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Chromatin-associated RNA (caRNA) is a vital component of the interphase nucleus; yet its distribution and role in the three-dimensional (3D) genome organization and function remain poorly understood. Here, we map caRNA’s spatial distribution on the 3D genome in human embryonic stem cells, fibroblasts, and myelogenous leukemia cells. We find that the relative abundance of trans-acting caRNA reflects the functional nuclear compartmentalization of the 3D genome, and the caRNA’s sequence features are predictive of its spatial localization. We observe extensive caRNA-genome interactions that span several hundred kilobases to several megabases. These caRNA domains correlate with chromatin loops and enhancer-promoter interactions. We report a tradeoff of caRNA’s promotive and suppressive roles to chromatin interactions. As a result, caRNA promotes chromatin interactions outside the anchors of chromatin loops, whereas caRNA exhibits an overall suppressive impact to chromatin interactions between loop anchors. Furthermore, caRNA suppresses chromatin loops’ number and strengths genome-wide, and this suppression depends on electric charge-mediated RNA interactions. These results reveal caRNA’s multifaced role in regulating 3D chromatin organization and highlight caRNA’s ability to modulate chromatin looping.

Introduction: We investigated the function of cell division cycle 6 (CDC6) on the prognosis in colorectal carcinoma (CRC). Methods: CDC6 protein expression levels in 121 patients with colorectal cancer and adjacent normal mucosa were detected by immunohistochemistry. Results: Compared to adjacent normal tissues, CDC6 mRNA level was overexpressed in CRC tissues. Moreover, CDC6 protein levels were expressed up to 93.39% (113/121) in CRC tissues in the cell nucleus or cytoplasm. However, there were only 5.79% (7/121) in normal mucosal tissues with nuclear expression. CDC6 expression was significantly correlated with TNM stage and tumor metastasis. The 5-year survival rate was lower in the high CDC6 expression group than the low group. After silencing of CDC6 expression in SW620 cells, cell proliferation was slowed, the tumor clones were decreased, and the cell cycle was arrested in G1 phase. In multivariate analysis, increased CDC6 protein expression levels in colon cancer tissues were associated with cancer metastasis, TNM stage, and patient survival time. Conclusion: CDC6 is highly expressed in CRC, and downregulation of CDC6 can slow the growth of CRC cells in vitro. It is also an independent predictor for poor prognosis and may be a useful biomarker for targeted therapy and prognostic evaluation.

Viral interfering RNA (viRNA) has been identified from several viral genomes via directly deep RNA sequencing of the virus-infected cells, including zika virus (ZIKV). Once produced by endoribonuclease Dicer, viRNAs are loaded onto the Argonaute (AGO) family proteins of the RNA-induced silencing complexes (RISCs) to pair with their RNA targets and initiate the cleavage of target genes. However, the identities of functional ZIKV viRNAs and their viral RNA targets remain largely unknown. Our recent study has shown that ZIKV capsid protein interacted with Dicer and antagonized its endoribonuclease activity, which requires its histidine residue at the 41st amino acid. Accordingly, the engineered ZIKV-H41R loss-of-function (LOF) mutant virus no longer suppresses Dicer enzymatic activity nor inhibits miRNA biogenesis in NSCs. By combining AGO-associated RNA sequencing, deep sequencing analysis in ZIKV-infected human neural stem cells (NSCs), and miRanda target scanning, we defined 29 ZIKV derived viRNA profiles in NSCs, and established a complex interaction network between the viRNAs and their viral targets. More importantly, we found that viRNA production from the ZIKV mRNA is dependent on Dicer function and is a limiting factor for ZIKV virulence in NSCs. As a result, much higher levels of viRNAs generated from the ZIKV-H41R virus-infected NSCs. Therefore, our mapping of viRNAs to their RNA targets paves a way to further investigate how viRNAs play the role in anti-viral mechanisms, and perhaps other unknown biological functions.

Background Hospitalisation of an older adult due to acute medical illness can result in adverse events and accelerate loss of independence despite recovery from the illness. Promoting mobility during hospitalisation can help to mitigate the risks of functional decline. Understanding the perspectives on the barriers and the maintenance of mobility is essential in the development of effective strategies. Aims To explore the perceptions of patients and their carers, as well as the nurses on promotion of mobility among hospitalised adult older patients. Methods A qualitative descriptive study design with purposive and convenience sampling approach was undertaken. A total of fourteen patients, six carers and ten nurses from a general medical ward of an acute care tertiary public hospital in Singapore were recruited over the period of November 2017 to February 2018. Face to face semi-structured interviews were conducted and audio-recorded. The interview transcripts were coded and an inductive content analysis approach was adopted to generate categories of sub-themes and themes through the open coding process. Results Four main themes were generated: (1) perceived and induced fear of falling; (2) challenges in initiation of mobility; (3) influence of premorbid status and physical condition on activity level; and (4) strategies to promote mobility. Conclusion Recognition of the importance of mobility as well as the detrimental effects of prolonged bed rest during hospitalization is an essential first step toward developing successful interventions to promote mobility in the Asian context. Improvements need to be made to help overcome the various barriers and challenges in the mobilization of the older patients in the acute care settings. Nurses and other members of the care team can help to increase the confidence of patients and among family carers (in providing assistance during mobility) by role modelling and provision of walking aids as well as risk-based education.

Aberrant expression of HOXC6 and HOXC4 is commonly detected in prostate cancer. The high expression of these transcription factors is associated with aggressive prostate cancer and can predict cancer recurrence after treatment. Thus, HOXC4 and HOXC6 are clinically relevant biomarkers of aggressive prostate cancer. However, the molecular mechanisms by which these HOXC genes contribute to prostate cancer is not yet understood. To begin to address the role of HOXC4 and HOXC6 in prostate cancer, we performed RNA-seq analyses before and after siRNA-mediated knockdown of HOXC4 and/or HOXC6 and also performed ChIP-seq to identify genomic binding sites for both of these transcription factors. Our studies demonstrate that HOXC4 and HOXC6 co-localize with HOXB13, FOXA1 and AR, three transcription factors previously shown to contribute to the development of prostate cancer. We suggest that the aberrantly upregulated HOXC4 and HOXC6 proteins may compete with HOXB13 for binding sites, thus altering the prostate transcriptome. This competition model may be applicable to many different human cancers that display increased expression of a HOX transcription factor.

High expression of the transcription factor ZFX is correlated with proliferation, tumorigenesis, and patient survival in multiple types of human cancers. However, the mechanism by which ZFX influences transcriptional regulation has not been determined. We performed ChIP-seq in four cancer cell lines (representing kidney, colon, prostate, and breast cancers) to identify ZFX binding sites throughout the human genome. We identified roughly 9000 ZFX binding sites and found that most of the sites are in CpG island promoters. Moreover, genes with promoters bound by ZFX are expressed at higher levels than genes with promoters not bound by ZFX. To determine if ZFX contributes to regulation of the promoters to which it is bound, we performed RNA-seq analysis after knockdown of ZFX by siRNA in prostate and breast cancer cells. Many genes with promoters bound by ZFX were down-regulated upon ZFX knockdown, supporting the hypothesis that ZFX acts as a transcriptional activator. Surprisingly, ZFX binds at +240 bp downstream from the TSS of the responsive promoters. Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq), we show that ZFX binds between the open chromatin region at the TSS and the first downstream nucleosome, suggesting that ZFX may play a critical role in promoter architecture. We have also shown that a closely related zinc finger protein ZNF711 has a similar binding pattern at CpG island promoters, but ZNF711 may play a subordinate role to ZFX. This functional characterization of ZFX provides important new insights into transcription, chromatin structure, and the regulation of the cancer transcriptome.

Summary: Prostate cancer (PCa) is the leading cancer among men in the United States, with genetic factors contributing to ∼42% of the susceptibility to PCa. We analyzed a PCa risk region located at 7p15.2 to gain insight into the mechanisms by which this noncoding region may affect gene regulation and contribute to PCa risk. We performed Hi-C analysis and demonstrated that this region has long-range interactions with the HOXA locus, located ∼873 kb away. Using the CRISPR/Cas9 system, we deleted a 4-kb region encompassing several PCa risk-associated SNPs and performed RNA-seq to investigate transcriptomic changes in prostate cells lacking the regulatory element. Our results suggest that the risk element affects the expression of HOXA13 and HOTTIP, but not other genes in the HOXA locus, via a repressive loop. Forced expression of HOXA13 was performed to gain further insight into the mechanisms by which this risk element affects PCa risk. : Luo et al. identify an 800-kb loop between a prostate cancer risk region and HOXA13. Deleting the risk region removes one anchor point of the repressive loop and upregulates HOXA13, leading to changes in the transcriptome, including overexpression of an oncogene (GATA2). Keywords: Hi-C, chromatin structure, transcriptional regulation, GWAS, CRISPR, HOX genes

Introduction: We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events, and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state. Materials and methods: We used Hoechst nuclear staining and fluorescence activated cell sorting to isolate the Hoechst-low drug resistant side population (SP) cells from the less drug resistant Hoechst-high non-side population (NSP) cells in bladder cancer cell lines. We performed RNA sequencing and AcceSssIble assay to profile RNA expression, global DNA methylation and chromatin accessibility in SP and NSP cells. Putative enhancer regions were identified using H3K27ac ChIPseq data in bladder cancer cell lines, which are publicly available. We next performed transcription factor motif analysis on accessible enhancers near genes overexpressed in SP cells relative to NSP cells. The differentially expressed candidate target transcription factor by RNA-seq was validated using qRT-PCR. Functional validation was done using CRISPR knock out and drug resistance assays. Results: A higher percentage of enhancers were differentially accessible between SP and NSP cells compared with the promoters. We identified 746 enhancers that are accessible only in SP cells and 465 enhancers only in NSP cells. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 was consistently overexpressed in bladder tumor tissues relative to normal bladder tissues in three independent studies in BaseSpace Correlation Engine. CRISPR knock-out of FOXC1 in multiple bladder cancer cell lines resulted in decreased cisplatin resistance. Conclusion: We found that FOXC1 is overexpressed and its motif is enriched on the accessible enhancers when bladder cancer cells shift to a drug-resistant tumorigenic phenotype. Conversely, FOXC1 knock-out attenuates cisplatin resistance in bladder cancer cells. These observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer. Citation Format: Yi-Tsung Lu, Tong Xu, Tien-Chan Hsieh, Maheen Iqbal, Zhifei Luo, Peggy J. Farnham, Suhn K. Rhie, Amir Goldkorn. FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1406.

The current widespread outbreak of Zika virus (ZIKV) infection has been linked to severe clinical birth defects, particularly microcephaly, warranting urgent study of the molecular mechanisms underlying ZIKV pathogenesis. Akt-mTOR signaling is one of the key cellular pathways essential for brain development and autophagy regulation. Here, we show that ZIKV infection of human fetal neural stem cells (fNSCs) causes inhibition of the Akt-mTOR pathway, leading to defective neurogenesis and aberrant activation of autophagy. By screening the three structural proteins and seven nonstructural proteins present in ZIKV, we found that two, NS4A and NS4B, cooperatively suppress the Akt-mTOR pathway and lead to cellular dysregulation. Corresponding proteins from the closely related dengue virus do not have the same effect on neurogenesis. Thus, our study highlights ZIKV NS4A and NS4B as candidate determinants of viral pathogenesis and identifies a mechanism of action for their effects, suggesting potential targets for anti-ZIKV therapeutic intervention.

Homeobox genes (HOX) are a large family of transcription factors that direct the formation of many body structures during early embryonic development. There are 39 genes in the subgroup of homeobox genes that constitute the human HOX gene family. Correct embryonic development of flies and vertebrates is, in part, mediated by the unique and highly regulated expression pattern of the HOX genes. Disruptions in these fine-tuned regulatory mechanisms can lead to developmental problems and to human diseases such as cancer. Unfortunately, the molecular mechanisms of action of the HOX family of transcription factors are severely under-studied, likely due to idiosyncratic details of their structure, expression, and function. We suggest that a concerted and collaborative effort to identify interacting protein partners, produce genome-wide binding profiles, and develop HOX network inhibitors in a variety of human cell types will lead to a deeper understanding of human development and disease. Within, we review the technological challenges and possible approaches needed to achieve this goal.

Summary Zika virus (ZIKV) causes microcephaly and disrupts neurogenesis. Dicer-mediated miRNA biogenesis is required for embryonic brain development and has been suggested to be disrupted upon ZIKV infection. Here we mapped the ZIKV-host interactome in neural stem cells (NSCs) and found that Dicer is specifically targeted by the capsid from ZIKV, but not other flaviviruses, to facilitate ZIKV infection. We identified a capsid mutant (H41R) that loses this interaction and does not suppress Dicer activity. Consistently, ZIKV-H41R is less virulent and does not inhibit neurogenesis in vitro or corticogenesis in utero. Epidemic ZIKV strains contain capsid mutations that increase Dicer binding affinity and enhance pathogenicity. ZIKV-infected NSCs show global dampening of miRNA production, including key miRNAs linked to neurogenesis, which is not observed after ZIKV-H41R infection. Together these findings show that capsid-dependent suppression of Dicer is a major determinant of ZIKV immune evasion and pathogenesis and may underlie ZIKV-related microcephaly.

Cosmic ray muon tomography is a novel technology to detect high-Z material. A prototype of TUMUTY with 73.6 cm x 73.6 cm large scale position sensitive MRPC detectors has been developed and is introduced in this paper. Three test kits have been tested and image is reconstructed using MAP algorithm. The reconstruction results show that the prototype is working well and the objects with complex structure and small size (20 mm) can be imaged on it, while the high-Z material is distinguishable from the low-Z one. This prototype provides a good platform for our further studies of the physical characteristics and the performances of cosmic ray muon tomography., 7 pages, 4 figures, SORMA XV

Introduction: Highly tumorigenic, drug resistant cancer stem-like cells play a key role in therapy resistance and disease progression. We and others have previously shown that cancer cells can transition cyclically in and out of this aggressive phenotype in a manner that is rapid and spontaneous and does not require new mutations, drug selection, or ectopic gene expression. Phenotypic plasticity has been linked to altered cellular metabolic states, so we explored whether cancer cells exhibit distinct metabolic shifts as they switch to and from a cancer stem-like phenotype. Materials and Methods: Bladder cancer cells (J82 cell line) were fractionated by Hoechst staining and FACS into a side population (SP) marked by high tumorigenicity and drug resistance, and a non-side population (NSP) lacking these characteristics. SP and NSP cells were profiled by Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics after labeling with [U-13C]-glucose or [U-13C]-glutamine, as well as by RNAseq. The results were analyzed by Metabolite Set Enrichment Analysis (MSEA) and Gene Set Enrichment Analysis (GSEA), respectively. Fluorescence Lifetime Imaging Microscopy (FLIM), which measures bound vs. free cellular NADH, was used to image SP and NSP cells separately or in co-culture with or without cisplatin. Results: SP cells exhibited LC-MS MSEA profiles marked by upregulated TCA cycle activity and increased glutamine usage relative to NSP cells. Consistent with this finding, SP cells also exhibited RNAseq GSEA profiles marked by upregulated oxidative phosphorylation relative to NSP cells. Distinct FLIM metabolic signatures were successfully assigned to SP and NSP cells and used to image these subpopulations over time. When grown separately or in co-culture, SP cells’ FLIM signature gradually shifted to an NSP signature. Conversely, NSP cells’ FLIM signature gradually shifted an SP signature. When treated with cisplatin, SP cells survived and retained an unchanged SP FLIM signature. In contrast, fewer NSP cells survived cisplatin treatment, and NSP cells that did survive shifted towards an SP FLIM signature. Conclusion: Spontaneous rapid transition to and from a cancer stem-like state involves a metabolic shift to and from oxidative phosphorylation and the TCA cycle. These distinct metabolic profiles can be tracked directly and noninvasively by FLIM, revealing that cancer cells spontaneously switch between the metabolic states over time but preferentially shift toward the cancer stem-like state when exposed to cisplatin. Thus, metabolic plasticity can be characterized, tracked and exploited therapeutically to disrupt cancer resistance and dissemination. Citation Format: Tong Xu, Cosimo Arnesano, Alireza Delfarah, Zhifei Luo, Kevin Delijani, Emmanuelle Hodara, Peggy Farnham, Nicholas A. Graham, Scott E. Fraser, Amir Goldkorn. Transition to and from a cancer stem-like state is marked by rapid metabolic shifts that are observable in real time using fluorescence lifetime imaging microscopy (FLIM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5282.

SUMMARY Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered β-transducin repeat-containing protein (β-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking IκBα degradation and thereby dampening nuclear factor κB (NF-κB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-κB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target., Graphical Abstract, In Brief Neuroinflammation drives pathology during brain injury. Zeng et al. show that TRIM9 is induced after ischemic insults in young mice, but not old mice, and promotes resolution of neuroinflammation. AAV-mediated TRIM9 therapy in aged mice restricts neuroinflammation and alleviates stroke damage, representing a potential therapeutic target for brain injury.

Recently, a novel bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), was isolated in central China. The virus can cause multi-clinical symptoms: severe fever, thrombocytopenia, leukocytopenia, with a mortality rate of ~10%. Several studies show that SFTSV could undergo rapid evolution via gene mutation and homologous recombination. However, as an important evolutionary force for segmented-genome viruses, reassortment has not been reported in SFTSV. In this study, we identified two SFTSV strains of which the S segment has different origin from M and L, suggesting that reassortment might be potential force driving rapid change of SFTSV. This result might shed new light on the evolutionary behavior of the novel virus.

Cosmic ray muon tomography is a developing method based on scattering theory which can be applied in nuclear material discrimination because of its sensitivity to high Z material in recent years[1]. Various muon tomography systems with different kinds of detectors have been set up, such as drift tube [2], Gem [3], RPC [4]/MRPC [5], scintillator strip [6] and drift chamber [7]. Because of the importance of muon energy in material scattering density reconstruction, muon spectrometer of at least two slabs whose size and atom number are known and track detectors has been constructed to provide an estimate of the momentum of each exit muon [8], which may increase the complexity and costs of muon tomography system. In addition to this kind of muon spectrometer, Time of Flight (TOF) method can also be applied to obtain energy. The MRPC detectors employed in TUMUTY (Tsinghua University Cube for Cargo Inspection) [5], developed by Tsinghua University, have both good spatial resolution and time resolution, motivating the thought of muon energy measurement with TOF method [9]. With the muon track and time of flight obtained from 6-layer MRPC detectors, the energy of each muon can be detected. However, as muon travels almost as fast as light, it is not easy to get the muon energy with high accuracy by TOF or muon spectrometer method. Fortunately, accurate muon energy is not necessary in muon tomography [10]. In this paper, the influence of muon energy on material discrimination has been studied and preliminary simulation results indicate that muons with low-energy have more significant influence on the distribution of scattering angles than high energy. Multilayer TOF method has been employed for muon energy segment and the RMS difference between materials of interest becomes larger with the low-energy muons acquired via this method in our simulation, making it easier to distinguish different materials.

With the increase of accelerator luminosity, the rate requirement for timing MRPC becomes important. Usually MRPCs are assembled with ordinary glass plates of 1.0 mm or thicker, but their rate capability is only about 200 Hz/cm2, which cannot afford to the future high rate requirement. DC model and a two dimensional resistor network model suggested that the rate capability of MRPC is largely dependent on the thickness of glass electrode. Three MRPCs based on three different thickness of thin float glass plates were assembled in our Lab to study the rate effects. The cosmic rays test result shows that those three MRPCs can work properly in a low rate environment. The X-rays test results suggested that the thinner the electrode the higher counting rate can be obtained by MRPC. Uniformly irradiation under a 1 GeV/c deuteron beams was carried out at the Nuclotron accelerator at the Joint Institute for Nuclear Research. MRPCs assembled with three thickness (0.7mm, 0.5mm and 0.35mm) glass can afford particle rate up to 500 Hz/cm2, 0.75 kHz/cm2 and 3.2 kHz/cm2, respectively. In the mean time, a time resolution of 75ps was remained. What's more, the results indicate that reduce the thickness of glass electrode is a promising way to improve the rate capability of MRPC.

Muon tomography with cosmic ray muon is a novel technology for high-Z material detection. Preliminary research on performance evaluation method and models for muon tomography is introduced. Cube model and TUCCI model are introduced to evaluate the material discrimination capability and detection capability of high-Z material hidden in cargo container, line-pair model and hole model are introduced to evaluate the spatial resolution of muon tomography. The relationship between image quality and exposure time is also discussed.

Cosmic-ray muon tomography based on multiple coulomb scattering is typically applied in detecting high-Z materials. The energy of muon also plays an important role in the distribution of the scattering angles, and the information of muon energy would be useful for the image reconstruction. In this paper, a simulation experiment has been conducted to check the feasibility of TOF method applied in measuring muon energy for muon tomography system. The simulation results have been discussed and some preliminary conclusions have also been drawn.

Muon tomography with cosmic ray muons is a novel technology for high-Z material detection. The tomographic imaging is based on multiple coulomb scattering of cosmic ray muons in matter, which requires large-scale, high-efficiency, high spatial resolution detectors for tracking of incoming and outgoing muons. From previous studies, the multi-gap resistive plate chamber (MRPC) would be an excellent and inexpensive choice for this application, which also offers the possibility of introducing energy information of muons by TOF measurement for image reconstruction of muon tomography. A prototype of muon tomography facility with 6 layers large-scale MRPC detectors has been designed and is under construction, the preliminary results of muon tomography and design details of the MRPC detectors and electronics were presented.

Muon tomography is an advanced technology to non-destructively detect high atomic number materials. It exploits the multiple Coulomb scattering information of muon to reconstruct the scattering density image of the traversed object. Because of the statistics of muon scattering, the measurement error of system and the data incompleteness, the reconstruction is always accompanied with a certain level of interference, which will influence the reconstructed spatial resolution. While statistical noises can be reduced by extending the measuring time, system parameters determine the ultimate spatial resolution that one system can reach. In this paper, an effective frequency-domain model is proposed to analyze the reconstructed spatial resolution of muon tomography. The proposed method modifies the resolution analysis in conventional computed tomography (CT) to fit the different imaging mechanism in muon scattering tomography. The measured scattering information is described in frequency domain, then a relationship between the measurements and the original image is proposed in Fourier domain, which is named as "Muon Central Slice Theorem". Furthermore, a preliminary analytical expression of the ultimate reconstructed spatial is derived, and the simulations are performed for validation. While the method is able to predict the ultimate spatial resolution of a given system, it can also be utilized for the optimization of system design and construction.