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Abstract The replacement of farmland by native hygro-plants is increasingly common globally within the context of wetland ecosystem restoration. Understanding the long-term effects of this replacement on the abundance and persistence of soil organic carbon (SOC) in mountain marshes is important for soil carbon management. Here, the restored plateau mountain marshes of Duliu River Wetland Provincial Nature Reserve, China was selected. The properties, soil moisture content (SMC), pH, texture, free-form iron oxides ( $$\:{\text{F}\text{e}}_{\text{d}}$$ ), amorphous iron oxides ( $$\:{\text{F}\text{e}}_{\text{o}}$$ ), mineral-associated organic carbon (MAOC), and iron-bound organic carbon (Fe-OC) were analyzed in topsoil samples (0 ~ 20 cm) during the restoration of rice paddies to Sphagnum palustre L. wetlands for 0, 2, 10, and 20 years. Natural Sphagnum wetlands were also used as the control. We found that marsh restoration increased SMC, $$\:{\text{F}\text{e}}_{\text{o}}$$ , $$\:{\text{F}\text{e}}_{\text{d}}$$ , $$\:{\text{F}\text{e}}_{\text{o}}$$ / $$\:{\text{F}\text{e}}_{\text{d}}$$ , SOC, MAOC, Fe-OC, Fe-OC/SOC, but decreased the MAOC/SOC ratio. MAOC/SOC ratio of marshes were expectedly lower than the proportion of labile SOC in total SOC during the restoration period. SMC, SOC, and MAOC were higher in the natural Sphagnum wetlands than in other habitats. Both SOC and Fe-OC/SOC were positively correlated with SMC, $$\:{\text{F}\text{e}}_{\text{o}}$$ , $$\:{\text{F}\text{e}}_{\text{d}}$$ , $$\:{\text{F}\text{e}}_{\text{o}}$$ / $$\:{\text{F}\text{e}}_{\text{d}}$$ , and Fe-OC, but negatively correlated with soil pH. MAOC/SOC was negatively correlated with SMC, $$\:{\text{F}\text{e}}_{\text{o}}$$ and $$\:{\text{F}\text{e}}_{\text{o}}$$ / $$\:{\text{F}\text{e}}_{\text{d}}$$ . These results emphasized the significance of reconverting rice paddies to marsh wetlands for increasing the sequestration of labile SOC and Fe-OC. Further studies are required to identify and quantify the organo-mineral stabilization mechanisms of SOC at the different SOC fractionations throughout the restoration period.

Biological monitoring using environmental DNA (eDNA) technology has expanded from micro- to macro-organisms. In aquatic eDNA studies, large volumes of water need to be filtered rapidly in the field, which requires development of effective eDNA collection devices. In this study, we introduce a novel portable eDNA collection system containing a GM dual-channel water filter and a DNA extraction kit adapted to large filter membranes (ø 100 mm). The water filter is powered by a high-capacity lithium battery (9,000 mA), which operates two peristaltic pumps and maintains a continuous filtration rate of up to 1 L/min for 5 h in outdoor settings. For sample collection, the optimum conditions are still water and turbidity below 8 nephelometric turbidity units. This allows for the filtration of 10 L of water within 10 min by use of a 0.22-μm filter. Metagenomic and 12S metabarcoding sequencing showed that the DNA extraction quality and species annotation accuracy of our custom DNA extraction kit, which was tailored for this system, rivaled the performance of established kits. The GM water filter’s enrichment mode gave consistent results with vacuum filtration, which greatly reduced the filtration time for large water samples, while accurately reproducing species annotations. This innovation streamlines the eDNA collection and annotation process and offers substantial benefits for biodiversity monitoring and conservation efforts.

Purpose: The aim of this study is to investigate the impact of vibration stimulation on gingival crevicular fluid biomarkers and orthodontic tooth movement. Methods: Forty patients were randomly assigned to receive therapy with an intraoral vibration device (n = 20, AcceleDent®) or no treatment (n = 20) at a university orthodontic clinic. The quantity of fluid in the gingival sulcus, biomarkers of each fluid in the gingival sulcus, and orthodontic tooth movement were analyzed at three-time intervals (T1, T2, T3) before and after therapy (T0). Results: The results showed that vibration treatment led to higher levels of osteoclast biomarkers (RNAKL, RANKL/OPG) and inflammatory biomarkers (TNF-, IL-11, IL-18) compared to the control group. Additionally, vibration treatment at T1, T2, and T3 significantly improved tooth mobility and GCF volume. The gingival crevicular fluid biomarker levels of the T0, T1, and T2 vibration groups, as well as IL-11, IL-18, TGF-1, and TNF-α vibration groups, were significantly higher than those of the control group at different time points. Conclusion: vibration therapy was found to be closely associated with bone-breaking cells and inflammatory factor levels.

Active matter is characterized by out-of-equilibrium behaviors, offering an attractive, alternative route for revolutionizing disease diagnostics and therapy. A better understanding of how active matter interacts with cell membranes is critical to elucidating the underlying physical mechanisms and broadening the potential biomedical applications. This review provides a conceptual framework on the physiochemical mechanisms underlying active matter-biomembrane interactions. We briefly introduce the physical models of active matter and lipid membranes, and summarize the typical phenomena emerging from various active matter, including artificial active particles, cellular cytoskeletons, bacteria, and membrane proteins. Moreover, the remaining challenges and future perspectives of such non-equilibrium systems in living organisms are discussed. The findings and fundamental principles discussed in this review shed light on the rational design of activity-mediated cellular interaction, and could trigger better strategies to design and develop novel functional systems and materials toward advantageous biomedical applications.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permitted BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

Purpose: Intravitreal injection of conbercept (IVC) is a novel anti-vascular endothelial growth factor (anti-VEGF) treatment for retinopathy of prematurity (ROP). This study aimed to assess the intraocular pressure (IOP) effect of IVC.Methods: All IVC surgeries were performed in the Department of Ophthalmology, Guangdong Women and Children Hospital, from January 2021 to May 2021. In this study, 30 eyes of 15 infants who received intravitreal injections of conbercept at a dose of 0.25 mg/0.025 mL were included. The IOP of all participants was measured prior to administering the injection and subsequently at 2 min, 1 h, 1 day, and 1 week thereafter.Results: We included 30 eyes (10 boys and 5 girls) with ROP. For the male group, the mean birth weight, mean gestational age at birth, and the mean time of postmenstrual age (PMA) at IVC treatment were 1,174.0 ± 446.0 g, 28.4 ± 3.0 weeks, and 37.1 ± 1.6 weeks, respectively; for the female group, they were 1,108 ± 285.5 g, 28.2 ± 2.5 weeks, and 36.8 ± 2.1 weeks, respectively. For the male group, the IOP at baseline, 2 min, 1 h, 1 day, and 1 week after IVC were 12.4 ± 1.5 mmHg, 49.0 ± 3.1 mmHg, 26.3 ± 2.5 mmHg, 13.4 ± 2.2 mmHg, and 11.6 ± 1.7 mmHg, respectively; for the female group, they were 10.7 ± 2.0 mmHg, 47.3 ± 3.2 mmHg, 26.4 ± 3.2 mmHg, 10.7 ± 1.8 mmHg, and 10.2 ± 1.8 mmHg, respectively. In both groups, the IOP immediately (2 min) after the operation was significantly higher than that at any other time point (p < 0.01). IOP values returned to the preoperative baseline level on the first day after surgery, with no significant difference compared with that before injection (p > 0.05). IOP continued to be maintained at the preoperative baseline level on the first week after surgery, with no significant difference compared with that before surgery (p > 0.05).Conclusion: Infants with ROP who received IVC experienced a sharp increase in the IOP immediately after injection, which decreased to below 30 mmHg after 1 h and maintain that level for 1 week or longer.

Introduction: Longxuetongluo Capsule (LTC) is wildly applied to treat ischemic stroke in clinical practice in China. However, the pharmacological mechanism of LTC on ischemic stroke is still unstated. Objective: Our research was designed to study the protective effect of LTC against cerebral ischemia–reperfusion (I/R) injury and reveal the underlying mechanism both in vivo and in vitro. Methods: PC12 cells treated with glucose deprivation/reperfusion (OGD/R) were used to simulate in vitro ischemia/reperfusion (I/R) injury. The cell viability, apoptosis rate, and protein expressions of PC12 cells were evaluated. In vivo validation of the protective effect of LTC was carried out by middle cerebral artery occlusion (MCAO)/reperfusion treatment, and the underlying mechanism of its anti-apoptosis ability was further revealed by immunohistochemistry staining and Western blotting. Results: In the current study, we observed that LTC effectively inhibited oxygen-glucose deprivation/reperfusion (OGD/R) induced apoptosis of PC12 cells through suppressing the cleavage of poly ADP-ribose polymerase (PARP), caspase-3, and caspase-9. Further investigation revealed that OGD/R insult remarkably triggered the endoplasmic reticulum stress responses (ER stress) to induce PC12 cell apoptosis. LTC treatment alleviated OGD/R induced ER stress by inhibiting the activation of protein kinase RNA (PKR)-like ER kinase (PERK)/eukaryotic translation initiation factor 2 (eIF2α) and inositol requiring enzyme 1 (IRE1)/tumor necrosis factor receptor-associated factor 2 (TRAF2) pathways. Additionally, LTC also restrained the OGD/R-induced PC12 cell apoptosis by reversing the activated mitogen-activated protein kinase (MAPK) through IRE1/TRAF2 pathway. Animal studies demonstrated LTC significantly restricted the infarct region induced by middle cerebral artery occlusion (MCAO)/reperfusion, the activation of ER stress and apoptosis of neuronal cells had also been suppressed by LTC in the penumbra region. Conclusion: LTC protects the cerebral neuronal cell against ischemia/reperfusion injury through ER stress and MAPK-mediated mechanisms.

Pu-erh tea as a popular post-fermented tea displays a significant effect on improving metabolic syndrome. Puerin III (P3) had already been identified as the characteristic chemical constituent in pu-erh tea. Nevertheless, the activity of P3 on glucose and lipid metabolism disorder is still unclear. In this study, the high-fat high-sucrose diet-induced ApoE−/− mice model was employed to assess the effects of P3 in vivo. Our study found that P3 dose-dependently ameliorated the plasma total cholesterol and fasting blood glucose (FBG) in the hypercholesterolaemic and hyperglycemic ApoE−/− mice. Our investigation in vitro revealed P3 decreased the FBG by inhibiting α-glucosidase. The study on HepG2 cells also confirmed that P3 promoted the clearance of cholesterol by increasing the low-density lipoprotein receptor (LDLR) expression. This research illustrated that P3 might be the active ingredient of Pu-erh tea and a promising therapeutic option to treat glucose and lipid metabolism disorders.

Abstract Background Numerous cytokines have been proven to participate in the pathogenesis of neovascular age-related macular degeneration (nAMD). The present study aimed to investigate the aqueous humor cytokine expression profile in nAMD patients before and after ranibizumab treatments in comparison to cataract patients. Methods This prospective study included 20 treatment-naïve nAMD eyes of 20 patients who received three consecutive monthly injections of ranibizumab. Aqueous humor samples were collected before the first (baseline), second (1 month later), and third (2 months later) injections. Controls were 20 age- and gender-matched cataract patients without any other ocular disease. The aqueous concentrations of 28 cytokines were measured using a multiplex bead assay. Central macular thickness (CMT) and maximum retinal thickness (MRT)-3 mm were measured by spectral domain optical coherence tomography (SD-OCT). The greatest linear diameter (GLD) was measured by fundus fluorescein angiography (FA). Results Three cytokines in aqueous humor, including angiogenin, interleukin-36β (IL-36β), and fibroblast growth factor-acidic (FGF-α) were significantly higher in nAMD patients in comparison to cataract patients, both before and after two consecutive monthly ranibizumab injections. Compared with the nAMD patients’ basal levels, two consecutive monthly ranibizumab injections effectively reduced the aqueous concentrations of VEGF-A and placental growth factor (PlGF), as well as the values of CMT, MRT-3 mm, and GLD. Conclusions Angiogenin, IL-36β, and FGF-α have higher expression levels in nAMD patients in comparison to cataract patients, both before and after 2 months of ranibizumab therapy. These cytokines may have correlations with the pathogenesis of nAMD.

Corneal graft melting is a severe complication of keratoplasty. This review is to summarize the incidence, the pathogenesis, the risk factors, the prognosis and the prevention of corneal graft melting after keratoplasty. We systematically searched PubMed, Web of Science and WanFang database to retrieve potentially eligible articles about relevant clinical reports and animal experiments. We read the full texts to identify eligible articles. The selection of studies and data extraction were performed independently by two reviewers. In conclusion, the pathogenesis of corneal graft melting is complicated, and many risk factors are closely related to corneal graft melting. Analysis of pathogenesis and risk factors of corneal graft melting can facilitate the development of targeted therapies to better guide clinical practice.

Objective Previous investigations indicated the anticancer activity of puerarin. The current study aimed to evaluate the effect and molecular mechanisms of puerarin in chemotherapy-resistant ovarian cancer cells. Methods We examined the effects of puerarin in platinum-resistant epithelial ovarian cancer cells in vitro and in vivo . We also analyzed the molecular mechanism underlying Wnt/β-catenin inhibition and sirtuin 1 (SIRT1) regulation following puerarin treatment. Results Our study demonstrated that puerarin effectively inhibited cell growth in vitro and in vivo by increasing apoptosis in ovarian cancer cells. More importantly, puerarin sensitized cisplatin-resistant ovarian cancer cells to chemotherapy. Puerarin treatment decreased SIRT1 expression, which attenuated the nuclear accumulation of β-catenin to inhibit Wnt/β-catenin signaling. In addition, SIRT1 overexpression diminished the effects of puerarin treatment on cisplatin-resistant ovarian cancer cells. Further analysis supported SIRT1/β-catenin expression as a candidate biomarker for the disease progression of epithelial ovarian cancer. Conclusions Puerarin increased the apoptosis of platinum-resistant ovarian cancer cells. The mechanism is partly related to the downregulation of SIRT1 and subsequent inhibition of Wnt/β-catenin signaling.

Abstract Background This study aims to optimize the therapeutic regimen for refractory obstructive meibomian gland dysfunction (o-MGD) patients by combining intraductal meibomian gland probing (MGP) and intense pulsed light (IPL) to enhance their positive effects and reduce their limitations. Methods This randomized, assessor blind study includes 45 patients (90 eyes) with refractory o-MGD who were divided into 3 groups via allocation concealment: IPL (group I, received an IPL treatment course: 3 times at 3-week intervals), MGP (group II, received MGP one time), and combined MGP-IPL (group III, MGP first followed by an IPL treatment course). Standard Patient Evaluation of Eye Dryness score (SPEED), tear break-up time (TBUT), corneal fluorescein staining (CFS), meibum grade, and lid margin finding results were assessed at baseline, 3 weeks after final treatment for groups I and III, 3 and 12 weeks after MGP for group II. Six months after final treatment, the SPEED and willingness to receive any treatment again were also collected for all groups. Paired Wilcoxon, Mann-Whitney U with Bonferroni correction, and Kruskal-Wallis tests were used for data analysis. Results For all 3 groups, all previously mentioned indexes improved significantly following treatment (P

The novel coronavirus pneumonia COVID-19 is characterized by all age susceptibility, which imposes a dramatic threat to the human species all over the world. According to current available data, the cytokine storm appears to be the most life-threatening symptom of severe COVID-19 cases accompanied with lung fibrosis. Galectin-3 (Gal-3), a member of soluble β-galactoside-binding lectin families, has been implicated as a key regulator in various inflammation conditions in addition to its well-documented roles in cancer. The pro-inflammatory activity of Gal-3 in the inflammatory response and lung fibrosis of COVID-19 has been proposed by emerging studies, which suggested that inhibition of Gal-3 may represent a novel treatment approach for COVID-19 patients. Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis. ICC accounts for 10–25% of primary liver cancers with limited therapeutic options, which has higher incidence in Asian countries, particularly in China. Cancer patients, including ICC patients, are highly vulnerable to COVID-19 due to their impaired immune system. It is thus undoubtedly a challenge for our oncology department to establish effective treatment strategies under the influence of the COVID-19 crisis. According to our management procedures in the COVID-19 era, emergency treatment will be applied to ICC patients who are under life-threatening conditions, despite the COVID-19 infection. To the best of our knowledge, the modulatory function of Gal-3 in ICC is still barely explored to date. In order to evaluate the therapeutic potential of Gal-3 for ICC patients or those comprised with COVID-19, we herein report our preliminary investigation into roles of Gal-3 in ICC. Our results exhibited that the expression of Gal-3 was significantly up-regulated in ICC tissues, and a significant correlation was observed between its overexpression and malignant progression of ICC cells. We further discussed the activity and possible molecular mechanisms of Gal-3 in ICC, which may pave the ways for further exploring the possibility of Gal-3 as a potential therapeutic target for treating ICC patients or those with COVID-19-related conditions.

Purpose. To perform fundus examinations of full-term and premature infants to identify common congenital ocular diseases and determine the incidence and additional risk factors in Ningbo, China. Methods. Fundus examinations were performed on newborns between January 2017 and July 2020 in Ningbo using a RetCam3 or PanoCam LT wide-field digital imaging system. The neonates’ birth weight, gestational age, gender, delivery mode, oxygen intake, and other conditions were recorded. We compared the incidence of ocular abnormalities in both full-term newborns and premature infants. Results. There were 23,861 newborns in this study comprising 12,605 (52.8%) male and 11,256 (47.2%) female infants, 20,938 full-term babies, and 2,923 premature babies. The average gestational age was 37.9 ± 5.6 weeks, and the average birth weight was 3,189 ± 417 g. Overall, we found ocular abnormalities in 6,645 (27.8%). The most common abnormality in full-term newborns was retinal hemorrhage (RH), which we found in 3,827 (18.3%) cases. Other diseases identified included familial exudative vitreoretinopathy (FEVR), retinoblastoma (RB), and congenital cataracts. The delivery method had a significant impact on the incidence of neonatal RH (P

The dry powder inhaler is a new form of drug delivery that is widely used as an alternative to traditional drug delivery methods, addressing the shortcomings of traditional drug delivery methods and obtaining better therapeutic results. This mode of delivery is also one of the most rational ways to treat pulmonary diseases such as chronic obstructive pulmonary disease (COPD). Curcumin, a natural polyphenol, has been shown to be effective in the treatment of COPD. In this study, different concentrations of curcumin ethanol solution were spray dried with mannitol as a carrier to obtain dry powder particles with different particle size distribution for the preparation of curcumin dry powder inhaler. The solubility and physicochemical properties were further characterized by differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy. The characterization results showed that the product obtained in the experiment had reasonable particle size distribution and excellent solubility properties, which were positive for the treatment of COPD or other pulmonary diseases.

In order to achieve the controlled release of curcumin, HPMC (hydroxypropyl methyl cellulose) was spray dried with curcumin and lactose. The spray-dried materials were pressed into tablets with a diameter of 8 mm, and their release characteristics in vitro were measured. In vitro experiments showed that the release of curcumin from the HPMC mixture was significantly slower due to the sustained-release property of HPMC as a typical excipient. The release profile of curcumin from the HPMC mixture was relatively stable for a controlled release. SEM images show that the HPMC co-spray-dried powders have crumpled surfaces due to the large molecular weight of HPMC. DSC, XRD, FTIR, N2 adsorption, and TGA have been measured for the spray-dried curcumin materials. This work indicates that HPMC can be used as a controlled-release excipient for curcumin preparations.

Abstract Background Oral administration of drugs is convenient and shows good compliance but it can be affected by many factors in the gastrointestinal (GI) system. Consumption of food is one of the major factors affecting the GI system and consequently the absorption of drugs. The aim of this study was to develop a mechanistic GI absorption model for explaining the effect of food on fenofibrate pharmacokinetics (PK), focusing on the food type and calorie content. Methods Clinical data from a fenofibrate PK study involving three different conditions (fasting, standard meals and high-fat meals) were used. The model was developed by nonlinear mixed effect modeling method. Both linear and nonlinear effects were evaluated to explain the impact of food intake on drug absorption. Similarly, to explain changes in gastric emptying time for the drug due to food effects was evaluated. Results The gastric emptying rate increased by 61.7% during the first 6.94 h after food consumption. Increased calories in the duodenum increased the absorption rate constant of the drug in fed conditions (standard meal = 16.5%, high-fat meal = 21.8%) compared with fasted condition. The final model displayed good prediction power and precision. Conclusions A mechanistic GI absorption model for quantitatively evaluating the effects of food on fenofibrate absorption was successfully developed, and acceptable parameters were obtained. The mechanism-based PK model of fenofibrate can quantify the effects of food on drug absorption by food type and calorie content.

Background/aims: The pathogenesis of intrahepatic cholestasis of pregnancy (ICP) is poorly understood. Objective: This study aimed to explore the possible effect of HDAC3 (histone deacetylase) on cytokines IL-18, IL-12 and TNF-α in ICP. Methods: Serum levels of cytokines IL-18, IL-12 and TNF-α, bile acids and hepatic function parameters were measured. The expression of HDAC3 in the placenta was determined by immunohistochemistry (IHC), western blotting and RT-PCR. Results: IL-18, IL-12 and TNF-α serum levels were significantly higher in the severe ICP group than in the mild ICP group and the control group, and the difference between the mild ICP group and control group was not significant. HDAC3 protein expression was identified in the nucleus of the placental trophoblast by IHC. HDAC3 mRNA and protein expression were significantly lower in the ICP groups (mild ICP and severe ICP groups) than in the control groups, and no significant difference was found between the mild ICP and severe ICP groups. Conclusions: The low expression of HDAC3 and overexpession of inflammatory cytokines (IL-18, IL-12 and TNF-α) in ICP may be involved in liver cell apoptosis. We suspect that HDAC3 may play an important role in the pathophysiology of ICP.

In the human fungal pathogen Cryptococcus neoformans, sex can benefit its pathogenicity through production of meiospores, which are believed to offer both physical and meiosis-created lineage advantages for its infections. Cryptococcus sporulation occurs following two parallel events, meiosis and differentiation of the basidium, the characteristic sexual structure of the basidiomycetes. However, the circuit integrating these events to ensure subsequent sporulation is unclear. Here, we show the spatiotemporal coordination of meiosis and basidial maturation by visualizing event-specific molecules in developing basidia defined by a quantitative approach. Monitoring of gene induction timing together with genetic analysis reveals co-regulation of the coordinated events by a shared regulatory program. Two RRM family regulators, Csa1 and Csa2, are crucial components that bridge meiosis and basidial maturation, further determining sporulation. We propose that the regulatory coordination of meiosis and basidial development serves as a determinant underlying the production of infectious meiospores in C. neoformans.

Macrophage-derived foam cell formation is considered to be a critical step in the development of atherosclerosis. In the present study, we evaluated the inhibitory effect of Ilex kudingcha triterpenoid-enriched extract (TS) on foam cell formation in THP-1 macrophage cells induced by aggregated low density lipoproteins (agLDL). The inhibition of low density lipoprotein receptor-related protein 1 (LRP1) expression after TS treatment is based on mRNA and protein levels in the macrophages. These inductions were sterol regulatory element-binding protein-2 (SREBP2) dependent. On the basis of the in vitro results, the anti-atherosclerotic activity of TS was investigated in ApoE−/− mice fed a high-fat diet, ApoE−/− mice treated with TS for 6 weeks appeared to have decreased aortic sinus lesions compared with the untreated mice. These findings indicate that TS may act as an anti-atherosclerotic agent.

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

BACKGROUND:Recent advances in stem cell technology afford an unlimited source of neural progenitors and glial cells for cell based therapy in central nervous system (CNS) disorders. However, current differentiation strategies still need to be improved due to time-consuming processes, poorly defined culture conditions, and low yield of target cell populations. METHODOLOGY/PRINCIPLE FINDINGS:This study aimed to provide a precise sequential differentiation to capture two transient stages: neural epithelia-like stem cells (NESCs) and oligodendrocytes progenitor cells (OPCs) derived from mouse embryonic stem cells (ESCs). CHIR99021, a glycogen synthase kinase 3 (GSK-3) inhibitor, in combination with dual SMAD inhibitors, could induce ESCs to rapidly differentiate into neural rosette-like colonies, which facilitated robust generation of NESCs that had a high self-renewal capability and stable neuronal and glial differentiation potentials. Furthermore, SHH combined with FGF-2 and PDGF-AA could induce NESCs to differentiate into highly expandable OPCs. These OPCs not only robustly differentiated into oligodendrocytes, but also displayed an increased migratory activity in vitro. CONCLUSIONS/SIGNIFICANCE:We developed a precise and reliable strategy for sequential differentiation to capture NESCs and OPCs derived from ESCs, thus providing unlimited cell source for cell transplantation and drug screening towards CNS repair.