Preliminary investigation of the diagnostic value of shear wave elastography in evaluating the testicular spermatogenic function in patients with azoospermia.

To assess the diagnostic value of shear wave elastography (SWE) for evaluating the histological spermatogenic function of azoospermic males, 91 patients with azoospermia who underwent standardised greyscale ultrasound and SWE examinations followed by testicular biopsy were retrospectively recruited. Spermatogenic function was classified by biopsy as normal testicular spermatogenesis (n = 61), hypospermatogenesis (n = 18), spermatogenesis arrest (n = 6) and Sertoli cell-only syndrome (n = 6). Significant differences in testicular size and SWE values were observed between these 4 groups (p < .01). The mean SWE value had good discrimination power (AUC = 0.79) with a cut-off value of 1.55 KPa, a sensitivity of 0.58, specificity of 0.85, positive predictive value (PPV) of 0.36 and negative predictive value (NPV) of 0.93. Testicular volume had an AUC of 0.75. With a cut-off value of 8.41 ml, the testicular volume had a sensitivity of 0.58, specificity of 0.92, PPV of 0.54 and NPV of 0.93. The mean SWE value and testicular volume efficiently discriminated patients with normal spermatogenesis and hypospermatogenesis from patients with Sertoli cell-only syndrome and spermatogenesis arrest.

Testicular quantitative ultrasound: A noninvasive monitoring method for evaluating spermatogenic function in busulfan-induced testicular injury mouse models.

Busulfan-induced testicular injury mouse models are commonly used for experiments on spermatogonial stem cell transplantation, treatments for azoospermia due to spermatogenic failure and preserving male fertility after chemotherapy. Here, we investigated the value of testicular quantitative ultrasound for evaluating spermatogenic function in this model. In this study, testicular ultrasound was performed on mice from day 0 to 126 after busulfan treatment (n = 48), and quantitative data, including the testicular volume, mean pixel intensity and pixel uniformity, were analysed. The results revealed that from day 0 to 36, the testicular volume was positively associated with the testicle-to-body weight ratio (r = .92). On day 63, the pixel uniformity, which remained stable from day 0 to 36, declined significantly compared with that on day 36 (p < .01). On day 126, when the whole progression of spermatogenesis could be observed in most tubules, the mean pixel intensity also returned to normal (p > .05). In conclusion, testicular quantitative ultrasound could be used as a noninvasive and accurate monitoring method for evaluating spermatogenic function in busulfan-induced testicular injury mouse models.

MAL-PDT inhibits oral precancerous cells and lesions via autophagic cell death.

BACKGROUND: Oral cancer is a common cancer with a high mortality rate. While surgery is the most effective treatment for oral cancer, it frequently causes deformity and dysfunction in the orofacial region. In this study, methyl aminolevulinate photodynamic therapy (MAL-PDT) as a prevention tool against progression of precancerous lesion to oral cancer was explored. METHODS: For in vitro studies, we evaluated the effects of MAL-PDT on viability of DOK oral precancerous cells by XTT, cell morphology by TEM, and intracellular signaling pathways by flow cytometry, Western blotting, and immunofluorescence. For in vivo study, DMBA was used to induce oral precancerous lesions in hamsters followed by MAL-PDT treatment. We measured tumor size and body weight weekly. After sacrifice, buccal pouch lesions were processed for H&E stain and immunohistochemistry analysis. RESULTS: MAL-PDT induced autophagic cell death in DOK oral precancerous cells. The autophagy-related markers LC3II and p62/SQSTM1 and autophagosome formation in DOK cells were increased after MAL-PDT treatment. In vivo, Metvix® -PDT treatment decreased tumor growth and enhanced LC3II expression in hamster buccal pouch tumors induced by DMBA. CONCLUSIONS: Our in vitro and in vivo results suggest that MAL-PDT may provide an effective therapy for oral precancerous lesions through induction of autophagic cell death.

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations.

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.

Rottlerin, BDNF, and the impairment of inhibitory avoidance memory.

RATIONALE AND OBJECTIVE: As a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor and a mitochondrial uncoupler, oncologists have extensively studied rottlerin. Neuroscientists, however, have accumulated scarce data on the role of rottlerin in affective and cognitive functions. Only two prior studies have, respectively, documented its antidepressant-like effect and how it impairs psychostimulant-supported memory. Whether or not rottlerin would affect aversive memory remains unknown. Hence, we sought to investigate the effects of rottlerin on aversive memory in the inhibitory avoidance (IA) task in mice. MATERIALS AND METHODS: Male C57BL/6J mice were trained to acquire the IA task. Rottlerin (5 mg/kg, i.p. or 3 µg bilaterally in the hippocampus) or the vehicle was administered before footshock training (acquisition), after footshock training (consolidation), after the memory reactivation (reconsolidation), and before the test (retrieval) in the IA task. RESULTS: Systemic and intrahippocampal rottlerin impaired the acquisition, consolidation, and retrieval of IA memory, without affecting the reconsolidation process. Rottlerin (5 mg/kg, i.p.) induced a fast-onset and long-lasting increase in the brain-derived neurotrophic factor (BDNF) protein levels in the mouse hippocampus. Systemic injection of 7,8-dihydroxyflavone (7,8-DHF, 30 mg/kg), a BDNF tropomyosin receptor kinase B (TrkB) agonist impaired IA memory consolidation, and treatment with K252a (5 µg/kg), a Trk receptor antagonist, reversed the suppressing effect of rottlerin on IA memory consolidation. CONCLUSION: Rottlerin impairs IA memory consolidation through the enhancement of BDNF signaling in the mouse hippocampus. Excessive brain BDNF levels can be detrimental to cognitive function. Rottlerin is likely to affect the original memory-associated neuroplasticity. Thus, it can be combined with exposure therapy to facilitate the forgetting of maladaptive aversive memory, such as post-traumatic stress disorder (PTSD).

Development of the CK-MB-1 trastuzumab-resistant HER2-positive breast cancer cell line and xenograft animal models.

BACKGROUND: Patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who fail to respond to anti-HER2 treatments have poor prognoses. Most trastuzumab-resistant breast cancer cell lines available from biobanks feature either phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) mutation or the loss of phosphatase and tensin homolog (PTEN). However, PIK3CA mutations and/or PTEN loss do not account for most trastuzumab-resistant tumors in humans. METHODS: Breast cancer cells were collected from one patient's malignant ascites. These cells were cultured and maintained to develop a stable cell line, which we named CK-MB-1. We used western blotting to evaluate protein expression. The PIK3CA status of CK-MB-1 cells was analyzed using Sanger sequencing and validated using next-generation sequencing. In vivo, CK-MB-1 xenograft tumor models were developed in zebrafish and immunodeficient mice. RESULTS: CK-MB-1 cells maintained the major characteristics of the parental tumor including HER2 positivity and estrogen receptor negativity. The HER2 gene amplification of CK-MB-1 cells was detected by fluorescence in situ hybridization. The integrity of PTEN was confirmed by its positive protein expression and the absence of gene mutations. No common PIK3CA mutation was detected. Compared with the findings in two other HER2-positive trastuzumab-resistant cell lines, CK-MB-1 cells exhibited greater resistance to trastuzumab, chemotherapeutics, and small-molecule drugs. Trastuzumab resistance in CK-MB-1 cells was confirmed in vivo using the NOD SCID mouse model. CONCLUSIONS: CK-MB-1 cells represent a stable HER2-positive trastuzumab-resistant breast cancer cell line. The resistance of CK-MB-1 cells does not originate from the PTEN or phosphoinositide 3-kinase signaling pathway, which can provide an alternative approach for potential drugs.

The facilitating effect of MK-801 on inhibitory avoidance memory via mTOR signaling in the mouse hippocampus.

Despite the widespread belief that MK-801 induces memory deficits associated with dementia and schizophrenia in animal models, data regarding the impairing effect of MK-801 on aversive memory have been inconclusive. In this study, we investigated the effect of MK-801 on multiple memory stages of the inhibitory avoidance task, as well as its underlying signaling mechanism in the mouse hippocampus. We successfully replicated a previous finding suggesting that systemic injection of MK-801 impaired memory acquisition, but we observed that an intrahippocampal infusion of MK-801 facilitated the same memory process. We also found that both systemic and intrahippocampal administration of MK-801 facilitated memory consolidation and memory retrieval of the inhibitory avoidance task. We demonstrated that MK-801-induced increases in shock sensitivity and locomotor activity in the pre-training regimen confounded the detrimental effect of MK-801 on memory acquisition, thereby reconciling the inconsistent results in previous studies. In addition, the memory-facilitating effect of MK-801 was found to be dependent on drug dose and shock intensity. We next showed that MK-801 induced a fast-onset increase in the extent of mammalian target of rapamycin (mTOR) phosphorylation in the hippocampus. Finally, we observed that rapamycin, an mTOR inhibitor, blocked both the MK-801-induced increases in phosphorylated mTOR and the facilitating effect of MK-801 on memory consolidation. These results indicate that hippocampal mTOR signaling mediates the facilitating effect of MK-801 on memory consolidation of the inhibitory avoidance task. These findings further imply that MK-801 indeed functions as a memory enhancer and that mTOR signaling serves as a therapeutic target for memory disorders.

Chlorination of soil-derived dissolved organic matter: Long term nitrogen deposition does not increase terrestrial precursors of toxic disinfection byproducts.

Terrestrial dissolved organic matter (DOM) in forested watersheds is a known precursor of disinfection byproducts (DBPs) in drinking water. Although the characteristics of terrestrial DOM may change with increasing nitrogen (N) deposition in forests, how these changes alter formation potential and toxicity of DBPs remains unexplored. We analyzed the speciation and toxicity of DBPs from chlorination of DOM derived from soils (O, A, and B horizons) in an experimental temperate forest with 22 years of N addition. With long-term N addition, the DOM reactivity toward the formation of trihalomethanes (from 27.7-51.8 to 22.8-31.1 µg/mg-dissolved organic carbon (DOC)) and chloral hydrate (from 1.25-1.63 to 1.14-1.36 µg/mg-DOC) decreased, but that toward the formation of haloketones increased (from 0.23-0.26 to 0.26-0.33 µg/mg-DOC). The DOM reactivity toward the formation of haloacetonitriles was increased in the deeper soil but reduced in the surface soil. The DBP formation potential of DOM draining from a certain area of forest soils (in µg-DBP/m2-soil) was estimated to be reduced by 20.3% for trihalomethanes and increased by 37.5% for haloketones and have minor changes for haloacetonitriles and chloral hydrate (both <7%). Furthermore, the DBPs from chlorination of the soil-derived DOM showed lowered microtoxicity with N addition possibly due to reduced brominated DBP formation. Overall, this study highlights that N deposition may not increase drinking water toxicity through altering terrestrial DOM characteristics.

Spectroscopic and molecular-level characteristics of dissolved organic matter in the Pearl River Estuary, South China.

Coastal populations are expanding globally, resulting in great anthropogenic impacts on the organic matter in estuaries and regional carbon cycles. However, the molecular-level characteristics of dissolved organic matter (DOM) within highly disturbed estuaries are still not well understood. Here, water samples collected during two seasons (wet and dry) from the subtropical Pearl River Estuary of China were analyzed using absorption and fluorescence spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to determine the spatiotemporal variations in the DOM characteristics. In the seaward direction, the abundances of chromophoric and fluorescent DOM decreased by greater percentages than the bulk dissolved organic carbon concentration. The spectroscopy and FT-ICR MS analyses collectively indicated seaward declines in the aromaticity of DOM and terrestrial DOM contributions from natural terrestrial markers and anthropogenic synthetic surfactants. In particular, the S content in DOM was much higher here than in previously reported estuaries, suggesting a strong anthropogenic impact on the estuarine DOM. Greater terrestrial and anthropogenic signatures in DOM were observed in the wet season than in the dry season. Importantly, this study implies that the terrestrial and anthropogenic contributions to DOM were strongly driven by season in the anthropogenically disturbed subtropical estuary.

Anti-Idiotype Vaccine Provides Protective Immunity Against Vibrio Harveyi in Grouper (Epinephelus Coioides).

Since anti-idiotype antibodies (anti-Id Abs) can display internal images similar to the epitopes of the original antigens, we aimed to produce an effective vaccine based on anti-Id Abs to protect grouper from Vibrio harveyi. Anti-Id IgG showing V. harveyi-like internal images was produced from rabbits immunized with the Id portion of grouper anti-V. harveyi antibodies and its Fab portion, anti-Id IgG (Fab), was then prepared to use as the anti-Id vaccine. The resulting anti-Id IgG (Fab) was intraperitoneally injected twice at a 21-day interval into grouper to evaluate its ability to induce effective anti-V. harveyi immunity and protection, in comparison with inactivated V. harveyi bacteria. We found that administration of grouper with anti-Id IgG (Fab) resulted in enhanced V. harveyi-specific serum titers, as well as lymphocyte proliferation. In addition, three weeks after boosting, 90% (18/20) of fish immunized with anti-Id IgG (Fab) survived at least 28 days after a lethal challenge of the heterologous, virulent strain of V. harveyi. The capability of this anti-Id IgG (Fab) to imitate the epitopes of V. harveyi antigens and effectively induce protective immunity would be advantageous for its application in developing an efficacious vaccine against V. harveyi for future farm use in fish.

Cut-off Serum Zinc Concentration Affecting the Appetite, Growth, and Nutrition Status of Undernourished Children Supplemented With Zinc.

BACKGROUND: Zinc supplementation has varied effects on the linear growth of children who exhibited stunted growth. MATERIALS AND METHODS: This observational study involved 761 undernourished children, aged 2-10 years, who received a 24-week course of 10-mg elemental zinc per day. The clinical parameters for evaluation included appetite, height, weight, and body mass index (BMI). Evaluation of the effect of zinc supplementation was stratified by the initial serum zinc concentration. RESULTS: The enrolled participants comprised 390 boys and 371 girls. The mean age was 5.63 years. The height-for-age, weight-for-age, and BMI-for-age z scores increased gradually during the study period. When compared with the children with a serum zinc concentration ≥75 µg/dL, the height, weight, weight-for-age, and BMI-for-age z scores increased significantly in the patients with serum zinc concentrations of <75 µg/dL after 12- and 24-week zinc supplementation (all P < .001). BMI, height-for-age z score, and appetite also increased significantly in patients with serum zinc concentrations of <75 µg/dL after 24-week zinc supplementation (P = .003, .019, and <.001, respectively). CONCLUSION: The findings of this study indicate that undernourished children with serum zinc concentrations of <75 µg/dL experienced greater increments in appetite and growth as a result of zinc supplementation.

CYT-Rx20 inhibits ovarian cancer cells in vitro and in vivo through oxidative stress-induced DNA damage and cell apoptosis.

PURPOSE: The ß-nitrostyrene family has been previously reported to possess anticancer property. However, the biological effects of ß-nitrostyrenes on ovarian cancer and the underlying mechanisms involved remain unclear. In the present study, we synthesized a ß-nitrostyrene derivative, CYT-Rx20 3'-hydroxy-4'-methoxy-ß-methyl-ß-nitrostyrene), and investigated its anticancer effects and the putative pathways of action in ovarian cancer. METHODS: The effects of CYT-Rx20 were analyzed using cell viability assay, reactive oxygen species (ROS) generation assay, FACS analysis, annexin V staining, immunostaining, comet assay, immunoblotting, soft agar assay, migration assay, nude mice xenograft study and immunohistochemistry. RESULTS: CYT-Rx20 induced cytotoxicity in ovarian cancer cells by promoting cell apoptosis via ROS generation and DNA damage. CYT-Rx20-induced cell apoptosis, ROS generation and DNA damage were reversed by thiol antioxidants. In addition, CYT-Rx20 inhibited ovarian cancer cell migration by regulating the expression of epithelial to mesenchymal transition (EMT) markers. In nude mice, CYT-Rx20 inhibited ovarian tumor growth accompanied by increased expression of DNA damage marker γH2AX and decreased expression of EMT marker Vimentin. CONCLUSIONS: CYT-Rx20 inhibits ovarian cancer cells in vitro and in vivo, and has the potential to be further developed into an anti-ovarian cancer drug clinically.

Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21-6.27 µm in diameter, showed 72%-83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p < 0.05, nested design) long-lasting GAPDH-specific immunity (serum titers and lymphocyte proliferation) than PLG-encapsulated rGAPDH (PLG-rGAPDH) microparticles. After an experimental challenge of V. harveyi, PLG-PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p < 0.05, chi-square test) than that induced by PLG-rGAPDH microparticles (67%). In conclusion, PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles.

A dynamic analysis of motorcycle ownership and usage: a panel data modeling approach.

This study aims to develop motorcycle ownership and usage models with consideration of the state dependence and heterogeneity effects based on a large-scale questionnaire panel survey on vehicle owners. To account for the independence among alternatives and heterogeneity among individuals, the modeling structure of motorcycle ownership adopts disaggregate choice models considering the multinomial, nested, and mixed logit formulations. Three types of panel data regression models--ordinary, fixed, and random effects--are developed and compared for motorcycle usage. The estimation results show that motorcycle ownership in the previous year does exercise a significantly positive effect on the number of motorcycles owned by households in the current year, suggesting that the state dependence effect does exist in motorcycle ownership decisions. In addition, the fixed effects model is the preferred specification for modeling motorcycle usage, indicating strong evidence for existence of heterogeneity. Among various management strategies evaluated under different scenarios, increasing gas prices and parking fees will lead to larger reductions in total kilometers traveled.

[Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells].

OBJECTIVE: To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. METHODS: The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. RESULTS: The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. CONCLUSION: In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.