Key Control Mechanism Research Articles

HIV-1 control in vivo is related to the number but not the fraction of infected cells with viral unspliced RNA.

In the absence of antiretroviral therapy (ART), a subset of individuals, termed HIV controllers, have levels of plasma viremia that are orders of magnitude lower than non-controllers (NC) who are at higher risk for HIV disease progression. In addition to having fewer infected cells resulting in fewer cells with HIV RNA, it is possible that lower levels of plasma viremia in controllers are due to a lower fraction of the infected cells having HIV-1 unspliced RNA (HIV usRNA) compared with NC. To directly test this possibility, we used sensitive and quantitative single-cell sequencing methods to compare the fraction of infected cells that contain one or more copies of HIV usRNA in peripheral blood mononuclear cells (PBMC) obtained from controllers and NC. The fraction of infected cells containing HIV usRNA did not differ between the two groups. Rather, the levels of viremia were strongly associated with the total number of infected cells that had HIV usRNA, as reported by others, with controllers having 34-fold fewer infected cells per million PBMC. These results reveal that viremic control is not associated with a lower fraction of proviruses expressing HIV usRNA, unlike what is reported for elite controllers, but is only related to having fewer infected cells overall, maybe reflecting greater immune clearance of infected cells. Our findings show that proviral silencing is not a key mechanism for viremic control and will help to refine strategies toward achieving HIV remission without ART.

DNA stable isotope probing reveals the impact of trophic interactions on bioaugmentation of soils with different pollution histories

BackgroundBioaugmentation is considered a sustainable and cost-effective methodology to recover contaminated environments, but its outcome is highly variable. Predation is a key top-down control mechanism affecting inoculum establishment, however, its effects on this process have received little attention. This study focused on the impact of trophic interactions on bioaugmentation success in two soils with different pollution exposure histories. We inoculated a 13C-labelled pollutant-degrading consortium in these soils and tracked the fate of the labelled biomass through stable isotope probing (SIP) of DNA. We identified active bacterial and eukaryotic inoculum-biomass consumers through amplicon sequencing of 16S rRNA and 18S rRNA genes coupled to a novel enrichment factor calculation.ResultsInoculation effectively increased PAH removal in the short-term, but not in the long-term polluted soil. A decrease in the relative abundance of the inoculated genera was observed already on day 15 in the long-term polluted soil, while growth of these genera was observed in the short-term polluted soil, indicating establishment of the inoculum. In both soils, eukaryotic genera dominated as early incorporators of 13C-labelled biomass, while bacteria incorporated the labelled biomass at the end of the incubation period, probably through cross-feeding. We also found different successional patterns between the two soils. In the short-term polluted soil, Cercozoa and Fungi genera predominated as early incorporators, whereas Ciliophora, Ochrophyta and Amoebozoa were the predominant genera in the long-term polluted soil.ConclusionOur results showed differences in the inoculum establishment and predator community responses, affecting bioaugmentation efficiency. This highlights the need to further study predation effects on inoculum survival to increase the applicability of inoculation-based technologies.9HmCi-USiFQRck8tFgyMqrVideo

Abstract 6958: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, reciprocally communicate with breast cancer cells to promote tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying this cell-to-cell communication, could unlock novel therapeutic avenues for inhibiting cellular plasticity, breast cancer progression and metastasis. The small GPI-anchored adaptor protein CRIPTO is a key player in this context though important molecular details remain to be elucidated. CRIPTO orchestrates TGF-β pathway signaling as well as growth factor-like signaling pathways that activate c-Src/MEK/AKT, in an autocrine and paracrine manner. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO’s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. We find that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. Further, by modeling tumor cell:fibroblast crosstalk in vitro, we have demonstrated mechanistically that CRIPTO expression in tumor cells mediates subsequent extracellular vesicle (EV) uptake by fibroblasts, as well as fibroblast SMAD pathway activation and priming for reciprocal signaling back to tumor cells to promote transcriptional reprogramming and cellular invasion.The significance of these findings lies in their elucidation of key EV control mechanisms that could be exploited to intervene in essential tumor cell:fibroblast crosstalk or to enhance targeting of therapeutic moieties based on EV architecture and function in cancer and/or normative regenerative processes. Citation Format: David W. Freeman, Brooke L. Gates, Mauri D. Spendlove, Benjamin T. Spike. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6958.

Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

Pharmacological effects of ginseng and ginsenosides on intestinal inflammation and the immune system.

Intestinal inflammatory imbalance and immune dysfunction may lead to a spectrum of intestinal diseases, such as inflammatory bowel disease (IBD) and gastrointestinal tumors. As the king of herbs, ginseng has exerted a wide range of pharmacological effects in various diseases. Especially, it has been shown that ginseng and ginsenosides have strong immunomodulatory and anti-inflammatory abilities in intestinal system. In this review, we summarized how ginseng and various extracts influence intestinal inflammation and immune function, including regulating the immune balance, modulating the expression of inflammatory mediators and cytokines, promoting intestinal mucosal wound healing, preventing colitis-associated colorectal cancer, recovering gut microbiota and metabolism imbalance, alleviating antibiotic-induced diarrhea, and relieving the symptoms of irritable bowel syndrome. In addition, the specific experimental methods and key control mechanisms are also briefly described.

Epstein-Barr virus induces germinal center light zone chromatin architecture and promotes survival through enhancer looping at the BCL2A1 locus.

Epstein-Barr virus has evolved with its human host leading to an intimate relationship where infection of antibody-producing B cells mimics the process by which these cells normally recognize foreign antigens and become activated. Virtually everyone in the world is infected by adulthood and controls this virus pushing it into life-long latency. However, immune-suppressed individuals are at high risk for EBV+ cancers. Here, we isolated B cells from tonsils and compare the underlying molecular genetic differences between these cells and those infected with EBV. We find similar regulatory mechanism for expression of an important cellular protein that enables B cells to survive in lymphoid tissue. These findings link an underlying relationship at the molecular level between EBV-infected B cells in vitro with normally activated B cells in vivo. Our studies also characterize the role of a key viral control mechanism for B cell survival involved in long-term infection.

Insulin-like growth factor binding protein-3 mediates hyperosmolar stress–induced mitophagy through the mechanistic target of rapamycin

Hyperosmolarity of the ocular surface triggers inflammation and pathological damage in dry eye disease (DED). In addition to a reduction in quality of life, DED causes vision loss and when severe, blindness. Mitochondrial dysfunction occurs as a consequence of hyperosmolar stress. We have previously reported on a role for the insulin-like growth factor binding protein-3 (IGFBP-3) in the regulation of mitochondrial ultrastructure and metabolism in mucosal surface epithelial cells; however, this appears to be context-specific. Due to the finding that IGFBP-3 expression is decreased in response to hyperosmolar stress invitro and in an animal model of DED, we next sought to determine whether the hyperosmolar stress-mediated decrease in IGFBP-3 alters mitophagy, a key mitochondrial quality control mechanism. Here we show that hyperosmolar stress induces mitophagy through differential regulation of BNIP3L/NIX and PINK1-mediated pathways. In corneal epithelial cells, this was independent of p62. The addition of exogenous IGFBP-3 abrogated the increase in mitophagy. This occurred through regulation of mTOR, highlighting the existence of a new IGFBP-3-mTOR signaling pathway. Together, these findings support a novel role for IGFBP-3 in mediating mitochondrial quality control in DED and have broad implications for epithelial tissues subject to hyperosmolar stress and other mitochondrial diseases.

The Role of Parkin in Rat Pancreatic Beta Cells Fate.

Parkin is a member of the mitochondrial quality control system that plays a major role in mitophagy. Although the loss of function mutations in the Parkin gene has been associated with the Familial Parkinson's phenotype, research in recent years points out that Parkin's function is not limited to neurodegenerative diseases. Parkin's function impressing key cellular quality control mechanisms, including the ubiquitin-proteasome and autophagy-lysosome systems, makes it an important player in the maintenance of cellular homeostasis. In this study, we investigated whether Parkin affects cell viability and ER stress responses under lipotoxic conditions in INS-1E cells. Our results may suggest that silencing Parkin may affect autophagy in addition to apoptosis. We also showed that Parkin may have a protective effect against lipo-toxic effects in INS-1E cells. Consistent with previous studies, we observed that stress responses were different for high and low palmitic acid doses. The Parkin being inhibited under high-dose PA treatment and active under low-dose PA treatment indicate that regulation of stress responses is controlled by environmental conditions. Our preliminary findings may suggest that in low lipotoxic conditions, Parkin affects the ER stress response by modulating Chop activity and Ca2+ release from the ER to the cytoplasm.

Measuring the nonselective effects of motor inhibition using isometric force recordings.

Inhibition is a key cognitive control mechanism humans use to enable goal-directed behavior. When rapidly exerted, inhibitory control has broad, nonselective motor effects, typically demonstrated using corticospinal excitability measurements (CSE) elicited by transcranial magnetic stimulation (TMS). For example, during rapid action-stopping, CSE is suppressed at both stopped and task-unrelated muscles. While such TMS-based CSE measurements have provided crucial insights into the fronto-basal ganglia circuitry underlying inhibitory control, they have several downsides. TMS is contraindicated in many populations (e.g., epilepsy or deep-brain stimulation patients), has limited temporal resolution, produces distracting auditory and haptic stimulation, is difficult to combine with other imaging methods, and necessitates expensive, immobile equipment. Here, we attempted to measure the nonselective motor effects of inhibitory control using a method unaffected by these shortcomings. Thirty male and female human participants exerted isometric force on a high-precision handheld force transducer while performing a foot-response stop-signal task. Indeed, when foot movements were successfully stopped, force output at the task-irrelevant hand was suppressed as well. Moreover, this nonselective reduction of isometric force was highly correlated with stop-signal performance and showed frequency dynamics similar to established inhibitory signatures typically found in neural and muscle recordings. Together, these findings demonstrate that isometric force recordings can reliably capture the nonselective effects of motor inhibition, opening the door to many applications that are hard or impossible to realize with TMS.

Strong-coupling magnetophononics: Self-blocking, phonon-bitriplons, and spin-band engineering

Magnetophononics, the modulation of magnetic interactions by driving infrared-active lattice excitations, is emerging as a key mechanism for the ultrafast dynamical control of both semiclassical and quantum spin systems by coherent light. We demonstrate that, in a quantum magnet with strong spin-phonon coupling, resonances between the driven phonon and the spin excitation frequencies exhibit an intrinsic self-blocking effect, whereby only a fraction of the available laser power is absorbed by the phonon. Using the quantum master equations governing the nonequilibrium steady states of the coupled spin-lattice system, we show how self-blocking arises from the self-consistent alteration of the resonance frequencies. We link this to the appearance of mutually repelling collective spin-phonon states, which in the regime of strong hybridization become composites of a phonon and two triplons. We then identify the mechanism and optimal phonon frequencies by which to control a global nonequilibrium renormalization of the lattice-driven spin excitation spectrum and demonstrate that this effect should be observable in ultrafast THz experiments on a number of known quantum magnetic materials.

Conserved multiepitope vaccine constructs: A potent HIV-1 therapeutic vaccine in clinical trials.

Despite the success of Antiretroviral Therapy (ART) in preventing HIV-1-associated clinical progression to AIDS, it is unable to eliminate the viral reservoirs and eradicate the HIV-1 infection. Therapeutic vaccination is an alternative approach to alter the HIV-1 infection course. It can induce effective HIV-1-specific immunity to control viremia and eliminate the need for lifelong ART. Immunological data from spontaneous HIV-1 controllers have shown that cross-reactive T-cell responses are the key immune mechanism in HIV-1 control. Directing these responses toward preferred HIV-1 epitopes is a promising strategy in therapeutic vaccine settings. Designing novel immunogens based on the HIV-1 conserved regions containing a wide range of critical T- and B-cell epitopes of the main viral antigens (conserved multiepitope approaches) supplies broad coverage of global diversity in HIV-1 strains and Human Leukocyte Antigen (HLA) alleles. It can also prevent immune induction to undesirable decoy epitopes theoretically. The efficacy of different novel HIV-1 immunogens based on the conserved and/or functional protective site of HIV-1 proteome has been evaluated in multiple clinical trials. Most of these immunogens were generally safe and able to induce potent HIV-1-specific immunity. However, despite these findings, several candidates have demonstrated limited efficacy in viral replication control. In this study, we used the PubMed and ClinicalTrial.gov databases to review the rationale of designing curative HIV-1 vaccine immunogens based on the conserved favorable site of the virus. Most of these studies evaluate the efficacy of vaccine candidates in combination with other therapeutics and/or with new formulations and immunization protocols. This review briefly describes the design of conserved multiepitope constructs and outlines the results of these vaccine candidates in the recent clinical pipeline.

An optogenetic toolkit for light-inducible antibiotic resistance

Antibiotics are a key control mechanism for synthetic biology and microbiology. Resistance genes are used to select desired cells and regulate bacterial populations, however their use to-date has been largely static. Precise spatiotemporal control of antibiotic resistance could enable a wide variety of applications that require dynamic control of susceptibility and survival. Here, we use light-inducible Cre recombinase to activate expression of drug resistance genes in Escherichia coli. We demonstrate light-activated resistance to four antibiotics: carbenicillin, kanamycin, chloramphenicol, and tetracycline. Cells exposed to blue light survive in the presence of lethal antibiotic concentrations, while those kept in the dark do not. To optimize resistance induction, we vary promoter, ribosome binding site, and enzyme variant strength using chromosome and plasmid-based constructs. We then link inducible resistance to expression of a heterologous fatty acid enzyme to increase production of octanoic acid. These optogenetic resistance tools pave the way for spatiotemporal control of cell survival.

Michael Lobban. Imperial Incarceration: Detention without Trial in the Making of British Colonial Africa.

Michael Lobban’s Imperial Incarceration: Detention without Trial in the Making of British Colonial Africa provides a rich and methodical examination of how the detention without trial of defiant or rebellious African leaders became a key mechanism for British imperial expansion and control throughout the nineteenth and early twentieth centuries. Lobban’s analysis begins by sketching out a central paradox: How could a nation that professed its commitment to the rule of law and the sacred common-law value of habeas corpus commit such apparently large-scale violations of these principles throughout Britain’s African empire? The answer, Lobban argues, lies in the ambiguity of what the “rule of law” itself meant, as well as the racialized nature of empire. As Lobban demonstrates, conceptions of the “rule of law” were defined by both a “substantive” tradition that sought to protect the rights of the individual against arbitrary state power and a “formalistic” tradition that believed any act done under the legal authority of parliament was legitimate (7–8). While the former position is typically associated with the renowned English jurist A. V. Dicey, Lobban argues that the “Diceyan” view of the rule of law could tolerate violations of due process so long as these were given legal “cover” through the legitimacy of legislative and other lawmaking bodies. Detention without trial was also much more palatable within the inherently unequal racial hierarchies of Western imperialism. Simply put, colonial and imperial officials were less concerned about protecting the rights of nonwhite imperial subjects.

Tunable Metasurfaces Based on Mechanically Deformable Polymeric Substrates

The emergence of metamaterials has presented an unprecedented platform to control the fundamental properties of light at the nanoscale. Conventional metamaterials, however, possess passive properties that cannot be modulated post-fabrication, limiting their application spectrum. Recent metasurface research has explored a plethora of active control mechanisms to modulate the optical properties of metasurfaces post-fabrication. A key active control mechanism of optical properties involves the use of mechanical deformation, aided by deformable polymeric substrates. The use of deformable polymeric substrates enables dynamic tuning of the optical properties of metasurfaces including metalenses, metaholograms, resonance, and structural colors, which are collectively relevant for biosensing and bioimaging. Deformable–stretchable metasurfaces further enable conformable and flexible optics for wearable applications. To extend deformable–stretchable metasurfaces to biocompatible metasurfaces, a fundamental and comprehensive primer is required. This review covers the underlying principles that govern the highlighted representative metasurface applications, encompassing stretchable metalenses, stretchable metaholograms, tunable structural colors, and tunable plasmonic resonances, while highlighting potential advancements for sensing, imaging, and wearable biomedical applications.

Mechanisms of control and resistance in the devolved work of public school principals in New South Wales

ABSTRACT Globally, New Public Management reforms have transformed work in many areas of the public sector. A key aspect of these reforms has been devolution: the provision of greater local autonomy over resources and decision making as part of a broader decentralisation strategy. Important questions about managers’ work in devolved settings, not in the least in public education, remain to be addressed. The 2012–18 ‘Local Schools, Local Decisions’ reform to public education in the Australian state of New South Wales was framed by familiar aims: to achieve ‘efficiency and effectiveness’ through greater local control. Recognising principals as the nexus of this ostensible local authority, principals’ work in the final stages of the reform’s implementation were investigated. Through the utilisation of labour process theory to examine tensions in this focal employment relationship, three key control mechanisms in the work of principals were identified: a reduction of the indeterminacy gap through principals’ management of resources; the degradation of work under devolution; and the legitimation of the state through principals’ work.

Targeting Deubiquitinating Enzymes (DUBs) That Regulate Mitophagy via Direct or Indirect Interaction with Parkin.

The quality control of mitochondria is critical for the survival of cells, and defects in the pathways required for this quality control can lead to severe disease. A key quality control mechanism in cells is mitophagy, which functions to remove damaged mitochondria under conditions of various stresses. Defective mitophagy can lead to a number of diseases including neurodegeneration. It has been proposed that an enhancement of mitophagy can improve cell survival, enhance neuronal function in neurodegeneration and extend health and lifespans. In this review, we highlight the role of deubiquitinating enzymes (DUBs) in the regulation of mitophagy. We summarise the current knowledge on DUBs that regulate mitophagy as drug targets and provide a list of small molecule inhibitors that are valuable tools for the further development of therapeutic strategies targeting the mitophagy pathway in neurodegeneration.

Enhanced cipher text-policy attribute-based encryption and serialization on media cloud data

PurposeThere are various system techniques or models which are used for access control by performing cryptographic operations and characterizing to provide an efficient cloud and in Internet of Things (IoT) access control. Particularly in cloud computing environment, there is a large-scale distribution of these traditional symmetric cryptographic techniques. These symmetric cryptographic techniques use the same key for encryption and decryption processes. However, during the execution of these phases, they are under the problems of key distribution and management. The purpose of this study is to provide efficient key management and key distribution in cloud computing environment.Design/methodology/approachThis paper uses the Cipher text-Policy Attribute-Based Encryption (CP-ABE) technique with proper access control policy which is used to provide the data owner’s control and share the data through encryption process in Cloud and IoT environment. The data are shared with the the help of cloud storage, even in presence of authorized users. The main method used in this research is Enhanced CP-ABE Serialization (E-CP-ABES) approach.FindingsThe results are measured by means of encryption, completion and decryption time that showed better results when compared with the existing CP-ABE technique. The comparative analysis has showed that the proposed E-CP-ABES has obtained better results of 2373 ms for completion time for 256 key lengths, whereas the existing CP-ABE has obtained 3129 ms of completion time. In addition to this, the existing Advanced Encryption Standard (AES) scheme showed 3449 ms of completion time.Originality/valueThe proposed research work uses an E-CP-ABES access control technique that verifies the hidden attributes having a very sensitive dataset constraint and provides solution to the key management problem and access control mechanism existing in IOT and cloud computing environment. The novelty of the research is that the proposed E-CP-ABES incorporates extensible, partially hidden constraint policy by using a process known as serialization procedure and it serializes to a byte stream. Redundant residue number system is considered to remove errors that occur during the processing of bits or data obtained from the serialization. The data stream is recovered using the Deserialization process.

Response of a turbulent separation bubble to zero-net-mass-flux jet perturbations

Separating turbulent boundary layers exhibit unsteady motions at various frequencies. Using numerical and analytical techniques we identified the distinct strong responses of the flow to actuations at its natural unsteady frequencies. The low-frequency motion of the separating shear layer can be modulated by small streamwise perturbations thus leading to a smaller mean separation region. The key control mechanisms are the strong structural changes dominated by two-dimensional roller vortices. The stress/load unsteadiness on the wall is a possibly harmful outcome of flow control.

Role of Chaperone-Mediated Autophagy in Ageing Biology and Rejuvenation of Stem Cells.

What lies at the basis of the mechanisms that regulate the maintenance and self-renewal of pluripotent stem cells is still an open question. The control of stemness derives from a fine regulation between transcriptional and metabolic factors. In the last years, an emerging topic has concerned the involvement of Chaperone-Mediated Autophagy (CMA) as a key mechanism in stem cell pluripotency control acting as a bridge between epigenetic, transcriptional and differentiation regulation. This review aims to clarify this new and not yet well-explored horizon discussing the recent studies regarding the CMA impact on embryonic, mesenchymal, and haematopoietic stem cells. The review will discuss how CMA influences embryonic stem cell activity promoting self-renewal or differentiation, its involvement in maintaining haematopoietic stem cell function by increasing their functionality during the normal ageing process and its effects on mesenchymal stem cells, in which modulation of CMA regulates immunosuppressive and differentiation properties. Finally, the importance of these new discoveries and their relevance for regenerative medicine applications, from transplantation to cell rejuvenation, will be addressed.

Subsets of Slow Dynamic Modes Reveal Global Information Sources as Allosteric Sites

Allostery is a key biological control mechanism, and dynamic information flow provides a perspective to describe allosteric interactions in causal relationships. Here, as a novel implementation of the Gaussian Network Model (GNM) based Transfer Entropy (TE) calculations, we show that the dissection of dynamic information into subsets of slow dynamic modes discloses different layers of multi-directional allosteric pathways inherent in a given protein structure. In these subsets of slow modes, the degree of collectivity (Col) in the information transfer of residues with their TE values (TECol score) identifies distinct residues as powerful effectors, global information sources; showing themselves with a high dynamic capacity to collectively disseminate information to others. As exemplified on aspartate transcarbamoylase (ATCase), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), and human transient receptor potential melastatin 2 (TRPM2) along with a dataset of 20 proteins, these specific residues are associated with known active and allosteric sites. These information source residues, which collectively control others and lead allosteric communication pathways, hint at plausible binding sites for structure-based rational drug design.