bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Staphylococcus aureus Biofilm and Planktonic Secreted Products on Myofibroblast Differentiation
Authors: Hall Preston M , Brennan Robert E , Vaughan Melville B
Published: November 2023

Wounds often become chronic by staying in the inflammatory stage of the healing process for too long. The ability of fibroblasts to differentiate into myofibroblasts is key for the normal progression of wound healing. Recent experimental evidence showed products secreted by planktonic S. aureus and S. aureus biofilms differentially affect viability and inflammatory cytokine production by human fibroblasts, along with human keratinocytes. Our goal was to determine whether media conditioned by S. aureus affected myofibroblast differentiation, using immunostaining techniques. We grew normal human fibroblasts on coverslips in media containing 1ng/ml TGF‐β plus or minus 100 μL/ml of the planktonic S. aureus secretions or the S. aureus biofilm secretions; these were compared to media‐only control. We found that products secreted by S. aureus, especially planktonic S. aureus, significantly decreased myofibroblast differentiation in three separate experiments across two different cell types which suggests that wound healing won't progress causing the wound to become chronic. Bacterial biofilms are difficult to treat, so promoting myofibroblasts may aid the healing of chronic wounds. These findings should spark future studies related to wound healing.

Support or Funding Information

3M Corporation and UCO Office of Research and Grants
Japanese herbal medicine, Juzentaihoto, suppress the lymphocyte infiltration in the aging mouse salivary glands
Authors: Nonaka Naoko , Nakamura Masanori
Published: November 2023

Xerostomia or dry mouth is a systemic disease resulting from the change in the composition of saliva or reduced saliva flow. The symptom is caused by various factors such as the dysfunction of salivary glands and nervous system, the side effect of medicines and so on. The amount of salivation also decreases with aging. This symptom is very common today. Artificial saliva is usually prescribed for the treatment of xerostomia. We previously indicated that pituitary adenylate cyclase activating polypeptide (PACAP) stimulated the increase of salivation and the concentration of epidermal growth factor in secreted saliva. Furthermore, we found the severe lymphocyte infiltration in salivary glands of old C57B/6 mice. Juzentaihoto (JTT) is one of the Kampo Japanese herbal medicines and caused the induction of IL‐12 and subsequent activation of natural killer cells. In this study, we have examined the effect of JTT on lymphocyte infiltration in the major salivary glands (parotid, submandibular and sublingual glands). Male C57BL/6 mice were bred for 2 years under SPF condition. To examine the effect of the JTT, feed with/without JTT was given to mice during the breeding period. At the indicated time, salivary glands were dissected and processed for histological examination. Histological examination clearly indicated the severe lymphocyte infiltration in parotid glands and submandibular glands, but not in sublingual glands of control mice. Histomorphometric analysis indicated that the area of the lymphocyte infiltration in control mice was 4.35 ± 1.67% in parotid glands and 8.58 ± 4.43 % in submandibular glands respectively. On the other hand, the ratio of the infiltration was reduced to 1.94 ± 1.10 % in parotid glands and 1.74 ± 0.59 % in submandibular glands of JTT‐treated group. These results expect the old C57BL/6 (male) mice as a model of autoimmune disease such as Sjogren's syndrome and suggest JTT might be the effective therapeutic drug for the salivary gland disease with aging or inflammation.

Support or Funding Information

Grant sponsors: Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant numbers: 20592148, 21592342 and 15K11022.
The Facial Nerve: The Path of Its Branches to the Muscles of the Infraorbital Region that Lift the Upper Lip in Relation to the Deep Fascia of the Midface
Authors: Amodeo Chiara Andretto , Delmas Vincent , Casasco Andrea , Keller Gregory S
Published: November 2023

The mimetic muscles are innervated by the facial nerve. The function of these muscles is very important because they partecipate to the eye protection, the speach and the nutrition. Furthermore from an evolutionary point of view the facial expressions are a major way of communication between human beings. Classic anatomy describes the path of the branches of the facial nerve in the midface, anterior to the parotid fascia, as running in the subcutaneous tissue, a recent study related to the fascial layers of this region has shown that a deep fascia exists in the infraorbital region, it continues from the temporal area and it covers the mimetic muscles that lift the upper lip. The aim of this study is to describe the path of the branches of the facial nerve in this facial area in relation to the deep fascia recently described.

Materials and Methods

24 hemifaces have been dissected at the Laboratoire d'Anatomie de Paris Descartes to study the relationship between the branches of the facial nerve and the deep fascia of the infraorbital region.

Results

This study demonstrates that the branches of the facial nerve that go to the muscles of the infraorbital region are always covered and protected by the deep fascia of this region. Some branches run deep to the muscles, on the contrary some run superficial to the zygomaticus major, minor, levaetor labii superioris etc and anastomize with other branches to reach the glabellar region, all these branches run deep to the deep fascia.

Conclusions

The existance of a deep fascia in the infraorbital region has been shown. This fascia covers and protects the branches of the facial nerve that run to the muscles that lift the upper lip, this finding changes the classic anatomical concept of the facial nerve running without protection in the subcutaneous tissue of the midface and it has important implications in both descriptive and applied anatomy.
Quantification of Telomerase Activity in Colonial Ascidians
Authors: Durham Megan , Beck Chelsey , Keeling Elena
Published: November 2023

Colonial ascidians are marine invertebrate chordates that reproduce sexually and asexually, and are also capable of whole body regeneration in response to damage. Telomerase is required to maintain chromosome length in all eukaryotic cells that divide indefinitely; it is therefore expressed in stem cells and is hypothesized to be necessary for both continuous asexual budding and for regeneration. Here, we investigate telomerase activity in the colonial ascidian Botrylloides. Telomerase activity was determined using a qPCR‐based assay and normalized by total protein. Results so far show that adult ascidian tissue has high levels of telomerase activity; this may be related to stem cell abundance and regeneration abilities of Botrylloides. Work in progress is examining telomerase activity in different tissues, including probable stem cell niches, as well as in different developmental and regeneration stages.

Support or Funding Information

CSU Council on Ocean Affairs, Science & Technology (COAST); Biological Sciences Department, California Polytechnic State University
Expression and Assembly of Active Human Telomerase in Escherichia coli
Authors: Hansen Jeffrey Lewis , Thiyagarajan Thirumagal , Larson Amy Kraft , Rideout April , Hansen Debra Tumbula
Published: November 2023

The assembly of human telomerase is a complicated process in which over a dozen different human proteins are implicated. These proteins include the Hsp90 heat shock complex, the box H/ACA snoRNP complex, and other proteins. The specific roles of these proteins in telomerase assembly are unclear. Telomerase expression and assembly in reported recombinant systems occurs either in eukaryotic cells or in eukaryotic cell extracts. We are studying human telomerase expression and assembly in a non‐eukaryotic system, Escherichia coli. In order to study the assembly of active human telomerase in E. coli cells, we developed a modular system of plasmids compatible for co‐transformation and co‐expression of various components of telomerase assembly. This approach differs from previous recombinant approaches because co‐expression allows for co‐translational and co‐transcriptional assembly. In fact, we detected telomerase activity by the trap assay in cell extracts from a small subset of our library of co‐expression strains. Surprisingly, we found that co‐expression of hTERT with hTR (the protein component and RNA component of telomerase) alone in E. coli is sufficient for the assembly of active telomerase, with the certainty that no other eukaryotic factors are present. We are now relying on the modular nature of our system to efficiently produce additional E. coli strains containing new combinations of plasmids in an effort to increase the yield of active telomerase.

Support or Funding Information

This research was originally supported by startup funding at the Medical University in South Carolina.

Currently support is through internal funding at A.T. Still University.
Aberrant Activation of the Kynurenine Pathway in Gliomas Increases Polymerase Kappa Expression and Promotes Genomic Instability and Chemoresistance
Authors: Bostian April , Maddukuri Leena , Reed Megan , Savenka Tatsiana , Hartman Jessica , Davis Lauren , Pouncey Dakota , Miller Grover , Eoff Robert
Published: November 2023

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and has a median survival time of 12 months, due in large part to poor responses to chemo‐ and radio‐therapy. These tumors exhibit high levels of DNA damage response activation before treatment and this likely plays a role in the inherent chemo‐ and radio‐resistance of GBM. Y‐family polymerases aid cells in bypassing genotoxic damage, but they are mis‐regulated in many cancers. Over‐expression of the Y‐family enzyme human polymerase kappa (hpol κ), which is up‐regulated in GBM patients, has been linked to an increase in genomic instability. The factors promoting over‐expression of hpol κ in GBM are unknown, but previous work implicates the aryl hydrocarbon receptor (AhR) in the regulation of hpol k levels. Up‐regulation of the AhR pathway through aberrant production of the endogenous ligand kynurenine is associated with malignant progression and shorter survival in glioma patients. We tested the hypothesis that the AhR signaling pathway transcriptionally regulates hpol κ levels in gliomas using in vitro cell culture models. Exposure to the AhR agonist 3‐methylcholanthrene increases hpol κ protein and mRNA levels 2–3‐fold in GBM cells. Furthermore, when the enzyme tryptophan 2,3‐dioxygenase (TDO), which is responsible for aberrant kynurenine production in gliomas, is inhibited by the small molecule 680C91, hpol κ expression decreased 20–60% in three GBM cell lines. We then examined the idea that stimulation of hpol k expression by the AhR pathway contributes to the inherent genomic instability seen in gliomas. Supporting our hypothesis, TDO inhibition leads to a decrease in spontaneous micronuclei formation in three GBM cell lines (33.6% DMSO to 12.9% 10 μM 680C91 in T98G cells; 12.3% DMSO to 8.7% 10 μM 680C91 in U87MG; and 25.3% DMSO to 20.1% 10 μM 680C91 in A172 cells). Additionally, treatment with an AhR antagonist also decreases micronuclei formation in T98G cells (33.6% DMSO to 17.2% 10 μM CH‐223191), as does knock down of hpol κ expression (25.5% for scr siRNA to 19.7% hpol κ). Furthermore, we performed epistasis assays which supported our hypothesis that TDO activity, AhR signaling, and hpol κ expression act in the same pathway to promote genomic instability in gliomas. Moreover, we found that TDO inhibition also significantly increases sensitivity of the T98G cells to various concentrations (10 – 100 μM) of cisplatin. Our study reveals how activation of AhR signaling could impact replication dynamics in gliomas and may provide insight into the mechanisms leading to the genomic instability and chemoresistance often inherent to this disease.

Support or Funding Information

This work was supported by NIH grant CA 183895 (to R.L.E.).
Identifying Y‐family DNA Polymerase Inhibitors to Improve Anti‐Cancer Drugs
Authors: Maddukuri Leena , Zafar Maroof Khan , Penthala Narsimha , Crooks Peter A , Eoff Robert L
Published: November 2023

Replication stress is a hallmark of cancer and many of the current chemo‐ and radio‐therapies act to limit tumor growth primarily though the induction of DNA damage in order to block replication. Translesion DNA polymerases (TLS pols), such as the Y‐family members pols h, k, i and Rev1, can attenuate the effects of genotoxic anti‐cancer drugs by facilitating bypass of otherwise cytotoxic DNA damage. Increased levels of hpol h expression has been implicated in platinum‐based drug resistance in ovarian cancer stem cells, as well as in patients suffering from metastatic stomach cancer. We have screened a series of N‐substituted indole thiobarbituric acid (ITBA) derivatives for inhibition of pol h activity using a fluorescence‐based activity assay. From these assays, we have identified several ITBA molecules that inhibit hpol h with low micromolar IC50 values. Ovarian cancer cells were co‐treated with the most potent hpol h inhibitors and cisplatin (CDDP) to determine if the combination treatment produced a synergistic effect on cell viability. Treating cells with a combination of PNR‐7‐02 and CDDP decreased OVCAR3 ovarian cancer cell survival in a synergistic fashion (combination index value of 0.40). As expected, transient knock‐down of hpol h sensitizes OVCAR3 cells to CDDP relative to control siRNA. Combining hpol h knock‐down with PNR‐7‐02 does not further sensitize OVCAR3 cells to CDDP, which indicates that both treatments act through the same pathway to influence cell viability in response to CDDP. This suggests that PNR‐7‐02 may specifically inhibit hpol h in cells. In addition, flow cytometry analysis of cells treated with CDDP and PNR‐7‐02 revealed enrichment of cells in G2/M phase compared to cells treated with CDDP alone. From this data, it appears that combining TLS pol inhibitors with CDDP has the potential to impede ovarian cancer cell growth by inhibiting hpol h‐catalyzed bypass of CDDP‐induced DNA damage and blocking exit from the G2/M phase of the cell cycle.

Support or Funding Information

(Supported by NIH grants CA183895 to RLE and CA140409 to PAC. This research was also supported by a grant from the Arkansas Breast Cancer Research Program, the UAMS CTSA and a Seeds of Science Award from the Winthrop P. Rockefeller Cancer Institute).
Effect of Inflammatory Cytokines on DNA Methylation and Demethylation
Authors: Winfield Jacquay , Esbitt Alexis , Seutter Sara F , Desai Biren , Abdo Mohamed , Vasconez Megan , Laidlaw William , Green Kevan , Shamseddin Seyed M , Borghaei Ruth C
Published: November 2023

Objective

To determine the effects of inflammatory cytokines and mediators on expression of DNMT and TET enzymes in human gingival fibroblasts isolated from patients with periodontitis as well as normal and transformed cell lines.

Methods

Expression levels of DNA methylatransferase (DNMT1 and 3a) and demethylase (TET1) were examined by RT‐PCR in cells treated for up to 24 hours with IL‐1, IL‐6 or prostaglandin E2. The role of PGE2 in IL‐1 suppression of DNMT3a expression was assessed by treating cells with IL‐1 in the presence and absence of NS‐398. Global levels of DNA methylation following treatment with IL‐1 were measured in an ELISA‐like assay.

Results

Treatment of HGF with IL‐1 caused a three‐fold increase in levels of DNMT1 mRNA over 24 hours, but a two‐fold decrease in DNMT3a mRNA in the same samples. Global levels of 5mC were increased in HGF treated with IL‐1 for 72 hours relative to untreated controls. PGE2 caused a dose‐dependent decrease in levels of DNMT3a similar to IL‐1, but also slightly decreased levels of DNMT1.

Conclusion

These data are consistent with the hypothesis that exposure to inflammatory cytokines or PGE2 alters levels and/or activity of enzymes involved in DNA methylation, resulting in global and possibly gene‐specific changes which can lead to changes in cellular characteristics over time. Further experiments will be aimed at testing the effects of these cytokines on expression of DNMT and TET enzymes in normal and transformed cell lines, and at elucidating mechanisms involved in cytokine regulation of these enzymes.

Support or Funding Information

This work has been supported by the Philadelphia College of Osteopathic Medicine Division of Research.
Sphingosine kinase 2 Generated Nuclear S1P is an Epigenetic Co‐Regulator of Pseudomonas aeruginosa‐Induced Lung Inflammation
Authors: Ebenezer David Lenin , Fu Panfeng , Berdyshev Evgeny , Natarajan Viswanathan
Published: November 2023

Pseudomonas aeruginosa (PA), a gram‐negative, opportunistic, aerobic bacilli is associated with rapid deterioration of the cystic fibrotic lung and is the significant cause of pneumonia in immunocompromised and mechanical ventilated patients. A distinctive feature of P. aeruginosa lung infection is the incessant inflammation‐driven respiratory failure that contributes to increased morbidity and mortality. Our research aims to define the epigenetic role of Sphingolipids in regulating immunological events in P. aeruginosa‐induced lung inflammation that has remained undefined so far. Sphingosine‐1‐Phosphate (S1P), is a bioactive sphingolipid that plays a key role in regulating signaling pathways modulating inflammatory responses in cells. Sphingosine Kinase (SphK) 1 & 2 generate intracellular S1P and Sphingosine‐1‐Phosphate Lyase (S1PL) and S1P Phosphatase (SPP) catalyze its degradation. Our murine model studies, as evident from decreased levels of inflammatory cytokines, proteins and cell counts in bronchoalveolar lavage and neutrophil migration to the alveolar space, suggest that knocking out SphK2, but not SphK1, offers protection against PA‐induced lung inflammation. PA infection also induced phosphorylation of SphK2 and subsequent translocation to the nucleus in mouse lung tissues and mouse lung epithelial cell line (MLE‐12). PA‐induced SphK2 phosphorylation resulted in increased acetylation of Histone H3 and H4 that correlated with increased IL‐6 gene expression at mRNA and protein levels. Moreover, PA infection decreased Histone Deacetylase (HDAC) activity in a time dependent manner in mouse alveolar type II cells and we validated SphK2 association with HDAC 1 & 2 by co‐immuno precipitation with nuclear HDAC repressor complex proteins. Nuclear fractions isolated from MLE‐12 cells exhibited significant levels of sphingosine and ceramide and stimulation with PA enhanced nuclear S1P in the presence of S1P lyase inhibitor, 4‐deoxypyridoxine. Interaction of sphingosine and S1P with HDAC1 and 2 in MLE‐12 nuclear fraction was confirmed by bio‐conjugate technique using biotin tagged sphingosine and S1P. Furthermore, using MLE‐12 nuclear fraction, we were able to demonstrate, that inhibition of nuclear HDAC activity by S1P is as potent as the inhibition obtained using Trichostatin A (TSA), a HDAC inhibitor. To examine the therapeutic potential of targeting SphK2‐S1P signaling axis in the treatment of PA‐induced lung inflammation, we evaluated ABC 294640, a specific SphK2 inhibitor in mice and observed reduction in PA‐induced inflammation. Taken together, these results emphasize the novel role of nuclear S1P in PA‐induced lung inflammation and targeting SphK2 could be a therapeutic option to treat bacterial infection of the lung.

Support or Funding Information

This work is supported by NIH/HLBI P01 HL98050 to VN.
The Role of the Actin‐binding Protein, Alpha Actinin 4, in Transcriptional Regulation by Glucocorticoid Receptors in Glomerular Podocytes
Authors: Zhao Xuan , Khurana Simran , Bruggeman Leslie , Kao Hung‐ying
Published: November 2023

Glomerular podocytes are highly specialized terminally differentiated cells that function as a filtration barrier in the kidney. Loss or damage to podocytes is associated with nephrotic syndromes, including focal segmental glomerulosclerosis (FSGS), a chronicle kidney disease characterized by proteinuria. Mutations in the actin‐crosslinking protein alpha actinin 4 (ACTN4) are associated with familial forms of FSGS. ACTNs are better known for their ability to bind actin filaments and modulate cytoskeletal organization. However, we have previously demonstrated that a fraction of ACTN4 is found in the nucleus and functions as a transcriptional co‐activator of some nuclear receptors and that ACTN4 coactivates NF‐κB transcription activity and is recruited to NF‐κB targeted promoters in cultured human podocytes (HPCs). Glucocorticoid therapy is a mainstay treatment option for many forms of nephrotic syndrome, although little is known about the mechanism by which the glucocorticoids ameliorate proteinuria and glomerular disease. Our data indicate that, similar to its action in most cell types, glucocorticoids induce nuclear translocation of glucocorticoid receptor (GR) and regulate target gene expression through transactivation and transrepression. In this study, we show that ACTN4 harbors a LXXLL nuclear receptor interacting motif and is capable of enhancing transcription activity by GR. ACTN4 interacts with GR in the nucleus of HPCs. Disruption of the receptor interacting motif, LXXLL significantly reduces its ability to potentiate transcriptional activity by GR. Stable knockdown of ACTN4 by shRNA in HPCs does not affect glucocorticoid‐induced nuclear translocation of GR, while it significantly reduces dexamethasone (Dex)‐mediated induction of GR target genes and the activity of a Glucocorticoid receptor element (GRE)‐driven reporter construct. Furthermore, loss of ACTN4 partially ablates transrepression of NF‐κB target genes in a gene‐dependent manner. Lastly, we demonstrate that FSGS‐linked ACTN4 mutants lose their ability to bind liganded GR and transcription potentiation activity. Taken together, our data suggest a role of ACTN4 in transcriptional regulation mediated by GR. We are currently dissecting the molecular basis of the interaction of ACTN4 and GR and investigating the mechanism by which ACTN4 works as a transcription co‐activator of GR.
Characterization of MarR family transcriptional regulator (BTH_I0021) from Burkholderia thailandensis
Authors: SABRIN AFSANA , Gupta Ashish , Grove Anne
Published: November 2023

Burkholderia thailandensis, a soil dwelling bacterium, is a Gram‐negative bacillus, which is structurally similar to pathogenic species such as Burkholderia pseudomallei but a much less virulent species. B. thailandensis encodes nine Multiple antibiotic resistance Regulator (MarR) homologs, which act as transcriptional regulators of multiple genes, some of which may be involved in virulence and pathogenesis. The genomic locus of one of the MarR homologs, BTH_I0021 (BtMarR), predicts that it regulates the expression of both an EmrB/QacA family drug resistance transporter (BTH_I0022) and a Resistance Nodulation Division (RND) efflux pump (BTH_I0019). Increased expression of EmrB/QacA transporter and RND efflux systems have been associated with enhanced resistance to a wide variety of antimicrobials. Using Electrophoretic Mobility Shift Assays we have shown that BtMarR binds to the intergenic DNA of its own gene and a divergently oriented gene encoding a conserved hypothetical protein (BTH_I0020) of unknown subcellular localization. Upon treatment with inorganic and organic oxidants, SDS PAGE revealed the formation of reversible oligomeric species of BtMarR. DNA binding is attenuated by oxidized protein suggesting structural changes due to disulfide bond formation between redox‐active cysteines. PAR (4‐(2‐Pyridylazo) resorcinol) assay illustrated that this protein binds divalent metals such as zinc. This raises the possibility that BtMarR controls gene expression in response to oxidative stress via cysteine oxidation and by metal binding. Attenuation of DNA binding in presence of oxidants may lead to increased expression of efflux systems. Since oxidative stress is an important host defense mechanism, this study should shed light on the understanding of adaptive responses of Burkholderia strains towards the oxidative burst that characterizes the initial host response to bacterial infection.

Support or Funding Information

This work was supported by National Institutes of Health grant 1R15GM107825 (to Anne Grove)
Antitumor DNA‐Damaging C‐1748 is a New Inhibitor of Autophagy that Triggers Apoptosis in Human Pancreatic Cancer Cell Lines
Authors: Borowa‐Mazgaj Barbara , Skwarska Anna , Augustin Ewa , Radomińska‐Pandya Anna , Mazerska Zofia
Published: November 2023

Despite the enormous progress that has been made over the past decades in diagnosis, treatment and prevention of many types of tumors, survival rates in pancreatic cancer still remain poor. Pancreatic cancer is one of the most malignant and chemoresistant tumors and the profound mechanism supporting these phenomena is the constitutively activated prosurvival autophagy. The antitumor 1‐nitroacridine derivative C‐1748 (9‐(2′‐hydroxyethylamino)‐4‐methyl‐1‐nitroacridine) belongs to a new set of DNA‐damaging agents developed in our laboratory and is currently ready for phase I clinical trials. Here, we have uncovered a new role for C‐1748 as a potent inhibitor of autophagy in four tested pancreatic cancer cell lines: Panc‐1, MiaPaCa‐2, BxPC‐3 and AsPC‐1. C‐1748 treatment at 0.1 – 1 μM significantly reduced the typical hallmarks of autophagy, such as conversion of soluble LC3‐I protein to its lipid‐bound LC3‐II isoform or development of acidic vesicular organelles (AVOs), and importantly shifted pancreatic cancer cells into apoptotic cell death. Co‐treatment with C‐1748 and wortmannin, an early‐stage autophagy inhibitor, had a synergistic effect on autophagy inhibition and potentiated C‐1748‐induced apoptosis. In particular, compared with exposure to C‐1748 alone, the combination treatment increased time‐ and dose‐dependent cytotoxic activity of C‐1748 with the most profound effect observed in BxPC‐3 cells. Moreover, along with decrease in autophagy, co‐treatment with wortmannin significantly elevated dysfunction of mitochondria, as manifested by a drop in mitochondrial membrane potential (ΔΨm), increased caspase‐3 activation, PARP cleavage, DNA fragmentation into apoptotic bodies and accumulation of lipid droplets arising due to the interruption of autophagy. In all studied cell lines, the level of apoptotic cells after dual exposure with C‐1748 and wortmannin increased by about 20% as compared to C‐1748 alone. Surprisingly, simultaneous exposure of pancreatic cancer cells to C‐1748 in combination with the late‐stage autophagy inhibitor, chloroquine, neither significantly increased the cytotoxic activity of C‐1748 nor ameliorated its apoptosis‐inducing potential. The synergistic effect of C‐1748 and wortmannin on the inhibition of autophagy and lack of such effect when C‐1748 was co‐administered with chlroquine, suggests that C‐1748, in addition to its DNA‐damaging properties may also affects early stages of autophagy. Further studies are undergoing to uncover the role of C‐1748 as a new inhibitor of autophagy, which is especially effective against pancreatic cancer.

Support or Funding Information

Research Grant from the Polish Ministry of Science and Higher Education NR 13‐0133‐10/2011 to BB‐M, AS, EA, ZM; DoD‐W81XWH1110795 to AR‐P and UAMS TRI ABCRP funds to AR‐P
Cholesterol‐Mediated Degradation of 7‐Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis
Authors: Sharpe Laura Jane , Prabhu Anika Vinayak , Luu Winnie , Brown Andrew John
Published: November 2023

Cholesterol is essential for normal embryogenesis, but in excess, it can be detrimental to human health. To achieve balanced cholesterol levels, enzymes involved in its synthesis possess many layers of regulation. Here, we investigated the post‐translational regulation of 7‐dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis, converting 7‐dehydrocholesterol to cholesterol. In the absence of functional DHCR7, accumulation of 7‐dehydrocholesterol and a lack of cholesterol production leads to the devastating developmental disorder, Smith‐Lemli‐Opitz Syndrome (SLOS). We found that statin treatment can ameliorate the low DHCR7 expression observed with common SLOS mutants. Furthermore, we show that DHCR7 protein is rapidly turned over and this degradation is induced by the cholesterol product itself, resulting in decreased DHCR7 protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7‐dehydrocholesterol, which in turn leads to an increased production of vitamin D. Thus, DHCR7 is an important enzymatic switch between cholesterol and vitamin D synthesis.

Support or Funding Information

National Health and Medical Research Council Grant (1060515), UNSW Goldstar Award, UNSW Career Advancement Fund
Development of Methionyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Robles Sara , Hu Yanmei , Bullard James M
Published: November 2023

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the enzymatic characterization and development of a screening platform based on methionyl tRNA synthetase (MetRS) from P. aeruginosa.

Results

MetRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The kinetic parameters for the interaction of P. aeruginosa MetRS with its three substrates (tRNA, ATP, and methionine) were determined. Initial velocities were determined for charging of tRNA using tRNAMet concentrations between 0.5 and 5 μM. The KM and Vmax was determined to be 29 μM and 3.0 μM min−1, respectively, resulting in an observed kcat of 1.9 sec−1. This resulted in a kcat/KM value of 0.067 s−1μM−1. The ATP:PPi exchange reaction was used to monitor interaction with ATP and methionine (Met). The KM, Vmax, observed kcat, and kcat/KM for interaction with ATP and Met was 515, 145, 24.2, 0.05 and 35.5, 223, 37.2, 1.05, all respectively. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa MetRS in a high throughput format. Using this assay, a natural product (800 compounds) and a synthetic compound (890 compounds) library was screened. Two compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM01F04 and BM01C11 inhibit the activity of P. aeruginosa MetRS with IC50 values of 26 and 70 μM, respectively and both of these compounds exhibited promising MICs against Gram+ bacteria and mutant forms of Gram‐ bacteria.

Conclusion

MetRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.

Support or Funding Information

Research was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and the Howard Hughes Medical Institute (HHMI) Precollege and Undergraduate Science Education Program grant #52007568.
Assembly of human translational elongation factor 1B is based on the interaction of the glutathione transferase‐like domains from alpha and gamma subunits
Authors: Choi Yoon Seo , Lee Hyun Joo , Kang Beom Sik
Published: November 2023

In the process of human translational elongation, eEF1A·GDP is exchanged to eEF1A·GTP by eEF1B, which consists of three subunits, α, β, and γ. Among them, α and β subunits have guanidine exchange function while eEF1Bγ can bind to both α and β subunits. The N‐terminal GST domain of eEF1Bγ is responsible for the interaction to the N‐terminal regions of eEF1Bα and β. We obtained the 2:2 complex of the N‐terminal eEF1Bα and γ domains, and determined the crystal structure of the complex at the resolution of 2.1 Å. eEF1Bγ has a canonical GST domain consisted GST‐N containing a four stranded β‐sheet and helical GST‐C subdomains while the N‐terminal region of the eEF1Bα forms a GST‐C subdomain. The tetrameric complex has an order of α‐γ‐γ‐α subunits. The interaction between γ and γ subunits is similar to that of canonical GST dimer. however, the interaction between α and γ subunits is using their GST‐C domains. Binding assay with α and γ subunits containing mutations at the binding interfaces confirmed the interfaces for α‐γ and γ‐γ subunit interactions. Although the N‐terminal domains of eEF1Bα and eEF1Bγ form a heterotetramer, gel filtration analysis of the full‐length complex indicate their 4:4 complex suggesting another interactions through other regions than the N‐terminal domains. Furthermore, these two subunits could form even a bigger complex (8:8:8) in the presence of eEF1Bβ subunit.

Support or Funding Information

KNU Creative BioResearch Group, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702‐701, Korea
Functional Analysis of DUF1 in Saccharomyces cerevesiae
Authors: Keuls Rachel , Lozzi Brittney , Anderson Philip , Erickson F. Les
Published: November 2023

The yeast DUF1 gene encodes a highly‐conserved, WD40‐repeat protein that interacts with and activates deubiquitinases (DUBs). DUBs carry out the process of deubiquitination, the removal of ubiquitin from tagged proteins. Animal homologues of DUF1 have been shown to form complexes with DUBs that have a variety of important functions. Human WDR48 activates DUBs that deubiquitinate key subunits in the Fanconi anemia DNA repair pathway. Caenorhabditis elegans WDR48 interacts with Usp46 and stabilizes Glutamate receptors (GLR‐1) to promote changes in locomotion behavior. Previous research has shown that in Saccharomyces cerevisiae, Duf1 interacts with Ubp9 and Ubp13, DUBs involved in the biosynthesis of mitochondrial subunits necessary for respiration. When a DUF1 mutant strain was grown at a higher temperature on a non‐fermentable carbon source, increased petite colony formation was observed. Thus, we hypothesize that Duf1 enhances the yeast's ability to cope with stress. Our interest lies in determining which cellular stress pathways require Duf1 in order to gain insight about its function. By exposing a DUF1 knockout mutant strain to different stress conditions and comparing its growth to the wild type parent strain, phenotypes may be revealed to provide a better understanding of the cellular activities of Duf1. Cell growth experiments were performed in 96‐well plates using a Spectramax i3 plate reader and cell number was plotted against time. Growth rate and maximum cell number were computed in R and differences between the knockout mutant and the wild type parent strain were determined using a pairwise Mann‐Whitney U test. When grown in minimal media, the knockout mutant strain grew faster than the wild type parent strain. The knockout mutant was also able to reach a larger maximum cell number than that of the wild type parent strain in minimal media containing high levels of glucose, a stressful growth situation. Conversely, the wild type parent strain grew better when minimal media was supplemented with high amounts of ethanol. This implies that the action of Duf1 as either a positive or negative regulator is dependent upon the type of stress experienced and that DUF1 may play a role in multiple cellular stress response pathways.

Support or Funding Information

This project is supported by an NSF Major Research Instrumentation grant and Salisbury University's Henson Undergraduate Research Fund.
Comparative Genomics of Novel Phage Leo1 Reveal a Promiscuous Cluster of Phage
Authors: Badran Sondos , Morales Nathanael , Schick Phil , Jacoby Brandon , Geiger Alyssa , Geiger Stefanie , Thummel Alysse , Lorenz Todd Christopher
Published: November 2023

Bacillus spp. are spore forming, gram positive, rod‐shaped microbes. Many Bacillus spp. have been associated with infections. For example, B. cereus is linked with gastrointestinal infections and food poisoning, B. thuringensis, a known insecticide, has recently been associated with periodontitis, and B. pumilus is related with food poisoning as well as contamination in clean rooms. One potential mechanism that could diminish harmful effects of Bacillus spp. would be to utilize bacteriophage. While there are approximately 1031 bacteriophages on earth's biosphere only a small portion have been studied. In fact, less than 2000 phage genomes have been sequenced and Bacillus bacteriophages have been insufficiently studied. Thus, there is a lack of understanding about their genomes. A novel phage, Phage Leo1, was found to infect a soil isolated B. pumilus. Genomic sequence of Phage Leo1 was examined using bioinformatics tools to determine that it has 74 genes, 31.08% have known associated functions while 68.92% of the coding regions are unknown. Phylogenetic analysis of genomes in a large collection of Bacillus phage were analyzed to determine the clustering of Phage Leo1 compared with relatives all belonging to the siphoviridae family of viruses. These viruses were limited to sizes ranging from 35,000 bp to 65,000 bp to help avoid long‐branch attractions. The phylogenetic analysis reveals that Phage Leo1 is a novel virus belonging to the H1 cluster and indicates a potential promiscuous nature to the B. thuringiensis and B. cereus phage clusters. This promiscuous nature of the two phages may indicate that the phages of B. thuringiensis and B. cereus have a wide host ranges and therefore, can infect other Bacillus spp. Further studies are required to observe the host ranges of these phages.
Optimization of Falling Drop Copper Sulfate Method to Quantify Hemoglobin Concentration
Authors: Sinha Rahul Kumar , Randolph Tim R
Published: November 2023

Many clinics in underdeveloped countries lack stable electricity, climate control, refrigeration, clean water, and properly trained staff, necessary to implement modern laboratory testing methods. Anemia is a common condition around the world and blood hemoglobin testing has proven to be a significant challenge in these clinics. Even simple methods of anemia assessment like microhematocrit, HemoCue and reflectance instruments require electricity or batteries and are cost prohibitive. This research aims to optimize the Falling Drop hemoglobin method previously developed in our laboratory at Saint Louis University, and in particular to assess the linearity between the descent time of a drop of blood and the hematocrit level of the sample. In accordance with our method, blood was collected in tubes coated with sodium fluoride/potassium oxalate and 44uL was delivered through a modified pipette tip onto a 4.58% solution of copper sulfate in a column created from a 10mL pipette. The descent time of a drop of blood through a column of copper sulfate was measured and recorded. Normal blood was mixed with autologous plasma to create eight sets of hematocrit levels ranging from 15% to 50% in 5% increments. Six sets of eight tubes were created and each hematocrit level was dropped 6 times through a column. A strong inverse correlation was assessed, with the lowest R2 = 0.8461 and the highest R2 = 0.9703. After plotting the mean of each hematocrit level for all 6 of the runs, the R2 = 0.9622. Other best fit models were also evaluated. Therefore, a correlation between blood drop descent time and hemoglobin concentration does exist, and this can be applied to optimize a method to determine the hemoglobin level of a blood sample.

Support or Funding Information

RKS is supported by the DeNardo Education and Research Foundation.
UV MUTAGENESIS ENHANCE FUNCTION OF CELLOBIOHYDROLASE I GENE OF ASPERGILLUS NIGER
Authors: Babalola Musa Oladayo , Magbagbeola Olubunmi Abiola , Ebuehi Albert Osaretin , Okochi Veronica Ihuaku
Published: November 2023

Background

The need for utilizing renewable resources to meet the future demand for fuel and other value added products has increased the attention on lignocellulosic wastes. Lignocelluloses are degraded by cellulase and other lignocellulolytic enzymes produced by fungi and bacteria. Mutagenic agents have been used to achieve improvement of these strains. There is however, paucity of data on the effect of these mutagenic agents on the structure and function of cellulase genes. The aim of this study was to carry out a structural analysis on selected cellulase genes obtained from ultraviolet light mutated Aspergillus niger.

Methods

Fungi were isolated from decomposing wood wastes. They were genotyped by amplifying the internally transcribed spacer (ITS) regions on their DNA, sequencing the amplicons and analyzing the sequences. They were genetically modified for enhanced cellulase production using ultraviolet rays. RNA of the mutated A. niger was reverse transcribed to complementary DNA (cDNA). The selected genes; aepI, aceI, cbhI and a laccase gene, Lacc1 were amplified and sequenced. The sequences were aligned and compared with referenced sequences which included those of Trichoderma reesei and Phanerochaete chrysosporium. The sequences were also translated into amino acid and in‐silico X‐ray crystallography structure of the active site of the enzyme was constructed using MEGA5 and JPred version of Jalview softwares.

Results

Bioinformatics analysis of the sequence of the cbh I gene of the A. niger mutant showed that it's enzyme had strong similarity with compared industrially beneficial fungi; Trichoderma reesei and Phanerochaete chrysosporium. Amino acid changes occurred between the 8th to the 92nd position on the chain‐A of the enzyme. Amino acids residues in its active site had low hydropathy index. Predicted structure of cellulase from A. niger mutant revealed that alteration occurred in the β‐pleated sheets of the enzyme.

Conclusion

Genetic modification of Aspergillus niger resulted in enhanced cellulase enzyme activity.

Support or Funding Information

Central Research Laboratory, College of Medicine, University of Lagos
Chemotherapeutic Potential of Averrhoa bililmbi Leaves Ethanolic Extracts Against Human Colorectal Carcinoma through Anti‐inflammatory Activity in Murine RAW 264.7 Macrophage Cells and Anti‐proliferative Activities Against HT‐29 Human Colorectal Carcinoma
Authors: Vergara Emil Joseph Sanvictores , Kim Yeon Soo , Lumbera Wenchie Marie Lara , Nguyen Thuy Linh , dela Cruz Joseph Flores , Hwang Seong Gu
Published: November 2023

Chronic inflammation and cancer are already known to be two intertwined processes and many studies have suggested that the hindrance of the former can serve as an effective preventive measure for the occurrence of colorectal carcinoma (CRC). The current study was done in order to evaluate both the anti‐inflammatory and cytotoxic properties of ethanol extracts of Averrhoa bilimbi leaves in light of its potential as a source of an effective chemotherapeutic agent against CRC. In this study, RAW 264.7 murine macrophage cells and HT‐29 human colorectal carcinoma cells were used to evaluate the anti‐inflammatory and anti‐tumor properties of the extract, respectively. Cell viability analysis, NO assay, RT‐PCR assay, Western blot analysis, flow cytometry analysis and antioxidant assays were used to evaluate the chemotherapeutic properties of the extract. It was shown that treatment with low doses (25, 50 and 100 μg/mL) of the extract has potent anti‐inflammatory activity through alterations in nuclear factor‐kappa B (NF‐KB) activity, nitric oxide (NO) production and cycloxygenase‐2 (COX‐2) release. The extract also shows potent cytotoxic activity against the cancer cell line used in the study. It was seen that the extract shows anti‐tumor properties through the promotion of apoptosis, through up regulation of the Smac/DIABLO‐caspase cascade, and cell cycle arrest, through up regulation of the tumor suppressor gene p21. Antioxidant screening showed that the extract possesses antioxidant activity supporting its anti‐inflammatory and anticancer activities. In conclusion, the current study shows that Averrhoa bilimbi can be a source of bioactive compounds which can be used as a potential chemotherapeutic agent against colorectal carcinoma by virtue of its chemopreventive (anti‐inflammatory) and chemotoxic (cytotoxic) properties.
The Anti‐Proliferative Effects of Curcumin Derivatives, Dimethoxycurcumin, Bisdemethoxycurcumin and Tetrahydrocurcumin, on DLD‐1 Colon Cancer Cells
Authors: Khatwani Natasha , Adeyeni Temitope , Ezekiel Uthayashanker
Published: November 2023

Phytochemicals are natural plant‐derived products that show preventative and therapeutic properties against a wide range of human diseases. Studies comparing the effects of Western and Eastern diets on disease outcome have shown that consumption of a diet rich in these phytochemicals proves effective in inhibiting colon cancer. Curcumin, the predominant hydrophobic polyphenolic compound that is isolated from the rhizomes of Curcuma longa, has attracted a lot of attention due to its diverse applications in traditional medicines as a potent anti‐inflammatory, antioxidant, antiangiogenic and anticancer agent. Despite its promising chemotherapeutic activity, preclinical and clinical studies of curcumin highlight its narrow in vivo therapeutic application due to its instability and limited bioavailability in human physiological conditions. For these reasons, we compared the chemotherapeutic effects of three curcumin derivatives, dimethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin, that studies have shown to have increased bioavailability and antiproliferative effects on various cancer cell lines, versus curcumin by measuring their antiproliferative effects on the DLD‐1 colon cancer cell line. The results showed that while bisdemethoxycurcumin (IC50 = 10 μM) showed similar effects and tetrahydrocurcumin (IC50 = 50 μM) showed less potent antiproliferative effects than curcumin, dimethoxycurcumin showed 6‐fold increased potency (IC50 = 2 μM) compared to curcumin. In previous experimental studies on curcumin we observed that the antiproliferative effects of the phytochemical were enhanced in combination with silymarin, the bioactive component of milk thistle. Given this promising synergistic interaction and given the range of results displayed in our current study of the curcumin derivatives, our future experiment will be focused on the comparative studies of the potential synergistic interactions of dimethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin with silymarin.

Support or Funding Information

NK and TA are supported by the DeNardo Education and Research Foundation Fellowship.