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Open Access Publications from the University of California

Scholarly Works (12 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2016)

Point-of-care technologies provide innovative solutions that improve treatment. Healthcare systems including some low-resource settings have begun implementing these technologies providing the convenience and reduction of large laboratory set-ups. Low-cost is one of the main driving components when it comes to point-of-care diagnostics. Paper-based microfluidics has generated a great amount of interest for the development of low-cost diagnostic and self-contained analytical devices. Satisfying the World Health Organization’s (WHO) recommended ASSURED criteria; Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment free, Deliverable, paper-based microfluidics have made point-of-care testing more accessible. Applications range from healthcare, food safety, and environmental monitoring, among others. What has in part attracted attention is the low-cost, ease-of-use, and adaptability of these paper devices. Compared to conventional microfluidic devices, the paper-based counterparts are able to utilize paper’s inherent wicking property to eliminate the external pumping needed to drive the fluid. Channels are easily formed by either selectively removing sections of the paper substrate or by pattering channel boundaries with a hydrophobic material.

In spite of the benefits and advantages described above, paper-based microfluidic technologies often lack the necessary sensitivity and sophistication available in conventional microfluidic devices. In order to be a competitive alternative, paper-based microfluidics require improvement and novel development of feasible detection methods. These methods will likely require increasingly complex chemistry and control of reagents. Thus, understanding imbibition as well as obtaining precise, accurate, and consistent fluid handling within the paper device will be crucial.

Although considerable knowledge exists on techniques to manipulate fluid within the paper channel, what is lacking are studies on how non-laboratory conditions (e.g. relative humidity) influence fluid flow. This presentation aims to address this gap with particular focus on the effects of relative humidity and channel width. A series of controlled imbibition experiments is reported using cellulose papers commonly used in the field of paper-based microfluidics. We show that both the imposed relative humidity and the channel width have critical design considerations in paper-based devices. Additionally, we compare three models, the Lucas-Washburn model, the Fries et al. (2008) model which incorporates evaporation, and a newly developed water saturation model that incorporates evaporation as well as residual water in the paper. We assess their accuracy in representing the experimental data and systematically evaluate the importance of evaporation and water saturation under a wide range of relative humidity conditions. The current study has created a library of paper-specific, imbibition-related properties for commonly used filter and chromatography papers for the first time.

Lastly, the effort of fluid manipulation is continued. A qualitative investigation on two-dimensional wax-bound channels is covered. The channels encompass the most basic geometry that may be present in complex fluidic designs; sudden expansion, contraction, and a box along the channel. It is found that these simple channel cross-sections can accelerate and decelerate fluid flow, therefore altering the time of fluid delivery.

Collectively, the success of this research will improve the development of future diagnostic and analytical paper devices producing a user-friendly and cost effective point-of-care alternative.

    Cover page: Imbibition in Paper-Based Microfluidic DevicesCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2017)
    The transcriptional factor MED12 is part of the essential mediator transcriptional complex that acts as a transcriptional coactivator in all eukaryotes. Missense gain-of-function mutations in human MED12 are associated with uterine leiomyomas, yet the role of MED12 deficiency in tumorigenesis and reproductive biology has not been fully explored. We generated a Med12 reproductive conditional knockout mouse model to evaluate its role in uterine mesenchyme, granulosa cells, and oocytes. Mice heterozygous for Med12 deficiency in granulosa cells and uterus (Med12fl/+ Amhr2-Cre) were subfertile, while mice homozygous for Med12 deficiency in granulosa cells and uterus (Med12fl/fl Amhr2-Cre) were infertile. Morphological and histological analysis of the Med12fl/fl Amhr2-Cre reproductive tract revealed atrophic uteri and hyperchromatic granulosa cells with disrupted expression of Lhcgr, Esr1, and Esr2. Med12fl/fl Amhr2-Cre mice estrous cycle was disrupted, and serum analysis showed blunted rise in estradiol in response to pregnant mare serum gonadotropin. Uterine atrophy was partially rescued by exogenous steroid supplementation with dysregulation of Notch1 and Smo expression in steroid supplemented Med12fl/fl Amhr2-Cre uteri, indicating intrinsic uterine defects. Oocyte-specific ablation of Med12 caused infertility without disrupting normal folliculogenesis and ovulation, consistent with maternal effects of Med12 in early embryo development. These results show the critical importance of Med12 in reproductive tract development and that Med12 loss of function does not cause tumorigenesis in reproductive tissues.
      Cover page: Med12 regulates ovarian steroidogenesis, uterine development and maternal effects in the mammalian egg
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2019)
      Gap junction (GJ) channels in invertebrates have been used to understand cell-to-cell communication in vertebrates. GJs are a common form of intercellular communication channels which connect the cytoplasm of adjacent cells. Dysregulation and structural alteration of the gap junction-mediated communication have been proven to be associated with a myriad of symptoms and tissue-specific pathologies. Animal models relying on the invertebrate nervous system have exposed a relationship between GJs and the formation of electrical synapses during embryogenesis and adulthood. The modulation of GJs as a therapeutic and clinical tool may eventually provide an alternative for treating tissue formation-related diseases and cell propagation. This review concerns the similarities between Hirudo medicinalis innexins and human connexins from nucleotide and protein sequence level perspectives. It also sets forth evidence of computational techniques applied to the study of proteins, sequences, and molecular dynamics. Furthermore, we propose machine learning techniques as a method that could be used to study protein structure, gap junction inhibition, metabolism, and drug development.
        Cover page: Gap Junction Channels of Innexins and Connexins: Relations and Computational Perspectives
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2015)
        Uterine leiomyomas are benign tumors that can cause pain, bleeding, and infertility in some women. Mediator complex subunit 12 (MED12) exon 2 variants are associated with uterine leiomyomas; however, the causality of MED12 variants, their genetic mode of action, and their role in genomic instability have not been established. Here, we generated a mouse model that conditionally expresses a Med12 missense variant (c.131G>A) in the uterus and demonstrated that this alteration alone promotes uterine leiomyoma formation and hyperplasia in both WT mice and animals harboring a uterine mesenchymal cell-specific Med12 deletion. Compared with WT animals, expression of Med12 c.131G>A in conditional Med12-KO mice resulted in earlier onset of leiomyoma lesions that were also greater in size. Moreover, leiomyomatous, Med12 c.131G>A variant-expressing uteri developed chromosomal rearrangements. Together, our results show that the common human leiomyoma-associated MED12 variant can cause leiomyomas in mice via a gain of function that drives genomic instability, which is frequently observed in human leiomyomas.
          Cover page: Med12 gain-of-function mutation causes leiomyomas and genomic instability
          • Article
          • Peer Reviewed
          UC Merced Previously Published Works (2024)
          Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.
            Cover page: Mechanism of DNA origami folding elucidated by mesoscopic simulations.
            • Article
            • Peer Reviewed
            LBL Publications (2018)
            Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. Here we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~ 6-14 nm resolution. The statistical analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.
              Cover page: Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2018)
              We performed whole exome sequencing to identify an unknown genetic cause of azoospermia and male infertility in a large Pakistani family. Three infertile males were subjected to semen analysis, hormone testing, testicular histology, ultrasonography, karyotyping, Y-chromosome microdeletion and CFTR testing. The clinical testing suggested a diagnosis of obstructive azoospermia (OA). To identify the cause, we performed whole exome sequencing (WES) for 2 infertile brothers and 2 fertile family members. For segregation analysis and variant confirmation, we performed Sanger sequencing. WES data analysis of the family revealed segregated variants in 3 candidate genes. We considered novel nonsense variant c.2440C > T(p.Arg814*) in X-linked gene ADGRG2 as biologically most plausible. It is predicted to truncate the protein by 204 amino acids (aa) at a key transmembrane domain. Adgrg2-knockout male mice show sperm loss due to obstructive fluid stasis, while ADGRG2 mutations cause OA in the infertile male patients. Our analysis of testicular histology reveals secondary severe reduction of spermatogenesis, consistent with human and knockout mouse phenotypes. The ADGRG2 nonsense mutation is absent in the largest population databases, ExAC and gnomAD. Analysis of the novel nonsense mutation in extended family members confirmed co-segregation of the mutation with OA in all affected males. The likely pathogenic nature of the mutation is supported by its truncation effect on the transmembrane domain and distinctive ultrasound results. The study demonstrates effectiveness of WES in discovering a genetic cause of azoospermia.
                Cover page: X-linked ADGRG2 mutation and obstructive azoospermia in a large Pakistani family
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2015)
                Social bee colonies can allocate their foraging resources over a large spatial scale, but how they allocate foraging on a small scale near the colony is unclear and can have implications for understanding colony decision-making and the pollination services provided. Using a mass-foraging stingless bee, Scaptotrigona pectoralis (Dalla Torre) (Hymenoptera: Apidae: Meliponini), we show that colonies will forage near their nests and allocate their foraging labor on a very fine spatial scale at an array of food sources placed close to the colony. We counted the foragers that a colony allocated to each of nine feeders containing 1.0, 1.5, or 2.0 M sucrose solution [31, 43, and 55% sucrose (wt/wt), respectively] at distances of 10, 15, and 20 m from the nest. A significantly greater number of foragers (2.6-5.3 fold greater) visited feeders placed 10 vs. 20 m away from the colony. Foraging allocation also corresponded to food quality. At the 10-m feeders, 4.9-fold more foragers visited 2.0 M as compared to 1.0 M sucrose feeders. Colony forager allocation thus responded to both differences in food distance and quality even when the travel cost was negligible compared to normal colony foraging distances (10 m vs. an estimated 800-1 710 m). For a nearby floral patch, this could result in unequal floral visitation and pollination.
                  Cover page: Colony foraging allocation is finely tuned to food distance and sweetness even close to a bee colony
                  • Article
                  • Peer Reviewed
                  UC Merced Previously Published Works (2020)
                  The specific detection in clinical samples of analytes with dimensions in the tens to hundreds of nanometers, such as viruses and large proteins, would improve disease diagnosis. Detection of these "mesoscale" analytes (as opposed to their nanoscale components), however, is challenging as it requires the simultaneous binding of multiple recognition sites often spaced over tens of nanometers. In response, we have adapted DNA origami, with its unparalleled customizability to precisely display multiple target-binding sites over the relevant length scale, to an electrochemical biosensor platform. Our proof-of-concept employs triangular origami covalently attached to a gold electrode and functionalized with redox reporters. Electrochemical interrogation of this platform successfully monitors mesoscale, target-binding-induced changes in electron transfer in a manner consistent with coarse-grained molecular dynamics simulations. Our approach enables the specific detection of analytes displaying recognition sites that are separated by ∼40 nm, a spacing significantly greater than that achieved in similar sensor architectures employing either antibodies or aptamers.
                    Cover page: An electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targets.
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2022)
                    DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.
                      Cover page: CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells
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