Serum thymidine kinase 1 protein concentrations and presence of its autoantibody as biomarkers for screening dogs with malignant tumors.

BACKGROUND: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. OBJECTIVES: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. ANIMALS: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. METHODS: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. RESULTS: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P < .0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games-Howell test, P < .0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592-0.675, P < .0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723-0.793, P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor-bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors.

Diisobutyl phthalate (DiBP)-induced male germ cell toxicity and its alleviation approach.

Diisobutyl phthalate (DiBP) is a commonly used plasticizer in manufacturing consumer and industrial products to improve flexibility and durability. Despite of the numerous studies, however, the direct mechanism underlying the male reproductive damage of DiBP is poorly understood. In this study, we investigated the male germ cell toxicity of DiBP using GC-1 spermatogonia (spg) cells. Our results indicated that DiBP exposure causes oxidative stress and apoptosis in GC-1 spg cells. In addition, DiBP-derived autophagy activation and down-regulation of phosphoinositide 3-kinase (PI3K)-AKT and extracellular signal-regulated kinase (ERK) pathways further inhibited GC-1 spg cell proliferation, indicating that DiBP can instigate male germ cell toxicity by targeting several pathways. Importantly, a combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine significantly reduced DiBP-induced male germ cell toxicity and restored proliferation. Taken together, the results of this study can provide valuable information to the existing literature by enhancing the understanding of single phthalate DiBP-derived male germ cell toxicity and the therapeutic interventions that can mitigate DiBP damage.

Development of a LED light therapy device with power density control using a Fuzzy logic controller.

The biological effects of a light-emitting diode (LED) light therapy device are determined by irradiation parameters, mainly wavelength and power density. However, using a battery to provide power causes a problem in the variation of LED power density during battery discharge. As a result, maintaining a stable LED power density, along with extending battery life and operating time, are the primary concerns in designing a LED light therapy device. The present study aims to introduce a LED light therapy device design with different LED color power density control. A Fuzzy logic, based on the relationship between LED power density and operating time, was proposed to control constant power density in this design. The experimental results demonstrate that by using the designed controller, the LED light therapy device's power density (40 mW/cm2, 50 mW/cm2, 60 mW/cm2 for red, blue, and green light, respectively) can be controlled. The newly designed LED light therapy device could be considered an advanced version with energy savings and stabilized LED power emitting property under a broad range voltage variation.

AniScan Using Extracellular Cyclic AMP-Dependent Protein Kinase A as a Serum Biomarker Assay for the Diagnosis of Malignant Tumors in Dogs.

The early detection of tumors improves chances of decreased morbidity and prolonged survival. Serum biomarkers are convenient to use and have several advantages over other approaches, such as accuracy and straightforward protocols. Reliable biomarkers from easily accessible sources are warranted for the development of cost-effective assays for routine screening, particularly in veterinary medicine. Extracellular c-AMP-dependent protein kinase A (ECPKA) is a cytosolic leakage enzyme. The diagnostic accuracy of detecting autoantibodies against ECPKA was found to be higher than that of ECPKA activity from enzymatic assays, which use a complicated method. Here, we investigated the diagnostic significance of measuring serum ECPKA autoantibody levels using an in-house kit (AniScan cancer detection kit; Biattic, Anyang, Korea). We used sera from 550 dogs, including healthy dogs and those with malignant and benign tumors. Serum ECPKA and immunoglobulin G were determined using the AniScan cancer detection kit. ECPKA autoantibody levels were significantly higher (p < 0.01) in malignant tumors than in benign tumors, non-tumor diseases, and healthy controls. On the basis of sensitivity and specificity values, AniScan ECPKA is a rapid and easy-to-use assay that can be applied to screen malignant tumors from benign tumors or other diseases in dogs.

Improved Depth-of-Field Photoacoustic Microscopy with a Multifocal Point Transducer for Biomedical Imaging.

In this study, a photoacoustic microscopy (PAM) system based on a multifocal point (MFP) transducer was fabricated to produce a large depth-of-field tissue image. The customized MFP transducer has seven focal points, distributed along with the transducer's axis, fabricated by separate spherically-focused surfaces. These surfaces generate distinct focal zones that are overlapped to extend the depth-of-field. This design allows extending the focal zone of 10 mm for the 11 MHz MFP transducer, which is a great improvement over the 0.48 mm focal zone of the 11 MHz single focal point (SFP) transducer. The PAM image penetration depths of a chicken-hemoglobin phantom using SFP and MFP transducers were measured as 5 mm and 8 mm, respectively. The significant increase in the PAM image-based penetration depth of the chicken-hemoglobin phantom was a result of using the customized MFP transducer.

Design, Fabrication, and Evaluation of Multifocal Point Transducer for High-Frequency Ultrasound Applications.

The present study illustrates the design, fabrication, and evaluation of a novel multifocal point (MFP) transducer based on polyvinylidene fluoride (PVDF) film for high-frequency ultrasound application. The fabricated MFP surface was press-focused using a computer numerical control (CNC) machining tool-customized multi-spherical pattern object. The multi-spherical pattern has five spherical surfaces with equal area and connected continuously to have the same energy level at focal points. Center points of these spheres are distributed in a linear pattern with 1 mm distance between each two points. The radius of these spheres increases steadily from 10 mm to 13.86 mm. The designed MFP transducer had a center frequency of 50 MHz and a -6 dB bandwidth of 68%. The wire phantom test was conducted to study and demonstrate the advantages of this novel design. The obtained results for MFP transducer revealed a significant increase (4.3 mm) of total focal zone in the near-field and far-field area compared with 0.48 mm obtained using the conventional single focal point transducer. Hence, the proposed method is promising to fabricate MFP transducers for deeper imaging depth applications.

Extracellular cyclic adenosine monophosphate-dependent protein kinase A autoantibody and C-reactive protein as serum biomarkers for diagnosis of cancer in dogs.

Protein kinase A, a cyclic adenosine monophosphate (AMP)-dependent enzyme, normally exists within mammalian cells; however, in cancer cells, it can leak out and be found in the serum. Extracellular cyclic AMP-dependent protein kinase A (ECPKA) has been determined to increase in the serum of cancer-bearing dogs. However, there have been no reports in the veterinary literature on serum ECPKA autoantibody (ECPKA-Ab) expression in dogs with cancer. The aim of this study was to evaluate ECPKA-Ab and C-reactive protein (CRP) as serum biomarkers for cancer in dogs. ECPKA-Ab and CRP levels were detected by an enzyme-linked immunosorbent assay in serum samples from dogs with malignant tumours (n = 167), benign tumours (n = 42), or non-tumour disease (n = 155) and from healthy control dogs (n = 123). ECPKA-Ab and CRP levels were significantly higher in the dogs with malignant tumours than in those with benign tumours or non-tumour diseases, as well as in the healthy controls (P < 0.001, Kruskal-Wallis test). There was a significant positive correlation between the neoplastic index, which was developed using ECPKA-Ab and CRP levels, and the presence of cancer in dogs (P < 0.001); the area under the receiver-operating characteristic curve was estimated to be >0.85 (P < 0.001). In conclusion, ECPKA-Ab is a potential serum biomarker for a broad spectrum of cancers. Combined measurement of CRP and ECPKA-Ab levels in serum improves the sensitivity and accuracy of a diagnosis of cancer in dogs.

Platelet-derived growth factor receptor-alpha positive cardiac progenitor cells derived from multipotent germline stem cells are capable of cardiomyogenesis in vitro and in vivo.

Cardiac cell therapy has the potential to revolutionize treatment of heart diseases, but its success hinders on the development of a stem cell therapy capable of efficiently producing functionally differentiated cardiomyocytes. A key to unlocking the therapeutic application of stem cells lies in understanding the molecular mechanisms that govern the differentiation process. Here we report that a population of platelet-derived growth factor receptor alpha (PDGFRA) cells derived from mouse multipotent germline stem cells (mGSCs) were capable of undergoing cardiomyogenesis in vitro. Cells derived in vitro from PDGFRA positive mGSCs express significantly higher levels of cardiac marker proteins compared to PDGFRA negative mGSCs. Using Pdgfra shRNAs to investigate the dependence of Pdgfra on cardiomyocyte differentiation, we observed that Pdgfra silencing inhibited cardiac differentiation. In a rat myocardial infarction (MI) model, transplantation of a PDGFRAenriched cell population into the rat heart readily underwent functional differentiation into cardiomyocytes and reduced areas of fibrosis associated with MI injury. Together, these results suggest that mGSCs may provide a unique source of cardiac stem/progenitor cells for future regenerative therapy of damaged heart tissue.

Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male's lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

Petasites japonicus Stimulates the Proliferation of Mouse Spermatogonial Stem Cells.

Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.

Effects of paracrine factors on CD24 expression and neural differentiation of male germline stem cells.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Characterization of EST Gene in the Bovine Corpus Luteum during the Estrous Cycle.

The objective of this study was to investigate the expression of bovine luteum expressed sequence tags (ESTs), vascular endothelial growth factor (VEGF), and tumor necrosis factor receptor 1 (TNFR1) and the presence of functional ESTs in the bovine corpus luteum (CL) during different stages of the estrus cycle. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a difference in the expression of ESTs during the CL stage. Concentration of ESTs in the CL tissue increased significantly from the mid-luteal stage and decreased thereafter. RT-PCR analysis showed higher levels of the EST genes in the CL of the mid-luteal stage than in other stages, and the same level of expression of VEGF. Immunohistochemistry analysis of the tissue from CL formation to regression showed low cytosol and aggregation of the nucleus. And activity caspase 3 (apoptosis detector) was most strongly detected in the CL1 stage of bovine. During the estrous cycle, the cytosol was magnified and differentiation of the nucleus was clearly manifested. The ESTs affected the CL, and the relationship between VEGF and TNFR1 played a pivotal role for CL development and activation, dependent on the stage of CL. These results suggest local production of ESTs, the presence of functional ESTs in the bovine CL, and that ESTs play a role in regulating the function of cell death in bovine CL.

Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells.

OBJECTIVE: To study the influence of sugars and establish a serum-free freezing method for the cryopreservation of spermatogonial stem cells (SSCs). DESIGN: Animal study. SETTING: University laboratory. ANIMAL(S): C57BL/6-TgEGFP, C57BL/6 mice. INTERVENTION(S): Germ cells enriched from testis cells were frozen using standard freezing medium containing sugars, including monosaccharides, disaccharides, and trisaccharides at 50, 100, and 200 mM, respectively. To study the feasibility of establishing a serum-free freezing method, fetal bovine serum was substituted with knockout serum replacement. MAIN OUTCOME MEASURE(S): Freeze-thawed germ cells were evaluated for recovery rate, proliferation capacity, and stem cell activity after transplantation to recipient testes. RESULT(S): Supplementation of freezing medium with 200 mM disaccharide is an effective method for cryopreservation of SSCs. Trehalose is the most effective cryoprotectant among all the sugars tested and only lactose was comparable to trehalose. Our proliferation and transplantation data show that serum-free freezing can be achieved in freezing medium supplemented with 200 mM trehalose, 10% knockout serum replacement, and 10% dimethyl sulfoxide (DMSO) for cryopreservation of SSCs. CONCLUSION(S): These findings raise the possibility of effectively banking frozen SSCs from various species, including humans, in a traditional serum-free medium for germ cell research and male infertility treatments.

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Efficient enhancement of lentiviral transduction efficiency in murine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

Enrichment of testicular gonocytes and genetic modification using lentiviral transduction in pigs.

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.