bluuebunny/crossref_metadata_2025 huggingface dataset:
Sexual Dimorphism in Facial Fluctuating Asymmetry among Hausa Population of Nigeria
Authors: Taura Magaji Garba , Adamu Lawan , Danborno Barnabas
Published: November 2023

The aim of the study was to investigate the sex differences in three fluctuating asymmetry (FA) indices, and to determine the side dominance in facial features among Hausas of Kano State Nigeria. A total of 283 participants comprising 147 males and 136 females of age range 18–25 years were randomly selected. Photometric method was used to capture the face. Three indices of asymmetry used include signed asymmetry (SA), absolute asymmetry (AA) and composite asymmetry (CA). Independent sample t test was used to test for sex differences in the variables and one sample t test was used to determine fluctuating asymmetry. The result showed that females tend to have statistically significant mean value in orbital width. For zygon to gnathion facial distance, a statistically significant higher mean value was observed in males. For the three indices of fluctuating asymmetry above, no significant sexual dimorphism in signed FA index was observed. However, males tend to have leftward FA in orbital width and zygon to gnathion distance. In absolute and composite FA, a significant sexual dimorphism was noticed only in zygon to gnathion distance, with females having higher mean value in AA and males having higher mean value in CA respectively. The facial parameters exhibited fluctuating type of asymmetry. In conclusion, there were no sex differences in the FA indices. However, left warded type of FA was exhibited in males of Hausa origin.

Support or Funding Information

None

Sexual dimorphism in signed asymmetry (SA) of the paired facial dimensions

image

One sample t test and normality test for evaluation of fluctuating asymmetry of facial dimensions

One sample t – test

Sex
Variables
Mean
SEM
t

Male
ps‐pi1
−0.11
0.12
−0.93*

ex‐en1
0.06
0.16
0.37*

zy‐gn1
−0.02
0.34
−0.07*

Female
Ps‐Pi1
0.08
0.12
0.63*

ex‐en1
0.08
0.16
0.53*

zy‐gnl
0.19
0.42
0.46*

P>0.05.

SEM: standard error of mean,

Shapiro‐Wilk test P > 0.05
Comparison Analysis of Metopism Prevalence in Sexes and Various Populations
Authors: Bliss Kaitlyn N , Russell Michelle L , Koons Aaron W , Mangus Kelsey R , Zdilla Matthew J
Published: November 2023

The metopic suture (MS) is a cranial suture located at the junction between the two primary intramembranous nuclei of the developing frontal bone. The MS first becomes evident at the end of the second month of fetal life and typically closes in infancy or early childhood. For reasons that remain unclear, the MS may persist into adulthood‐ a condition commonly referred to as metopism. The prevalence of metopism is significant from both a developmental and clinical perspective. Alone, persistent metopic sutures may mimic midline fractures; however, metopism may also be related to several craniofacial defects including frontal sinus anomalies, cleft lip, and cleft palate. This study analyzed 505 crania from varied populations. Of the 505 crania studied, 13 crania demonstrated metopism (1.01%). Out of the total population, 0.99% observed were European (Anglo‐American, Swedish, German, Irish, Finnish, European, Spaniard, Etruscan, Sclavonian, and Celtic), 0.40% were East Asian (Japanese and Chinese), 0.40% were Egyptian, 0.20% were Bengali, and 0.60% were from other populations. Within the total population, 0.99% were males with metopism and 1.60% were females.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.14.13]
Student Generated Structure Lists: Bringing Self‐Directed Learning into the Anatomy Laboratory
Authors: Dunham Stacey M
Published: November 2023

Self‐directed learning (SDL) opportunities are a necessary component of medical education and identified as an approach to developing lifelong learning skills. The anatomy laboratory is one place that SDL can be incorporated in the medical curriculum. This study examined the content generated by a group of approximately 40 first year medical students in a yearlong human gross anatomy course. Students were required to generate their own anatomy structure list based on the instructions provided in the class dissector. Structure lists were to indicate the anatomical structures to be identified in the day's dissection. This required students to thoughtfully and carefully review the dissection instructions for the lab procedure prior to attending the laboratory session. Rather than simply list the structures, students were asked to classify structures into primary, secondary, and tertiary groups and provide an explanation for their chosen organizing principle. Organizing principals used by students included: size based divisions, clinical relevance, body systems, and locations. While some students consistently exhibited principles of self‐directed learning in the work submitted, the lack of a grade based assignment led other students to submit work of limited value in preparing for the dissection.

Support or Funding Information

This study was not funded.
Glutamic acid 446 and Arginine 447 in heat shock protein 70 (hsp70) are critical for regulating superoxide dismutase‐2 (SOD2) function
Authors: Afolayan Adeleye James , Teng Ru Jeng , Konduri Girija Garnesh
Published: November 2023

Background

Stress‐inducible hsp70 interacts with SOD2 after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the amino acid residues in hsp70 that are critical for SOD2 interactions remain unknown.

Methods and results

We map the SOD2‐binding site in hsp70 by making a series of hsp70 mutants (GST‐tagged 1–400, 393–537 and 537–641) and tested for their ability to bind SOD2. The GST‐tagged hsp70 mutants and EGFP‐tagged wild type SOD2 were co‐expressed in HEK293T cells. Using pull down assays, we showed that SOD2 preferentially bound to the hsp70 mutant corresponding to amino acid 393–537 region in hsp70 (Fig. 1). SOD2 did not bind to the other regions tested. This exclusivity suggests that SOD2 associates with the 393hsp70537 domain. To better define the binding site, nine overlapping decoy peptides (p1–p9) were made spanning the entire length of the 393hsp70537 region, where SOD2 is suspected to bind to hsp70. The peptides were screened to determine which one could inhibit SOD2/hsp70 interactions. Out of the nine decoy peptides tested, p4 (YSDNOPGVLIQVYEGERAMT: amino acid residues 423–443) and p5 (GERAMTRDNNLLGRFELSGIP; amino acid residues 439–459) significantly disrupted SOD2 association with hsp70 in endothelial cell lysates (Fig 2A). Since GERAMT is common to both p4 and p5 peptides, we speculated that this sequence may represent the minimal portion responsible for disrupting SOD2 interactions with hsp70. To test this hypothesis, we added the GERAMT peptide alone or GERAMT‐less P4 and P5 peptides (Δp4 and Δp5) individually to aliquots of cell lysates. Colorimetry analysis revealed that GERAMT‐treated lysates had ~3‐fold decrease in SOD2 activity compared to the GERAMT‐less peptides (Fig 2B). Small peptides containing the GERAMT sequence decreased the activity of SOD2 in vitro and in vivo. To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine (A) in the GERAMT‐binding site in a step‐wise fashion and tested the effects of mutations on SOD2‐hsp70 interactions. Substitutions of alanine for glutamic acid (E446A) and arginine (R447A) inhibited the ability of hsp70 to bind SOD2 and decreased SOD2 activity, more than other substitutions (Fig 3).

Conclusions

Together, these findings indicate that the GERAMT sequence is critical for the chaperone‐mediated regulation of SOD2 antioxidant function and that E446 and R447 cooperate with other residues in the GERAMT‐binding site for proper hsp70 chaperone functions.

Support or Funding Information

Funding supports from Department of Pediatrics and Children's Research institute (CRI) at the Medical College of Wisconsin and 1K08HL133379‐01 (Adeleye Afolayan)
Investigating the regulation of recA in the emerging pathogen Acinetobacter baumannii
Authors: Ching Carly , Gozzi Kevin , Heinemann Bjorn , Godoy Veronica G
Published: November 2023

Acinetobacter baumannii (Ab) is an emerging multidrug‐resistant, opportunistic pathogen. Ab survives desiccation, remaining on hospital surfaces affecting immunocompromised individuals. Ab antibiotic resistance acquisition has been linked to its DNA damage response (DDR). DDR genes, including error‐prone DNA polymerases, are involved in DNA damage and desiccation induced mutagenesis. In Escherichia coli, RecA, the cells' main recombinase, binds single stranded DNA, the signal of DNA damage, forming the nucleoprotein filament (RecA*), which is endowed with co‐protease activity. RecA* promotes cleavage and inactivation of LexA, the global DDR repressor, resulting in expression of DDR genes. In Ab, this well‐known circuitry does not exist in part because Ab lacks a functional LexA. We have shown that conserved DDR genes in Ab form two phenotypic subpopulations in response to DNA damage: one with low and another with high expression. The DDR in Ab is dependent on RecA. Thus, we are focused on understanding the mechanism underlying recA regulation to begin to understand the regulatory network that underlies this bimodality.

We have found that the recA gene contains a cis‐acting element in its 5′ untranslated region (UTR). In‐vitro transcription demonstrated that this 5′UTR contains secondary structure. A plasmid‐borne reporter was constructed that contains the cis‐regulatory element driving a fluorescent reporter. Mutants of the cis‐regulatory element that deletes putative regulatory sequences or predicted in silico secondary structure were constructed using site‐directed mutagenesis. Using fluorescent microscopy of the reporter strains, we have shown that the cis‐acting regulatory element and its structure are important for bimodal expression of recA, while the promoter contains elements for DNA Damage sensing. Furthermore, we found that the recA 5′UTR modulates transcript levels and subsequently RecA in the cell. Disrupting the structure of the 5′UTR results in a significant decrease of UV survival and antibiotic resistance acquisition. This provides evidence that in Ab, two levels of regulation mediate recA. Remarkably, the Ab promoter and UTR sequence is capable of responding not only in Ab but also in other bacterial species as observed through similar fluorescent reporters induced with DNA damaging agents. Bimodal DDR gene expression may provide a strategy for survival and plasticity in an ever‐changing environment. These findings may also provide insight into the evolution of other highly antibiotic resistant bacteria lacking conserved DDR gene networks

Support or Funding Information

NIH Grant (GM088230) to VG
The Epsilon Subunit of DNA Polymerase III in the Bacterial Response to Quinolones
Authors: Whatley Zakiya , Sy Nene , DiDomenico Sarah , Finck Amanda
Published: November 2023

Quinolones are widely prescribed, broad‐spectrum antibacterial drugs. Despite their widespread use, the molecular mechanisms of quinolone cytotoxicity are not clearly understood. Quinolones target topoisomerases, preventing the resealing of otherwise transient DNA breaks. Poisoned topoisomerase, or stabilized cleavage complexes, are not sufficient for cell death. The goal of this study is to determine how stabilized cleavage complexes are converted into irreversible double strand DNA breaks (DSBs) that accumulate and drive quinolone cytotoxicity.

Previous studies identified an unexpected connection between dnaQ and quinolone‐induced DNA damage in Escherichia coli. The ɛ subunit of DNA polymerase III, encoded by dnaQ, is an exonuclease providing the 3′ ‐> 5′ proofreading activity of the core. The absence of ɛ causes higher mutation rates, slow growth, constitutive SOS, and a defective SOS response following quinolone treatment. We found that these phenotypes are separable, which implies that ɛ has multiple, separable roles, aside from proofreading.

We explore the role of ɛ‐β interaction in quinolone‐induced DSB generation. Using two different types of cell lysis in conjunction with pulsed‐field gel electrophoresis, we distinguished between latent breaks at the cleavage complex and overt chromosomal breaks due to additional processing. We provide evidence that supports the replication run‐off model of DSB generation, whereby a stronger ɛ‐β interaction causes the replicase to physically interact with stabilized cleavage complexes rather than stalling or disassembling.

Support or Funding Information

Gettysburg College Research & Professional Development Grant & Howard Hughes Medical Institute ‐ Cross‐Disciplinary Sciences Institute at Gettysburg College
Argonaute2 Cooperates with LaminB to Repress Transcription at Lamin‐associated Domains in Drosophila melanogaster
Authors: Nazer Ezequiel , Chinen Madoka , Dale Ryan , Lei Elissa
Published: November 2023

Argonaute proteins are commonly known as core components of RNA silencing pathways. However, Argonaute proteins have also been shown to possess nuclear functions, such as regulation of transcription, splicing and chromatin architecture. Previous work showed that Drosophila AGO2 functions directly on euchromatin to promote enhancer‐promoter interaction at the homeotic Abd‐B locus independently of the RNA interference (RNAi) pathway. ChIP‐seq analysis revealed that AGO2 binds thousands of sites in the genome, raising the possibility that AGO2 could modulate global chromatin architecture. To identify factors that can function with AGO2 to regulate transcription, we performed immunoaffinity purification of AGO2 from nuclear extracts followed by mass spec analysis. Interestingly, we found that LaminB is enriched among the top AGO2‐associated proteins. Reciprocal co‐immunoprecitation validated the specificity of this interaction, and biochemical fractionation assays confirmed that both proteins reside in chromatin and nuclear matrix fractions. To directly assess the global role of both proteins in transcription, we performed nascent RNA‐Seq upon depletion of either AGO2 or LaminB in Kc167 cells. We found that both proteins co‐repressed a highly significant number of genes, particularly those located at the borders of Lamin‐associated domains (LADs). In order to assess the physiological role of AGO2 in transcriptional regulation, we performed mRNA‐Seq in null versus RNA slicing catalytic activity mutant female larvae. Strikingly, we observed de‐repressed transcription of spermatogenesis genes in the absence of AGO2, independent of its catalytic activity. One of the de‐repressed genes is nht, which encodes a key upstream activator of spermatogenesis gene expression. Null mutation of nht suppresses the up‐regulation of spermatogenesis genes observed in AGO2 null mutants, suggesting that AGO2 acts upstream of nht to silence the spermatogenesis gene program. Given that nht is located within a LAD harboring flanking AGO2 chromatin binding sites, we hypothesized that AGO2 and LaminB could modulate chromatin topology to repress nht. Circular chromosome conformation capture assays (4C‐Seq) using the nht promoter as bait showed a decrease in the frequency of interactions within the LAD upon AGO2 or LaminB knockdown. We conclude that both proteins may repress transcription at LAD borders by regulating chromatin architecture.
The role of CCCTC binding factor (CTCF) in epithelial to mesenchymal transition (EMT)
Authors: Bhattacharya Atrayee , Hur Junguk , Dhasarathy Archana
Published: November 2023

The process of Epithelial to Mesenchymal transition (EMT) plays a major role in cancer metastasis. TGF‐β or EGF signaling pathways can trigger EMT, which involves a cell state transition from an attached, epithelial state to a migratory, mesenchymal phenotype. In addition, massive reorganization of chromatin occurs during EMT, coupled with significant gene expression changes. However, the underlying cause for what triggers these chromatin structure changes remains unknown. The well‐studied transcription factor CTCF is known to regulate the 3D structure of chromatin, and reorganize the genome. Recently, it was shown in Drosophila cells that CTCF recruits Smad proteins to their gene targets following addition of TGF‐β. EMT master regulatory protein SNAIL is among the key targets of Smad proteins. Therefore, we hypothesized that CTCF plays a role in TGF‐β‐induced EMT, both at the transcriptional level, and at the genome architectural level, leading to acquisition of an invasive phenotype.

To test our hypothesis, we induced EMT in normal murine mammary gland (NMuMG) cells followed by chromatin immunoprecipitation (ChIP), to determine CTCF enrichment at the SNAIL promoter. We found TGF‐β, but not EGF, dependent enrichment of CTCF at the promoter and upstream regions of SNAIL relative to the control‐untreated cells. Correspondingly, we noted a TGF‐β dependent increase in expression of SNAIL mRNA, which is diminished when coupled with siRNA knockdown of CTCF. Further, CTCF is associated with the promoter and upstream regions of the SNAIL gene in metastatic cancer cell lines with high endogenous SNAIL expression. Taken together, we propose a role for CTCF in direct transcriptional regulation of genes involved in EMT. We are currently investigating the functional role of CTCF in EMT.

Support or Funding Information

This work was supported by award 5P20GM104360 from the National Institutes of Health to AD.
Post‐transcriptional modulation of aENaC mRNA stability in alveolar epithelial cells: involvement of conserved domains on its 3′ untranslated region
Authors: Gagnon Frédéric , Migneault Francis , Berthiaume Yves , Dagenais André
Published: November 2023

The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells (AEC) plays a major role for lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that different conditions affecting alveolar epithelial cells induce the rapid breakdown of αENaC mRNA by post‐transcriptional modulation of its stability. Since the 3′ untranslated region (3′UTR) of mRNA are known to modulate transcript stability, a sequence analysis was performed comparing αENaC 3′UTR of different species to find conserved homologous domains. Five highly conserved sequences (CS) were identified in the 3′UTR portion of αENaC mRNA, suggesting a role for these sequences in modulation of its stability. Rat αENaC cDNA with full 3′UTR sequences were cloned in pTRE‐tight vector of the TET‐off system. After transfection of AEC by electroporation, transcription of the αENaC TET‐off clone was inhibited with doxycycline. The level of transcript remaining after different time points was evaluated by RT‐PCR. Using this technique, the half‐life (T1/2) of the transcript was evaluated to be ~100min. To study the impact of αENaC 3′UTR sequences and the role of the different CS in modulation of αENaC mRNA stability, 3′UTR deletion mutants were designed by gradually removing the 3′ distal portion of the 3′UTR sequence deleting gradually CS1 (263bp), CS1–2 (454bp), CS1–3 (515bp) and CS1–5 (complete 3′UTR; 894bp). All the deletion mutants affected αENaC mRNA stability. The resulting T1/2 were respectively of 203 min (CS1), 300 min (CS1–2), 48 min (CS1–3) and 375 min (no 3′UTR). These results suggest that there is a complex stabilizing and destabilizing interplay between cis‐elements present in different regions of 3′UTR to modulate αENaC mRNA stability. Because large portions of 3′UTR were removed in these mutants, deletion mutants targeted to remove specifically the different CS were tested to assess the role of each of these domains in transcript stability. A deletion mutant removing only CS1 (31 bp) shows the same T1/2 than the first sequential deletion mutant (T1/2 202 min). The CS2 and CS3 domains are present in close tandem. This is the same for CS4–CS5. Deleting CS2–3 (82 bp) increases T1/2 to 176 min while deletion of CS4–5 (74 bp) increases the stability of the transcript to 220 min. Altogether, our results show that CS play an important role for destabilization of αENaC mRNA since their removal increases αENaC mRNA T1/2. These elements could play an important role in the rapid breakdown of αENaC mRNA during inflammation and injury in the lung.
The Role of mRNA Degradation in Dynamic Nitrogen Environments in Saccharomyces cerevisiae
Authors: Rodriguez‐Tirado Carla Sofia , Gresham David , Abdul‐Rahman Farah
Published: November 2023

Studying the reprogramming of gene expression is fundamental to understanding cellular growth, development and stem cell differentiation. In Saccharomyces cerevisiae cells undergo reprogramming of gene expression when they are grown in nitrogen‐poor conditions and then switched to nitrogen‐rich conditions. This is controlled by the nitrogen catabolite repression (NCR) response. In a previous study, NCR transcripts were shown to undergo accelerated degradation when the cells where switched from growth on proline in a nitrogen‐poor environment to growth in a nitrogen‐rich environment upon the addition of glutamine. This study aims to determine the benefit of accelerated mRNA degradation in dynamic nitrogen environments by studying the effects of mutants of NCR repressors during this transition. We suspect that knocking out the repressors will stop the accelerated degradation once glutamine is added. This will in turn affect the growth rates and cause the mutants to have longer lag phase, a period where there is no detectable change in the growth. The general amino acid permease (GAP1) was used in representation of NCR transcripts to study the effects. This gene encodes a permease that senses the presence of amino acids substrates to regulate localization to the plasma membrane when needed. We identified mutants where GAP1 is stabilized in a nitrogen‐rich environment by using knock‐outs (KO) of the NCR repressor genes Ure2, Dal80 and Gzf3. The Ure2KO mutant showed depression of GAP1 in nitrogen‐rich conditions, but it caused a growth defect when the cells were grown in nitrogen‐poor conditions. In addition, we tested the effect of the GAP1 5′ untranslated region (UTR) mutant on growth dynamics because it has been shown to stabilize the GAP1 transcript during the transition from nitrogen‐poor environment to a nitrogen‐rich environment. A T‐test was performed to see if there was any difference between the growth rates of the wildtype and the 5′UTR deletion. Turns out that there was no significant difference between them before (p value=0.13) and after the glutamine addition (p value= 0.15), despite there being a notable distinction between the slopes. When the graphs for the nitrogen up shift were generated a lag phase was detected and it appeared to be an hour longer in the mutant. Which indicates that accelerated degradation is an important process in dynamic nitrogen environments.

Support or Funding Information

NIH‐MARC Program undergraduate fellowship (5T34GM00781‐37)
Structural and Biochemical Analyses of Alcohol Dehydrogenase E Enzymes from Entamoeba invadens IP‐1, E. invadens VK‐1:NS and E. dispar
Authors: Gabrielle Matthew , Leito Joshua , Espinosa Avelina
Published: November 2023

The Entamoeba lineage belongs to the Amoebozoa, one of six major divisions of eukaryotes. Entamoeba trophozoites, similar to other protists, actively capture ingest and digest bacteria thorough anaerobic pathways that process diverse energy sources. The bifunctional alcohol/aldehyde dehydrogenase enzyme in Entamoeba histolytica (EhADH2) belongs to the ADHE iron dependent family, and is essential for trophozoite growth and survival. EhADH2 catalyzes the conversion of acetyl Co‐A to acetaldehyde and the final reduction of acetaldehyde to ethanol by its separate ADH and ALDH domains respectively. Several Entamoeba spp have homologous genes (ADHE) which may have similar structure and function in the glycolytic pathway to that of E. histolytica. This study aims to clone, express, and sequence the genes that encode for ADHE in three Entamoeba spp. The sequencing identifies evolutionary relationships between the homologs. The transformation of ADHE genes into E. coli cells will be used for protein purification and kinetic assays for comparison with Entamoeba histolytica ADHE (EhADH2) previously studied by this lab. These kinetic assays will provide Km values for both substrates and information on chemical inhibitors of the ADHE proteins. Inhibitors of this enzyme have promising anti‐amoebic capabilities for drugs due to ADHE necessity during anaerobic respiration. Anaerobic pathogens have evolved adaptive metabolic enzymes that differ from vertebrates, and are ideal targets for novel compounds. We are interested in furthering our knowledge about the bifunctional alcohol/aldehyde dehydrogenase EhADH2 enzyme (required for E. histolytica growth and survival) and its adaptations to develop improved/non‐toxic strategies to manage amebiasis in humans.
Synthesis, Antimicrobial Activity and Molecular Docking Studies of Novel Bioactive Fused Heterocyclic Systems
Authors: Odai Sevdije Govori , Haziri Arben , Ibrahimi Hamide , Ademi Kastriot , Neziraj Nexhat
Published: November 2023

Current study based on the synthesis of new 1,2,4‐triazole compounds containing purine moiety: 9H‐[1,2,4]triazolo[4,3‐g] purin‐5‐amine I and 5‐amino‐3H‐[1,2,4]triazolo[4,3‐g]purine‐3‐thione II.Synthesized compounds were characterized by IR,NMR, and elemental analysis. The synthesized compounds have been screened for in vitro antibacterial activity against Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 and Klebsiella pneumonie PR 23/07 by cup plate method at concentrations 1,3,5 mg/ml. DMF was used as solvent control. The antibacterial activity of the synthesized compounds was found to be mild to moderate antibacterial activity. The synthesized compounds (I–II) were subjected to molecular docking studies of the Glucosamine 6‐Phosphate Synthase. The in silico molecular docking study results showed that, the synthesized compounds may be consider as good inhibitors of GlcN‐6. Also results showed similarity with in vitro tests against bacteria.
Carbohydrate‐linked cisplatin analogue: reactivity studies with RNA and DNA
Authors: Gamage Supuni Duneeshya Thalalla , Muthunayake Nisansala , Sonousi Amr , Crich David , Chow Christine
Published: November 2023

With the discovery of cisplatin in the 1960s, it has been widely studied as a precursor for anticancer drug development. Despite its effectiveness against certain cancers, clinical usage of cisplatin is restricted by a number of side effects and resistance. In the past decade, scientists have been exploring biologically important ligands such as sugar derivatives in the hope of overcoming such challenges. Attachment of a sugar moiety could facilitate lower accumulation of platinum drugs in the body as well as enhance cellular uptake. In this study, a carbohydrate‐linked cisplatin analogue, cis‐dichloro[(2‐β‐D‐glucopyranosidyl)propane‐1,3‐diamine]platinum (1) has been studied. The aim is to evaluate the binding targets and mechanism of action compared to cisplatin. Compound 1 was synthesized and a reactivity study was carried out with deoxy‐/ribonucleosides and nucleic acids using high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC‐MS), and matrix‐assisted laser‐desorption ionization mass spectrometry (MALDI‐MS). A study was done to analyze the effects of compound 1 on DNA duplex structure by measuring the bending angles. Further probing studies on ribosomal RNA showed compound 1 localization on rRNA. Overall, this work allows us to compare ligand binding and subsequent effects on nucleic acids through a series of chemical and biochemical techniques.

Support or Funding Information

Wayne State University‐Faculty Schaap Fellowship
Effect of bleaching products on the collagen of teeth
Authors: Cavallaro Christina , Schiliro Chelsea , Keenan Kelly
Published: November 2023

Americans spend an estimated 1.4 billion dollars on over‐the‐counter bleaching products which whiten teeth. The active ingredient is hydrogen peroxide which can damage proteins. The dentin contains an estimated 90% of its protein in the form of collagen and the effect of bleaching on collagen has not been determined. In this project, the non‐collagen proteins from teeth were extracted with EDTA and the remains of the teeth were dialyzed against water and then pulverized with liquid nitrogen. The resulting powder was re‐suspended and treated with pepsin to produce fragments. The protein level of these fragments was tested using the Lowry assay and individual fragments were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Individual fragments were removed and hydrolyzed to produce amino acids which were separated and identified using reverse phase high performance liquid chromatography. This was done on untreated teeth as well as teeth that had received either one or three rounds of tooth whitening with an over‐the‐counter product.

Support or Funding Information

The authors wish to thank the NAMS and Provost Travel Funds and the Provost Opportunity funds.
A Biosensing Soft Robot: Integrating Chemical and Optical Responsive Synthetic Cells with Soft Robotics
Authors: Justus Kyle , Lewis Daniel , Majidi Carmel , LeDuc Philip , Tan Cheemeng
Published: November 2023

Soft robotics and synthetic biology have shown tremendous potential mimic living organisms and exploit inherent biological machinery respectively. By combining these principles, sensory and control capabilities of the soft devices can be expanded through utilization of complex genetic controls in synthetic cells. Here, we take the first step towards such bio‐opto‐fluidic systems by constructing a hybrid device that consists of soft materials, synthetic bacteria, fluidic systems and electronics. Specifically our device consists of a series of circular wells and channels patterned into a flexible polydimethylsiloxane (PDMS) layer for culturing synthetic Escherichia coli that express green fluorescent proteins (GFP) and a flexible electronic layer housing an LED circuit designed to excite the GFP within the biomaterial layer. The PDMS chamber has high translucence that allows for optical control of synthetic bacteria, and high viscoelasticity that provides mechanical versatility, allowing it to function within a pneumatically actuated soft machine when sealed within a permeable flexible membrane. The pore sizes allow for the permeation of small molecules while retaining larger objects such as cells, a result validated through size‐varying fluorescent bead assays. We have also shown compatibility with an optogenetic genetic construct within the bacteria, which produces GFP in the presence of a chemical inducer and ceases production when exposed to blue light. Lastly, we have demonstrated the chemical sensing capabilities of the approach by designing a soft gripper which features chemo‐sensitive surface reliant on synthetic cells and is able to detect IPTG infused within hydrogels. Our work would have wide impact on the development of the next‐generation bio‐opto‐fluidic devices and the integration of synthetic biological systems with soft electronic materials.
Elucidation of molecular signaling battles between the eukaryotic host and a bacterial pathogen
Authors: Orth Kim , de Souza Santos Marcela , Salomon Dor
Published: November 2023

The marine bacterium Vibrio parahaemolyticus is the worldwide leading cause of seafood‐borne acute gastroenteritis. For decades, this pathogen has been studied exclusively as an extracellular bacterium. However, recent results have revealed the pathogen's ability to invade and replicate within host cells. Microbial killing by host cells is largely a function of phagocytosis followed by generation of reactive oxygen species (ROS) to kill the engulfed microbes. The phagocytic NADPH oxidase (NOX2) complex, which is composed of at least five proteins, generates these superoxides at the site of engulfment, which activates proteolytic enzymes that mediate killing and immune signaling. The GTPase Rac1/2 is essential for the function of the complex as its activation, localization to the membrane, and binding to p67 activates the complex. To maintain its existence inside a eukaryotic cell, the pathogen manipulates the intracellular host cell environment to suit its needs and survive. We have discovered a novel posttranslational mechanism that prevents the localization of Rac to the membrane, thereby, disabling the ability of the host to produce ROS and cripple the invading bacteria.

Support or Funding Information

NIHAID, NIHGMS, Welch Foundation, Once Upon a Time..., HHMI
Methionine sulfoximine reduces proinflammatory cytokine release by murine macrophages
Authors: Peters Tyler , Jambekar Amruta , Brusilow William
Published: November 2023

Methionine sulfoximine (MSO) is an amino acid that irreversibly inhibits glutamine synthetase (GS). Several studies have linked aberrant GS activity with pathological states, and GS inhibition by MSO has been extensively reviewed. We have previously shown that IP injection of MSO (50mg/kg) increases survival and decreases pro‐inflammatory cytokine levels in a mouse model of acute liver failure. We are trying to characterize this activity, and we present evidence that MSO directly modulates the innate immune response by decreasing cytokine secretion by macrophages. Primary rat kupffer cells and mouse peritoneal macrophages were treated with MSO prior to addition of 1ug/mL LPS. A cytokine array analysis demonstrated appreciable decreases of TNFα and IL‐6 in the supernatants from cells treated with MSO compared to controls. These data were confirmed by ELISA quantification showing that MSO significantly reduced the levels of TNFα and IL‐6 at both 4 hours and 6 hours after LPS treatment in both cell culture models (p<0.05). We are currently attempting to establish a link between GS inhibition and cytokine secretion, but because these studies used a racemic mixture of LS and LR MSO, we have also treated macrophages with the purified LR isomer, which does not inhibit GS. Preliminary results show that the LR isomer does affect the cytokine response, suggesting an additional target for MSO besides glutamine synthetase.
Repurposing P‐glycoprotein Inhibitors as Modifiers of Sphingolipid Metabolism— Therapeutic Implications in Cancer
Authors: Cabot Myles C
Published: November 2023

Chemotherapy efflux transporters such as P‐glycoprotein (P‐gp), a major player in multidrug resistance, remain attractive targets in laboratory and clinical studies in cancer therapy. Research in this area has largely been directed towards limiting the efflux capacity of these pumps with the goal of attaining lethal chemotherapy concentrations within tumor cells. One common avenue for reducing chemotherapy efflux is via the introduction of P‐gp inhibitors; however, whereas this goal remains promising, clinical use of a myriad of inhibitors has provided little progress over decades of trials. Herein we reveal that classical first, second, and third generation P‐gp inhibitors, in the single digit micromolar range, block sphingolipid (SL) metabolism at the juncture of ceramide glycosylation, a prominent ceramide detoxification pathway in cancer cells; this action effectively curbs the tumor‐suppressor properties of ceramide. Herein we sought to determine whether P‐gp inhibitors could be repurposed to circumvent the blunting effects elicited by ceramide glycosylation. Our work demonstrates that co‐administration of a ceramide therapeutic with a P‐gp inhibitor results in synergistic tumor cell killing. For example, in instances wherein short‐chain ceramides (C6‐ceramide) were supplied to the culture medium or when ceramide‐generating drugs like fenretinide were used, the inclusion of P‐gp inhibitors such as tamoxifen, cyclosporin A, biricodar, tariquidar, and the high‐affinity, specific P‐gp inhibitor zosuquidar, effectively depressed ceramide glycosylation in cancer cells by as much as 85%, resulting in magnification and perpetuation of ceramide's cancer‐suppressor activities. The C6‐ceramide‐P‐gp‐inhibitor‐containing regimens compared to the single agents, were more effective in inducing apoptosis in cellular models of human acute myeloid leukemia, including multidrug resistant HL‐60/VCR cells, colorectal, ovarian, and prostate cancer, and melanoma, with upstream mechanisms of action that enlisted mitochondrial dysfunction, ROS generation, inhibition of glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins. Although multidrug resistance modulators, when paired with chemotherapy drugs, have demonstrated little success clinically, the data herein suggest that repurposing these agents as modifiers of SL metabolism in ceramide‐centric drug regimens could provide novel therapeutic approaches, independent of drug resistance status, in treatment of cancer.

Support or Funding Information

National Institutes of Health (NCI) grant number 5 P01 CA171983
Phosphatidylinositol 3,5‐Bisphosphate acts as a Novel Regulator of Calcium Transport During Saccharomyces Cerevisiae vacuolar fusion
Authors: Miner Gregory E. , Fratti Rutilio A.
Published: November 2023

Phosphoinositides (PIs) are a group of lipids that are differentially phosphorylated to provide specific protein‐lipid interactions that are necessary in maintaining eukaryotic homeostasis. In membrane traffic, different PIs can interact with nucleotide exchange factors, tethering molecules and SNARE proteins ultimately leading to membrane fusion and transfer of cargo material. Our work has shown that phosphatidylinositol 3‐phosphate (PI3P), PI4P and PI(4,5)P2 are essential for the fusion of yeast vacuoles/lysosomes. However, little is known about the role of PI(3,5)P2 in the fusion pathway. Others have shown that PI(3,5)P2 is necessary for fission to occur during osmotic shock that is coupled to the efflux of vacuolar Ca2+ into the cytoplasm. This is thought to occur through the interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Ca2+ efflux has also been linked to vacuole fusion and occurs upon the formation of SNARE complexes between partner vesicles. Our data show that increasing levels of PI(3,5)P2 (through the addition of exogenous short chain (diC8) lipid) led to inhibition of vacuolar fusion. Importantly, sub‐inhibitory concentrations of diC8‐PI(3,5)P2 completely blocked Ca2+ efflux without affecting fusion. This suggests that PI(3,5)P2 plays opposing roles in fission, fusion, and Ca2+ transport. It further suggests that the Ca2+ efflux channel used during fusion is different than Yvc1. Finally, these data indicate that Ca2+ efflux is not required for vacuolar fusion and may be a parallel response. To further elucidate the role of PI(3,5)P2 in fusion we examined the effect of inhibiting the PI3P 5‐kinase, Fab1 using the PIKfyve (human homolog of Fab1) inhibitor Apilimod. In accord with our other data, treatment of vacuoles with Apilimod led to enhanced Ca2+ efflux, illustrating that PI(3,5)P2 negatively regulates fusion‐linked Ca2+ efflux, however the mechanism through which this occurs remains unknown. In addition to effects on Ca2+ transport, PI(3,5)P2 has been shown to enhance vacuolar H+ pumping through its association with the V‐ATPase. This is further supported by our data showing that Apilimod inhibited H+ pumping. Taken together we now hypothesize that PI(3,5)P2 may affect the activity of the H+/Ca2+ exchanger, Vcx1, due to its effect on both H+ pumping and Ca2+ efflux.

Support or Funding Information

NIGMS GM101132
Monitoring Organelle‐Specific Responses to Amphotericin B in Mammalian Cells and Candida albicans Biofilms
Authors: Tourville Claudia , Rigden Gabrielle , Lewis David , Hartsel Scott
Published: November 2023

The use of indwelling medical devices such as catheters or dentures in combination with immunosuppressive therapies increase the body's susceptibility to fungal infections as a pathway to life threatening systemic infections. In particular, Candida albicans, an opportunistic pathogen existing in the normal microbial flora can produce serious systemic infections. This species is unique in its ability to exist in two phases: a yeast form and hyphal biofilm. Once it advances into its biofilm formation, Candida albicans is resistant towards drug treatment, making it immensely difficult to treat in clinical settings. Confocal laser microscopy using organelle‐specific fluorescent probes provides an internal view of Candida albicans along the stages of development and drug response. We have developed and deployed a series of our own naphthalimide‐based fluorescent probes targeted to mitochondria, lysosomes, and the Golgi apparatus to monitor organelle vitality in real time. The brightness, chemical and photo‐stability of our organelle probes allows us to image live cells in their appropriate culture media to observe cellular responses to drug treatments. The resistance to photobleaching makes them useful for longer periods of laser exposure as well as 3D imaging. We have investigated the various liposomal as well as micellar formulations of Amphotericin B including Fungizone, Abelcet, and Ambisome. All formulations of Amphotericin B display some level of toxicity, but lipid formulations such as Abelcet and Ambisome have shown a great reduction in human toxicity without significantly sacrificing their antifungal activity. Using our organelle probes in vitro, we are able to monitor the changes in specific organelles in both model human cells and Candida albicans yeast and hyphal cells to pinpoint the intracellular responses to Amphotericin B in both the target and host. Thus far we have found that Amphotericin B, especially Fungizone, leads to depolarization of mitochondria in some human cell lines while having little acute effect on mitochondria in Candida albicans biofilms. Comparable Amphotericin B doses in the forms of Abelcet and Ambisome, on the other hand, seem to allow for the maintenance of mitochondrial membrane potential in human cells as seen by persistent polarization following multiple hours of incubation. We are currently investigating the time‐dependent relationship between organelle vitality and the differing Amphotericin B formulations with all our probes and cell lines to provide insight as to how Amphotericin B affects mammalian, yeast and biofilm cells differently. Gaining this intracellular perspective of cell health will open doors for the optimization of antifungal drugs through pinpointing their effective mechanisms against fungi while minimizing cellular casualties resulting from organelle decline.

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Office of Research and Sponsored Programs‐UW‐Eau Claire
A Potential Role for Mitochondrial Ca2+ Uptake During B Cell Activation
Authors: Torres‐Quintanilla Alejandro , González‐Castillo Elena , García‐Rivas Gerardo
Published: November 2023

Our understanding of the role played by mitochondria during immune cell activation has passed from just an energy‐producing organelle to an active modulator of cell signaling. Ca+2 handling and redox homeostasis are intracellular signals modulated by mitochondria. For example, mitochondrial Reactive Oxygen Species (mROS) have recently been described as signaling molecules that fine‐tune activation pathways by allowing full nuclear translocation of NFAT in T cells. Moreover, mitochondria have received special attention as buffers of cytosolic Ca+2 dynamics during activation, through MCU‐dependent Ca+2 uptake. Here, we suggest that mitochondria participate during B cell activation through Ca+2 overload. Primary murine B cells were purified and subjected to BCR‐dependent activation using anti‐IgM antibody and BCR‐independent activation using thapsigargin. Thapsigargin induces store operated Ca+2 entry by emptying endoplasmic reticulum Ca+2 storage. After 7hrs of stimulation, flow cytometry was used to determine activation through CD69 surface expression and mROS levels using a mitochondrial superoxide fluorescent probe. Mitochondrial Ca+2 content and uptake was determined in permeabilized lymphocytes using Fluo‐4 and Ca+2 Green 5N after activation. We found that after BCR‐independent stimulation, CD69 surface expression was equivalent to activation via BCR. Importantly, pretreatment with a mitochondrial antioxidant reduced CD69 surface expression after stimulation with both anti‐IgM or thapsigargin. This result suggested that mitochondrial Ca+2 uptake occurs during activation to trigger mROS production. Lastly, we found that activated cells show higher contents of mitochondrial Ca+2. Together, these results suggest that B cell activation induces an important uptake of Ca+2 by mitochondria, which might participate in the activation cascade by increasing mROS production.

Support or Funding Information

ATQ is supported by CONACyT MSc scholarship
Differential Effects of 1,25D3‐MARRS Expression on Mammary Gland Growth and Development Depend on the Vitamin D3 Dose
Authors: Wilkin Allison , Trinh Thao , Edson Michael , Meckling Kelly
Published: November 2023

1,25 dihydroxyvitamin D3 (1,25D3) is one of the most potent biologically active forms of vitamin D3. Primary activities include regulation of blood calcium and phosphate homeostasis and a plethora of other effects including cell growth inhibition, induction of differentiation, and anti‐cancer effects, depending on the target tissue. Most of the activities of 1,25D3 continue to be ascribed to the classic vitamin D receptor despite the identification of another membrane receptor for 1,25D3. The membrane‐associated rapid response steroid‐binding (MARRS) receptor is receiving more attention as a novel vitamin D receptor working through rapid, non‐genomic pathways. The purpose of this study was to explore the role of MARRS in the mammary gland using a conditional knockout mouse model and a vitamin D3 dietary intervention. 1,25D3‐MARRS expression was reduced in epithelial cells of mammary glands (MG) using the Cre/loxp system. Dams were weaned onto semi‐purified diets with 10,000 IU/kg, 1,000 IU/kg, or 0 IU/kg of 1,25D3 for 4 weeks before being set‐up to breed. 4th MGs were collected from 6‐week old female MMTV‐Cre mice (n=73). MGs were whole‐mounted and stained with carmine alum. Tissue growth was assessed by counting the number of terminal end buds (TEB) of alveolar branches, measuring the length of the longest ductal extension, and measuring total MG area covered ducts. Whole body weight was similar between all groups. There was a significant interaction between genotype and diet. Knockout mice on the 1,000 IU/kg diet had significantly fewer TEBs (p=0.016) and reduced ductal growth and extension (p=0.037, p=0.023 respectively) compared to controls on the same diet. Mice on the 0 IU/kg diet had similar TEB counts across all genotypes. Knockout mice on the 10,000IU/kg diet had significantly more TEBs than the other mice of the same diet (p=0.042), and more extensive growth and coverage of alveolar branches than knockouts on the 1,000 IU/kg diet. These results suggest that there is an effect of 1,25D3‐MARRS that is dependent on vitamin D3 dose. Because the dose‐response is not linear in this animal model, it suggests caution when making recommendations for vitamin D3 nutrition in animals, and possibly humans as well.

Support or Funding Information

The Natural Sciences and Engineering Research Council of Canada