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The HIV-1 Antisense Transcript AST Regulates Viral Expression via Interaction with Multiple Host Factors
Get PDFGrowing evidence suggests that the HIV-1 antisense gene plays a critical role in HIV-1 infection. Antisense transcript (AST), the RNA product of the antisense gene, has been reported to promote HIV latency. The ability of AST to reinforce viral suppression makes it a potential tool to achieve a functional cure for HIV-1 in the “block and lock” strategy. Therefore, it is important to fully understand its mechanisms of action.
In this study, we validated molecular interactions between AST and host proteins characterized by a previous mass spectrometry analysis, which are polypyrimidine tract binding protein (PTBP1), SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 2 (SMARCD2, a core subunit of the BAF complex), and TAR DNA-binding protein 43 (TDP-43). All these factors have been reported to be capable of regulating HIV-1 expression. However, due to time constraints, we focused on investigating whether the activities of PTBP1 and BAF complex were mediated by their interaction with AST. To gain a preliminary understanding of how the interactions between AST and cellular proteins might affect HIV-1 expression, we reduced the expression of these proteins or impaired their functions in Jurkat-derived clonal cell lines infected with a latent, replication defective HIV-1 (JE4) with and without ectopic overexpression of AST (JE4AST).
In the PTBP1 knockdown experiment through lentiviral delivery of short hairpin RNAs (shRNAs), we observed a non-specific reactivation of HIV-1 expression. Therefore, alternative knockdown methods that do not lead to HIV-1 reactivation are required. In the BAF pharmacologic inhibition experiment, we found that treatment with the BAF inhibitor, pyrimethamine, caused latency reversal in JE4 cells but not JE4AST cells. This result suggests that in JE4AST, higher levels of AST facilitate and stabilize the recruitment of the BAF complex at the proviral promoter region, thus reinforcing a status of transcriptional silencing. Additionally, we found that domain A (nucleotide 376-926) of AST may harbor the structure essential for BAF complex binding. In conclusion, our results provide novel insights to support the involvement of AST in promoting HIV-1 latency via interaction with multiple host cell factors
Research Integrity: The Need for a Research Misconduct Definition Within a Global Scope
Get PDFResearch misconduct (RM) is the act of willingly falsifying, duplicating, or plagiarizing research, results, or peer-reviewed literature. It is heavily penalized and can have career-ending consequences for those who engage in it. Transgressions related to RM can be traced through investigative processes set in place by institutions of higher learning that receive federal research funds. RM can also be tracked through the retractations of peer-reviewed articles, which are the result of fraudulent research. In the last few years, RM has exponentially become a problem worldwide. Previous research suggests that this problem might be due to cultural differences and how RM is perceived worldwide. Since a global RM definition does not exist, this project aimed to examine how RM definitions around the world, understand how these culturally intersect, and how these can be put together in an RM definition that accounts for cultural differences. In order to achieve this, RM definitions from government agencies, universities, scientific organizations, and peer-reviewed articles were extracted. These definitions were organized into two tables. In Table 1, government agencies, universities, and scientific organizations were organized and categorized as official definitions. The rest were definitions obtained from scientific articles. The two tables were visually compared, and assessments were drawn from the comparisons made. Such comparisons suggest that most of the examined literature utilizes a similar version of the RM definition. In addition, the table evaluations suggest that there are too many variables to be considered. Therefore, these can be better examined by a more extensive global task force composed of researchers, research administrators, students, advocates, and research integrity officers
NOVEL VERTICAL AND HORIZONTAL TRANSISTOR DESIGNS FOR PHOTOVOLTAIC NANOMATERIALS CHARACTERIZATION
Get PDFIn recent years, Lead Sulfide (PbS) Colloidal Quantum Dots (CQDs) have
emerged as a material of interest due to their versatility and scalability in
photovoltaic applications. Synthesis methods for CQDs make it possible to
change the semiconductor’s electrical bandgap as a function of quantum
dot diameter, meaning that CQDs can be tuned to absorb light at different
wavelengths. This desirable property of quantum dots has led PbS CQDs to
be integrated into various electronic devices, such as photodetectors, light-emitting diodes, and solar cells.
Analyzing the different electrical properties of semiconductors in these
devices is critical for achieving highly efficient device performance. For solar
cell applications, organic and inorganic ligands are often used to crosslink
quantum dots in solution-processed semiconducting thin films. If CQD solar
cells made from these crosslinked films are to approach the single junction Shockley–Queisser efficiency limit, then a complete understanding of
parameters such as charge carrier mobility and transport mechanisms in these films must be pursued.
Field Effect Transistors (FETs) provide an avenue for semiconductor
characterization through transconductance measurements. The effects of the gate and drain bias on current flow in the transistor’s semiconductor channel
can be analyzed to yield figures of merit for evaluating a semiconductor’s
performance in analog, digital, and photovoltaic circuits.
This thesis reports on work to design and fabricate quantum dot field
effect transistors which are then used to characterize PbS CQD films with
Ethanedithiol (EDT) ligands. Two FET architectures – vertical and planar – are fabricated and tested, each providing unique insights toward further quantum
dot film optimization. It is found that the PbS-EDT film exhibits strong ptype
behavior in air, with hole mobility of 1 × 10−3 and 1 × 10−4 cm2V−1s−1
reported for vertical and planar FETs, respectively. Other differences in device properties, and their implications for future CQD film applications, are also
discussed in this work
AN EXPRESSIVE THEORY OF PLANT-BASED DIETS
Get PDFAnimal welfare has been a central argument for the adoption of plant-based diets, with the utilitarian animal welfare argument (AWA) contending that minimizing animal suffering in
industrial animal agriculture morally obligates people to avoid animal products. However, the enormous scale and nature of modern animal agriculture make it difficult to assign culpability for animal suffering to any one individual. This is the basis of what is called the
causal impotence objection, which represents a significant threat to the advancement of the plant-based movement. Beyond the threat of this objection, plant-based arguments
that center animal welfare are problematic as they overlook the wide array of human harms involved in the production of animal products. As such, the moral reasons to reject animal-based dietary choices come to include reasons based in human welfare. This paper argues that choosing to consume animal products despite available plant-based options expresses an attitude of nonchalance towards injustice and a devaluation of the interests of others. Such expressions, where alternative choices exist, constitute a moral wrong termed an expressive wrong. To avoid expressive wrongs, people ought to make more plant-based choices. This is the basis of the expressive theory of plant-based diets outlined in this paper
Cyberspace, Cyber Sovereignty, and Cyber Conflict: The Digital Wild West
Get PDFThis thesis addresses the current debates on cyber sovereignty, state cyber policy, and the role of international law in mitigating cyber conflict. Specifically, how do individual states redefine and reassert their physical sovereignty within the
borderless confines of global networking? Examining the internal mechanisms of state authority, to what extent do state action and technological innovation interact for effective cyber policy? Additionally, within the external environment, do international
laws, norms, and agreements temper state authority in cyberspace and mitigate cyber conflict amongst states? Comparative case studies of the United States (US) and the People’s Republic of China (PRC) and a policy evaluative approach demonstrate that the US and PRC categorize issues in cyberspace according to their ideological
foundations. Nevertheless, similarities between the states exist in methodology.
The first chapter on cyber sovereignty reveals that the US and PRC have
implemented traditional expressions of state power, notably restrictions, within
cyberspace according to their national interests. Furthermore, an analysis of cyber directives from the Biden administration in the US and the Made in 2025 policy advocated by the PRC highlights that a balance of national policymaking and technological innovation comprises the formula for successful cybersecurity. The third
chapter expresses the shortcomings of international solutions for cyber conflict, namely arguments of jus ad bellum. This thesis demonstrates the present criteria for jus ad bellum and its inapplicability toward cyber conflict. A successful framework of international law for cyberspace must be modeled on a multistakeholder, multilateral basis. An ad-hoc coalition for adjudicating issues in cyberspace is currently the most
substantial remedy for mitigating conflict.
The significant implications of this subject are that (1) cybersecurity is vital to state security in the 21st century, (2) states are the primary actors in cyberspace, (3) cyberspace is multistakeholder, (4) coalition building is currently the best alternative in the absence of international law. Future work on this subject area is needed regarding
emerging technologies such as 5G networking and artificial intelligence (AI).
Moreover, new theorization is necessary for a digitized world that juxtaposes all
involved actors in an unprecedented manner
FROM LOCAL TO STATEWIDE: ANALYZING BULLYING TRENDS IN NEW JERSEY SCHOOLS WITH EMPHASIS ON DISTRICT AFFLUENCE
No full textThis study explores the problem of harassment, intimidation, and bullying (HIB) within a K-8 school district in New Jersey, and provides a statewide analysis of HIB trends, particularly in relation to affluence. Using a networked ecological systems theory framework and quantitative analysis, the research examines how the number of HIB incidents has changed over time, the frequency of cases based on perceived characteristics, the involvement of students with 504 plans or IEPs, and the distribution of HIB incidents by location, and the larger impact of affluence on reporting. Key findings reveal significant patterns in HIB incidents, including a notable increase in bullying incidents targeting students with disabilities, a higher frequency of in-person bullying occurring in classrooms and hallways, and a trend where school districts with higher affluence reported fewer bullying incidents. The results show the critical need for comprehensive anti-bullying strategies that address systemic issues such as understaffing and equitable access to resources. Recommendations include the implementation of an AI chatbot to address social stigma and help overcome socioeconomic barriers
CULTIVATING TEACHER INNOVATIVE BEHAVIOR THROUGH GENERATIVE SYSTEMS LEADERSHIP
Get PDFThere is a lack of innovative instructional practices which cultivate in students the necessary skills and competencies to tackle challenges in the 21st century (Rajaram, 2021). Despite a general understanding among educators of the importance of 21st-century competencies, there has been either overt resistance from teachers or token adoption of innovation, where teachers may leave their day-to-day practices unchanged (Karavas-Doukas, 1998; Lu & Campbell, 2021). This mixed methods study conducted at a private IB school in Hong Kong investigated the barriers which inhibited teacher innovative behavior.
Guided by ecological systems theory (Bronfenbrenner, 1994), the research supports existing literature by identifying barriers to teacher innovative behavior, such as limited effective collaboration within multicultural teams, absence of team psychological safety, and a lack of shared decision-making. This study also contributed new insights by identifying several additional barriers to teacher innovative behavior, including time constraints, resistance to excessive change, insufficient support from leadership, and a lack of trust in leadership. Significantly, this research highlighted the potential for leadership to serve as a key catalyst for promoting teacher innovative behavior.
To address the various challenges that hinder the promotion of teacher innovative behavior, the researcher designed an applied project—a Professional Development (PD) program on Generative Systems Leadership— to tackle low psychological safety within teams and limited effective collaboration in a multicultural work setting, both exacerbated by perceptions of topdown leadership. The 6-month long program consists of two modules: the first covers systems thinking, systems sensing and generative dialogue, and real-life application. Upon completion of the first module, participants have the option to form Communities of Practice (CoP) in-person or online, where they collaboratively address specific Problems of Practice. Over time, it is anticipated that these efforts will cultivate a generative learning culture, which in turn is expected to boost innovative behavior among teachers, ultimately contributing to the development of students' innovative competencies
CO-TRANSCRIPTIONAL REGULATION OF mRNA TURNOVER BY THE BACILLUS SUBTILIS glmS RIBOZYME
No full textThe glmS ribozyme riboswitch, located in the 5′ untranslated region of the Bacillus subtilis glmS messenger RNA (mRNA), regulates cell wall biosynthesis through ligand-induced self-cleavage and decay of the glmS mRNA by RNase J1. Although self-cleavage of the refolded glmS ribozyme has been studied extensively, it is not known how early the ribozyme can respond to metabolite during transcription and how efficiently RNase J1 degrades the cleaved mRNA.
Here, I combine single-molecule fluorescence with kinetic modeling to show that self-cleavage can occur during transcription before the ribozyme is fully synthesized. Moreover, co-transcriptional folding of the RNA at a physiological elongation rate allows the ribozyme catalytic core to bind ligand without the downstream peripheral stability domain. Dimethyl sulfate footprinting further revealed how slow sequential folding favors formation of the native core structure through fraying of misfolded helices and nucleation of a native pseudoknot.
After ribozyme cleavage, initiation of mRNA turnover is the rate-limiting step for regulation. Using biochemical and single-molecule FRET methods, I showed that RNA structures at or near the 5’ end of the ribozyme interfere with RNase J1 loading and processivity. Thus, the presence of tertiary structure immediately downstream of the cleavage site may slow down RNA turnover. However, RNase J1 biochemical assays with MgCl2 titration revealed that the 5’ structure of the ribozyme is not fully stabilized at physiological magnesium ion levels, allowing RNase J1 to initiate degradation. Furthermore, by using 3’ truncated ribozymes in the RNase J1 turnover assay, which mimics degradation of nascent ribozymes, I observed an improved RNase J1 activity compared to turnover of the full-length ribozyme. This indicates that early ribozyme self-cleavage during transcription may benefit mRNA turnover, as it exposes the mRNA to RNase J1 before the ribozyme core is stabilized by downstream domains. By combining the turnover assay with primer extension analysis, I also demonstrated that once RNase J1 degrades the first new nucleotides, it becomes fully processive, resolving RNA structures and rapidly degrading the remaining glmS mRNAs to complete gene repression. My results emphasize the importance of co-transcriptional folding of RNA tertiary structure for cis-regulation of mRNA stability
SINGLE-CHAIN VARIABLE FRAGMENT AFFINITY TUNING CAN OPTIMIZE ANTI-AML CAR-NK CELL FUNCTIONALTIY
No full textBackground: Relapsed/refractory acute myeloid leukemia (AML) carries a poor prognosis. Immunotherapy approaches hold great promise, but their application is limited by AML disease heterogeneity and the lack of a tumor-specific target antigen. Natural Killer (NK) cells have powerful intrinsic antileukemic activity that can be redirected towards AML blasts with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequence of CAR binding affinity on CAR-NK cell activation, cytolytic synapse formation, and anti-tumor activity.
Methods: We characterized in detail primary NK-cell populations expressing a panel of variant affinity AML (CD123)-specific CARs that contain antibody single-chain variable fragments (scFvs) targeting two distinct epitopes (26292 and 7G3). 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library and previously reported 7G3 affinity variants were used. The resulting CAR-NK cell panels were studied through in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models.
Results: 26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in primary NK cells and conferred antigen specific activation in vitro, as measured by proximal cytokine secretion and short-term cytotoxicity studies. High-resolution imaging analysis demonstrated 7G3 CAR-mediated increase in cytotoxic granule convergence at the immunological synapse with greater high affinity CAR-NK cell clustering and resultant AML target cell death in a short-term time lapse. In contrast, low affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with killing of a high antigen density target cell line compared to a low antigen density cell line. Of note, high affinity 26292-based CAR-NK cells targeted to an alternate epitope of the same antigen exhibited enhanced antigen density discrimination in short-term cytotoxicity assays. In longer term assays, low affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low affinity 7G3 CAR-NK cells in two xenograft disease models with enhanced tumor control evident in a subcutaneous bilateral flank tumor model.
Conclusions: 26292 and 7G3 CARs with a range of CD123 binding affinities are highly expressed in primary NK cells, and the resulting CAR-NK cells demonstrate antigen-specific activation and cytotoxicity. Affinity-based differences in functional activation and anti-tumor activity are dependent on time course and are scFv/epitope specific. These data support the role of affinity tuning as a component of CAR-NK cell design to fully optimize cellular therapy efficacy
AN OVERVIEW OF TOOLING REQUIREMENTS FOR THE MANUFACTURING OF CLASS 8 TRUCKS
Get PDFThe manufacturing of Class 8 trucks comes with intricate tooling requirements that demand specific attention to fixture design, optimization, and fastening solutions. Mack Trucks' Tooling and Equipment (T&E) Engineering Department has been at the forefront of addressing these challenges. In this essay, we explore the department's collaborative participatory action research approach to designing fixtures that cater to assembly needs while considering ergonomic concerns and environmental impact. We take a closer look at the case studies derived from co-op experiences, which include the design of fixtures for brake pneumatic manifold assembly, protective covers for hub flanges, and a fixture table for lube jockey module preparation. The methodology adopted involves comprehensive data collection, iterative design processes, and additive manufacturing techniques for prototype fabrication. The study's systematic approach aims to enhance manufacturing efficiency, quality, and cost-effectiveness within the Class 8 truck industry. Through this study, we hope to provide practical insights into tooling department operations specific to Class 8 truck manufacturing, bridging gaps in existing literature and providing valuable perspectives for academic discussions. Ultimately, this study highlights the significance of addressing tooling challenges to drive innovation and sustainability in the manufacturing sector, with direct implications for the practical operations of the stakeholders