Characterization and Genome Analysis of the Delftia lacustris Strain LzhVag01 Isolated from Vaginal Discharge.

Delftia has been separated from freshwater, sludge, and soil and has emerged as a novel opportunistic pathogen in the female vagina. However, the genomic characteristics, pathogenicity, and biotechnological properties still need to be comprehensively investigated. In this study, a Delftia strain was isolated from the vaginal discharge of a 43-year-old female with histologically confirmed cervical intraepithelial neoplasm (CIN III), followed by whole-genome sequencing. Phylogenetic analysis and average nucleotide identity (ANI) analysis demonstrated that it belongs to Delftia lacustris, named D. lacustris strain LzhVag01. LzhVag01 was sensitive to ß-lactams, macrolides, and tetracyclines but exhibited resistance to lincoamines, nitroimidazoles, aminoglycosides, and fluoroquinolones. Its genome is a single, circular chromosome of 6,740,460 bp with an average GC content of 66.59%. Whole-genome analysis identified 16 antibiotic resistance-related genes, which match the antimicrobial susceptibility profile of this strain, and 11 potential virulence genes. These pathogenic factors may contribute to its colonization in the vaginal environment and its adaptation and accelerate the progression of cervical cancer. This study sequenced and characterized the whole-genome of Delftia lacustris isolated from vaginal discharge, which provides investigators and clinicians with valuable insights into this uncommon species.

In vitro biofilm formation of Gardnerella vaginalis and Escherichia coli associated with bacterial vaginosis and aerobic vaginitis.

Objective: To determine the optimum biofilm formation ratio of Gardnerella vaginalis (G. vaginalis) in a mixed culture with Escherichia coli (E. coli). Methods: G. vaginalis ATCC14018, E. coli ATCC25922, as well as five strains of G. vaginalis were selected from the vaginal sources of patients whose biofilm forming capacity was determined by the Crystal Violet method. The biofilm forming capacity of E. coli in anaerobic and non-anaerobic environments were compared using the identical assay. The Crystal Violet method was also used to determine the biofilm forming capacity of a co-culture of G. vaginalis and E. coli in different ratios. After Live/Dead staining, biofilm thickness was measured using confocal laser scanning microscopy, and biofilm morphology was observed by scanning electron microscopy. Results: The biofilm forming capacity of E. coli under anaerobic environment was similar to that in a 5% CO2 environment. The biofilm forming capacity of G. vaginalis and E. coli was stronger at 106:105 CFU/mL than at other ratios (P<0.05). Their thicknesses were greater at 106:105 CFU/mL than at the other ratios, with the exception of 106:102 CFU/mL (P<0.05), under laser scanning microscopy. Scanning electron microscopy revealed increased biofilm formation at 106:105 CFU/mL and 106:102 CFU/mL, but no discernible E. coli was observed at 106:102 CFU/mL. Conclusion: G. vaginalis and E. coli showed the greatest biofilm forming capacity at a concentration of 106:105 CFU/mL at 48 hours and could be used to simulate a mixed infection of bacterial vaginosis and aerobic vaginitis in vitro.

Routine Workflow of Spatial Proteomics on Micro-formalin-Fixed Paraffin-Embedded Tissues.

In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed paraffin-embedded (FFPE) tissues are an important material in spatial proteomics, in which fixed tissues are excised using laser capture microdissection (LCM), followed by protein identification with mass spectrometry. For a satisfied spatial proteomics upon FFPE tissues, the excision area is expected to be as small as possible, and the identified proteins are countered upon as much as possible. For a general laboratory for spatial proteomics, a routine workflow is required, not relying on any special device, and is easily operating. In view of these challenges in technology, we initiated a technology evaluation throughout the entire procedure of proteomic analysis with micro-FFPE tissues. In contrast to the protocols reported previously, several innovations in technology were proposed and conducted, such as removal of destaining, decross-linking with "hang-down", solution simplification for peptide generation and balancing to excision area, and capture rate of micro-FFPE tissues. After optimization of all the necessary steps, a routine workflow was established, in which the minimized area for protein identification was 0.002 mm2, while the excision area for a consistent proteomic analysis was 0.05 mm2. Using the developed workflow and collecting the micro-FFPE tissues continuously, for the first time, a spatial proteomic atlas of mouse brain was preliminarily constructed, which exhibited the typical characteristics of spatial-dependent protein abundance and functional enrichment.

The fungal quorum-sensing molecule, farnesol, regulates the immune response of vaginal epithelial cells against Candida albicans.

BACKGROUND: Farnesol is a Candida-secreted quorum-sensing molecule of great interest as a potential antifungal agent for serious and hardly curable infections-candidiasis, especially vulvovaginal candidiasis (VVC). METHODS: The effect of farnesol on cellular morphology and viability and evaluated the production of Th1 (IL-2), Th2 (IL-4), proinflammatory (IL-6), chemotactic (IL-8), and Th17 (IL-17) cytokines in the culture supernatants of vaginal epithelial cell line (VK2) were evaluated. Moreover, we tested the inhibitory effect of farnesol on C. albicans adhesion. Scanning electron microscopy was conducted to observe any VK2 cell ultrastructural changes. RESULTS: Only low concentrations (≤ 50 µmol/L) of farnesol did not affect the morphology and viability of the VK2 cells (P > 0.05). Farnesol reduced the adhesion of C. albicans to the VK2 cells. When treated with farnesol, statistical elevated levels of both IL-4 and IL-17 secreted by the infected VK2 cells were present in the culture supernatants (P < 0.05). CONCLUSIONS: Farnesol acts as a stimulator to up-regulate the Th17-type innate immune response, as well as Th2-type humoral immunity following C. albicans infection. Further research is required to select the optimal therapeutic dose to develop efficacious and safe mucosal immune adjuvant for treating VVCs.

Antimicrobial Effects of Sophora flavescens Alkaloids on Metronidazole-Resistant Gardnerella vaginalis in Planktonic and Biofilm Conditions.

Bacterial vaginosis (BV) is a common infectious disease of the lower female reproductive tract, which is characterized by the augmentation of anaerobic bacteria. Gardnerella (G.) vaginalis plays a predominant role in BV recurrence relating to its higher virulence potential and biofilm formation ability. With the increased proportion of metronidazole-resistant G. vaginalis, controlling resistance to metronidazole and finding more effective drugs became a major concern. In this study, 30 clinical strains were cultured from the vaginal secretions of BV patients, followed by PCR and 16S rDNA sequencing identification. According to the CLSI guidelines for anaerobic drug sensitivity testing, 19 strains were identified as metronidazole-resistant (minimum inhibitory concentration, MIC ≥ 32 µg/mL), of which 4 clinical strains were observed to be strong biofilm producer and the final minimum biofilm inhibitory concentration (MBIC) of metronidazole was increased to 512 µg/mL. Sophora flavescens Alkaloids (SFAs), a traditional chinese medicine, could not only inhibit the growth of metronidazole-resistant G. vaginalis in planktonic (MIC: 0.3125-1.25 mg/mL), but also eliminate the biofilm formation (MBIC: 0.625-1.25 mg/mL). In the high-magnification scanning electron, it was observed that the morphology of biofilm changed from a thick to flaky shape and was nearly depleted. These results indicate that SFAs could not only inhibit the growth of metronidazole-resistant G. vaginalisin planktonic and biofilm levels, but also destroyed the biofilm morphology and microstructure, which may contribute to the prevention of BV recurrence.

Eye movement characteristics in a mental rotation task presented in virtual reality.

Introduction: Eye-tracking technology provides a reliable and cost-effective approach to characterize mental representation according to specific patterns. Mental rotation tasks, referring to the mental representation and transformation of visual information, have been widely used to examine visuospatial ability. In these tasks, participants visually perceive three-dimensional (3D) objects and mentally rotate them until they identify whether the paired objects are identical or mirrored. In most studies, 3D objects are presented using two-dimensional (2D) images on a computer screen. Currently, visual neuroscience tends to investigate visual behavior responding to naturalistic stimuli rather than image stimuli. Virtual reality (VR) is an emerging technology used to provide naturalistic stimuli, allowing the investigation of behavioral features in an immersive environment similar to the real world. However, mental rotation tasks using 3D objects in immersive VR have been rarely reported. Methods: Here, we designed a VR mental rotation task using 3D stimuli presented in a head-mounted display (HMD). An eye tracker incorporated into the HMD was used to examine eye movement characteristics during the task synchronically. The stimuli were virtual paired objects oriented at specific angular disparities (0, 60, 120, and 180°). We recruited thirty-three participants who were required to determine whether the paired 3D objects were identical or mirrored. Results: Behavioral results demonstrated that the response times when comparing mirrored objects were longer than identical objects. Eye-movement results showed that the percent fixation time, the number of within-object fixations, and the number of saccades for the mirrored objects were significantly lower than that for the identical objects, providing further explanations for the behavioral results. Discussion: In the present work, we examined behavioral and eye movement characteristics during a VR mental rotation task using 3D stimuli. Significant differences were observed in response times and eye movement metrics between identical and mirrored objects. The eye movement data provided further explanation for the behavioral results in the VR mental rotation task.

Age-related differences in upper limb motor performance and intrinsic motivation during a virtual reality task.

BACKGROUND: In recent years, virtual reality (VR) has evolved from an alternative to a necessity in older adults for health, medical care, and social interaction. Upper limb (UL) motor skill, is an important ability in manipulating VR systems and represents the brain's regulation of movements using the UL muscles. In this study, we used a haptic-feedback Virtual Box and Block Test (VBBT) system and an Intrinsic Motivation Inventory (IMI) to examine age-related differences in UL motor performance and intrinsic motivation in VR use. The findings will be helpful for the development of VR applications for older adults. METHODS: In total, 48 young and 47 older volunteers participated in our study. The parameters including VBBT score, number of velocity peaks, velocity, grasping force and trajectory length were calculated to represent the task performance, manual dexterity, coordination, perceptive ability and cognitive ability in this study. RESULTS: Age-related differences could be found in all the parameters (all p <  0.05) in VR use. Regression analysis revealed that the task performance of young adults was predicted by the velocity and trajectory length (R2 = 64.0%), while that of older adults was predicted by the number of velocity peaks (R2 = 65.6%). Additionally, the scores of understandability, relaxation and tiredness were significantly different between the two groups (all p <  0.05). In older adults, the understandability score showed large correlation with the IMI score (|r| = 0.576, p <  0.001). In young adults, the correlation was medium (|r| = 0.342, p = 0.017). No significant correlation was found between the IMI score and VBBT score (|r| = 0.142, p = 0.342) in older adults, while a medium correlation (|r| = 0.342, p = 0.017) was found in young adults. CONCLUSIONS: The findings demonstrated that decreased smoothness in motor skills dominated the poor VR manipulation in older adults. The experience of understandability is important for older adults' intrinsic motivation in VR use.

PCGEM1 promotes cell proliferation and migration in endometriosis by targeting miR-124-3p-mediated ANTXR2 expression.

BACKGROUND: Endometriosis, a common gynaecological disease in women, affects 10% of women of childbearing age. Among infertile women, this proportion is as high as 30-50%. Despite the high prevalence of endometriosis, the pathogenesis of endometriosis is still unclear. METHODS: In the present study, bioinformatics analysis and molecular and animal experiments were employed to explore the functions of PCGEM1 in the pathogenesis of endometriosis. We established an endometriosis rat model and isolated endometrial stromal cells (ESCs) and primary normal ESCs (NESCs). Bioinformatics analysis was adopted to study the roles of PCGEM1 in promoting the pathogenesis of endometriosis. Luciferase reporter assays and RNA pull-down assays were carried out to study the mechanism by which PCGEM1 regulates ANTXR2. RESULTS: Our results indicated that PCGEM1 promoted the motility and proliferation of ectopic endometrial cells, and the underlying mechanism was due to the direct binding of PCGEM1 to miR-124-3p to modulate ANTXR2 expression. CONCLUSION: PCGEM1 can influence endometrial stromal cell proliferation and motility and may be a novel therapeutic target for endometriosis.

Prevalence of Mycoplasma genitalium and Chlamydia trachomatis in Chinese female with lower reproductive tract infection: a multicenter epidemiological survey.

BACKGROUND: Chlamydia trachomatis and Mycoplasma infections have been regarded as severe challenges to public health worldwide because their potential risk of leading to serious reproductive complications. C. trachomatis is the most common sexually transmitted bacterial infections and the prevalence has been increasing in recent years. As a newly discovered pathogen, Mycoplasma genitalium has gradually been recognized as important sexually transmitted infection and even been called a "new chlamydia". There are no official epidemiological data of M. genitalium in China especially in women with lower reproductive tract infection. This work aims to understand the prevalence and risk factors of M. genitalium and C. trachomatis in women with lower reproductive tract infections and to provide reference for the formulation of health policy in China. METHODS: This study was conducted in the gynecological clinics of 12 hospitals geographically located in different regions in China. Women with purulent cervical secretions or abnormal vaginal microecology were included as the research group, and those with normal vaginal microecology and cervical secretions were included as the control group. A total of 2190 participants were recruited in this project including 1357 of research group and 833 of control group. All participants were required to complete questionnaires, whose vaginal discharge were collected for vaginal microecology test and cervical discharge for detection of M. genitalium and C. trachomatis. RESULTS: The prevalence of C. trachomatis and M. genitalium were 7.1% (96/1357) and 3.8% (51/1357), respectively in research group. The prevalence of C. trachomatis and M. genitalium varied in different regions. Infection rates of C. trachomatis and M. genitalium were higher in women with abnormal vaginal microecology (C.t P = 0.038, M.g P = 0.043), especially in women with bacterial vaginosis and mixed vaginitis, of which C. trachomatis showed statistical differences (bacterial vaginosis, P = 0.035; mixed vaginitis, P = 0.0001) and M. genitalium was close to statistical differences (bacterial vaginosis, P = 0.057; mixed vaginitis, P = 0.081). Alcoholism and abnormal vaginal microecology were positively correlated with both C. trachomatis and M. genitalium infection. Increasing age, being married and multi-parity were negatively correlated with C. trachomatis infection. There is a positive correlation between multiple sexual partners, diversed styles of sex and C. trachomatis infection. CONCLUSIONS: Women with lower genital dysbiosis have an increased risk of C. trachomatis and M. genitalium. The overall prevalence of M. genitalium is lower than that of C. trachomatis, while they have similarities in the characteristics of infection. Although M. genitalium is not routinely screened as C. trachomatis in young women, attention should be paid to M. genitalium infection in young women with abnormal vaginal microecology or having childbearing needs.

Kangfuxiaoyanshuan alleviates uterine inflammation and adhesion via inhibiting NF-κB p65 and TGF-ß/MMP-2 signaling pathway in pelvic inflammatory disease rats.

Background and aims: Pelvic inflammatory disease (PID) is infection-induced inflammation of the female upper reproductive tract that results in high fever, ectopic pregnancy, infertility, and varying degrees of chronic pelvic pain. Recent clinical studies have shown that Kangfuxiaoyanshuan (KFXYS), a Traditional Chinese Medicine (TCM) formulation, may short the course of the disease and reduce the occurrence of PID sequelae, but its pharmacological action and potential mechanism have not been fully elucidated. Here, we aimed to investigate the therapeutic effects and mechanism of KFXYS in rats with PID. Materials and Methods: A PID rat model was constructed through endometrial mechanical injury and pathogen infection. The rectal temperature was measured during the 14-days course of treatment, and the white blood cell (WBC) count in the blood and the levels of cytokines (IFN-γ, IL-1ß, IL-4, TNF-α) in the serum were evaluated by ELISA. Hematoxylin and eosin (HE) staining was performed to analyze pathological changes, and transmission electron microscopy (TEM) was used to observe ultrastructural changes. The p-p65/p65 protein expression was evaluated by western blotting and the levels of MMP-2 and TGF-ß in adhesion tissues were assessed by immunohistochemistry. Results: KFXYS lowered the rectal temperature and the WBC counts in the blood in the acute stage of PID and alleviated inflammatory cell infiltration of the uterus, especially when combined with levofloxacin. KFXYS significantly decreased the levels of proinflammatory cytokines (IFN-γ, IL-1ß, IL-4) and adhesion-related factors (TNF-α) and protected the ultrastructure of endometrial epithelial cells. Mechanistically, KFXYS inhibited the NF-κB activation by decreasing phosphorylation of p65, thus the alleviation of inflammation further reduced the expression of TGF-ß and MMP-2, and inhibited the occurrence of uterine adhesions. Conclusion: These results revealed that KFXYS alleviated pelvic inflammation and effectively inhibits inflammation-associated adhesion, which indicated the potential role of KFXYS for treatment of PID and the prevention of PID sequelae.

Integrated bioinformatic analysis of dysregulated microRNA-mRNA co-expression network in ovarian endometriosis.

INTRODUCTION: Ovarian endometriosis is a frequently occurring gynecological disease with large socioeconomic impact. Accumulating evidence has suggested that aberrant miRNA-mRNA interactions are involved in the pathogenesis and progression of ovarian endometriosis. This study aims to identify key miRNAs in ovarian endometriosis by using integrated bioinformatic analysis of a dysregulated miRNA-mRNA co-expression network. MATERIAL AND METHODS: Expression profiling of miRNA and mRNA in three normal endometria and five pairs of ectopic/eutopic endometria from patients with ovarian endometriosis was determined by high-throughput sequencing techniques. The data were then integrated with the public sequencing datasets (GSE105764 and GSE105765) using a non-biased approach and a miRNA-mRNA co-expression regulatory network was constructed by in-depth bioinformatic analysis. RESULTS: The constructed miRNA-mRNA network included 87 functionally DEMs, 482 target mRNAs and 1850 paired miRNA-mRNA regulatory interactions. Specifically, five miRNAs (miR-141-3p, miR-363-3p, miR-577, miR-767-5p, miR-96-5p) were gradually decreased and two miRNAs (miR-493-5p, miR-592) were gradually increased from normal endometria to eutopic endometria, and then ectopic endometria tissues. Importantly, miR-141-3p, miR-363-3p and miR-96-5p belonged to the miR-200 family, miR-106a-363 cluster and miR-183/96/182 cluster, respectively. Their target mRNAs were mainly associated with cell adhesion, locomotion and binding, which are suggested to play vital regulatory roles in the pathogenesis of ovarian endometriosis. CONCLUSIONS: Integrated bioinformatic analysis of the miRNA-mRNA co-expression network defines the crucial roles of the miR-200 family, miR-106a-363 cluster and miR-183/96/182 cluster in the pathogenesis of ovarian endometriosis. Further in-depth functional studies are needed to unveil the molecular mechanisms of these miRNAs, and may provide clues for the optimization of therapeutic strategies for ovarian endometriosis.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

The role of low-frequency oscillations in three-dimensional perception with depth cues in virtual reality.

Currently, vision-related neuroscience studies are undergoing a trend from simplified image stimuli toward more naturalistic stimuli. Virtual reality (VR), as an emerging technology for visual immersion, provides more depth cues for three-dimensional (3D) presentation than two-dimensional (2D) image. It is still unclear whether the depth cues used to create 3D visual perception modulate specific cortical activation. Here, we constructed two visual stimuli presented by stereoscopic vision in VR and graphical projection with 2D image, respectively, and used electroencephalography to examine neural oscillations and their functional connectivity during 3D perception. We find that neural oscillations are specific to delta and theta bands in stereoscopic vision and the functional connectivity in the two bands increase in cortical areas related to visual pathways. These findings indicate that low-frequency oscillations play an important role in 3D perception with depth cues.

Plastome Phylogenomics of Aucuba (Garryaceae).

Aucuba (Garryaceae), which includes approximately ten evergreen woody species, is a genus endemic to East Asia. Their striking morphological features give Aucuba species remarkable ornamental value. Owing to high levels of morphological divergence and plasticity, species definitions of Aucuba remain perplexing and problematic. Here, we sequenced and characterized the complete plastid genomes (plastomes) of three Aucuba species: Aucuba chlorascens, Aucuba eriobotryifolia, and Aucuba japonica. Incorporating Aucuba plastomes available in GenBank, a total of seven Aucuba plastomes, representing six out of ten species of Aucuba, were used for comparative plastome analysis, phylogenetic analysis and divergence time estimation in this study. Comparative analyses revealed that plastomes of Aucuba are highly conserved in size, structure, gene content, and organization, and exhibit high levels of sequence similarity. Phylogenetic reconstruction based on 68 plastid protein-coding genes strongly supported the monophyly of Garryales, Garryaceae and Aucuba. Aucuba eriobotryifolia was sister to the other Aucuba species examined, consistent with its unique fused anther locule. The divergence time of Aucuba was estimated to be approximately late Miocene. Extant Aucuba species derived from recent divergence events associated with the establishment of monsoonal climates in East Asia and climatic fluctuations.

Erratum to "MiRNA-10b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-ß/SMAD2 Signaling Pathway".

[This corrects the article on p. 1058 in vol. 9, PMID: 30574418.].

The genomic origins of the Bronze Age Tarim Basin mummies.

The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000-2800 BC from the Dzungarian Basin and 13 individuals dating to around 2100-1700 BC from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early-Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria-Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert.

Highly degenerate plastomes in two hemiparasitic dwarf mistletoes: Arceuthobium chinense and A. pini (Viscaceae).

MAIN CONCLUSION: The leafless and endophytic habitat may significantly relax the selection pressure on photosynthesis, and plastid transcription and translation, causing the loss/pseudogenization of several essential plastid-encoding genes in dwarf mistletoes. Dwarf mistletoes (Arceuthobium spp., Viscaceae) are the most destructive plant parasites to numerous conifer species worldwide. In this study, the plastid genomes (plastomes) of Arceuthobium chinense Lecomte and A. pini Hawksworth and Wiens were sequenced and characterized. Although dwarf mistletoes are hemiparasites capable of photosynthesis, their plastomes were highly degenerated, as indicated by the smallest plastome size, the lowest GC content, and relatively very few intact genes among the Santalales hemiparasites. Unexpectedly, several essential housekeeping genes (rpoA, rpoB, rpoC1, and rpoC2) and some core photosynthetic genes (psbZ and petL), as well as the rpl33 gene, that is indispensable for plants under stress conditions, were deleted or pseudogenized in the Arceuthobium plastomes. Our data suggest that the leafless and endophytic habit, which heavily relies on the coniferous hosts for nutrients and carbon requirement, may largely relax the selection pressure on photosynthesis, as well as plastid transcription and translation, thus resulting in the loss/pseudogenization of such essential plastid-encoding genes in dwarf mistletoes. Therefore, the higher level of plastome degradation in Arceuthobium species than other Santalales hemiparasites is likely correlated with the evolution of leafless and endophytic habit. A higher degree of plastome degradation in Arceuthobium. These findings provide new insights into the plastome degeneration associated with parasitism in Santalales and deepen our understanding of the biology of dwarf mistletoes.

Long noncoding RNA LYPLAL1-AS1 regulates adipogenic differentiation of human mesenchymal stem cells by targeting desmoplakin and inhibiting the Wnt/ß-catenin pathway.

Long noncoding RNAs are crucial factors for modulating adipogenic differentiation, but only a few have been identified in humans. In the current study, we identified a previously unknown human long noncoding RNA, LYPLAL1-antisense RNA1 (LYPLAL1-AS1), which was dramatically upregulated during the adipogenic differentiation of human adipose-derived mesenchymal stem cells (hAMSCs). Based on 5' and 3' rapid amplification of cDNA ends assays, full-length LYPLAL1-AS1 was 523 nt. Knockdown of LYPLAL1-AS1 decreased the adipogenic differentiation of hAMSCs, whereas overexpression of LYPLAL1-AS1 enhanced this process. Desmoplakin (DSP) was identified as a direct target of LYPLAL1-AS1. Knockdown of DSP enhanced adipogenic differentiation and rescued the LYPLAL1-AS1 depletion-induced defect in adipogenic differentiation of hAMSCs. Further experiments showed that LYPLAL1-AS1 modulated DSP protein stability possibly via proteasome degradation, and the Wnt/ß-catenin pathway was inhibited during adipogenic differentiation regulated by the LYPLAL1-AS1/DSP complex. Together, our work provides a new mechanism by which long noncoding RNA regulates adipogenic differentiation of human MSCs and suggests that LYPLAL1-AS1 may serve as a novel therapeutic target for preventing and combating diseases related to abnormal adipogenesis, such as obesity.

Lnc13728 facilitates human mesenchymal stem cell adipogenic differentiation via positive regulation of ZBED3 and downregulation of the WNT/ß-catenin pathway.

BACKGROUND: Obesity has received increasing attention because of its widespread worldwide occurrence and many threats to health. Human adipose-derived mesenchymal stem cells (hADSCs) are a critical source of adipocytes. Long noncoding RNAs (lncRNAs) play pivotal roles in cell fate determination and differentiation. The objective of the present study was to identify and investigate the function and regulatory mechanism of lncRNAs on adipogenic differentiation of hADSCs. METHODS: We used lncRNA arrays to identify the prominent differentially expressed lncRNAs before and after hADSC adipogenic differentiation and verified their biological function through antisense oligonucleotide knockdown or lentivirus overexpression. The adipogenic differentiation of hADSCs was assessed by oil red O staining as well as the mRNA and protein levels of adipogenic marker genes through qRT-PCR and western blot. Bioinformatic tool LncPro and immunofluorescence was performed to uncover the interaction between lnc13728 and ZBED3. WNT/ß-catenin signaling pathway was evaluated by western blot and immunofluorescence. RESULTS: The lncRNA arrays showed that lnc13728 expression was significantly upregulated after hADSC adipogenic differentiation and was correlated positively with the expression of the adipogenesis-related genes in human adipose tissue. Lnc13728 knockdown in hADSCs suppressed the expression of the adipogenesis-related genes at both mRNA and protein level and weakened lipid droplet production. Accordingly, lnc13728 overexpression enhanced hADSC adipogenic differentiation. Beyond that, lnc13728 co-localized with ZBED3 in the cytoplasm and regulated its expression positively. Downregulating ZBED3 had a negative effect on adipogenic differentiation, while the expression of WNT/ß-catenin signaling pathway-related proteins was upregulated. CONCLUSIONS: Lnc13728 promotes hADSC adipogenic differentiation possibly by positively regulating the expression of ZBED3 which plays a role in inhibiting the WNT/ß-catenin pathway.