Browsing by Author "Zhang, Lujuan"

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    Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly
    (Elsevier, 2022-04-30) Zhang, Lujuan; Fujiki, Robert Brinton; Brookes, Sarah; Calcagno, Haley; Awonusi, Oluwaseyi; Kluender, Keith; Berry, Kevin; Venkatraman, Anumitha; Maulden, Amanda; Sivasankar, M. Preeti; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Background/Objectives: While voice-related therapeutic interventions are often researched preclinically in the porcine model, there are no well-established methods to induce porcine glottic phonation. Described approaches such as training animals to phonate for positive reinforcement are time-consuming and plagued by inherent variability in the type of phonation produced and contamination of background noise. Thus, a reliable method of assessing glottic phonation in the porcine model is needed. Methods: In this study, we have created a novel pulley-based apparatus with harness for “pig-lifting” with surrounding acoustic insulation and high-directional microphone with digital recorder for recording phonation. Praat and Matlab were used to analyze all porcine vocalizations for fundamental frequency (F0), intensity, duration of phonation and cepstral peak prominence (CPP). Glottic phonation was detected using F0 (≥ 2000 hz), duration (≥.3 seconds) and researcher perceptual judgment. Partial-glottic phonations were also analyzed. Reliability between researcher judgment and acoustic measures for glottic phonation detection was high. Results: Acoustic analysis demonstrated that glottic and partial-glottic phonation was consistently elicited, with no formal training of the minipigs required. Glottic vocalizations increased with multiple lifts. Glottic phonation continued to be elicited after multiple days but became less frequent. Glottic and partial-glottic phonations had similar CPP values over the 6 experimental days. Conclusion: Our cost-effective, reliable method of inducing and recording glottic phonation in the porcine model may provide a cost effective, preclinical tool in voice research.
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    Impact of Needle Selection on Survival of Muscle-Derived Cells When Used for Laryngeal Injections
    (Longdom Publishing, 2023) Awonusi, Oluwaseyi; Harbin, Zachary J.; Brookes, Sarah; Zhang, Lujuan; Kaefer, Samuel; Morrison, Rachel A.; Newman, Sharlé; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Objective: To describe how differing injector needles and delivery vehicles impact Autologous Muscle-Derived Cell (AMDC) viability when used for laryngeal injection. Methods: In this study, adult porcine muscle tissue was harvested and used to create AMDC populations. While controlling cell concentration (1 × 107 cells/ml), AMDCs including Muscle Progenitor Cells (MPCs) or Motor Endplate Expressing Cells (MEEs) were suspended in either phosphate-buffered saline or polymerizable (in-situ scaffold forming) type I oligomeric collagen solution. Cell suspensions were then injected through 23- and 27-gauge needles of different lengths at the same rate (2 ml/min) using a syringe pump. Cell viability was measured immediately after injection and 24- and 48-hours post-injection, and then compared to baseline cell viability prior to injection. Results: The viability of cells post-injection was not impacted by needle length or needle gauge but was significantly impacted by the delivery vehicle. Overall, injection of cells using collagen as a delivery vehicle maintained the highest cell viability. Conclusion: Needle gauge, needle length, and delivery vehicle are important factors that can affect the viability of injected cell populations. These factors should be considered and adapted to improve injectable MDC therapy outcomes when used for laryngeal applications.
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    Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study
    (Wiley, 2021-10) Brookes, Sarah; Zhang, Lujuan; Puls, Theodore J.; Kincaid, John; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Objective/hypothesis: There are currently no treatments available that restore dynamic laryngeal function after hemilaryngectomy. We have shown that dynamic function can be restored post hemilaryngectomy in a rat model. Here, we report in a first of its kind, proof of concept study that this previously published technique is scalable to a porcine model. Study design: Animal study. Methods: Muscle and fat biopsies were taken from three Yucatan minipigs. Muscle progenitor cells (MPCs) and adipose stem cells (ASCs) were isolated and cultured for 3 weeks. The minipigs underwent a left laterovertical partial laryngectomy sparing the left arytenoid cartilage and transecting the recurrent laryngeal nerve. Each layer was replaced with a tissue-engineered implant: 1) an acellular mucosal layer composed of densified Type I oligomeric collagen, 2) a skeletal muscle layer composed of autologous MPCs and aligned oligomeric collagen differentiated and induced to express motor endplates (MEE), and 3) a cartilage layer composed of autologous ASCs and densified oligomeric collagen differentiated to cartilage. Healing was monitored at 2 and 4 weeks post-op, and at the 8 week study endpoint. Results: Animals demonstrated appropriate weight gain, no aspiration events, and audible phonation. Video laryngoscopy showed progressive healing with vascularization and re-epithelialization present at 4 weeks. On histology, there was no immune reaction to the implants and there was complete integration into host tissue with nerve and vascular ingrowth. Conclusions: This pilot study represents a first in which a transmural vertical partial laryngectomy was performed and successfully repaired with a customized, autologous stem cell-derived multi-layered tissue-engineered implant.
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    Novel anticancer agents based on targeting the trimer interface of the PRL phosphatase
    (AACR Publications, 2016-08-15) Bai, Yunpeng; Yu, Zhi-Hong; Liu, Sijiu; Zhang, Lujuan; Zhang, Ruo-Yu; Zeng, Li-Fan; Zhang, Sheng; Zhang, Zhong-Yin; Biochemistry and Molecular Biology, School of Medicine
    PRL oncoproteins are phosphatases overexpressed in numerous types of human cancer. Elevated levels of PRL associate with metastasis and poor clinical outcomes. In principle, PRL phosphatases offer appealing therapeutic targets, but they remain underexplored due to the lack of specific chemical probes. In this study, we address this issue by exploiting a unique property of PRL phosphatases, namely, that they may function as homotrimers. Starting from a sequential structure-based virtual screening and medicinal chemistry strategy, we identified Cmpd-43 and several analogs which disrupt PRL1 trimerization. Biochemical and structural analyses demonstrate that Cmpd-43 and its close analogs directly bind the PRL1 trimer interface and obstruct PRL1 trimerization. Cmpd-43 also specifically blocks the PRL1-induced cell proliferation and migration through attenuation of both ERK1/2 and Akt activity. Importantly, Cmpd-43 exerted potent anticancer activity both in vitro and in vivo in a murine xenograft model of melanoma. Our results validate a trimerization-dependent signaling mechanism for PRL and offer proof-of-concept for trimerization inhibitors as candidate therapeutics to treat PRL-driven cancers
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    Phosphatase of regenerating liver 2 (PRL2) deficiency impairs Kit signaling and spermatogenesis
    (ASBMB, 2014-02-07) Dong, Yuanshu; Zhang, Lujuan; Bai, Yunpeng; Zhou, Hong-Ming; Campbell, Amanda M.; Chen, Hanying; Yong, Weidong; Zhang, Wenjun; Zeng, Qi; Shou, Weinian; Zhang, Zhong-Yin; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The Phosphatase of Regenerating Liver (PRL) proteins promote cell signaling and are oncogenic when overexpressed. However, our understanding of PRL function came primarily from studies with cultured cell lines aberrantly or ectopically expressing PRLs. To define the physiological roles of the PRLs, we generated PRL2 knock-out mice to study the effects of PRL deletion in a genetically controlled, organismal model. PRL2-deficient male mice exhibit testicular hypotrophy and impaired spermatogenesis, leading to decreased reproductive capacity. Mechanistically, PRL2 deficiency results in elevated PTEN level in the testis, which attenuates the Kit-PI3K-Akt pathway, resulting in increased germ cell apoptosis. Conversely, increased PRL2 expression in GC-1 cells reduces PTEN level and promotes Akt activation. Our analyses of PRL2-deficient animals suggest that PRL2 is required for spermatogenesis during testis development. The study also reveals that PRL2 promotes Kit-mediated PI3K/Akt signaling by reducing the level of PTEN that normally antagonizes the pathway. Given the strong cancer susceptibility to subtle variations in PTEN level, the ability of PRL2 to repress PTEN expression qualifies it as an oncogene and a novel target for developing anti-cancer agents.
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    PRL2/PTP4A2 phosphatase is important for hematopoietic stem cell self-renewal
    (Wiley, 2014-07) Kobayashi, Michihiro; Bai, Yunpeng; Dong, Yuanshu; Yu, Hao; Chen, Sisi; Gao, Rui; Zhang, Lujuan; Yoder, Mervin C.; Kapur, Reuben; Zhang, Zhong-Yin; Liu, Yan; Department of Pediatrics, Indiana University School of Medicine
    Hematopoietic stem cell (HSC) self-renewal is tightly controlled by cytokines and other signals in the microenvironment. While stem cell factor (SCF) is an early acting cytokine that activates the receptor tyrosine kinase KIT and promotes HSC maintenance, how SCF/KIT signaling is regulated in HSCs is poorly understood. The protein tyrosine phosphatase 4A (PTP4A) family (aka PRL [phosphatase of regenerating liver] phosphatases), consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, represents an intriguing group of phosphatases implicated in cell proliferation and tumorigenesis. However, the role of PTP4A in hematopoiesis remains elusive. To define the role of PTP4A in hematopoiesis, we analyzed HSC behavior in Ptp4a2 (Prl2) deficient mice. We found that Ptp4a2 deficiency impairs HSC self-renewal as revealed by serial bone marrow transplantation assays. Moreover, we observed that Ptp4a2 null hematopoietic stem and progenitor cells (HSPCs) are more quiescent and show reduced activation of the AKT and ERK signaling. Importantly, we discovered that the ability of PTP4A2 to enhance HSPC proliferation and activation of AKT and ERK signaling depends on its phosphatase activity. Furthermore, we found that PTP4A2 is important for SCF-mediated HSPC proliferation and loss of Ptp4a2 decreased the ability of oncogenic KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation. Thus, PTP4A2 plays critical roles in regulating HSC self-renewal and mediating SCF/KIT signaling.
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    Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors
    (Wiley, 2017-04) Kobayashi, Michihiro; Nabinger, Sarah; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro- Ohtan, Yumi; Pear, Warren S.; Carlesso, Nadia; Yoder, Mervin C.; Kapur, Reuben; Kaplan, Mark H.; Lacorazza, H. Daniel; Zhang, Zhong-Yin; Liu, Yan; Pediatrics, School of Medicine
    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors.
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    PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805
    (Taylor & Francis, 2023) Bai, Yunpeng; Yu, Guimei; Zhou, Hong-Ming; Amarasinghe, Ovini; Zhou, Yuan; Zhu, Peipei; Li, Qinglin; Zhang, Lujuan; Meke, Frederick Nguele; Miao, Yiming; Chapman, Eli; Tao, W. Andy; Zhang, Zhong-Yin; Dermatology, School of Medicine
    Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about their physiological substrates. VCP is implicated in lysophagy via collaboration with specific cofactors in the ELDR complex. However, how the ELDR complex assembly is regulated has not been determined. Moreover, the functional significance of the penultimate and conserved Tyr805 phosphorylation in VCP has not been established. Here, we use an unbiased substrate trapping and mass spectrometry approach and identify VCP/p97 as a bona fide substrate of PTP4A2. Biochemical studies show that PTP4A2 dephosphorylates VCP at Tyr805, enabling the association of VCP with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy, the autophagic clearance of damaged lysosomes. Functionally, PTP4A2 is required for cellular homeostasis by promoting lysophagy through facilitating ELDR-mediated K48-linked ubiquitin conjugate removal and autophagosome formation on the damaged lysosomes. Deletion of Ptp4a2 in vivo compromises the recovery of glycerol-injection induced acute kidney injury due to impaired lysophagy and sustained lysosomal damage. Taken together, our data establish PTP4A2 as a critical regulator of VCP and uncover an important role for PTP4A2 in maintaining lysosomal homeostasis through dephosphorylation of VCP at Tyr805. Our study suggests that PTP4A2 targeting could be a potential therapeutic approach to treat cancers and other degenerative diseases by modulating lysosomal homeostasis and macroautophagy/autophagy. Abbreviations: AAA+: ATPases associated with diverse cellular activities; AKI: acute kidney injury; CBB: Coomassie Brilliant Blue; CRISPR: clustered regularly interspaced short palindromic repeats; ELDR: endo-lysosomal damage response; GFP: green fluorescent protein; GST: ‎glutathione S-transferase; IHC: immunohistochemistry; IP: immunoprecipitation; LAMP1: lysosomal-associated membrane protein 1; LC-MS: liquid chromatography-mass spectrometry; LGALS3/Gal3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; PLAA: phospholipase A2, activating protein; PTP4A2: protein tyrosine phosphatase 4a2; PUB: NGLY1/PNGase/UBA- or UBX-containing protein; PUL: PLAP, Ufd3, and Lub1; TFEB: transcription factor EB; UBXN6/UBXD1: UBX domain protein 6; UPS: ubiquitin-proteasome system; VCP/p97: valosin containing protein; VCPIP1: valosin containing protein interacting protein 1; YOD1: YOD1 deubiquitinase.
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    Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis
    (SpringerNature, 2016-09-26) Bai, Yunpeng; Zhou, Hong-Ming; Zhang, Lujuan; Dong, Yuanshu; Zeng, Qi; Shou, Weinian; Zhang, Zhong-Yin; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The PRL phosphatases are oncogenic when overexpressed but their in vivo biological function is less well understood. Previous gene deletion study revealed a role for PRL2 in spermatogenesis. We report here the first knockout mice lacking PRL1, the most related homolog of PRL2. We found that loss of PRL1 does not affect spermatogenesis and reproductive ability of male mice, likely due to functional compensation by the relatively higher expression of PRL2 in the testes. However, PRL1-/-/PRL2+/- male mice show testicular atrophy phenotype similar to PRL2-/- mice. More strikingly, deletion of one PRL1 allele in PRL2-/- male mice causes complete infertility. Mechanistically, the total level of PRL1 and PRL2 is negatively correlated with the PTEN protein level in the testis and PRL1+/-/PRL2-/- mice have the highest level of PTEN, leading to attenuated Akt activation and increased germ cell apoptosis, effectively halting spermatozoa production. These results provide the first evidence that in addition to PRL2, PRL1 is also required for spermatogenesis by downregulating PTEN and promoting Akt signaling. The ability of the PRLs to suppress PTEN expression underscores the biochemical basis for their oncogenic potential.
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    Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage
    (Wiley, 2018-04) Zhang, Hongji; Voytik-Harbin, Sherry; Brookes, Sarah; Zhang, Lujuan; Wallace, Joseph; Parker, Noah; Halum, Stacey; Biomedical Engineering, School of Engineering and Technology
    OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement.