Plasma Neurofilament Light Chains as Blood-Based Biomarkers for Early Diagnosis of Canine Cognitive Dysfunction Syndrome.

The number of elderly dogs is increasing significantly worldwide, and many elderly dogs develop canine cognitive dysfunction syndrome (CCDS). CCDS is the canine analog of Alzheimer's disease (AD) in humans. It is very important to develop techniques for detecting CDDS in dogs. Thus, we used the detection of neurofilament light chains (NfL) in plasma as a blood-based biomarker for the early diagnosis of canine Alzheimer's disease using immunomagnetic reduction (IMR) technology by immobilizing NfL antibodies on magnetic nanoparticles. According to the 50-point CCDS rating scale, we divided 36 dogs into 15 with CCDS and 21 without the disease. The results of our IMR assay showed that the plasma NfL levels of dogs with CCDS were significantly increased compared to normal dogs (p < 0.01). By plasma biochemical analysis, we further confirmed that the liver and renal dysfunction biomarkers of dogs with CCDS were significantly elevated compared to normal dogs (p < 0.01-0.05). On the basis of our preliminary study, we propose that IMR technology could be an ideal biosensor for detecting plasma NfL for the early diagnosis of CCDS.

Therapeutic Effects of Plant Extracts of Anoectochilus roxburghii on Side Effects of Chemotherapy in BALB/c Breast Cancer Mice.

Breast cancer is the most common cancer in women, and chemotherapy is an effective treatment. However, chemotherapy often causes adverse side effects such as cardiotoxicity, myelosuppression, immunodeficiency, and osteoporosis. Our study focused on the alleviating effects of Anoectochilus roxburghii extracts (AREs) on the adverse side effects of chemotherapy in mice with breast cancer. We individually evaluated the antioxidant capacity and cytotoxicity of the AREs using DPPH and MTT assays. We also examined the effects of the AREs on intracellular F-actin, reactive oxygen species (ROS), and the mitochondrial membrane potential (MMP) of 4T1 cancer cells before and after doxorubicin (DOX) treatment. Our results showed that ARE treatment enhanced the effects of DOX chemotherapy by promoting cell morphology damage, oxidative stress, and ROS generation, as well as by reducing MMP in the 4T1 breast cancer cells. By using BALB/c mice with breast cancer with DOX treatment, our results showed that the DOX treatment reduced body weight, blood pressure, and heart rate and induced myelosuppression, immunodeficiency, cardiotoxicity, and osteoporosis. After oral ARE treatment of BALB/c mice with breast cancer, the chemotherapeutic effects of DOX were enhanced, and the adverse side effects of DOX chemotherapy were alleviated. Based on the above results, we suggest that AREs can be used as an adjuvant reliever to DOX chemotherapy in BALB/c mice with breast cancer.

Extracellular domain of semaphorin 5A serves a tumor­suppressing role by activating interferon signaling pathways in lung adenocarcinoma cells.

Semaphorin 5A (SEMA5A), which was originally identified as an axon guidance molecule in the nervous system, has been subsequently identified as a prognostic biomarker for lung cancer in nonsmoking women. SEMA5A acts as a tumor suppressor by inhibiting the proliferation and migration of lung cancer cells. However, the regulatory mechanism of SEMA5A is not clear. Therefore, the purpose of the present study was to explore the roles of different domains of SEMA5A in its tumor­suppressive effects in lung adenocarcinoma cell lines. First, it was revealed that overexpression of full length SEMA5A or its extracellular domain significantly inhibited the proliferation and migration of both A549 and H1299 cells using MTT, colony formation and gap closure assays. Next, microarray analyses were performed to identify genes regulated by different domains of SEMA5A. Among the differentially expressed genes, the most significant function of these genes that were enriched was the 'Interferon Signaling' pathway according to Ingenuity Pathway Analysis. The activation of the 'Interferon Signaling' pathway was validated by reverse transcription­quantitative PCR and western blotting. In summary, the present study demonstrated that the extracellular domain of SEMA5A could upregulate genes in interferon signaling pathways, resulting in suppressive effects in lung adenocarcinoma cells.

Chinese Herbal Medicine Formula Guizhi Li-Zhong Tang as an Alternative to Antibiotic Feed Additives for Preventing Pneumonia in Piglets through Antioxidant, Anti-Inflammatory, and Antiapoptotic Protection.

Weanling piglets often develop respiratory diseases such as pneumonia because they encounter substantial environmental stress. This study investigated an alternative herbal feed additive, Guizhi Li-Zhong Tang (GLZ), for preventing pneumonia in weanling piglets. An in vitro experiment demonstrated that GLZ has high antioxidant capacity and low cytotoxicity toward Kupffer cells. In addition, GLZ treatment can alleviate lipopolysaccharide (LPS)-induced damage in Kupffer cells. A total of 94 4-week-old piglets were randomly divided into three groups, which received sham treatment, 0.2% Tilmicosin antibiotic (TAB) treatment, or 0.2% GLZ treatment. Piglets receiving the GLZ treatment had a higher survival rate and higher immunoglobulin G levels but lower allergy-related eosinophil levels and cough incidence than did piglets receiving the sham or 0.2% TAB treatments. Through immunohistochemistry and Western blot analysis, we discovered that piglets receiving the 0.2% GLZ treatment had significantly higher expression of antioxidant-related SOD2 and lower expression of oxidative-stress-related 3-NT (p < 0.01), inflammation-related TNF-α (p < 0.01) and NF-κB (p < 0.05), and apoptosis-related caspase-3 (p < 0.01) in lung tissue than did piglets receiving the sham or 0.2% TAB treatment. Therefore, GLZ treatment is promising as an alternative to antibiotic medicine for weanling piglets because of its protective antioxidative, anti-inflammatory, and antiapoptotic effects in lung tissue.

Awareness regarding and vaccines acceptability of human papillomavirus among parents of middle school students in Zunyi, Southwest China.

The objective of this study was to investigate the parent cognition of information regarding the human papillomavirus (HPV) and their willingness toward HPV vaccination of their middle-school-aged children in Zunyi, Guizhou Province, China.The results provide a basis for improving the awareness concerning HPV-related information as a key vaccination strategy for implementing the HPV vaccine in the local context. Methods include the random cluster sampling method and questionnaires to survey parents. General descriptive and single-factor analyses were used to assess cognition to determine factors influencing vaccine willingness. Of 1,074 parents, 28.2% (302) and 38.0% (408) had heard of HPV and its vaccine before the survey, and when given HPV-related information, 73.9% (794) parents were willing to vaccinate their children. Reasons why parents did or did not want the vaccination were surveyed, with lack of sufficient knowledge about HPV and its vaccine being the primary reason to refuse vaccination. Concerns about safety, effectiveness, and perceiving low risk are the biggest obstacle in promoting vaccination. When the price is <1000, most parents (56.1%) are willing to vaccinate their children; thus, cost is also one of the concerns. Therefore, strategies for improving public awareness regarding the risk of cervical cancer and confidence in vaccination must be considered by policymakers.If the national authority confirms that the vaccine is safe and effective, the vaccine should be included in the national immunization program to increase publicity, address safety concerns, and allow for price regulation.

Regulatory Mechanisms and Functional Roles of Hypoxia-Induced Long Non-Coding RNA MTORT1 in Breast Cancer Cells.

Long non-coding RNAs (lncRNAs) have been found to participate in multiple genetic pathways in cancer. Also, mitochondria-associated lncRNAs have been discovered to modulate mitochondrial function and metabolism. Previously, we identified oxygen-responsive lncRNAs in MCF-7 breast cancer cells under different oxygen concentrations. Among them, a novel mitochondria-encoded lncRNA, mitochondrial oxygen-responsive transcript 1 (MTORT1), was chosen for further investigation. Nuclear, cytoplasmic, and mitochondrial fractionation assays were performed to evaluate the endogenous expression levels of MTORT1 in breast cancer cells. In vitro proliferation and migration assays were conducted to investigate the functions of MTORT1 in breast cancer cells by knockdown of MTORT1. RNA immunoprecipitation and luciferase reporter assays were used to examine the physical binding between MTORT1 and microRNAs. Our results showed that MTORT1 had low endogenous expression levels in breast cancer cells and was mainly located in the mitochondria. Knockdown of MTORT1 enhanced cell proliferation and migration, implying a tumor suppressor role of this novel mitochondrial lncRNA. MTORT1 served as sponge of miR-26a-5p to up-regulate its target genes, CREB1 and STK4. Our findings shed some light on the characterization, function, and regulatory mechanism of the novel hypoxia-induced mitochondrial lncRNA MTORT1, which functions as a microRNA sponge and may inhibit breast cancer progression. These data suggest that MTORT1 may be a candidate for therapeutic targeting of breast cancer progression.

Application of digital holographic tomography in antitumor effect of cantharides complex on 4T1 breast cancer cells.

The study focuses on a methodology providing noninvasive monitoring and evaluation of the antitumor effect of traditional Chinese medicine, cantharides complex (canth), on 4T1 breast tumor cells. Digital holographic tomography (DHT) and developed data post-processing algorithms were used for quantitative estimation of changes in optical and morphological parameters of cells. We calculated and compared data on the refractive index, thickness, and projected area of 4T1 breast tumor cells in control untreated specimens and those treated with doxorubicin hydrochloride (DOX), canth, and their combinations. Post-treatment changes in cellular morphology recorded by DHT demonstrated that the two drugs led to noticeably different morphological changes in cells that can be presumably associated with different pathways of their death, apoptosis, or necrosis. The effect of combined treatment with these two drugs strongly depended on their relative concentrations and could lead to changes characteristic either for DOX or for canth; however, being more profound than those obtained when using each drug solely. The results obtained by DHT are in a good correspondence with commonly used cell viability analysis and immunofluorescent analysis of changes in cellular cytoskeleton.

Alternative antibiotic feed additives alleviate pneumonia with inhibiting ACE-2 expression in the respiratory system of piglets.

Weaning piglets often suffer from pneumonia during growth, so in general, antibiotics are used by owners to make piglets grow smoothly. However, antibiotics may be accompanied by many side effects such as gastrointestinal discomfort and allergies. The aim of this study was to develop an alternative antibiotic herbal veterinary medicine for alleviating pneumonia in weanling piglets. As observed in the pig ranches, many weanling piglets suffer from the pneumonia and also show high expression of angiotensin-converting enzyme 2 (ACE-2) in their respiratory and intestinal tracts. ACE inhibitors have been reported that can decrease pneumonia risk through their main mechanism of action. Thus, we also try to find alternative antibiotic feed additives that can reduce expression of ACE-2 in piglets. We selected the Guizhi Li-Zhong Tang Extract Granules (GLZ) as a natural product for piglets. Then, we compared the serum immunoglobulin levels of piglets with sham, tilmicosin antibiotic (TAB), and GLZ treatments. Our results showed that piglets with GLZ treatment had significantly a higher expression of immunoglobulin A and immunoglobulin G but a lower expression of immunoglobulin E than those with sham and TAB treatments. In addition, piglets with GLZ treatment showed obviously low pneumonia incidence than those with sham and TAB treatments. Similarly, piglets with GLZ treatment showed significantly lower expressions of ACE-2 in their tracheal, bronchial, and lung tissues than those with sham and TAB treatments. GLZ seems to be an alternative ACE inhibitor that can decrease pneumonia risk through inhibiting ACE-2 expression and alleviating allergies in their respiratory systems. Thus, we suggest that GLZ can be an alternative antibiotic feed additive for weaning piglets.

MPTP toxicity causes vocal, auditory, orientation and movement defects in the echolocation bat.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can damage dopaminergic neurons in the substantia nigra in many mammals with biochemical and cellular changes that are relatively similar to those observed in Parkinson's disease. Our study examined whether MPTP-treated echolocation bats can cause changes in bat echolocation system. By considering ultrasound spectrums, auditory brainstem-evoked potentials and flight trajectories of normal bats, we observed that the vocal, auditory, orientation and movement functions of MPTP-treated bats were significantly impaired, and they exhibited various symptoms resembling those in patients with Parkinson's disease. Our immunohistochemistry and western blot analyses further indicated that expression of vocal-related FOXP2 in the superior colliculus, auditory-related otoferlin in the inferior colliculus, dopamine synthesis-related aromatic l-amino acid decarboxylase in the substantia nigra and dopamine receptor in the striatum was significantly decreased. Furthermore, protein expression related to inflammation, oxidative stress and apoptosis in the substantia nigra was significantly increased in MPTP-treated bats. These results indicate that inflammation, oxidative stress and apoptosis may be instrumental in dopaminergic neurodegeneration in the substantia nigra. The vocal, auditory and orientation and movement dysfunctions of MPTP-treated bats are relatively consistent with symptoms of Parkinson's disease.

Establishment of an Immunocompetent Metastasis Rat Model with Hepatocyte Cancer Stem Cells.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality. Cancer stem cells (CSCs) are responsible for the maintenance, metastasis, and relapse of various tumors. The effects of CSCs on the tumorigenesis of HCC are still not fully understood, however. We have recently established two new rat HCC cell lines HTC and TW-1, which we isolated from diethylnitrosamine-induced rat liver cancer. Results showed that TW-1 expressed the genetic markers of CSCs, including CD133, GSTP1, CD44, CD90, and EpCAM. Moreover, TW-1 showed higher tolerance to sorafenib than HTC did. In addition, tumorigenesis and metastasis were observed in nude mice and wild-type rats with TW-1 xenografts. Finally, we combined highly expressed genes in TW-1/HTC with well-known biomarkers from recent HCC studies to predict HCC-related biomarkers and able to identify HCC with AUCs > 0.9 after machine learning. These results indicated that TW-1 was a novel rat CSC line, and the mice or rat models we established with TW-1 has great potential on HCC studies in the future.

Chinese Veterinary Medicine B307 Promotes Cardiac Performance and Skeletal Muscle Contraction via Enhancing Intracellular Calcium Levels and Neural Electrical Activity in Animal and Cell Models.

The study mainly investigated the effects of Chinese veterinary medicine B307 in cardiac and motor functions in animal models of pigeons and mice. Related cellular mechanisms were also studied in the neuroblastoma cell model of SH-SY5Y. Cardiac functions of pigeons and mice were examined by using moorFLPI Laser color Doppler imager and M-mode echocardiography, and motor functions were examined by using muscle electrical stimulation and force recording in the isolated breast muscle. Intracellular calcium levels and electrical activity of SH-SY5Y cells were examined by using Fura 2-AM fluorescence and MED64 system separately. Our results in vivo found that those pigeons under oral B307 treatment obviously enhanced subcutaneous microcirculation and contractile force and prolonged fatigue time in their breast muscles. Those mice under oral B307 treatment obviously elevated ejection fraction and cardiac output in their hearts. Our results in vitro showed that those SH-SY5Y cells under B307 treatment obviously increased intracellular calcium mobilization and electrical activities. These results revealed that improvement of cardiac and motor functions under B307 treatments may be caused by increasing electrical activities and intracellular calcium levels in neuromuscular cells and a similar mechanism may also occur in muscle cells. Thus, we suggested that B307 can be a functional Chinese veterinary medicine for flying pigeons.

Oral treatment with the Chinese herbal supplements B307 enhances muscle endurance of ICR mice after exhaustive swimming via suppressing fatigue, oxidative stress, and inflammation.

Exhaustive exercise may damage muscles due to oxidative stress and inflammation and cause muscle fatigue and soreness. The study investigated the effects of Chinese herbal supplements (CHS) B307 on muscle endurance after exhaustive swimming (ES). Thirty-two male ICR mice were randomly divided into 4 groups: Sham + ES, pretreatment of CHS B307 + ES (Pre + ES), post-treatment of CHS B307 + ES (Post + ES), and dual treatment of CHS B307 + ES (Dual + ES). All mice were subjected to ES in the form of a forced swimming test. Then, we compared ES time (EST) as the index of muscular endurance. Also, we examined the fatigue, oxidative stress, inflammation, and damage in the muscle tissue among these groups by using immunohistochemistry (IHC), chemiluminescence, and biochemical analysis. Our results revealed that those mice of Pre + ES and Dual + ES groups had remarkably better EST than those mice of Sham + ES and Post + ES groups. Those mice with oral treatment of CHS B307(Pre + ES, Post + ES, and Dual + ES groups) showed significantly reduced leukocyte counts in the urine, and reduced levels of reactive oxygen species (ROS), neutrophils, and lactic acid in the blood than those mice of Sham + ES. In addition, those mice with oral treatment of CHS B307 (Pre + ES, Post + ES, and Dual + ES groups) showed significant alleviation of oxidative stress, inflammation, and damage in the muscle tissue than those mice of Sham + ES. Thus, we suggested that CHS B307 can be a functional sports supplement because it can enhance muscle endurance after exhaustive swimming via suppressing fatigue, oxidative stress, and inflammation.