bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of the Vascular Disrupting Agent OXi8006 on Activated Endothelial Cell Signaling and Tumor Growth Delay in a Breast Cancer Mouse Model Treated with the Corresponding Phosphate Prodrug Salt OXi8007
Authors: Odutola Samuel O. , Strecker Tracy E. , Taylor Emily A. , Schwartze Jeffrey , Gerberich Jeni , MacDonough Matthew T. , Chaplin David J. , Liu Li , Mason Ralph P. , Pinney Kevin G. , Trawick Mary Lynn
Published: November 2023

Tubulin‐binding, small‐molecule vascular disrupting agents (VDAs) are anticancer agents that rapidly depolymerize microtubules in the activated, tumor endothelium causing a pronounced shutdown in blood flow while leaving normal tissues intact. VDAs offer significant therapeutic advantage against solid tumors by selectively targeting pre‐established tumor vasculature and are mechanistically distinct from antiangiogenic agents. The indole‐based tubulin‐binding compound OXi8006 and its phosphate prodrug OXi8007 have shown promise as VDAs although important questions regarding features of their mechanism of action remain. We have shown that OXi8006 and OXi8007 disrupted microtubules in activated human umbilical vein endothelial cells (HUVECs) and caused extensive reorganization of the cytoskeletal network. The pathway involved an increase in phosphorylation of non‐muscle myosin light chain and focal adhesion kinase, resulting in increased actin stress fiber and focal adhesion formation via the intramolecular G protein switch RhoA and RhoA dependent kinase (ROCK) [Strecker et al. Cancer Lett. 369, 229 (2015)].

An important unresolved question is the connection between microtubule disruption and the downstream effects of VDA treatment. In this study we demonstrated the role of GEF‐H1, a protein that is able to activate RhoA but is inactive when bound to microtubules. GEF‐H1 is released upon microtubule disruption and can activate RhoA. The downstream effects of RhoA activation upon OXi8006 treatment were abrogated by siRNA‐mediated knockdown of GEF‐H1. We also showed the importance of activation of the LIM kinase/cofilin pathway in actin stress fiber formation with VDA treatment. LIM kinase is phosphorylated and activated by RhoA kinase and resulted in phosphorylation of cofilin, thus inhibiting the ability of cofilin to disassemble actin filaments. Treatment of HUVECs with LIM kinase inhibitor significantly diminished the actin stress fiber formation observed with OXi8006 treatment alone. In MDA‐MB‐231 breast cancer cells, OXi8006 treatment reduced cell migration. The water soluble prodrug OXi8007 caused significant tumor growth delay in an MDA‐MB‐231 breast cancer xenograft mouse model. The results of this study elucidated important aspects of the mechanism of tubulin‐binding VDA action, and confirmed the potent anticancer effects of the VDA prodrug OXi8007.
Differential Sedimentation of Linear versus Circular DNAs Using Divalent Metal Cations
Authors: England Corbin James , Grey Tanner , Lewis Kevin
Published: November 2023

Molecular biologists routinely purify and concentrate DNA and RNA molecules by precipitation with ethanol, a method that does not discriminate between the different structural forms of each nucleic acid. In the current project we have assessed the ability of monovalent Group I and divalent Group II metal cations to selectively precipitate DNAs with different shapes and sizes out of aqueous solutions. Particular emphasis was placed on separation of circular plasmid DNAs from contaminating linear chromosomal DNA fragments. This was done by mixing Group 1 or Group 2 metals with DNA at various pH values then spinning the solutions in a microcentrifuge. The absorbance was then measured at 260 nm. Group 1 metal ions proved to be ineffective at sedimenting large chromosomal DNA fragments. Group 2 metal cations (Mg2+, Ca2+, Sr2+ and Ba2+) sedimented linear DNAs strongly, but only at high pH values (10–12). Ca2+ was chosen for further experiments because it is not a cofactor for nucleases like Mg2+ and it is found naturally within cells, unlike Sr2+ and Ba2+. Preliminary studies involving manipulation of parameters such as metal and buffer concentrations identified conditions that allowed chromosomal DNA to be sedimented approximately 10 times more efficiently than supercoiled plasmid DNA. Current experiments are seeking to maximize this difference in precipitation efficiencies, with a focus on sedimenting linear chromosomal DNA while leaving supercoiled plasmid DNA in solution.

Support or Funding Information

Texas State University
Online Learning Modules Based on Spacing and Testing Effects Improve Medical Student Performance on Anatomy Examinations
Authors: Baatar Dolgor , Ricks Elizabeth , Gest Thomas
Published: November 2023

Recent studies demonstrated that spaced education enhances learning in various educational settings. However, the efficacy of spaced education for learning anatomy remains unknown. Spaced education is based on two main findings of the cognitive research: spacing effect (knowledge retains better when acquired in small pieces at spaced intervals) and testing effect (repeated testing improves knowledge retention). The aim of this study was to determine whether the use of a spaced education online learning tool improves medical student performance on written anatomy examinations.

The online educational platform Cerego (http://cerego.com) was used to create a learning module (set) on the anatomy of the head and neck which is one of the most challenging topics for students as judged by low performance on related examination questions. One hundred and six first‐year medical students were provided with a set one week before the corresponding anatomy laboratory session. The Cerego platform allows assigning various goal levels for each set. Progress to the goal (0–100%) is measured by the frequency of use and the accuracy of responses to quiz questions. For this set, the goal level was set to 1 (achievable in one week). The relationship between the set usage data and the anatomy examination scores was analyzed. Student perceptions of the set were assessed through a survey.

More than half of the Class (59 students) have used the set. Based on the percent of progress to the goal, the users were designated as High Users (19 students who had 50% or more progress to the goal) and Low Users (40 students who had less than 50% progress to the goal). Students who did not use the set were designated as Non‐Users (n=47). The student anatomy rank (average score of anatomy questions from 3 previous summative examinations) was not significantly different between the 3 groups. High Users performed significantly better than Non‐Users on anatomy examination questions related to the material included in the set. Performance on anatomy questions unrelated to the set (Control items) was not different between the groups. Set use was associated with improved overall anatomy grades of students who fell into the bottom half of the Class by the anatomy rank. Surveys have shown that the majority of students found Cerego sets useful and effective. Lack of time was reported as the most common barrier to using the set.

This study shows that the use of online anatomy learning tools which utilize spacing and testing effects is associated with improved medical student performance on anatomy examinations. These results support the increased use of such learning tools as an adjunct to anatomy instruction in medical schools.
Using Metabolite Profiling to Understand the Consequences of Iron‐Source Choices by Staphylococcus aureus
Authors: DuBois Jennifer , Celis Arianna
Published: November 2023

Bacteria inhabiting different environments are presented with different sources of nutritional iron. Major metabolic decisions are often made based on what sources of iron are available, and how limited those sources might be. For Staphylococcus aureus, heme iron is preferred under pathogenic conditions, where the bacteria survive by lysing red blood cells and consuming hemoglobin. We therefore hypothesize that Staphylococcus aureus may regulate a whole variety of metabolic decisions based on the sensed availability of heme. To test this hypothesis and examine the fate of heme and non‐heme iron sources, we are using NMR‐ and mass spectrometric based methods to show how whole‐cell metabolite pools change in response to changes in iron sources. Results from initial studies will be presented.

Support or Funding Information

NIH R01 GM‐090260
Application of unknowns in organismal biology laboratories to encourage inquiry‐based learning
Authors: Guenther Merrilee , Bennett Kyle , Arriola Paul
Published: November 2023

The study of anatomy and morphology is often mistakenly seen as an exercise in memorization. Rather, morphological study is a unique opportunity for students to build their observational skill set. Observational skills are an important complement to experimental skills and crucial to students considering clinical professions. The Elmhurst Biology Department recognized the need to change the culture of certain lab experiences in order to achieve this goal and reinforce the mission of the department.

The Elmhurst College Biology Department is currently utilizing a tool in the laboratory that applies concept‐based learning to building observational skills in the context of organismal and morphological education. The use of unknowns is a standard in many experimental exercises in science labs. The introduction of specimen‐based unknowns in organismal courses has encouraged the development of critical thinking skills as well as improved the mastery of anatomical content. This approach has been applied to courses across the biology curriculum, including nursing anatomy and physiology (BIO 107/108 Human Anatomy and Physiology), general biology for majors (BIO 201 General Biology II), and upper level major biology electives (BIO 330 Comparative Chordate Anatomy, BIO 430 Advanced Human Anatomy). This method has been applied in individual laboratory sessions and has also served as the basis for a semester long dissection project in the Comparative Chordate Anatomy course.

The initial impact of this strategy has been apparent in terms of student engagement. Qualitatively, it has been observed that students are utilizing the complete lab period and increasing the time spent observing each specimen. The strategy appears to encourage more proactive, inquiry‐based learning by the students. By applying this approach in introductory courses, it has the potential to impact behavior in upper level courses.

Preliminary attempts to assess this laboratory method were carried out in Human Anatomy and Physiology I (BIO 107) and II (BIO 108), introductory courses aimed at nursing majors, non‐biology majors pursuing health professions, and biology majors. Experimental sections were given anatomical unknowns, while the control groups were not. To control for the impact of the amount of time spent studying on student performance, all groups were required to remain in lab for the same period of time. The use of unknowns in the experimental sections was replaced with a brief instructor led review of the material in the control sections, providing a contrast of active and passive learning approaches. On lab practical exams, the experimental group outperformed the control group throughout the semester.
Arl4A interacts with Robo1 to promote cell migration via up‐regulating Cdc42 activation
Authors: Lee Fang‐Jen Scott , Chiang Tsai‐Shin , Chen Chieh‐Hsin , Tsai Meng‐Chen , Jang Li‐Ting
Published: November 2023

Cell migration is a highly regulated event that is initiated by protrusion of the cell membrane and actin reorganization. Robo1, a single‐pass transmembrane receptor, is crucial for neuronal guidance and cell migration via activating Cdc42 GTPase. ADP‐ribosylation factor (Arf)‐like 4A (Arl4A), one of Arf small GTPases, functions in cell morphology, cell migration, and actin cytoskeleton remodelling; however, the molecular mechanisms for Arl4A in cell migration is not clear. Here, we report that Arl4A binding to Robo1 modulates cell migration via promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP‐dependent manner and residual 1394‐1399 of Robo1 is both required and sufficient for this interaction. Arl4A‐Robo1 interaction is essential for Arl4A‐induced cell migration and Cdc42 activation. We also showed that Arl4A binding to Robo1 decreases the association of a Cdc42‐GAP srGAP1 to Robo1. Furthermore, Slit2/Robo1 binding decreases Arl4A‐Robo1 interaction in vivo. Thus, our study reveals a novel mechanism that Arl4A participates in Slit2/Robo1 signaling on modulating cell motility via up‐regulating Cdc42 activation.

Support or Funding Information

NHRI‐EX106‐10601B1
Using 3D Paper Model Constructions and Wikki Stix&[trade] to Teach the Intricacies of the Pterygopalatine Fossa
Authors: Hatcher April , Naze Garrett , Hazzard Matt , MacPherson Brian
Published: November 2023

Purpose

The objective of this study was to create various model representations of the pterygopalatine fossa. This small, deep area within the facial skeleton is difficult to conceptualize as the various openings into and out of the fossa are not clearly visible from inspection of the skull alone. The traditional method for teaching this region to the dental students at our university has been to use a two‐dimensional representation wire‐diagram that emphasizes the various fiber types that are transmitted through the fossa.

Methods

Using materials easily accessible to educators, such as paper and craft supplies, a team of anatomists, clinicians, and medical illustrators collaborated to design 3D models representing the complex region of the pterygopalatine fossa.

Results

Two 3D models were created. Each used Wikki Stix, wax‐infused threads of various colors, to represent the sensory and autonomic fibers in the fossa. One kinesthetic model is a stylized inverted‐cone representation of the fossa that is pre‐labeled. The openings in the model were cut using a Cricut Explore One™ Machine. First‐year dental students were instructed how to arrange the Wikki‐Stix through the foramina of the fossa and how to fold the three‐dimensional cone model to incorporate these fibers. A second 3D kinesthetic model for teaching was developed alongside the simple model. The more complex 3D paper model used intervals of coronal, transverse, and sagittal sections through the skull to create the corresponding regions of communication with the pterygopalatine fossa, such as the cranial, oral, and nasal cavities and the orbit. These planes were then assembled into a free‐standing product. This model included black and white shadings of landmark structures such as the hard palate for the greater and lesser palatine nerves and the nasal cavity for the sphenopalatine (nasopalatine) nerves. This model was used to emphasize the spatial relationships of the nerves to approximate spaces within the cranium and skeleton. Dental students also crafted the Wikki‐Stix in context with the detailed 3D paper model to arrange the fibers in their target areas.

Conclusions

It is possible to produce 3D models of complex anatomical structures using basic materials. These models may be easily adopted by fellow educators as complex and expensive 3D printing tools are not required. Future research is needed to determine if these 3D models improve student outcomes compared to traditional teaching methods.
The Bacterial Chloride/Proton Antiporter: A Molecular Machine
Authors: Shannon Dan , Glass Jake , Hall Ben , Peters Kyle , Hardesty Jake
Published: November 2023

E coli has a voltage gated homodimer chloride transporter (ClC‐ec1) that is energized by a proton gradient. The transport of chloride ions is essential for controlling pH levels within a bacterial cell. In the past 10–15 years the crystal structure of CLC‐ec1 has been solved, which led to the realization of how little is known of chloride transport. The Moeller High School CBM SMART Team, working with Dr. Thomas Beck from the University of Cincinnati, used 3‐D modeling and printing technology to examine structure‐function relationships of CLC‐ec1. CLC‐ec1 is part of the Chloride Channel (ClC) family of molecules. The CLC family contains both chloride transporters and chloride channels. The difference being that a transporter is defined by having two ion selectivity gates and a channel having only one. CLC‐ec1 is an antiporter having two voltage gates, 1) a centrally located Tyrosine residue and 2) a Glutamate residue located on the extracellular membrane. These two residues function to block the movement of chloride ions into a cell. CLC‐ec1 exchanges 2 extracellular chloride ions for one intracellular proton. The proton is required to neutralize the slightly negative charge of each of the two gates, creating an environment for Chloride to pass across. CLC‐ec1's primary function is to modulate the pH gradient in E coli. When pH levels in E coli are too low (as occurs on a trip through the stomach) ClC‐ec1 is activated to reverse the pH gradient. To accomplish this CLC‐ec1 uses free energy from the uneven gradients to move protons and chloride ions across its membrane. CLC‐ec1 is homologous in both structure and function to its mammalian counterparts. This gives us a better picture of the functioning of the chloride channel in modulating the transmembrane potential of nerve and muscle cells, the acidification of vesicles, and the alteration of membrane permeability to chloride and other anions. It also leads us to understanding dysfunctional chloride transport, such as occurs in cystic fibrosis and nephrolithiasis. Questions remain, however, about the functioning of the central tyrosine and the mechanism of selectivity for and against other anions.
Heterogeneity Pattern of Apoptosis Mediators in Cervical Carcinoma Cells
Authors: García‐Aguiar Israel , Del Moral‐Hernández Oscar , Martínez‐Castillo Macario , Bonilla‐Moreno Raúl , Villegas‐Sepúlveda Nicolás
Published: November 2023

Alternative splicing is one important regulatory posttranscriptional mechanism for control of gene expression. It produces several mature messenger RNAs (mRNAs) from single immature pre‐messenger RNA (pre‐mRNA) by using alternatively donor or acceptor splicing sites. Viruses use this posttranscriptional mechanism to increase their coding capacity, because they usually contain small genomes. The infection with the Human Papillomavirus (HPV), mainly with the types 16 (HPV‐16) and 18 (HPV‐18), is etiologically related to cervical cancer. The HPV transcripts are bicistronic or polycistronic and contain suboptimal splicing sites, which favor alternative splicing in its intron‐1; it is located in the E6/E7 oncogenes which are responsible for the transformation of host epithelial cells. Interestingly, alternatively spliced forms of the E6/E7 oncogenes are heterogeneously expressed in tumor cells and the mechanism producing the heterogeneity is still unknown. In our laboratory, it has been observed that the E6/E7 heterogeneous profile might result of differential use of acceptor sites in different cellular contexts, for example, cervical carcinoma cells with differential expression of splicing factors. Cancer cells are heterogeneous and show differential expression profiles in some metabolic markers. This fact raises the question whether the E6/E7 splicing profile heterogeneity is produced by the same mechanisms which produce tumor cell heterogeneity. Besides, some proteins involved in apoptosis are also regulated by alternative splicing; this mechanism produces alternative proteins with altered subcellular localization, as well as antagonist or dominant‐negative functions, which affect the apoptotic cell signaling and produces the phenotype of apoptosis‐resistant cancer cells. Thus, our goal was to investigate the variation in the expression profile of apoptotic mediators among cervical carcinoma cell lines. In this work we investigate the expression of alternative splicing forms of proteins involved in apoptosis, by using RT‐PCR and western blot assays in four carcinoma cell lines: C33‐A (HPV‐), CaSki (HPV‐16), HeLa (HPV‐18) and SiHa (HPV‐16). Our results revealed that some of the alternative splicing forms of the apoptotic markers were expressed differentially in both, HPV negative or positive cervical carcinoma cells; the differentially expressed alternative transcripts not presented exact correlation with the level of proteins that they encode; all these results suggested that the expression heterogeneity was produced by a complex mechanism involving several steps of post‐transcriptional control.

Support or Funding Information

This work was supported in part by funds allocated to NVS by CINVESTAV and by CONACYT
Regulation of MAP kinase signaling by c‐Myc mediates the apoptotic response to vinca alkaloids
Authors: Gristock Rachel A , Wales Christina TK , Jacobs Aaron T
Published: November 2023

c‐Myc is a regulator of gene expression with dichotomous roles in cancer growth and viability. It is an oncogene that promotes cellular proliferation and is often over‐expressed in cancer. But c‐myc also sensitizes cancer cells to the actions of certain chemotherapeutics, including doxorubicin, cisplatin, and vinca alkaloids, among others, and is a potentially useful marker for predicting clinical responses to drug therapy. Our goal was to investigate the significance of c‐myc in mediating the apoptotic response to vinca alkaloids, and to determine the signaling pathways that relate c‐myc expression to the regulation of apoptosis. Experiments were performed in the colorectal cancer cell lines RKO and HCT‐116. Cells were transfected either with a negative control or c‐myc siRNA, then treated with vinca alkaloids (vinblastine, vincristine). Treatment with vinca alkaloids induced apoptosis in the range of 5–10 nM, as evidenced by caspase 3, 9 and PARP cleavage, and by fluorogenic assay of caspase‐3/7 activity. Vinca alkaloid treatment also caused robust JNK‐1/2 phosphorylation, which has been previously reported to promote apoptosis to these agents. Our data show that c‐myc is critical for JNK‐1/2 activation. Silencing c‐myc inhibits the phosphorylation of JNK‐1/2 on Thr183/Tyr185 in vinca alkaloid‐treated cells, and causes a corresponding reduction in drug‐induced apoptosis. The importance of JNK‐1/2 signaling was confirmed with the biochemical inhibitor SP600125, which attenuated apoptosis and increased the IC50 of vinca alkaloids by more than 20‐fold. Conversely to its effects on JNK‐1/2, silencing c‐myc enhanced the phosphorylation of ERK‐1/2, which promotes cell survival and resistance to apoptosis. Accordingly, inhibition of ERK‐1/2 with U0126 lowered the IC50 and restored apoptosis in c‐myc silenced cells. We therefore propose a model in which c‐myc facilitates JNK‐1/2 activation and suppresses ERK‐1/2, and demonstrate that when c‐myc expression is reduced, resistance to vinca alkaloid‐initiated apoptosis is acquired.

Support or Funding Information

Grant support: NIH Grant P20GM103466
Analysis of the Expression of E6^E7, a Short Splicing Variant of HPV‐16‐E6 in the HPV Negative Cervical Carcinoma Cells C33‐A
Authors: Vaisman Carolina Eugenia , Bonilla‐Moreno Raúl , Villegas‐Sepúlveda Nicolás
Published: November 2023

Human Papillomavirus (HPV) type 16 (HPV‐16) is the most important of the high risk HPV (hrHPVs) implicated in cervical cancer development. It is found in more than 60% of the cervical tumors. The E6/E7 oncogenes are responsible for the transformation of the epithelial cells; they targeted multiple proteins in the host cells, including p53 and other apoptosis mediators. The E6/E7 transcripts of the hrHPVs are first produced as bicistronic or polycistronic messages and then alternatively spliced. The alternative E6/E7 transcripts are expressed heterogeneously in the tumor cells and the impact of this differential splicing profile is not yet deeply investigated. Four transcripts are produced from the unspliced HPV16 E6/E7 pre‐mRNA: the unspliced E6/E7 RNA itself, which is the only source of the E6 oncoprotein, the E6*I/E7 and E6*II/E bicistronic transcripts, encoding two short forms of E6 transcript (E6*I and E6*II, respectively) and the E7 oncoprotein, lastly, a minor monocistronic transcript, namely E6^E7 which is formed by an in‐frame fusion of the 5′ end terminus of E6 plus the 3′ terminus of the E7; it potentially produces a hybrid protein from both the E6 and E7 oncoproteins, such fused protein could affect target of both the E6 and E7 oncoproteins. The E6^E7 transcript is also heterogeneously expressed in tumor cells but its function is poorly understood.

We aim to understood the function of the HPV16 E6^E7 protein and its role in tumor cell behavior.

We transfected stably HPV‐negative C33‐A cell line with a construct containing the monocistronic E6^E7 cDNA fused to the green fluorescent protein (GFP). Several stably transfected clones that expressed the fused protein differentially were selected, by using cell sorting. Proteins extracts were obtained from these clones and used for western blot analysis to investigated apoptosis effectors and members of the inhibitor apoptosis proteins (IAPs).

The results showed that the transfected clones expressing different levels of the E6^E7 fused protein displayed similar levels of the IAP proteins analyzed so far. These results suggest that there is no relation between the expression level of GFP reporter gen in E6^E7 transfected C33A cells and IAPs. Conversely, the expression of the constructs containing the E6, E6*1 or E6*II cDNAs fused to the GFP protein produced an altered expression of the same IAP proteins.

Support or Funding Information

CONACYT, CINVESTAV
BET Inhibition Suppresses Gastric Cancer Cell Proliferation by Inducing Cell Senescence
Authors: Dong Xingchen
Published: November 2023

The bromodomain and extra‐C terminal domain (BET) proteins are important in mediating cell cycle control, inflammatory cytokine production and developmental programs. BET proteins bind to acetylated histone or non‐histone proteins and serve as transcriptional co‐regulators of gene expression. Small molecules targeting bromodomains of BET proteins, BET inhibitors (BETi), possess strong anti‐tumor activities and have emerged as potential therapeutics in cancer and inflammatory diseases. However, BETi target all the BET family proteins, including Brd2, Brd3, Brd4 and Brdt. The contribution of each BET protein and the detailed mechanism of BETi‐mediated inhibition of cancer cell proliferation remain to be determined.

In this study, we demonstrate that BETi JQ1 suppresses the tumorigenicity of gastric cancer cells by targeting Brd4‐mediated microRNA expression. JQ1 inhibits gastric cancer cell proliferation, migration, and invasion by inducing cell senescence. Depletion of Brd4 but not other BET proteins recapitulates JQ1‐induced cell senescence. Furthermore, we demonstrate that knockdown of Brd4 or treatment of cells with JQ1 increase p21 level through down‐regulation of miR‐106b‐5p. Overexpression of miR‐106b‐5p prevents JQ1‐induced p21 expression and cell senescence, whereas miR‐106b‐5p inhibition up‐regulates p21 and induces cell senescence. Finally, we show that JQ1 abolishes the binding of Brd4 to the regulatory element of miR‐106b‐5p to inhibit the expression miR‐106b‐5p.

Our results identify a novel function of Brd4 in cancer cell proliferation by modulating cell senescence via miR‐106b‐5p/p21 axis and provide new insights into using BETi as a potential anti‐cancer drug.
Targeting potassium channels in cancer: A novel strategy for therapeutic intervention
Authors: Gentile Saverio
Published: November 2023

Breast cancer (BC) is the second leading cause of cancer‐related death in woman worldwide and accounts for more than 40.000 lives every year in the USA due to disease progression. Although the considerable success rate achieved in treating primary tumors, treatment of metastatic BC (mBC) have been severely hindered by the difficulty in targeting proteins governing important steps of the metastatic cascade, limited drug availability, side effects and drug resistance in BC patients. Therefore, finding novel therapeutic agents targeting specific molecular pathways involved in mBC are urgently needed.

During our investigation to identify new and critical targets in BC, we found a novel role for the potassium ion channel Kv11.1 in BC cells and xenograft tumors. Our preliminary experiments suggest that stimulation of Kv11.1 activity with the small molecule NS1643 induces a strong inhibition of tumor growth and metastasis of BC. However, NS1643 did not produce any effects in non‐transformed breast cells and was well tolerate in animal suggesting that NS1643 molecule could be considered efficacious and safe anticancer drug.

Failing of anticancer therapies is mostly due to our limited understanding of biochemical signaling in cancer cells. In this work we describe the biochemical signaling linking activation of Kv11.1 potassium channel to the tumor suppressor mechanism senescence which include a unique calcium‐dependent and p53‐independent upregulation of tumor‐suppressor p21 and novel degradation mechanism of tumor markers such as cyclin E2.

Interestingly, investigating on the possible mechanism linking Kv11.1 stimulation to inhibition of metastasis revealed that Kv11.1 agonists strongly inhibit cancer cell motility. Our experiments revealed that NS1643 strongly down‐regulated epithelial to mesenchymal transition (EMT). Although it has been well established that EMT is a process by which cells gain migratory properties and strongly contributes to metastasis, still very little is known about the biomolecular mechanism that regulates EMT.

We describe for the first time the mechanism linking activity of Kv11.1 potassium channel to suppression of EMT markers and increased cell‐cell contact.

Current therapeutic approaches against metastatic BC can aim only to ameliorate some of the symptoms and their clinical efficacy is strongly limited by the severity of their side effects. We think that this study offers an unprecedented opportunity to design a novel therapeutic approach against mBC in which Kv11.1 activators can be used to suppress cancer cell growth and dissemination.
Inhibition of Human Prostate Cancer Growth by Mesenchymal Stem Cells Delivering MiR‐16
Authors: Jones Eyone , Mazirka Pavel , McNurlan Margaret A , Brink Peter , Caso Giuseppe
Published: November 2023

We have previously shown that miR‐16 has suppressive effects on the growth of human prostate cancer cells (PC3). However, one of the limitation for the use of inhibitory microRNAs (miRs) or small interfering RNAs (siRNA) for cancer therapy is effective delivery to cancer cells in vivo. The aim of this study was to evaluate the use of human mesenchymal stem cells (hMSC) as a potential in vivo delivery system for miRs to cancer cells.

Human xenograft tumors were established by s.c. injection of 1 × 106 human prostate cancer cells (PC3) in immunocompromised nude mice. When tumors reached a measureable size, mice were randomized to receive hMSC previously transfected with miR‐16 (n=10, Treated) or saline (n=10, Control). 1 × 106 hMSC or saline were injected intratumorally. Tumor growth was assessed over a period of 3 weeks.

Tumors in the group treated with hMSC transfected with miR‐16 showed a slower growth rate. After 3 weeks the average tumor volume was 40% lower in the treated group compared to controls (1.2 ± 0.22 vs. 2.04 ± 0.46 cm3). The increment in tumor size over baseline (pre‐treatment) values was 5.0 fold in the treated and 8.4 fold in the control group (P<0.05). Tumor weight was also significantly lower in treated animals (0.82 ± 0.15 vs. 1.2 ± 0.16 g, P < 0.05).

In summary, treatment of established xenograft tumors derived from human prostate cancer cells (PC3) with hMSC previously transfected with miR‐16 significantly slowed tumor growth. The results confirmed that miR‐16 can suppress tumor growth and indicate that hMSC could represent a suitable system for in vivo delivery.

Support or Funding Information

Supported by the Stony Brook Foundation TRO‐Catacosinos Cancer Translational Researcher Award, and by the Dept. of Surgery, Stony Brook University Medical Center (Small Grant Program)
Subpopulations of Dopamine Transporters Show Enrichment in Phosphorylation or Palmitoylation
Authors: Storandt Michael H , Hovde Moriah J , Stanislowski Daniel J , Vaughan Roxanne A , Foster James D
Published: November 2023

The dopamine transporter (DAT) is a key regulator of duration and intensity of dopaminergic signaling through its uptake of dopamine from the extraneuronal space, and DAT dysregulation has been hypothesized to play a role in dopaminergic diseases such as autism, attention deficit hyperactivity disorder, and drug addiction. DAT consists of 12 transmembrane spanning domains that form the substrate permeation pathway, and cytoplasmic N‐ and C‐termini that contain sites for regulatory post‐translational modifications. Phosphorylation of the N‐terminus occurs on Ser7 and Thr53, which leads to decreased transport Vmax, and S‐palmitoylation occurs on Cys580 at the membrane‐cytoplasm interface of the most C‐terminal transmembrane helix, which leads to increased transport Vmax. We previously showed that palmitoylation and phosphorylation occur in a reciprocal manner, with high phosphorylation promoting reduced palmitoylation and visa versa, consistent with their opposing functional outcomes, but it has not been established if these modifications can coexist on a single molecule of DAT or if they are mutually exclusive. To address this issue, we isolated palmitoylated DAT by acyl‐biotin exchange (ABE) and neutravidin affinity chromatography, followed by phospho‐specific immunodetection of Thr53 phosphorylation. Our results indicate that only a small percentage of total DATs are simultaneously phosphorylated and palmitoylated, indicating that the modifications are predominantly mutually exclusive. We also found that S7A DATs display increased palmitoylation, consistent with previous findings, but also show a trend towards increased Thr53 phosphorylation, indicating complex interplay between all three of these modifications. These results support the presence of communication between subregions of the N‐terminus as well as between N‐ and C‐termini, and highlight the mechanistic integration of these post‐translational modifications in the establishment of dopamine reuptake capacity.

Support or Funding Information

National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)
Immunohistochemical Assessment of Hassall's Corpuscles in Human Foetal Thymuses
Authors: Sahni Suhalika S , Gupta Richa , Gupta Tulika , Aggarwal Anjali
Published: November 2023

Objective

Hassall's corpuscles are unique component of the thymic medulla, which participate in the physiological activities of the thymus. Although, they have role in maturation and differentiation of thymocytes, their function still remains an enigma. Present study proposes immunohistochemical criteria for assessing structural and functional characteristics of Hassall's corpuscles using antibodies against cytokeratin markers in human fetuses.

Methods

Hassall's corpuscles of 30 aborted human fetuses of various gestational ages (12 to 31 weeks) were studied by using CK5, CK8 and CK14 antibodies.

Results

Hassall's corpuscles exhibited positivity for CK5, CK8 from 13 weeks onwards, while CK14 positivity was seen after 16 weeks. CK8 and CK14 positivity was seen around the periphery, while CK5 positivity was observed both at peripheral and central part of Hassall's corpuscles. Two major growth spurts were observed after 16 and 22 weeks of gestation.

Conclusions

Demonstration of CK5, CK8 and CK14 (markers for simple and stratified epithelium) in Hassall's corpuscles from thymic medulla of human fetuses support the hypothesis that thymus undergoes sequential stages of differentiation similar to the epidermal tissue. This study enriches the information about structural and functional aspects of Hassall's bodies. This knowledge is crucial and can be used to treat various immunodeficiency diseases of skin and thymic tissue by acquiring progenitor stem cells from fetal thymic tissue.

Support or Funding Information

Director PGIMER, Chandigarh
Looking beyond phalangeal length and curvature: Functional correlation between manual phalangeal articular and collateral ligamentous morpohology and anthropoid locomotor adaptations
Authors: Deane Andrew S. , Tocheri Matthew , Wiles Kathleen
Published: November 2023

Primates exhibit more diversity in locomotor behavior than any other mammalian order. Discrete locomotor adaptations are characterized by distinct patterns of phalangeal orientation with dissimilar patterns of mechanical loading. Phalangeal shaft length and curvature are frequently used to infer locomotion in fossil anthropoids given the direct and repetitive contact between the hand and substrate; however, most fossil anthropoid phalanges are fragmentary or incomplete. Given that articular epiphyses and associated collateral ligaments are subject to the same locomotor behaviour‐specific mechanical loading that influences longitudinal shaft curvature and length, it is reasonable to predict that the proximal and distal articular surface morphology and collateral ligament fossae (CLF) are also influenced, at least in part, by locomotion.

We test the hypothesis that anthropoid phalangeal articular and collateral ligament fossae (CLF) morphology (i.e. contour curvature, size) varies with respect to locomotion and that more terrestrial primates experiencing higher compressive loads will have larger CLF's and increased mediolateral articular contour curvatures to resist mediolateral joint displacement. Segmented 3D models of proximal and distal articular surfaces and CLF's were obtained for the proximal phalanges (digits 2–5) of 16 locomotor diverse anthropoid genera (n=234 individuals). Direct comparisons among individual taxa and locomotor groupings reveal that articular and CLF morphology is a composite function of size, phylogeny, and locomotion but that more terrestrial primates have greater mediolateral articular contour curvatures and larger, deeper CLF's relative to more arboreal primates who have less mediolaterally restrictive joints with increased dorsoventral curvatures and smaller CLF's.
Epstein‐Barr Viral microRNAs Coordinately Repress Human Transcripts in Inflammatory and Apoptotic Pathways
Authors: Kolakada Divya , Katrak Callahan , Riley Kasandra
Published: November 2023

Epstein–Barr virus (EBV) infects 95% of the world population and is involved in the pathogenesis of Burkitt's (B cell) lymphoma, nasopharyngeal carcinoma, and other cancers in humans. In various states of latent infection and disease, EBV differentially expresses at least 49 mature microRNAs (miRNAs) from two major genomic clusters.

To identify potential miRNA‐mRNA pairs, we previously employed the high‐throughput sequencing and crosslinking immunoprecipitation (HITS‐CLIP) method to globally identify mRNA targets of both host and viral miRNAs expressed in unperturbed, EBV‐transformed B cells. Among the human transcripts regulated by EBV miRNAs (n = 1664), the most significantly enriched were involved in the regulation of transcription (n = 328) and apoptosis (n = 132). Detailed bioinformatic exploration of these transcripts predicted that the expression of ten or more key inflammatory/apoptosis proteins may be regulated by one or more EBV miRNAs.

In the present study, we combined HITS‐CLIP and bioinformatic predictions with luciferase reporter assays and Western blot analyses to identify and experimentally validate at single‐nucleotide resolution the 3′‐untranslated regions of select apoptotic and inflammatory transcripts bound by EBV miRNAs. Our findings suggest that certain EBV miRNAs co‐target specific transcripts to deregulate the host signaling machinery in a very complex manner during latent infection.

Support or Funding Information

K.R. is supported by Critchfield Research Grants, the Rollins Student‐Faculty Collaborative Scholarship Program, the Edward W. and Stella C. Van Houten Memorial Fund, and the Herbert E. Hellewege Research Fund.
Identification and characterization of population heterogeneity of Vibrio cholerae in vivo
Authors: Nguyen Y Nhu , Shull Lauren Marie , Camilli Andrew
Published: November 2023

Vibrio cholerae is a facultative pathogen that is found naturally in aquatic environments around the world. Upon ingestion of contaminated water or food, V. cholerae can colonize the human small intestine and induce profuse secretory diarrhea known as rice water stool (RWS). During the course of the infection V. cholerae undergoes through various transcriptional changes to adapt to the shifting environment. In the early stage of infection, bacteria rapidly replicate and increase expression of key virulence factors. The middle stage of infection is characterized by upregulation of the chemotaxis and flagellar genes. This allows V. cholerae to exit the luminal fluid of the small intestine. Finally in the late stage of infection, chemotaxis becomes repressed as V. cholerae is shed in the RWS. Studies have shown that V. cholerae exiting the mammalian host can enter into a hyperinfectious state. These hyperinfectious bacteria have been implicated to be major contributors in household transmission of the disease. Factors that promote hyperinfectivity are still largely unknown.

Other studies have demonstrated that host‐passed bacteria upregulate important late genes that prepare V. cholerae transition into the aquatic environment. For example, several of these late genes are involved in metabolism and are shown to be important for V. cholerae survival in pond water. One such gene is glpK, which encodes for a glycerol kinase and plays an important role in glycerol metabolism. By quantitative real time PCR, previous work has shown that mice‐passaged V. cholerae had an increased expression of glpK by 10‐ to 14‐fold. However, using recombination‐based in vivo expression technology (RIVET), our lab demonstrated that only 27% of the bacterial population exiting the host induces glpk expression. This suggests that glpK is highly expressed during the late stage of infection but only in a quarter of the population. These results led to the idea that the population of V. cholerae leaving the host may be heterogeneous, in which one subset of the population undergoes preparation for survival in the aquatic environment while the other remains hyperinfectious and therefore, can be more fit for rapid transmission to the next host. To examine this heterogeneous population in vivo, we generated a chromosomal GFP‐transcriptional fusion to glpK in the wave three Haiti V. cholerae strain. Using this strain we infected infant rabbits, which serve as an animal model for cholera in humans, and showed that only a subset of the host‐passed vibrios express GFP or glpK. This is consistent with previous RIVET studies. We will use fluorescence‐activated cell sorting (FACS) to separate the two populations and perform RNA‐seq to study their transcriptomes. Analysis of the subpopulation will provide further understanding of the mechanism of transmission and dissemination of V. cholerae.
Characterizing the mechanism of trogocytosis in Entamoeba histolytica
Authors: Feeney Shea E. , Ralston Katherine S.
Published: November 2023

E. histolytica (histo‐: tissue; lytic‐: dissolving) is a eukaryotic pathogen that can invade and destroy intestinal tissue. Tissue damage is likely to result from direct, contact‐dependent killing of human cells by amoebae. An improved understanding of this process would allow us to define the mechanism of pathogenesis. Our lab discovered that amoebae kill human cells by ingesting “bites,” an unusual strategy for cell warfare termed trogocytosis (trogo‐: nibble). The mechanism underlying trogocytosis, i.e. how an amoeba extracts bites from a living human cell, is unknown. Interestingly, amoebae ingest dead human cells whole via phagocytosis (phago‐: devour). While some aspects are shared with phagocytosis, nibbling is different from eating entire cells. Therefore, I hypothesize that, while some aspects are likely shared with phagocytosis, amoebic trogocytosis also requires signaling machinery distinct from phagocytosis. I aim to identify and characterize genes required for E. histolytica trogocytosis by using two complementary approaches: transcriptomics analysis and forward genetics. There is evidence that both phagocytosis and trogocytosis are transcriptionally regulated in E. histolytica, thus I am using RNA‐seq analysis to examine differential gene expression in amoebae undergoing trogocytosis versus phagocytosis. My preliminary results with imaging flow cytometry have established a method to quantify and optimize the number of amoebae performing either trogocytosis or phagocytosis respectively, setting the stage for RNA‐seq analysis. Preliminary studies for the forward genetics selection approach provide proof of principle that ingestion mutants can be isolated by selection. Results from these approaches will help to define the mechanism for trogocytosis. Since trogocytosis appears to be a fundamental form of eukaryotic cell‐cell interaction that is exaggerated and exploited for cell killing by microbes, a deeper understanding of the mechanism will have broad impact.

Support or Funding Information

NIH MCB T32 GM007377; NIH NIAID K22 Career Transition Award Al108814
Elucidation of Juglone Synthesis in Black Walnut
Authors: McCoy Rachel M. , Ye Ya‐Jin , Widhalm Joshua R.
Published: November 2023

Juglone has the potential to serve as a novel, natural product‐based herbicide. Juglone is an allelopathic naphthoquinone secreted by black walnut trees and other members of the Juglandaceae family. Metabolic engineering strategies to harness juglone as a new bio‐based herbicide are hampered by a lack of knowledge about its biosynthesis. Labeling data suggest that juglone is produced via 1,4‐naphthoquinone (1,4‐NQ) from 1,4‐dihydroxynaphthoic acid (DHNA), which is also an intermediate in the synthesis of the photosynthetic naphthoquinone phylloquinone (PhQ; vitamin K1). We, therefore, hypothesize that juglone is biosynthesized via a sequential decarboxylation and hydroxylation of DHNA derived from the PhQ pathway. Our first goal is to completely elucidate the plant pathways to synthesize DHNA. In Arabidopsis thaliana, all pathway enzymes to synthesize DHNA have been characterized except for DHNA‐CoA synthase. Based on homology to menB, the Escherichia coli DHNA‐CoA synthase gene involved in menaquinone (vitamin K2) biosynthesis, we have identified an Arabidopsis DHNA‐CoA synthase that coexpresses with other PhQ pathway genes. Efforts are currently underway to test if the candidate encodes a functional DHNA‐CoA synthase by functional complementation of the E. coli menB− mutant and reverse genetic approaches in Arabidopsis. Our next goal is to functionally screen DHNA decarboxylase and 1,4‐NQ hydroxylase candidate genes identified by transcriptome mining in black walnut through transient coexpression in tobacco to reconstitute the juglone pathway, relying on endogenous DHNA pools. We envision that our results will one day enable production of juglone in resistant, engineered crop systems, circumventing the need to apply synthetic herbicides.