Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.     

Exosomes Are Unlikely Involved in Intercellular Nef Transfer

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.     

Studies of Antibiotic Resistance of Beta-Lactamase Bacteria under Different Nutrition Limitations at the Single-Cell Level

Drug resistance involves many biological processes, including cell growth, cell communication, and cell cooperation. In the last few decades, bacterial drug resistance studies have made substantial progress. However, a major limitation of the traditional resistance study still exists: most of the studies have concentrated on the average behavior of enormous amounts of cells rather than surveying single cells with different phenotypes or genotypes. Here, we report our study of beta-lactamase bacterial drug resistance in a well-designed microfluidic device, which allows us to conduct more controllable experiments, such as controlling the nutrient concentration, switching the culture media, performing parallel experiments, observing single cells, and acquiring time-lapse images. By using GFP as a beta-lactamase indicator and acquiring time-lapse images at the single-cell level, we observed correlations between the bacterial heterogeneous phenotypes and their behavior in different culture media. The feedback loop between the growth rate and the beta-lactamase production suggests that the beta-lactamase bacteria are more resistant in a rich medium than in a relatively poor medium. In the poorest medium, the proportion of dormant cells may increase, which causes a lower death rate in the same generation. Our work may contribute to assaying the antibiotic resistance of pathogenic bacteria in heterogeneous complex media.     

High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1–10 μg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.     

Thinprep plus Papanicolaou Stain Method Is More Sensitive than Cytospin-Coupled Wright Giems Stain Method in Cerebrospinal Fluid Cytology for Diagnosis of Leptomeningeal Metastasis from Solid Tumors

Background

The present study was designed to determine whether the Thinprep plus Papanicolaou stain (Thinprep) method is more sensitive than the Cytospin-coupled Wright-Giemsa (WG) stain (Cytospin) method in diagnosis of leptomeningeal metastasis (LM) from malignant solid tumors in cerebrospinal fluid (CSF). We also explored if the Thinprep method could be used in the differential diagnosis of the type of primary tumor cells based on the morphology of tumor cells in CSF samples.

Methods

The morphological features of tumor cells in fresh CSF samples were analyzed using both methods. The tumor cell detection rates were compared between the two methods.

Results

Using the Thinprep method, we found that each type of tumor cells in the CSF samples had specific identifiable morphological features linked to their primary cancer origins, such as adenocarcinomas originated from the lungs, breast, and stomach, and lung squamous cell carcinomas, small cell lung cancer, large-cell neuroendocrine lung cancer, hepatocellular carcinoma, and malignant melanoma. In a retrospective study with 88 LM patients, cancer cells were detected in 80 out of the 88 CSF samples. In the comparative study with 45 LM patients, the initial detection rate of the Thinprep method was significantly higher than that of the Cytospin method (73.3% vs. 57.8%, P<0.01). The cell morphology was better preserved and subcellular structures were clearer using the Thinprep method, compared to the Cytospin method.

Conclusions

The Thinprep method is more sensitive and suitable for LM diagnosis in CSF in patients with malignant solid tumors than the Cytospin method. The Thinprep method may facilitate primary tumor detection and help design early treatment regimens for LM patients with tumors of unknown primary origin.     

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum.     

Technology Resource,Distribution, and Development Characteristics of Global Influenza Virus Vaccine: A Patent Bibliometric Analysis

Influenza virus vaccine (IVV) is a promising research domain that is closely related to global health matters, which has been acknowledged not only by scientists and technology developers, but also by policy-makers. Meanwhile, patents encompass valuable technological information and reflect the latest technological inventions as well as the innovative capability of a nation. However, little research has examined this up-and-coming research field using patent bibliometric method. Thus, this paper (a) designs the technology classification system and search strategy for the identification of IVV; and (b) presents a longitudinal analysis of the global IVV development based on the European Patent Office (EPO) patents. Bibliometric analysis is used to rank countries, institutions, inventors and technology subfields contributing to IVV technical progress. The results show that the global trends of IVV are a multi-developing feature of variety but an uneven technical resource distribution. Although the synthetic peptide vaccine is a comparatively young field, it already demonstrates the powerful vitality and the enormous development space. With the worldwide competition increasing, all nations especially China should be looking to increase devotion, enhance capability and regard effectiveness of technological innovation.     

Anti-Inflammatory Effects of Ang-(1-7) in Ameliorating HFD-Induced Renal Injury through LDLr-SREBP2-SCAP Pathway

The angiotensin converting enzyme 2-angiotensin-(1–7)-Mas axis (ACE2-Ang-(1–7)-Mas axis) is reported to participate in lipid metabolism in kidney, but its precise effects and underlying mechanisms remain unknown. We hypothesized that Ang-(1–7) reduces lipid accumulation and improves renal injury through the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr-SREBP2-SCAP) system by suppressing inflammation in high fat diet (HFD)-fed mice. In this study, male C57BL/6 mice were randomized into four groups: STD (standard diet)+saline, HFD+saline, HFD+Ang-(1–7) and STD+Ang-(1–7). After 10 weeks of feeding, mice were administered Ang-(1–7) or saline for two weeks. We found that high inflammation status induced by HFD disrupted the LDLr-SREBP2-SCAP feedback system. Treatment of mice fed a high-fat diet with Ang-(1–7) induced significant improvement in inflammatory status, following the downregulation of LDLr, SREBP2 and SCAP, and then, decreased lipid deposition in kidney and improved renal injury. In conclusion, the anti-inflammatory effect of Ang-(1–7) alleviates renal injury triggered by lipid metabolic disorders through a LDLr- SREBP2-SCAP pathway.     

Endovascular Management of Post-Irradiated Carotid Blowout Syndrome

Purpose

To retrospectively evaluate the clinical and technical factors related to the outcomes of endovascular management in patients with head-and-neck cancers associated with post-irradiated carotid blowout syndrome (PCBS).

Materials and Methods

Between 2000 and 2013, 96 patients with PCBS underwent endovascular management. The 40 patients with the pathological lesions located in the external carotid artery were classified as group 1 and were treated with embolization. The other 56 patients with the pathological lesions located in the trunk of the carotid artery were divided into 2 groups as follows: group 2A comprised the 38 patients treated with embolization, and group 2B comprised the 18 patients treated with stent-graft placement. Fisher’s exact test was used to examine endovascular methods, clinical severities, and postprocedural clinical diseases as predictors of outcomes.

Results

Technical success and immediate hemostasis were achieved in all patients. The results according to endovascular methods (group 1 vs 2A vs 2B) were as follows: technical complication (1/40[2.5%] vs 9/38[23.7%] vs 9/18[50.0%], P = 0.0001); rebleeding (14/40[35.0%] vs 5/38[13.2%] vs 7/18[38.9%]), P = 0.0435). The results according to clinical severity (acute vs ongoing PCBS) were as follows: technical complication (15/47[31.9%] vs 4/49[8.2%], P = 0.0035); rebleeding (18/47[38.3%] vs 8/49[16.3%], P = 0.0155). The results according to post-procedural clinical disease (regressive vs progressive change) were as follows: alive (14/21[66.7%] vs 8/75[10.7%], P<0.0001); survival time (34.1±30.6[0.3–110] vs 3.6±4.0[0.07–22] months, P<0.0001).

Conclusion

The outcomes of endovascular management of PCBS can be improved by taking embolization as a prior way of treatment, performing endovascular intervention in slight clinical severity and aggressive management of the post-procedural clinical disease.     

TOX Acts an Oncological Role in Mycosis Fungoides

Mycosis fungoides (MF) is a low-grade lymphoma characterized by clonal expansion of atypical CD4+ skin-homing T lymphocytes. Herein, we examined the role of thymocytes selection associated HMG-box (TOX), a gene previously found to be unregulated in MF skin biopsies, in MF pathogenesis. TOX encodes a high-mobility group family (HMG) domain DNA binding nuclear protein, which regulates the differentiation of developing T-cells. First, we confirmed that TOX expression levels in MF were increased compared with those in benign inflammatory dermatitis (BID) and normal skin. In addition, TOX level increased with the progression MF from patch stage to tumor stage. Overexpression of TOX accelerated the proliferation and migration of MF cell lines in vitro, which were blocked by AKT inhibitors. In conclusion, our study confirmed that TOX was highly expressed in MF lesions and accelerates the proliferation and migration of MF. TOX is a diagnostic marker for MF and may play a pathogenic role in disease progression.