Publications by authors named "Michelle Ho"

53 Publications

Family Members' Perceptions of Their Psychological Responses One Year Following Pediatric Intensive Care Unit (PICU) Hospitalization: Qualitative Findings From the Caring Intensively Study.

Front Pediatr 2021 7;9:724155. Epub 2021 Sep 7.

School of Nursing, Faculty of Health, Dalhousie University, Halifax, NS, Canada.

PICU hospitalization can have a profound impact on child survivors and their families. There is limited research on children's long-term recovery within the context of the family following critical illness. This study aimed to explore children's and parents' perceptions of long-term psychological and behavioral responses within the context of the family one year following PICU hospitalization. Caring Intensively is a mixed methods multi-site prospective cohort study that aims to examine children's psychological and behavioral responses over a 3-year period following PICU hospitalization. In this study, part of the qualitative arm of Caring Intensively, an interpretive descriptive design was used to explore children's recovery one year post-discharge. Purposive sampling was used to select 17 families, including 16 mothers, 6 fathers, and 9 children. Semi-structured, audio-recorded interviews were conducted. Data were analyzed iteratively using the constant comparison method. Families described efforts to readapt to routine life and find a new normal following PICU hospitalization. Finding a New Normal consisted of four major themes: (1) Processing PICU Reminders and Memories, (2) Changing Perceptions of Health and Illness, (3) We Are Not the Same, and (4) Altered Relationships. Participants described significant emotional and behavioral changes during the year following discharge. The psychological impact of individual family members' experiences led to changes in their sense of self, which affected family dynamics. PICU memories and reminders impacted participants' perceptions of childhood health and illness and resulted in increased vigilance. Parents and siblings demonstrated increased concern for the child survivor's health, and the experience of long absences and new or altered caregiving roles resulted in changes in relationships and family dynamics. PICU hospitalization impacted the psychological well-being of all family members as they sought to re-establish a sense of normalcy one year following discharge. Parent and child experiences and responses were closely interconnected. Findings highlight the importance of increased follow-up care aimed at supporting the family's psychological recovery.
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http://dx.doi.org/10.3389/fped.2021.724155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452961PMC
September 2021

N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes.

Virology 2021 Aug 27;563:107-115. Epub 2021 Aug 27.

Department of Bioengineering, Rice University, Houston, TX, 77005, USA; Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, 77005, USA; Department of Biosciences, Rice University, Houston, TX, 77005, USA; Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX, 77005, USA. Electronic address:

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.
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http://dx.doi.org/10.1016/j.virol.2021.08.010DOI Listing
August 2021

Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein.

ACS Pharmacol Transl Sci 2021 Aug 29;4(4):1349-1361. Epub 2021 Jul 29.

Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, Pennsylvania 19104-4205, United States.

Coronavirus disease 2019 (COVID-19) is caused by the newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the highly contagious nature of SARS-CoV-2, it has infected more than 137 million individuals and caused more than 2.9 million deaths globally as of April 13, 2021. There is an urgent need to develop effective novel therapeutic strategies to treat or prevent this infection. Toward this goal, we focused on the development of monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike) present on the surface of virus particles as well as virus-infected cells. We isolated anti-SARS-CoV-2 Spike mAbs from animals immunized with a DNA vaccine. We then selected a highly potent set of mAbs against SARS-CoV-2 Spike protein and evaluated each candidate for their expression, target binding affinity, and neutralization potential using complementary ACE2-blocking and pseudovirus neutralization assays. We identified a total of 10 antibodies, which specifically and strongly bound to SARS-CoV-2 Spike, blocked the receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) interaction, and neutralized SARS-CoV-2. Furthermore, the glycomic profile of the antibodies suggested that they have high Fc-mediated effector functions. These antibodies should be further investigated for elucidating the neutralizing epitopes on Spike for the design of next-generation vaccines and for their potential in diagnostic as well as therapeutic utilities against SARS-CoV-2.
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http://dx.doi.org/10.1021/acsptsci.1c00058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353887PMC
August 2021

Genetic regulation of nonsense-mediated decay underlies association with risk of severe COVID-19.

medRxiv 2021 Jul 13. Epub 2021 Jul 13.

Genomic regions have been associated with COVID-19 susceptibility and outcomes, including the chr12q24.13 locus encoding antiviral proteins OAS1-3. Here, we report genetic, functional, and clinical insights into genetic associations within this locus. In Europeans, the risk of hospitalized vs. non-hospitalized COVID-19 was associated with a single 19Kb-haplotype comprised of 76 variants included in a 95% credible set within a large genomic fragment introgressed from Neandertals. The risk haplotype was also associated with impaired spontaneous but not treatment-induced SARS-CoV-2 clearance in a clinical trial with pegIFN-λ1. We demonstrate that two exonic variants, rs10774671 and rs1131454, affect splicing and nonsense-mediated decay of . We suggest that genetically-regulated loss of expression contributes to impaired spontaneous clearance of SARS-CoV-2 and elevated risk of hospitalization for COVID-19. Our results provide the rationale for further clinical studies using interferons to compensate for impaired spontaneous SARS-CoV-2 clearance, particularly in carriers of the risk haplotypes.
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http://dx.doi.org/10.1101/2021.07.09.21260221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288155PMC
July 2021

Surgical and Radiological Management of Complicated Uterine Leiomyoma Aided by 3D Models in a Patient with Fibrodysplasia Ossificans Progressiva.

Am J Case Rep 2021 Jun 10;22:e931614. Epub 2021 Jun 10.

Department of Anesthesiology, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA.

BACKGROUND Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of the connective tissue. Over time, patients with FOP experience decreased range of motion in the joints and the formation of a second skeleton, limiting mobility. Patients with FOP are advised to avoid any unwarranted surgery owing to the risk of a heterotopic ossification flare-up. For patients who do require a surgical procedure, a multidisciplinary team is recommended for comprehensive management of the patient's needs. CASE REPORT A 27-year-old woman with FOP underwent a hysterectomy for removal of a suspected necrotic uterine fibroid. To aid in presurgical planning and management, patient-specific 3-dimensional (3D) models of the patient's tracheobronchial tree, thorax, and lumbosacral spine were printed from the patient's preoperative computed tomography (CT) imaging. The patient required awake nasal fiberoptic intubation for general anesthesia and transversus abdominus plane block for regional anesthesia. Other anesthesia modalities, including spinal epidural, were ruled out after visualizing the patient's anatomy using the 3D model. Postoperatively, the patient was started on a multi-modal analgesic regimen and a course of steroids, and early ambulation was encouraged. CONCLUSIONS Patients with FOP are high-risk surgical patients requiring the care of multiple specialties. Advanced visualization methods, including 3D printing, can be used to better understand their anatomy and locations of heterotopic bone ossification that can affect patient positioning. Our patient successfully underwent supracervical hysterectomy and bilateral salpingectomy with no signs of fever or sepsis at follow-up.
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http://dx.doi.org/10.12659/AJCR.931614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207543PMC
June 2021

Permeabilizing Cell Membranes with Electric Fields.

Cancers (Basel) 2021 May 10;13(9). Epub 2021 May 10.

Molecular Imaging Program at Stanford, Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The biological impact of exogenous, alternating electric fields (AEFs) and direct-current electric fields has a long history of study, ranging from effects on embryonic development to influences on wound healing. In this article, we focus on the application of electric fields for the treatment of cancers. In particular, we outline the clinical impact of tumor treating fields (TTFields), a form of AEFs, on the treatment of cancers such as glioblastoma and mesothelioma. We provide an overview of the standard mechanism of action of TTFields, namely, the capability for AEFs (e.g., TTFields) to disrupt the formation and segregation of the mitotic spindle in actively dividing cells. Though this standard mechanism explains a large part of TTFields' action, it is by no means complete. The standard theory does not account for exogenously applied AEFs' influence directly upon DNA nor upon their capacity to alter the functionality and permeability of cancer cell membranes. This review summarizes the current literature to provide a more comprehensive understanding of AEFs' actions on cell membranes. It gives an overview of three mechanistic models that may explain the more recent observations into AEFs' effects: the voltage-gated ion channel, bioelectrorheological, and electroporation models. Inconsistencies were noted in both effective frequency range and field strength between TTFields versus all three proposed models. We addressed these discrepancies through theoretical investigations into the inhomogeneities of electric fields on cellular membranes as a function of disease state, external microenvironment, and tissue or cellular organization. Lastly, future experimental strategies to validate these findings are outlined. Clinical benefits are inevitably forthcoming.
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http://dx.doi.org/10.3390/cancers13092283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126200PMC
May 2021

Landscape of humoral immune responses against SARS-CoV-2 in patients with COVID-19 disease and the value of antibody testing.

Heliyon 2021 Apr 17;7(4):e06836. Epub 2021 Apr 17.

Vaccine & Immunotherapy Center, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.

A new pandemic is ongoing in several parts of the world. The agent responsible is the newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The symptoms associated with this virus are known as the coronavirus disease-2019 (COVID-19). In this review, we summarize the published data on virus specific antibodies in hospitalized patients with COVID-19 disease, patients recovered from the disease and the individuals who are asymptomatic with SARS-CoV-2 infections. The review highlights the following: i) an adjunct role of antibody tests in the diagnosis of COVID-19 in combination with RT-PCR; ii) status of antibodies from COVID-19 convalescent patients to select donors for plasma therapy; iii) the potential confounding effects of other coronaviruses, measles, mumps and rubella in antibody testing due to homology of certain viral genes; and iv) the role of antibody testing for conducting surveillance in populations, incidence estimation, contact tracing and epidemiologic studies.
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http://dx.doi.org/10.1016/j.heliyon.2021.e06836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052472PMC
April 2021

Immunotherapy of prostate cancer using novel synthetic DNA vaccines targeting multiple tumor antigens.

Genes Cancer 2021 22;12:51-64. Epub 2021 Mar 22.

Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA, USA.

Prostate cancer is a prevalent cancer in men and consists of both indolent and aggressive phenotypes. While active surveillance is recommended for the former, current treatments for the latter include surgery, radiation, chemo and hormonal therapy. It has been observed that the recurrence in the treated patients is high and results in castration resistant prostate cancer for which treatment options are limited. This scenario has prompted us to consider immunotherapy with synthetic DNA vaccines, as this approach can generate antigen-specific tumor-killing immune cells. Given the multifocal and heterogeneous nature of prostate cancer, we hypothesized that synthetic DNA vaccines targeting different prostate specific antigens are likely to induce broader and improved immunity who are at high risk as well as advanced clinical stage of prostate cancer, compared to a single antigen approach. Utilizing a bioinformatics approach, synthetic enhanced DNA vaccine (SEV) constructs were generated against STEAP1, PAP, PARM1, PSCA, PCTA and PSP94. Synthetic enhanced vaccines for prostate cancer antigens were shown to elicit antigen-specific immune responses in mice and the anti-tumor activity was evident in a prostate tumor challenge mouse model. These studies support further evaluation of the DNA tools for immunotherapy of prostate cancer and perhaps other cancers.
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http://dx.doi.org/10.18632/genesandcancer.214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045963PMC
March 2021

F5-Atlanta: A novel mutation in F5 associated with enhanced East Texas splicing and FV-short production.

J Thromb Haemost 2021 07 20;19(7):1653-1665. Epub 2021 May 20.

Aflac Cancer and Blood Disorders Center, Emory University/Children's Healthcare of Atlanta, Atlanta, Georgia, USA.

Background: Elucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B-domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half-life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls.

Objective: We identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5-Atlanta (F5-ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression.

Methods & Results: Assessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV-short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5-ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells.

Conclusions: These findings support a mechanistic model consisting of cis-acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV-short and TFPIα levels.
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http://dx.doi.org/10.1111/jth.15314DOI Listing
July 2021

Design and Printing of a Low-Cost 3D-Printed Nasal Osteotomy Training Model: Development and Feasibility Study.

JMIR Med Educ 2020 Nov 17;6(2):e19792. Epub 2020 Nov 17.

Health Design Lab, Thomas Jefferson University, Philadelphia, PA, United States.

Background: Nasal osteotomy is a commonly performed procedure during rhinoplasty for both functional and cosmetic reasons. Teaching and learning this procedure proves difficult due to the reliance on nuanced tactile feedback. For surgical simulation, trainees are traditionally limited to cadaveric bones, which can be costly and difficult to obtain.

Objective: This study aimed to design and print a low-cost midface model for nasal osteotomy simulation.

Methods: A 3D reconstruction of the midface was modified using the free open-source design software Meshmixer (Autodesk Inc). The pyriform aperture was smoothed, and support rods were added to hold the fragments generated from the simulation in place. Several models with various infill densities were printed using a desktop 3D printer to determine which model best mimicked human facial bone.

Results: A midface simulation set was designed using a desktop 3D printer, polylactic acid filament, and easily accessible tools. A nasal osteotomy procedure was successfully simulated using the model.

Conclusions: 3D printing is a low-cost, accessible technology that can be used to create simulation models. With growing restrictions on trainee duty hours, the simulation set can be used by programs to augment surgical training.
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http://dx.doi.org/10.2196/19792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708083PMC
November 2020

Pediatric Project ECHO: Implementation of a Virtual Medical Education Program to Support Community Management of Children With Medical Complexity.

Hosp Pediatr 2020 12 2;10(12):1044-1052. Epub 2020 Nov 2.

Child Health Evaluative Sciences.

Objectives: Health care providers (HCPs) require ongoing support to meet the evolving care needs of children with medical complexity (CMC). Project Extension for Community Healthcare Outcomes (ECHO) is a model for delivering technology-enabled medical education and cultivating a community of practice. In this study, we focused on developing, implementing, and evaluating the first ECHO program dedicated to the care of CMC. Specific objectives were to evaluate the program feasibility (participation and acceptability) and impact on perceived HCP knowledge, self-efficacy, and clinical practice after 6 months.

Methods: A needs assessment was conducted to inform an interprofessional CMC curriculum. This curriculum was delivered through monthly virtual TeleECHO clinics (didactic and case-based learning) from January 2018 to 2020. The program was available at no cost to HCPs throughout Ontario. Surveys were distributed at baseline and 6 months to assess program acceptability, knowledge, self-efficacy, and practice impact by using 7-point Likert scales. Descriptive and inferential data analyses were conducted.

Results: Twenty-four clinics were completed with a mean of 19 ± 6 attendees. Acceptability scores ( = 27) ranged from 5.0 ± 1.1 to 6.4 ± 0.6. Participants reported an improvement in their knowledge and self-efficacy across all probed topics and skills ( values ranged from <.001 to .006). These knowledge and self-efficacy scores related to "complex care support," "feeding support," and "respiratory support." The majority of participants reported positive or very positive practice impacts, including enhanced ability to provide quality care to CMC.

Conclusions: Project ECHO is a feasible and acceptable model for virtual education of interprofessional HCPs in managing CMC. This program has the potential to increase system capacity to provide quality care to CMC close to home.
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http://dx.doi.org/10.1542/hpeds.2020-0067DOI Listing
December 2020

A novel synthetic DNA vaccine elicits protective immune responses against Powassan virus.

PLoS Negl Trop Dis 2020 10 29;14(10):e0008788. Epub 2020 Oct 29.

Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

Powassan virus (POWV) infection is a tick-borne emerging infectious disease in the United States and North America. Like Zika virus, POWV is a member of the family Flaviviridae. POWV causes severe neurological sequalae, meningitis, encephalitis, and can cause death. Although the risk of human POWV infection is low, its incidence in the U.S. in the past 16 years has increased over 300%, urging immediate attention. Despite the disease severity and its growing potential for threatening larger populations, currently there are no licensed vaccines which provide protection against POWV. We developed a novel synthetic DNA vaccine termed POWV-SEV by focusing on the conserved portions of POWV pre-membrane and envelope (prMEnv) genes. A single immunization of POWV-SEV elicited broad T and B cell immunity in mice with minimal cross-reactivity against other flaviviruses. Antibody epitope mapping demonstrated a similarity between POWV-SEV-induced immune responses and those elicited naturally in POWV-infected patients. Finally, POWV-SEV induced immunity provided protection against POWV disease in lethal challenge experiments.
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http://dx.doi.org/10.1371/journal.pntd.0008788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595275PMC
October 2020

Clinical situations for which 3D printing is considered an appropriate representation or extension of data contained in a medical imaging examination: adult cardiac conditions.

3D Print Med 2020 Sep 23;6(1):24. Epub 2020 Sep 23.

Department of Radiology and The Ottawa Hospital Research Institute, University of Ottawa, Ottawa, ON, Canada.

Background: Medical 3D printing as a component of care for adults with cardiovascular diseases has expanded dramatically. A writing group composed of the Radiological Society of North America (RSNA) Special Interest Group on 3D Printing (SIG) provides appropriateness criteria for adult cardiac 3D printing indications.

Methods: A structured literature search was conducted to identify all relevant articles using 3D printing technology associated with a number of adult cardiac indications, physiologic, and pathologic processes. Each study was vetted by the authors and graded according to published guidelines.

Results: Evidence-based appropriateness guidelines are provided for the following areas in adult cardiac care; cardiac fundamentals, perioperative and intraoperative care, coronary disease and ischemic heart disease, complications of myocardial infarction, valve disease, cardiac arrhythmias, cardiac neoplasm, cardiac transplant and mechanical circulatory support, heart failure, preventative cardiology, cardiac and pericardial disease and cardiac trauma.

Conclusions: Adoption of common clinical standards regarding appropriate use, information and material management, and quality control are needed to ensure the greatest possible clinical benefit from 3D printing. This consensus guideline document, created by the members of the RSNA 3D printing Special Interest Group, will provide a reference for clinical standards of 3D printing for adult cardiac indications.
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http://dx.doi.org/10.1186/s41205-020-00078-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510265PMC
September 2020

Photocatalytic trifluoromethoxylation of arenes and heteroarenes in continuous-flow.

Beilstein J Org Chem 2020 15;16:1305-1312. Epub 2020 Jun 15.

Micro Flow Chemistry and Synthetic Methodology, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Het Kranenveld, Bldg 14 - Helix, 5600 MB Eindhoven, The Netherlands.

The first example of photocatalytic trifluoromethoxylation of arenes and heteroarenes under continuous-flow conditions is described. Application of continuous-flow microreactor technology allowed to reduce the residence time up to 16 times in comparison to the batch procedure, while achieving similar or higher yields. In addition, the use of inorganic bases was demonstrated to increase the reaction yield under batch conditions.
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http://dx.doi.org/10.3762/bjoc.16.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308607PMC
June 2020

Neutralization of hepatitis B virus by a novel DNA-encoded monoclonal antibody.

Hum Vaccin Immunother 2020 09 28;16(9):2156-2164. Epub 2020 May 28.

Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA.

Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common "a determinant region" of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directing antibody expression. delivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cells . Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.
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http://dx.doi.org/10.1080/21645515.2020.1763686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553714PMC
September 2020

An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription.

Virology 2020 07 25;546:127-132. Epub 2020 Apr 25.

Department of Bioengineering, Rice University, United States. Electronic address:

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.
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http://dx.doi.org/10.1016/j.virol.2020.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395364PMC
July 2020

Results and Long-Term Functional Outcomes of Rib Fracture Fixation: A Case Series in Singapore and a Review of Indications for Surgical Fixation.

Ann Acad Med Singap 2020 Feb;49(2):93-97

Division of Plastic, Reconstructive and Aesthetic Surgery, Department of General Surgery, Tan Tock Seng Hospital, Singapore.

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February 2020

Estimating the impact of divergent mating phenology between residents and migrants on the potential for gene flow.

Ecol Evol 2019 Apr 12;9(7):3770-3783. Epub 2019 Mar 12.

Department of Ecology and Evolutionary Biology University of Toronto Toronto ON Canada.

Gene flow between populations can allow the spread of beneficial alleles and genetic diversity between populations, with importance to conservation, invasion biology, and agriculture. Levels of gene flow between populations vary not only with distance, but also with divergence in reproductive phenology. Since phenology is often locally adapted, arriving migrants may be reproductively out of synch with residents, which can depress realized gene flow. In flowering plants, the potential impact of phenological divergence on hybridization between populations can be predicted from overlap in flowering schedules-the daily count of flowers capable of pollen exchange-between a resident and migrant population. The accuracy of this prospective hybridization estimate, based on parental phenotypes, rests upon the assumptions of unbiased pollen transfer between resident and migrant active flowers. We tested the impact of phenological divergence on resident-migrant mating frequencies in experiments that mimicked a single large gene flow event. We first prospectively estimated mating frequencies two lines of selected or early and late flowering. We then estimated realized mating frequencies retrospectively through progeny testing. The two estimates strongly agreed in a greenhouse experiment, where procedures ensured saturating, unbiased pollination. Under natural pollination in the field, the rate of resident-migrant mating, was lower than estimated by phenological divergence alone, although prospective and retrospective estimates were correlated. In both experiments, differences between residents and migrants in flowering schedule shape led to asymmetric hybridization. Results suggest that a prospective estimate of hybridization based on mating schedules can be a useful, although imperfect, tool for evaluating potential gene flow. They also illustrate the impact of mating phenology on the magnitude and symmetry of reproductive isolation.
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http://dx.doi.org/10.1002/ece3.5001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468075PMC
April 2019

Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue.

Mol Ther 2019 03 29;27(3):611-622. Epub 2019 Jan 29.

Department of Bioengineering, Rice University, 6100 Main St., Houston, TX 77005, USA. Electronic address:

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.
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http://dx.doi.org/10.1016/j.ymthe.2019.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404099PMC
March 2019

Protective immunity by an engineered DNA vaccine for Mayaro virus.

PLoS Negl Trop Dis 2019 02 7;13(2):e0007042. Epub 2019 Feb 7.

Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA, United States of America.

Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge.
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http://dx.doi.org/10.1371/journal.pntd.0007042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366747PMC
February 2019

A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9-mediated somatic recombination.

Lab Invest 2019 07 6;99(8):1233-1244. Epub 2019 Feb 6.

Department of Translational Molecular Pathology and Sheikh Ahmed Pancreatic Cancer Research Center, UT MD Anderson Cancer Center, Houston, TX, USA.

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic Kras allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.
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http://dx.doi.org/10.1038/s41374-018-0171-zDOI Listing
July 2019

Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass.

Nat Biotechnol 2018 08 4;36(7):606-613. Epub 2018 Jun 4.

Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA.

Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
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http://dx.doi.org/10.1038/nbt.4153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349032PMC
August 2018

The effect of Chinese martial arts Tai Chi Chuan on prevention of osteoporosis: A systematic review.

J Orthop Translat 2018 Jan 26;12:74-84. Epub 2017 Jun 26.

Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Special Administrative Region.

Background/objective: Tai Chi Chuan (TCC) is suggested to have beneficial effects on the musculoskeletal system. The aim of this systematic review is to evaluate the evidence of the effect of TCC on bone mineral density (BMD) and its potential for prevention of osteoporosis.

Methods: A literature search was conducted using PubMed, Embase, and Cochrane databases from inception to January 2017. Randomized controlled studies, case-control trials, prospective cohort studies, and cross-sectional studies which evaluated the effect of TCC on BMD were selected without any subject or language restriction.

Results: Nine articles met the inclusion criteria, including seven randomized controlled trials (RCTs), one case-control trial (CCT), and one cross-sectional study, encompassing a total of 1222 participants. Five studies showed statistically significant improvements in BMD after TCC, three studies showed nonsignificant intergroup differences, and one study provided no statistical evaluation of results. The studies with nonsignificant results tended to have a shorter total duration of TCC practice. Apart from dual-energy X-ray absorptiometry (DXA), two studies additionally used peripheral quantitative computed tomography (pQCT) which showed statistically significant positive effects of TCC on preventing osteoporosis.

Conclusion: TCC is beneficial to BMD and may be a cost-effective and preventive measure of osteoporosis. This beneficial effect is better observed in long-term TCC practice.

The Translational Potential Of This Article: The beneficial effect of TCC on BMD is suggested to be clinically translated to its potential for early rehabilitation and prevention of secondary osteoporosis in patients after surgical treatment of common osteoporotic fractures. The length of practicing TCC, the form and style of TCC, and the types of patient suitable for TCC are to be investigated in future studies.
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http://dx.doi.org/10.1016/j.jot.2017.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866477PMC
January 2018

Paper #4: Referral Patterns and Prevalence of Sleep Abnormalities in Children with EOS.

Spine Deform 2017 Nov;5(6):441-442

Polysomnograms (PSGs) are not used in most spine centers for children with EOS. There are no standards for PSG referrals, and the yield from PSGs is unknown. We describe referrals for PSGs in 96 children > 5 years old with EOS, their lung functions, types of EOS, and frequency of abnormal PSGs.
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http://dx.doi.org/10.1016/j.jspd.2017.09.007DOI Listing
November 2017

TFEB-mediated activation of the lysosome-autophagy system affects the transduction efficiency of adeno-associated virus 2.

Virology 2017 10 5;510:1-8. Epub 2017 Jul 5.

Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main St, MS-362, Houston, TX 77005, USA; Department of Bioengineering, Rice University, 6500 Main St, Suite 1030, Houston, TX 770030, USA; Department of BioSciences, Rice University, 6100 Main St, MS-140, Houston, TX 77005, USA; Systems, Synthetic and Physical Biology Program, Rice University, 6100 Main St, MS-180, Houston, TX 77005, USA. Electronic address:

Adeno-associated virus (AAV)-mediated gene transfer is an appealing therapeutic option due to AAV's safety profile. Effective delivery of AAV's genetic cargo to the nucleus, however, requires evasion of host cell barriers, including cellular clearance mechanisms mediated by the lysosome-autophagy system. We used AAV serotype 2 to monitor the autophagic response to cellular internalization of AAV and to characterize the effect of AAV-induced activation of autophagy on transgene expression. We found AAV2 internalization to induce activation of transcription factor EB, a master regulator of autophagy and lysosomal biogenesis, and upregulation of the lysosome-autophagy system. We showed that AAV2-induced activation of autophagy parallels a reduction in transgene expression, but also an increase in autophagic clearance of protein aggregates. These results can inform the design of AAV vectors with autophagy-modulating properties for applications ranging from the design of efficient gene delivery vectors to the treatment of diseases characterized by accumulation of autophagic cargo.
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http://dx.doi.org/10.1016/j.virol.2017.06.030DOI Listing
October 2017

Musculoskeletal Injuries Resulting from Use of Hoverboards: Safety Concerns With an Unregulated Consumer Product.

Clin Pediatr (Phila) 2018 Jan 22;57(1):31-35. Epub 2017 Jan 22.

1 Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Hoverboards were recently introduced to the US consumer market and experienced rapid popularity. Given the high frequency of musculoskeletal injury with other wheeled recreation devices, we sought to analyze hoverboard injuries in children. A retrospective review of patients with musculoskeletal injury related to hoverboard use was performed at a tertiary care children's hospital. From November 2015 to January 2016, 2.3% of all fractures were related to hoverboards. Common injury mechanisms were fall (79%) and finger entrapment between wheel and wheel-well (10%). The most frequently fractured sites included the distal radius (43%) and phalanx (17%). Common surgical procedures were nailbed repair and pinning for Seymour fracture and percutaneous pinning for distal radius fracture. There exists high risk for distal radius fractures from falls and phalanx fractures from finger entrapment between the wheel and wheel-well. Hoverboard safety can be improved with regular use of wrist guards and improved wheel-well design.
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http://dx.doi.org/10.1177/0009922816687327DOI Listing
January 2018

The paleoclimate context and future trajectory of extreme summer hydroclimate in eastern Australia.

J Geophys Res Atmos 2016 Nov 31;121(21):12820-12838. Epub 2016 Oct 31.

School of Ecosystem and Forest Sciences, University of Melbourne, Richmond, Victoria, Australia.

Eastern Australia recently experienced an intense drought (Millennium Drought, 2003-2009) and record-breaking rainfall and flooding (austral summer 2010-2011). There is some limited evidence for a climate change contribution to these events, but such analyses are hampered by the paucity of information on long-term natural variability. Analyzing a new reconstruction of summer (December-January-February) Palmer Drought Severity Index (the Australia-New Zealand Drought Atlas; ANZDA, 1500-2012 CE), we find moisture deficits during the Millennium Drought fall within the range of the last 500 years of natural hydroclimate variability. This variability includes periods of multi-decadal drought in the 1500s more persistent than any event in the historical record. However, the severity of the Millennium Drought, which was caused by autumn (March-April-May) precipitation declines, may be underestimated in the ANZDA because the reconstruction is biased towards summer and antecedent spring (September-October-November) precipitation. The pluvial in 2011, however, which was characterized by extreme summer rainfall faithfully captured by the ANZDA, is likely the wettest year in the reconstruction for Coastal Queensland. Climate projections (RCP 8.5 scenario) suggest that eastern Australia will experience long-term drying during the 21 century. While the contribution of anthropogenic forcing to recent extremes remains an open question, these projections indicate an amplified risk of multi-year drought anomalies matching or exceeding the intensity of the Millennium Drought.
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http://dx.doi.org/10.1002/2016JD024892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956229PMC
November 2016

High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.

Sci Rep 2016 Sep 22;6:33697. Epub 2016 Sep 22.

Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct "biased sequences" and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the "biased sequences" was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.
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http://dx.doi.org/10.1038/srep33697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031971PMC
September 2016

AptaTRACE Elucidates RNA Sequence-Structure Motifs from Selection Trends in HT-SELEX Experiments.

Cell Syst 2016 07;3(1):62-70

National Center of Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD 20894, USA. Electronic address:

Aptamers, short RNA or DNA molecules that bind distinct targets with high affinity and specificity, can be identified using high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX), but scalable analytic tools for understanding sequence-function relationships from diverse HT-SELEX data are not available. Here we present AptaTRACE, a computational approach that leverages the experimental design of the HT-SELEX protocol, RNA secondary structure, and the potential presence of many secondary motifs to identify sequence-structure motifs that show a signature of selection. We apply AptaTRACE to identify nine motifs in C-C chemokine receptor type 7 targeted by aptamers in an in vitro cell-SELEX experiment. We experimentally validate two aptamers whose binding required both sequence and structural features. AptaTRACE can identify low-abundance motifs, and we show through simulations that, because of this, it could lower HT-SELEX cost and time by reducing the number of selection cycles required.
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http://dx.doi.org/10.1016/j.cels.2016.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042215PMC
July 2016

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

Mol Ther 2016 09 29;24(9):1561-9. Epub 2016 Jul 29.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.

Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.
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http://dx.doi.org/10.1038/mt.2016.148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113113PMC
September 2016
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