐3. The ability of MFT to formylate Met‐tRNAMet was monitored by measurement of CuSO4 hydrolysis of formylated tRNAMet. We also developed a pseudo‐natural mRNA that allows for monitoring protein synthesis. This mRNA contains high‐usage codons corresponding to tRNA anti‐codons recognized by each of the aaRS proteins present. The assay contains, mRNA, the initiation factors, MFT, elongation factors, ribosomes, aaRS, and amino acids corresponding to each of the aaRS proteins and [3H]Leu. The presence of four codons encoding leucine at the 3′ end of the transcript allows monitoring of full length peptide. Upon completion of synthesis the nascent peptide remains bound to the ribosome and activity is monitored using scintillation proximity assay (SPA) technology. The concentrations of each enzymatic component used in the system was arrived at through sequential rounds of optimization; the concentration of each component represents the concentration just below the saturation point of the titration. This system will be used for screening for chemical compounds that inhibit protein synthesis in P. aeruginosa.

Support or Funding Information

Research was supported by NIH grant 1SC3GM098173‐01A1 and partial student support was from the NIH UTPA RISE program grant # 1R25GM100866‐01 and from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017).
Functional Cooperativity Between the Trigger Factor Chaperone and the ClpXP Degradation System
Authors: Houry Walid A
Published: November 2023

Comprehensive bioinformatic analyses demonstrated the conservation of the close proximity of the tig gene coding for the ribosome‐associated chaperone trigger factor (TF) chaperone and the genes coding for the ClpXP proteolytic complex suggesting the possible functional association between the protein folding and the protein degradation systems. Genome‐wide genetic interaction studies further supported such an observation. The effect of TF on ClpXP‐dependent degradation varied depending on the nature of the substrate. For some substrates, TF slowed down their degradation by ClpXP, while for others it had no effect. Surprisingly, for a third class of substrates, TF increased the degradation rate. The ClpXP‐dependent degradation of the λ phage replication protein λO was significantly enhanced by TF in vitro and in vivo. TF was found to interact with both ClpX and λO with similar affinities and to enhance the ClpXP‐mediated degradation of ribosome‐stalled λO nascent chains. Experiments suggest that TF transfers λO to ClpX for eventual degradation by the ClpP serine protease and that this could occur cotranslationally. Globally, it is estimated that TF enhances the degradation of about 2% of newly synthesized E. coli proteins. This reveals a new role for TF in facilitating the degradation of newly translated proteins and the potential presence of cotranslational degradation in bacteria.

Support or Funding Information

Canadian Institutes of Health Research
Testing Specificity of Commercially Available Nicastrin Antibodies
Authors: Hatter Jessica , Dries Daniel
Published: November 2023

A protein complex known as γ‐secretase is involved in the development of Alzheimer's disease, which affects cognitive functions such as learning and memory. Nicastrin, a component of γ‐secretase, is the substrate receptor in the protein complex. In a previous research project, the expression of the nicastrin protein was tested by immunofluorescence in a line of nicastrin knockout cells. However, the antibodies stained structures in both wild‐type and knockout cells. This result suggested either of two possibilities: the cells were not nicastrin knockout cells, or the antibodies were nonspecific to nicastrin. Genotyping by polymerase chain reaction (PCR) determined that the nicastrin gene itself was, in fact, knocked out of the cells. To determine if the nicastrin antibodies are specific, western blots were run using the same antibodies with lysates from both wild‐type and knockout cells. By revealing cross‐reactive bands in both lysates, western blots will also aid in determining why the antibodies bind to both knockout and wild‐type cells in immunofluorescence microscopy. Therefore, determining the specificity of commercially available antibodies will better inform both ourselves and the scientific community about using these antibodies for experiments.

Support or Funding Information

Funding contributed by the International Healthspan Institute.
Transcriptome Analysis of Conditional Knockout Mice
Authors: Biefeld Chelsea , Sztroin Kelcie , Zhu Yi , Yu Gang , Dries Daniel
Published: November 2023

Gamme‐secretase is an intramembrane protein that cleaves type I transmembrane proteins. It is known to be involved in Alzheimer's disease and neurodevelopment. Previous studies have investigated the role of gamma‐secretase role in neurons however, it has seldom been considered in glial cells. Here, a conditional knockout mouse was made in which gamma‐secretase was rendered nonfunctional in oligodendrocytes. These mice displayed schizophrenic‐ and compulsive‐like phenotypes. In order to identify genes and pathways that elicit these phenotypes, RNA‐Seq was used to perform transcriptome analysis searching for differentially expressed genes in the experimental mice. Gene ontology programs were used to evaluate the genes and identify biological processes that may play a role in the behavioral aspects of these mice.

Support or Funding Information

We would like to thank the Howard Hughes Medical Institute, the National Institutes of Health (Grant R01 AG029547), and the International Healthspan Institute for providing funding.
Effects of cinnamon extract on glycogen synthase kinase 3 phosphorylation of tau
Authors: Donley Jena , Hurt William , Stockert Amy
Published: November 2023

Recently correlations have been suggested between abnormal glucose metabolism and the development of Alzheimer's disease. These suggested links stem from the similar portions of the insulin signaling cascade in muscle, adipose tissue, and brain. Although the downstream targets of the insulin signaling pathway differ in these three tissues, the initial signals remain the same. Compounds in cinnamon have been shown to reduce blood glucose levels by functioning in what is typically referred to as an “insulin mimic” fashion. Cinnamon has shown increases in expression levels of some glucose transporters (GLUTs) as well as an increase in GLUT translocation to the surface of the cell membrane. Observations of both of these effects suggests that cinnamon effects multiple cellular processes, potentially insulin signaling and microtubule mediated translocation of the GLUTs. Studies have also demonstrated that cinnamon extract inhibits tau aggregation, a process highly affected by the phosphorylation of tau protein. It is believes that the aggregation of tau affects the formation and function of the microtubules, thus eliminating the ability to effectively translocate damaged proteins included amyloid beta aggregates. Research has suggested that glycogen synthase kinase 3 (GSK3) is one of the enzymes responsible for phosphorylation of the tau protein found in microtubules. In order to determine is the compounds in cinnamon alter the GSK3 dependent phosphorylation of the tau protein in vivo, HEK293 cells were transfected with GSK3 and tau using transfection reagent. The cells were grown in the presence and absence of cinnamon extract for 48 hours. Following growth and treatment, cells were lysed and fractionated, run on an SDS‐Page gel, transferred and Western blotted with antibodies reactive with phosphorylated tau and non‐phosphorylated tau. In order to determine the effects of cinnamon treatment on tau aggregation, protein isolates of the cells were collected, immune‐precipitated in order to isolate tau protein. The isolated protein was then solubilized, disaggregated by short incubations in a sonicating bath, and then allowed to re‐aggregate with addition of heparin or other aggregation agent. Aggregation was monitored at 350 nm spectrophotometrically over several hours. For comparison, recombinant tau was reacted with heparin in the presence or absence of cinnamon extract and monitored spectrophotometrically. Results suggest that cinnamon extract plays a role in minimizing GSK3 phosphorylation of tau, perhaps by limiting activation of GSK3 by protein kinase A dependent phosphorylation.
The molecular basis for cross‐species prion transmission
Authors: Kurt Timothy , Jiang Lin , Alderson Nazilla , Liu Jun , Eisenberg David , Sigurdson Christina
Published: November 2023

Background

A key event in the pathogenesis of prion diseases is the conversion of the cellular prion protein, PrPC, into a misfolded, self‐templating conformer called PrPSc. The mechanism by which PrPSc templates the misfolding of PrPC is unknown. We previously demonstrated that the amino acid sequence within key segments of PrPC has a profound impact on conversion by PrPSc. For instance, two critical residues, E168 and S170, impede cross‐seeding of human PrPC by chronic wasting disease (CWD) prions from elk and deer1. Interestingly, bank voles, a small European rodent, are highly susceptible to prions from diverse species including humans, elk and mice, and have been referred to as the “universal acceptor for prions”2. The molecular basis for the remarkable susceptibility of bank voles to prion infection is unknown..

Hypothesis

We hypothesize that the bank vole PrPC sequence is susceptible to prion cross‐seeding due to key amino acids that are within key adhesive segments of the protein that are critical for prion aggregation. Further, we hypothesize that substituting the key bank vole residues into the PrPC from other species will enable conversion of PrPC from any species.

Methods

I used an in vitro prion conversion assay called cell‐lysate protein misfolding cyclic amplification (PMCA) to probe the effects of amino acid substitutions on the conversion of bank vole PrPC. PrPSc from elk, mice and sheep was used to seed conversion of PrPC. Data were measured by semi‐quantitative western blotting and analyzed by ANOVA and t‐test.

Results and Conclusions

We found that asparagine and glutamine amino acids are enriched among the key bank vole residues, suggesting that asparagine ladders and polar zippers may underlie the high susceptibility of bank vole PrPC to prion conversion. These data provide important insight into the structure of infectious prions, as the key residues must comprise an interface for interaction with PrPSc. Interestingly, we also found evidence suggesting that an interaction between residues in the N‐terminal disordered domain and the globular domain of PrPC impacts prion conversion. This finding has not been previously reported and indicates that the N‐terminal domain, which is not considered part of the β–sheet core of the misfolded prion, plays an important role in prion conversion. In addition, we have confirmed that heterologous amino acids residues may promote prion cross‐seeding, an important finding that is relevant to understanding cross‐species prion transmission and zoonotic disease risk.

Support or Funding Information

This work was supported by NIH grant NS069566 and NS076896 (CJS) and 1 K01 0D019919‐01 (TDK).
Effect of rLosac (Lonomia obliqua stuart‐factor activator) on skin wound healing
Authors: de Andrade Sonia Aparecida , Sato Ana Claudia , Alvarez‐Flores Miryam Paola , Bosch Rosemary , Chudzinski‐Tavassi Ana Marisa
Published: November 2023

Belonging to the hemolin family, Losac is a factor X activator, found on bristles of the Lonomia obliqua. Its features cytoprotective activity and modulates the expression of extracellular matrix molecules in a fibroblast culture. To study the effect of rLosac on skin wound healing in rats. Approval # 1254/14 by Comissão de Ética no Uso de Animais do Instituto Butantan. Twenty Wistar (Rattus norvergicus albinus) male rats, aged 6–8 weeks, weight 120 – 150 g obtained from Biotério Central do Instituto Butantan. Rats were submitted to oral preemptive analgesia 200 mg/kg paracetamol and kept on post‐surgery procedures. The animals were anesthetized by intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine. Four full‐thickness excisional 4 sq mm punches were created through the skin. The wounds of the animals were treated topically with 63.80 μg/mL rLosac (L1), 127.60 μg/mL of rLosac (L2) or saline solution (SS). Zero, 3, 7, 14 and 21 days after skin injury, the wounds were measured using a digital caliper rule and the rats euthanized with excess anesthetic. The areas corresponding to the lesion were removed with scalpel and analyzed. The three independent treatment groups were analyzed through the Kruskall‐Wallis test on days 0, 3, 7, 14 and 21. Regarding the initial area (12.56 sq mm), after three days of treatment the wound was reduced by 63.5 % on L1 group, 75.0 % on L2, and 51.25 % on SS. After seven days, the initial area was reduced by 72.76 % on SS; 87.5 % on L1 and 94.75% on L2. After 14 and 21 days of treatment, just scars were observed. The rLosac accelerates wound healing. The gelatinolytic activity from MMP‐2 increased on days zero, 3 and 14 on the wound treated with rLosac. However, on day 7 there was a decreased activity. MMP‐2 degrades basement membrane collagens and demots denatured structural collagens. Proteolytic degradation of the extracellular matrix is an essential feature of repair and remodeling during wound healing. On day zero, L2 featured two more intense spots, possibly corresponding to the α1 and α2 chains of the type I collagen when compared to control. There is also an approximately 210 KDa spot, probably corresponding to the β chain. On day three the spots remained more intense in L1 and L2 when compared to SS and to day zero. On days 7, 14 and 21 there was a significant increase on the intensity of the spots, possibly corresponding to the α1 and from the α2 type I collagen, with the α1 chain more intense when compared to the α2 chain. These initial results have shown that the healing time of the wounds treated with rLosac is significantly smaller when compared to the control, which suggests the potential benefit of this protein on wound healing.

Support or Funding Information

Support by FAPESP
Structural determinants of human tryptophan 2,3‐dioxygenase (TDO) protein degradation
Authors: Kim Sungmi , Karkashon Shay , Yeh Syun‐Ru , Correia Maria Almira
Published: November 2023

The hepatic hemoprotein tryptophan 2,3‐dioxygenase (TDO) catalyzes the irreversible oxidation of L‐tryptophan (Trp) to N‐formyl‐L‐kynurenine. It critically regulates the flux of Trp through the kynurenine and serotonin pathways, thereby controlling systemic Trp levels. TDO has been implicated in various diseases, including neurodegenerative diseases and cancers, and thus is an important target for drug discovery. Despite its importance, very little is known about the regulation of human TDO (hTDO) protein turnover required for its cellular homeostasis. Herein we report that hTDO turnover occurs via ubiquitin‐linked proteasomal degradation (UPD) pathway, and we define the structural features critical therein. Trp, the preferred substrate is known to “induce” TDO enzyme via stabilization. Structural analyses reveal that Trp binds hTDO at both the active site and an exo‐site that also binds aMethylTrp (aMeTrp). 35S‐pulse‐chase analyses in HepG2 cells revealed that exo‐site aMeTrp‐binding prolonged the half‐life of parent wild‐type (WT) hTDO 2‐fold. Site‐directed mutagenesis of exo‐site residues considerably accelerated hTDO degradation, despite aMeTrp presence. By contrast, deletion of hTDO N‐ and/or C‐terminus (ΔN, ΔC, ΔN/ΔC) greatly stabilized the protein in the order ΔN/ΔC>ΔC>ΔN, consistent with a processive proteolytic process. The extreme hTDO C‐terminus is rich in acidic (Asp/Glu) and phosphorylatable (Ser) residues. Relative to the WT, mutation of these Asp/Glu‐residues to Gly stabilized hTDO, whereas that of Ser to Gly had no effect. When the exo‐site mutation was incorporated into the hTDOΔN/ΔC mutant, its stability was lost, being degraded just as rapidly as the exo‐site hTDO‐mutant. Collectively these findings reveal that although the exo‐site along with the N‐ and C‐termini are important structural features in hTDO turnover, the structural integrity of the exo‐site Trp‐binding is a critical determinant of its UPD.

Support or Funding Information

NIH grants GM44037, DK26506, and GM086482.
p97 is Critical for Cardiac Proteostasis and Dysregulation of p97 Activity Causes Cardiomyopathy
Authors: Brody Matthew J , Karch Jason , Molkentin Jeffery D
Published: November 2023

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is a protein quality control process whereby proteins that cannot be properly folded in the ER are retrotranslocated to the cytosol for degradation by the ubiquitin proteasome system. p97 is a AAA‐ATPase that forms a ring‐shaped hexameric structure on the cytosolic side of the ER membrane and provides the energy to retrotranslocate misfolded substrates from the ER for ultimate ubiquitin‐dependent degradation. Mutations in p97 are known to cause protein aggregation‐based diseases including pathologies of the nervous system, skeletal muscle, and heart. However, the role of p97 in cardiomyocytes has not been examined. Here, we generated cardiac‐specific transgenic mice overexpressing wildtype p97 or a p97K524A mutant with deficient ATPase activity. Transgenic mice overexpressing wildtype p97 exhibit normal cardiac structure and function while mutant p97 overexpressing mice develop age‐dependent cardiomyopathy at baseline and greatly exacerbated cardiomyopathy in response to pressure overload. Mechanistically, both wiltype and mutant p97 overexpressing hearts upregulate the protein levels of Sel1l, an ERAD adaptor protein, while p97K524A overexpressing hearts additionally upregulate the ERAD complex components OS‐9, Herp, and the ER‐resident prolyl isomerase cyclophilin B. Importantly, hearts overexpressing p97K524A but not wildtype p97 have elevated levels of ubiquitinated proteins, indicating that p97 activity is required for efficient clearance of proteins targeted for proteasomal degradation. Moreover, overexpression of wildtype but not mutant p97 in cardiomyocytes enhanced ERAD activity and protected from ER stress induced cell death. These studies identify a critical role for p97 in cardiac proteostasis and demonstrate that dysregulation of p97 activity causes cardiomyopathy.

Support or Funding Information

This work was supported by a National Research Service Award (F32HL124698) from the National Institutes of Health to M.J.B. and grants from the Howard Hughes Medical Institute and National Institutes of Health (R01HL105924) to J.D.M.
Skeletal Muscle Wasting and Inflammatory Regulation by Ovarian Function and IL‐6 in Tumor Bearing Mice
Authors: Reszczynski Olivia , Saum Alison , Hetzler Kimbell , Carson James
Published: November 2023

Cancer cachexia is a progressive wasting condition involving the loss of muscle and fat, as well as biochemical abnormalities due to chronic underlying disease such as cancer. IL‐6, a pro‐inflammatory cytokine, has been shown to be a major driver of cancer cachexia in several mouse models, including the male ApcMin/+ (Min) mouse, and humans. Studies regarding cachexia and IL‐6 have largely been conducted in male mice; however, our lab has noted sex differences in cachexia development and IL‐6 response using the ApcMin/+ mouse. Inflammation related to IL‐6 signaling is associated with myofiber wasting in the male ApcMin/+ mouse, but it has been unexplored in the female. Though it has been seen that hypogonadism is a factor in cachexia in males, research has not been conducted to determine the effect of hypogonadism in the female. Given estrogen's known anti‐inflammatory effects, it is possible that ovarian endocrine function plays a role in the differential female IL‐6 response. The purpose of this study is to determine the effect of ovarian function and the role of IL‐6 in the muscle wasting seen during progression of cachexia. The hypothesis was that ovarian function loss (OVX) with IL‐6 overexpression would cause an increase in muscle inflammation, leading to increased myofiber wasting and decreased myofiber area. ApcMin/+ mice were divided among four groups: control (n=13), sham+IL‐6 overexpression (n=10), OVX+vector (n=10), OVX+IL‐6 (n=8). Tibialis anterior (TA) muscles were sectioned and mounted on slides. H&E staining was performed to determine myofiber cross‐sectional area (CSA). A greater proportion of small (<1500 μm), and smaller proportion of large (>2000μm) fibers were found in sham+IL‐6 mice vs. control mice (p<0.001), as well as in OVX+IL‐6 versus OVX+vector mice (p<0.001), indicating that IL‐6 overexpression induces a shift towards smaller fibers independent of ovarian function. Membrane receptor gp130, required for IL‐6 intracellular signaling, protein expression increased with IL‐6 overexpression and attenuated by OVX. OVX also decreased STAT3 phosphorylation, an intracellular mediator of IL‐6 signaling. Increased inflammation related to IL‐6 signaling is associated with muscle wasting in the female ApcMin/+ mouse and is attenuated by loss of ovarian function.

Support or Funding Information

The authors would like to acknowledge Justin Hardee, Dennis Fix, Song Gao, and Brandon VanderVeen for technical assistance. This research was funded by NIH grant NCI R01CA121249‐01.
Understanding The Lipid Mediated Oligomerisation In Membrane Proteins
Authors: Gupta Kallol , Robinson Carol
Published: November 2023

A quarter of the human proteome is composed of membrane proteins (MP). Nevertheless, less than 2% of known protein structures are of MPs and less than a quarter of those contain any modelled lipids. To date, no multimeric MP structures exist in the protein structure database with lipids at the protein‐protein interface. This highlights the limitation of crystallography in determining the identity of lipid molecules, owing to their conformational flexibility and high mobility. The structural and functional effects resulting from the interactions between MPs and their surrounding lipids have been the subject of constant debate. A major facet of these discussions is the role of lipids in regulating the oligomeric states of MP assemblies. To address this an extensive bioinformatics analysis of all MP structures led to a set of model systems with weak interfaces, potentially suggesting lipid mediation. Exploiting the ability of native‐mass spectrometry (nMS) to simultaneously observe the binding of lipids to MP and their impact on oligomeric states we have developed for the first time a model for lipid‐induced oligomerisation of a MP in atomistic detail and set up a general platform to study this phenomenon in other MPs. nMS analysis of these MPs revealed a defined mass addition of lipids to the oligomers. Among the target systems, for the bacterial amine transporter LeuT, it was observed that selective removal of specific lipids leads to complete de‐oligomerisation, establishing the essential role of lipids towards maintaining the dimer. For the bacterial semi‐SWEET, a homolog of eukaryotic sugar transporters, equilibrium between the monomer and the functionally active dimeric state was observed. Added lipids were found to specifically bind to and stabilize the active state, elucidating a mechanism through which an organism may select out an active oligomeric state of a membrane protein by modulating the interacting lipids in the membrane. Through an integrative lipid‐analysis, using a novel high‐energy platform that allows MS/MS analysis of MPs, we identified bound phospholipids and cardiolipins. Analysis of delipidated proteins, as well as mutants that lack dimeric interactions and the probable lipid binding sites, proposed through MD simulation, coupled with integrated lipid analysis and live cell imaging, an atomistic model was revealed of how lipids can act as plugs to bridge weakly bound oligomers. The approach, relying on the unique ability of MS to determine the oligomeric states of MPs, with precise identification of bound endogenous lipids, provides a platform that will further the understanding of how lipids can modulate the functional states of oligomeric membrane proteins and trigger specific downstream cellular pathways.

Support or Funding Information

KG is a fellow of the 1851 Royal Commission and a Junior Research Fellow at the St Catherine's College, University of Oxford
Using a Toxicogenomic Approach to Understand How Silver Poisons the Bacterial Cell
Authors: Lemire Joe , Chatfield‐Reed Kate , Chua Gordon , Turner Raymond J.
Published: November 2023

Introduction

The emergence of the post‐antibiotic era has left us desperate for alternative strategies to combat infectious disease. Historically, silver has been used to prevent and control microbial infections. Silver is now seeing widespread use in a variety of medical devices including bandages, catheters, and endotracheal tubes; as well as numerous household items. Yet, we still don't mechanistically understand how silver poisons the microbial cell.

Objectives

Recently, our research group demonstrated that the higher oxidation states of silver have an enhanced capacity to eradicate laboratory and antibiotic‐resistant strains of bacteria – both planktonic bacteria as well as biofilms. Our current research objective is to understand the mechanistic details of how silver exerts its toxicity on the bacterial cell.

Methodology

To address the mechanisms of how silver kills bacterial cells, we used a high‐throughput, toxicogenomic approach ‐ screening a knockout collection of E.coli K12 (the Keio collection) for Ag‐sensitive/resistant mutants. Following our toxicogenomic screen, markerless deletion mutants were generated from statistically significant hits using the Red Recombinase system. The markerless genetic mutants were then subjected to phenotypic and biochemical assays to verify their resistance or sensitivity profile to silver treatment.

Results

Although silver is thought to exert its toxicity by disrupting thiol metabolism and cellular redox‐status, our results demonstrate that silver poisons E.coli through a variety of previously unestablished mechanisms. These include interfering with genes involved in maintaining cell wall integrity, toxin/antitoxin systems, nutrient catabolism, and quorum sensing.

Conclusion

Here we demonstrate how a toxicogenomic approach was used to determine the mechanistic manner by which silver poisons the bacterial cell. To that end, the robust nature of this methodology can be used to screen other metals, toxins, and antimicrobials to determine their mechanisms of action. Mechanisms that are desperately needed to develop novel antimicrobial strategies.

Support or Funding Information

This work has been supported by the Natural Science and Engineering Council of Canada. J.L. is supported by a Banting Postdoctoral Fellowship and an Alberta Innovates Health Solutions Postdoctoral Fellowship Award.
Absorption Mechanisms of Poly I:C RNA Onto Zinc Oxide Nanoparticles: Maximizing The Payload
Authors: Ramani Meghana , DeLong Robert K
Published: November 2023

Development of stable RNA‐nanoparticle complexes will advance RNA therapeutics. Given the complexity of bionano interactions, it is crucial to develop efficient RNA loading techniques, to optimize RNA payload. Here we investigated the loading of anti‐cancer RNA (Poly I:C) using two different sizes of ZnO (14, 100 nm) and three different loading techniques; 1) Centrifugation, 2) Incubation 3) Reannealing. The amount of RNA binding per mg nanoparticle was quantitated using the absorption of RNA at 260 nm by UV‐visible absorption spectroscopy. In order to evaluate the number of binding sites and the binding kinetics, the samples were characterized by fluorescence spectroscopy using the modified Stern‐Volmer equation. In general, the RNA payload per mg nanoparticle was observed to be higher for 14 nm nanoparticles than the 100 nm due to larger surface area. Also, reannealing was most efficient with maximum payload of 20μg per mg of the 14 nm nanoparticles. Reannealing enhances the binding constant (kb = 6.8×103 g−1L) as well as the number of binding sites (n = 5) available on the surface of ZnO for one RNA. Taken together the data suggest three different mechanisms of RNA interaction with nanoparticle: physisorption, aggregation, and chemisorption. TEM analysis confirms an RNA corona surrounding the nanoparticle. This is the first example we are aware of an RNA corona surrounding an un‐functionalized inorganic non‐polymeric nanoparticle. Based on our spectroscopy analysis comparing these three techniques, reannealing poly I:C to 14 nm ZnO nanoparticle, demonstrates the highest payload and suggests a possible process for designing the optimal delivery system.

Support or Funding Information

This work was supported by NCI 7R15CA139390‐03
Analysis of Myogenic Markers in Duchenne's Muscular Dystrophy Cell Models
Authors: Harnois Melissa , Pincus Mark , Powers Elaine , Badarinarayana Vasu
Published: November 2023

The goal of the project was to compare the in vitro differentiation potential of Duchenne's Muscular Dystrophy (DMD) and non‐disease patient derived myoblasts. The differentiation of myoblasts into myotubes as well as the maturity of myotubes was assessed by morphology, immunohistochemistry (IHC) analysis of myotubes markers as well as transcript profiles of myogenesis markers. Human myoblasts derived from three non‐disease and three DMD patients were evaluated in multiple time‐course studies. Morphological evaluation as well as IHC analysis indicated that the DMD patient derived myoblasts have diminished capacity to differentiate and form mature myotubes. Gene expression profiling also revealed significantly reduced basal transcript levels of myogenic markers in DMD patient derived cells and impaired induction of these transcripts during differentiation.

Support or Funding Information

Sanofi

Tucson Innovation Center, Oro Valley, AZ