Mixed Wolbachia infections resolve rapidly during in vitro evolution.

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.

Mixed Wolbachia infections resolve rapidly during in vitro evolution.

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species, tissues, and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.

A Structural Proteome Screen Identifies Protein Mimicry in Host-Microbe Systems.

Host-microbe systems are evolutionary niches that produce coevolved biological interactions and are a key component of global health. However, these systems have historically been a difficult field of biological research due to their experimental intractability. Impactful advances in global health will be obtained by leveraging in silico screens to identify genes involved in mediating interspecific interactions. These predictions will progress our understanding of these systems and lay the groundwork for future in vitro and in vivo experiments and bioengineering projects. A driver of host-manipulation and intracellular survival utilized by host-associated microbes is molecular mimicry, a critical mechanism that can occur at any level from DNA to protein structures. We applied protein structure prediction and alignment tools to explore host-associated bacterial structural proteomes for examples of protein structure mimicry. By leveraging the Legionella pneumophila proteome and its many known structural mimics, we developed and validated a screen that can be applied to virtually any host-microbe system to uncover signals of protein mimicry. These mimics represent candidate proteins that mediate host interactions in microbial proteomes. We successfully applied this screen to other microbes with demonstrated effects on global health, Helicobacter pylori and Wolbachia , identifying protein mimic candidates in each proteome. We discuss the roles these candidates may play in important Wolbachia -induced phenotypes and show that Wobachia infection can partially rescue the loss of one of these factors. This work demonstrates how a genome-wide screen for candidates of host-manipulation and intracellular survival offers an opportunity to identify functionally important genes in host-microbe systems.

Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α.

BACKGROUND AND OBJECTIVE: This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and ß-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. RESULTS: TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that ß-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 µg/ml, 20 µg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. CONCLUSIONS: Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

OBJECTIVES: To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. MATERIALS AND METHODS: Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. RESULTS: The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. CONCLUSIONS: Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs.

KDM6A promotes chondrogenic differentiation of periodontal ligament stem cells by demethylation of SOX9.

OBJECTIVES: KDM6A has been demonstrated critical in the regulation of cell fates. However, whether KDM6A is involved in cartilage formation remains unclear. In this study, we investigated the role of KDM6A in chondrogenic differentiation of PDLSCs, as well as the underlying epigenetic mechanisms. METHODS: KDM6A shRNA was transfected into PDLSCs by lentivirus. The chondrogenic differentiation potential of PDLSCs was assessed by Alcian blue staining. Immunofluorescence was performed to demonstrate H3K27me3 and H3K4me3 levels during chondrogenesis. SOX9, Col2a1, ACAN and miRNAs (miR-29a, miR-204, miR-211) were detected by real-time RT-PCR. Western blot was performed to evaluate SOX9, H3K27me3 and H3K4me3. RESULTS: The production of proteoglycans in PDLSCs was decreased after knockdown of KDM6A. Depletion of KDM6A inhibited the expression of SOX9, Col2a1, ACAN and resulted in increased H3K27me3 and decreased H3K4me3 levels. EZH2 inhibitor rescued the chondrogenic potential of PDLSCs after knockdown of KDM6A by regulating H3K27me3. Additionally, miR-29a, miR-204 and miR-211 were also involved in the process of PDLSCs chondrogenesis. CONCLUSIONS: KDM6A is required in chondrogenic differentiation of PDLSCs by demethylation of H3K27me3, and EZH2 inhibitor could rescue chondrogenesis of PDLSCs after knockdown of KDM6A. It could be inferred that upregulation of KDM6A or application of EZH2 inhibitor might improve mesenchymal stem cell mediated cartilage regeneration in inflammatory tissue destruction such as osteoarthritis.

A large-inner-diameter multi-walled carbon nanotube-based dual-drug delivery system with pH-sensitive release properties.

A novel dual-drug delivery system (DDDS) for cancer chemotherapy has been established by employing highly purified and mildly oxidized large-inner-diameter multi-walled carbon nanotubes (LID-MWCNTs) as the vector. The LID-MWCNTs were modified with the antitumor drugs, cisplatin (CDDP) and doxorubicin (DOX). CDDP was encapsulated inside the nanotube vectors by a wet-chemical approach while DOX was attached to the external surfaces through non-covalently interaction. The loading efficiencies of CDDP and DOX were as high as 84.56 and 192.67%, respectively. Notably, after CDDP was encapsulated inside the nanotubes, a three-level blocking strategy, which included polyethylene glycol, folic acid and DOX, was employed to block the CDDP exits at different levels. The pH-sensitive release profile of CDDP was demonstrated using a modified characterization method, as well as that of DOX. Finally, the anticancer activity of the DDDS on MCF-7 cells was tested and a synergistic effect was recorded. This work is part of our LID-MWCNTs based drug delivery system studies, and provides a basis for developing a novel comprehensive antitumor treatment that combines chemotherapy and photothermal therapy.

Incorporation of cisplatin into PEG-wrapped ultrapurified large-inner-diameter MWCNTs for enhanced loading efficiency and release profile.

The efficacy of carbon nanotubes based drug delivery systems (DDSs) has long been compromised by low drug loading efficiency and rapid release profile, especially for drugs encapsulated inside nanotubes. To address these deficiencies, the large-inner-diameter multi-walled carbon nanotubes (LID-MWCNTs) were employed to synthesize an innovative cisplatin (CDDP)-loaded DDS. A multi-step purification and oxidation procedure was developed to achieve ultrapurified oxidized LID-MWCNTs prior to entrapping CDDP. High molecular weight polyethylene glycol (PEG) was grafted onto the nanotubes to partially block the exit paths of drugs. As assessed by TGA and ICP-OES analyses, CDDP loading efficiency of resulting DDS was as high as 100.12%, and sustained release profile was obtained. Finally, the anticancer activity on CAL-27 cells was evaluated, and enhanced inhibition effect (IC50 values 3.93 µg/ml) was recorded. The successful inner-cavity-loading of LID-MWCNTs also provides a basis for establishing more complicated multi-functional DDSs.

Cesarean section en caul and asphyxia in preterm infants.

We reviewed the outcome for 211 women undergoing a planned en caul (within intact membranes) cesarean section and for 836 control women with conventional lower segment section, in the period 2001-2010 at a university-affiliated hospital in China, where the former technique has been practiced. Of the intended en caul sections there were 141 successful deliveries (66.8%), and 70 that failed and were converted to conventional lower segment cesarean section. Maternal blood loss was similar for both operation types, but the rate of asphyxia was significantly lower among preterm infants delivered by the en caul method than in the control cases. Multivariate logistic regression revealed that the volume of amniotic fluid, a low Bishop score and high birthweight were associated with failed en caul deliveries. Cesarean section en caul can be a safer option than lower segment section when preterm delivery is required.

[Sex hormone-binding globulin of gestational diabetes mellitus pregnant women with well-controlled glucose and pregnancy outcomes].

OBJECTIVE: To explore the relationship between sex hormone-binding globulin (SHBG) of gestational diabetes mellitus (GDM) pregnant women with well-controlled glucose and pregnancy outcomes. METHODS: Two hundred and fifty-one GDM pregnant women of 24 - 28 weeks in Shengjing Hospital of China Medical University were recruited from Mar. 2005 to Mar. 2010. Two hundred and sixteen cases of GDM with well-controlled glucose were defined as glycemic satisfied group, and they were treated by diet therapy (169 cases) or insulin therapy (47 cases). Thirty-five cases with unsatisfied glucose were defined as glycemic unsatisfied group. One hundred and ninety-two healthy pregnant women of 24 - 28 weeks were defined as healthy control group. Serum SHBG and homeostasis model analysis of insulin resistance (HOMA-IR) at 24 - 28 weeks and above 36 weeks were measured. GDM was diagnosed by "two-step" method according to the National Diabetes Data Group (NDDG) criteria. The pregnancy outcomes and complications of the three groups were recorded. RESULTS: (1) Comparison of pregnancy outcomes and complications:glycemic satisfied group was less likely to develop hypertensive disorders in pregnancy (10.6%), premature birth (8.3%), large for gestational age (LGA) (8.8%), neonatal asphyxia (3.7%) and neonatal hypoglycemia (2.3%) compared to glycemic unsatisfied group (42.9%, 34.3%, 31.4%, 22.9% and 11.4%, respectively). And the difference was statistically significant (P < 0.05 or P < 0.01). There was no significant difference for incidence of polyhydramnios, pueperal infection, postpartum hemorrhage, neonatal hyperbilirubinemia between the two groups (P > 0.05). When compared to healthy control group (7.3%, 2.1%, 4.2%, 2.1% and 1.6%), no significant difference was found for incidence of premature birth (8.3%), pueperal infection (3.2%), postpartum hemorrhage (5.1%), neonatal asphyxia (3.7%) and neonatal hypoglycemia (2.3%, P > 0.05). (2) Comparison of results of 24 - 28 weeks and above 36 weeks: serum SHBG of glycemic satisfied group [(384 ± 88), (457 ± 48) nmol/L] was significantly higher than that of glycemic unsatisfied group [(313 ± 45), (401 ± 73) nmol/L]; HOMA-IR of glycemic satisfied group (5.3 ± 1.1, 5.5 ± 1.1) was significantly lower than that of glycemic unsatisfied group (7.0 ± 1.3, 7.6 ± 1.7; P < 0.01). Serum SHBG of glycemic satisfied group was significantly lower than that of healthy control group [(492 ± 95), (565 ± 40) nmol/L]; and HOMA-IR of glycemic satisfied group (5.3 ± 1.1, 5.5 ± 1.1) was significantly higher than that of healthy control group (3.6 ± 0.6, 3.9 ± 0.5; P < 0.01). FPG of glycemic satisfied group [(5.84 ± 0.28), (5.16 ± 0.13) mmol/L] was significantly lower than that of glycemic unsatisfied group [(6.13 ± 0.16), (5.68 ± 1.14) mmol/L;P < 0.01]. FINS of glycemic satisfied group [(20.4 ± 2.1), (24.1 ± 4.2) mmol/L] was significantly lower than that of glycemic unsatisfied group [(24.7 ± 4.5), (29.9 ± 2.7) mmol/L; P < 0.01]. (3) Correlation analysis. Between 24-28 weeks, SHBG was negatively correlated with HOMA-IR in the three groups (r = -0.952, P < 0.01); and SHBG was negatively correlated with HOMA-IR in glycemic satisfied group (r = -0.903, P < 0.01). CONCLUSIONS: Well-controlled glucose can not completely improve maternal and fetal outcomes of GDM pregnant women. High insulin resistance and low serum SHBG can influence pregnancy outcomes.