Rate Of Outgrowth Research Articles

From Germination to Stagnation: Sodium Diacetate's Impact on Clostridium perfringens Spores

ABSTRACTSodium diacetate is recognized for its high efficiency as a preservative, demonstrating strong antibacterial properties that help extend the shelf life of food products. However, it is still unclear how it influences spores. This research investigated the influence of sodium diacetate on the germination and outgrowth stage in the revival process of Clostridium perfringens (C. perfringens) spores. The germination and outgrowth rate of spores after sodium diacetate treatment were measured. The changes of spore inner membrane were monitored by laser confocal microscope, electron scanning microscope and macromolecular leakage. The interaction between sodium diacetate and DNA was investigated by EB fluorescence probe, and the finding was verified by the outgrowth of spores in ham sausage. An investigation into the effects of sodium diacetate treatment on the biological activity, inner membrane permeability, morphology, and DNA integrity of spores devoid of both the coat and cortex revealed that sodium diacetate significantly hindered the transition of germinated spores into vegetative cells. In the presence of sodium diacetate, C. perfringens spores underwent germination, characterized by the hydrolysis of the spore cortex and the disassembly of the spore coat. As the spores advanced into the outgrowth phase, sodium diacetate penetrated their structure, causing damage to the inner membrane and compromising DNA integrity. Moreover, sodium diacetate was demonstrated to effectively inhibit spore outgrowth in ham sausage. This study provided theoretical guidance and references for the application of sodium diacetate in food to control the germination and outgrowth of spore‐forming bacteria.

Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Brief Electrical Stimulation Promotes Recovery after Surgical Repair of Injured Peripheral Nerves.

Injured peripheral nerves regenerate their axons in contrast to those in the central nervous system. Yet, functional recovery after surgical repair is often disappointing. The basis for poor recovery is progressive deterioration with time and distance of the growth capacity of the neurons that lose their contact with targets (chronic axotomy) and the growth support of the chronically denervated Schwann cells (SC) in the distal nerve stumps. Nonetheless, chronically denervated atrophic muscle retains the capacity for reinnervation. Declining electrical activity of motoneurons accompanies the progressive fall in axotomized neuronal and denervated SC expression of regeneration-associated-genes and declining regenerative success. Reduced motoneuronal activity is due to the withdrawal of synaptic contacts from the soma. Exogenous neurotrophic factors that promote nerve regeneration can replace the endogenous factors whose expression declines with time. But the profuse axonal outgrowth they provoke and the difficulties in their delivery hinder their efficacy. Brief (1 h) low-frequency (20 Hz) electrical stimulation (ES) proximal to the injury site promotes the expression of endogenous growth factors and, in turn, dramatically accelerates axon outgrowth and target reinnervation. The latter ES effect has been demonstrated in both rats and humans. A conditioning ES of intact nerve days prior to nerve injury increases axonal outgrowth and regeneration rate. Thereby, this form of ES is amenable for nerve transfer surgeries and end-to-side neurorrhaphies. However, additional surgery for applying the required electrodes may be a hurdle. ES is applicable in all surgeries with excellent outcomes.

The effect of vitamin D on neurite length and neurite elongation velocity during development

AbstractBackgroundStudies have shown that vitamin D plays an important role in the brain (1). In an animal model of nerve trauma, vitamin D2 (ergocalciferol) was shown to be a potent neuromodulatory compound that increases axogenesis, axon diameter, and physiological maturation (2). In addition, the administration of high‐dose vitamin D3 (cholecalciferol) has been demonstrated to enhance the number of preserved or newly formed axons at the proximal end, axon diameter at the distal end, and neurite myelination at both the distal and proximal ends in another animal model of nerve trauma (3). Vitamin D has been shown to increase neurite outgrowth when added to cultured hippocampal cells. However, when removed from the diet of pregnant rats, decreased expression of NGF (neuron growth factor (NGF) was observed in the brains of newborns and adults. Recently, vitamin D has been shown to increase the expression of microtubule‐associated protein‐2 (MAP2), axonal growth‐associated protein‐43 (GAP43), and synapsin‐1 protein in rat cortical neurons (4). Based on this knowledge, we sought to determine whether vitamin D treatment during development might affect neurite length, and elongation velocity.MethodIn our study, primary cortical neuron culture was performed using 16‐day‐old rat embryos. 10−8 M 1,25‐dihydroxyvitamin D3 was applied 24 hours after the culture. Following vitamin D administration, neurite length measurements were made at 30‐second intervals for 30 min and at half‐hour intervals for 12 h under a live cell imaging microscope at 20X magnification.ResultThe results of our study showed that 30 min after vitamin D administration, neurite length and elongation velocity significantly increased in the vitamin D group compared to the control group (p = 0.0029 and p = 0.0310, respectively). In addition, the neurite outgrowth rate and neurite length increased in the vitamin D group 12 h after vitamin D administration compared to the control group (p = 0.0275, p = 0.0398, respectively).ConclusionOnly a few research have examined at how vitamin D affects neurite outgrowth. As a result of vitamin D administration in primary cortical neurons during the developmental phase, our study’s results demonstrated that neurite length and velocity increase in the first 12 hours.

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

Extracellular Vesicle MicroRNAs From Corneal Stromal Stem Cell Enhance Stemness of Limbal Epithelial Stem Cells by Targeting the Notch Pathway.

The limbal niche supports the self-renewal of limbal epithelial stem cells (LESCs). The corneal stromal stem cell (CSSC) is an important component in the niche that regulates the LESC phenotype. However, the intercellular communication between LESCs and CSSCs has yet to be elucidated. A traditional two-dimensional (2D) system, a direct three-dimensional (3D) system, and an indirect 3D coculture system of LESCs and CSSCs were used to elucidate the paracrine pathway effect of CSSCs on LESCs. To reveal the impact of CSSC derived extracellular vesicles (CSSC-EVs) on LESCs, GW4869 and CSSC-EVs were added separately to the LESC culture medium. The outgrowth rate, cell density, differentiation, and stemness maintenance were compared among these methods. The miRNAs in the CSSC-EVs were sequenced, and the targeted Notch pathway was further confirmed by RT‒qPCR and Western blotting. Compared with 2D culture, both the direct and indirect 3D coculture systems yielded a higher outgrowth rate and expression of stem cell markers of LESCs. The phenotypes of LESCs cultivated using the two coculture approaches were also comparable. Nevertheless, GW4869 inhibited the effect of CSSCs on LESCs, and the addition of CSSC-EVs to the 2D culture system could increase cell density, and the proportion of p63αbright cells, which indicated that CSSC-EVs were crucial in regulating LESCs. Furthermore, the EV-AlixKD with reduced miRNA partly lost its regulating function. The abundant miRNAs in CSSC-EVs, such as hsa-miR-663b, hsa-miR-16-5p, and hsa-miR-1290, target the Notch pathway. The LESCs transfected with miR-663b had higher p63 expression via downregulating of the Notch pathway. CSSC-EV played an important role in promoting LESC proliferation and stemness maintenance by targeting Notch signaling via miRNAs, which will increase our understanding of the limbal niche and provide a potential new approach for LESC culture and the treatment of corneal epithelial disorders.

Guided axon outgrowth of neurons by molecular gradients generated from femtosecond laser-fabricated micro-holes

ObjectiveTransplantation of scaffold-embedded guided neurons has been reported to increase neuronal regeneration following brain injury. However, precise axonal integration between host and transplant neurons to form functional synapses remains a major problem. Thus, a high-precision tool to actuate neuronal axon outgrowth in real-time conditions is required to attain robust axon regeneration. This study aims to establish a microfluidic platform for precise and real-time axon outgrowth guidance. Methods: A microfluidic device with a 4 μm thick thin-glass sheet as the neuron culture substrate is fabricated. Surface of the glass sheet is chemically modified to facilitate neuron attachment. Femtosecond (fs) laser is used to engrave the glass sheet to achieve micro-holes, where netrin-1 is released for directing the movement of the neuronal axon. Results: Numerical simulation and experimental data demonstrate that netrin-1 gradient is formed after it passes through the micro-hole. The neuronal response results show the outgrowth rate of the axon is significantly increased by netrin-1 gradient. Furthermore, a majority of neuronal axons exhibit guided outgrowth characterized by positive turning angles of axon displacement in the direction of netrin-1 gradients. Conclusion: Integrating fs laser and microfluidic device facilitates controlled and instantaneous axon outgrowth in a non-invasive manner. Significance: The developed real-time microfluidic platform shows potential in the application for on-site neuronal transplantation, which is significant for the treatment of a range of neurological disorders and injuries.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor β (TGFβ), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response.

An environmentally responsive root system is crucial for plant growth and crop yield, especially in sub-optimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density whilst simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture. Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here we describe a search for proteins that regulate root system architecture by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co-purifying proteins. Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here we show that, in Arabidopsis, AZG1 is a cytokinin import protein that co-localizes with and stabilizes PIN1, linking auxin and cytokinin transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1-dependent under salt stress. This report therefore identifies a potential point for auxin:cytokinin crosstalk which shapes root system architecture in response to NaCl.

Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1A4V mutation

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by degeneration of upper and lower motor neurons that leads to muscle weakness, paralysis, and death, but the effects of disease-causing mutations on axonal outgrowth of neurons derived from human induced pluripotent stem cells (iPSC)-derived motor neurons (hiPSC-MN) are poorly understood. The use of hiPSC-MN is a promising tool to develop more relevant models for target identification and drug development in ALS research, but questions remain concerning the effects of distinct disease-causing mutations on axon regeneration. Mutations in superoxide dismutase 1 (SOD1) were the first to be discovered in ALS patients. Here, we investigated the effect of the SOD1A4V mutation on axonal regeneration of hiPSC-MNs, utilizing compartmentalized microfluidic devices, which are powerful tools for studying hiPSC-MN distal axons. Surprisingly, SOD1+/A4V hiPSC-MNs regenerated axons more quickly following axotomy than those expressing the native form of SOD1. Though initial axon regrowth was not significantly different following axotomy, enhanced regeneration was apparent at later time points, indicating an increased rate of outgrowth. This regeneration model could be used to identify factors that enhance the rate of human axon regeneration.

Repetitive Bleomycin-Based Electrochemotherapy Improves Antitumor Effectiveness in 3D Tumor Models of Conjunctival Melanoma.

Conjunctival melanoma (CM) is associated with a high rate of local recurrence and poor survival rate. Novel therapeutic options are needed to reduce recurrence rate. The objective of the study was to demonstrate the improved effectiveness of electrochemotherapy (ECT) on CM using repetitive application. Tumor spheroids of three CM cell lines (CRMM1, CRMM2, CM2005.1) were treated repetitively with ECT using the chemotherapeutic agent bleomycin on days 3, 5, and 7 of culture. Application of bleomycin alone and electroporation alone served as controls. The cytotoxic effect was analyzed on day 10 compared to untreated control using an independent t-test. The spheroid outgrowth rate was measured. CM tumor spheroid size (median value: 78%, SD: 32%) and viability (median value: 11%, SD: 11%) were dramatically reduced after repetitive ECT treatment (p-value < 0.001). Decreased proliferation capacity (down to 8%) and an increase of apoptotic cells were observed. In most repetitive ECT-treated spheroids, no viable or proliferating cells were detected. Only 33-40% of repetitive ECT-treated spheroids exhibited single outgrowing cells with a delay of time up to 38 days. Repetitive ECT application effectively induces cytotoxic effects in CM spheroids by inducing apoptosis, inhibiting proliferation and decreasing the percentage of surviving tumor cells. Thus, repetitive ECT results in improved antitumor effectiveness in CM and could be an alternative therapy option.

Identification of Genes Associated with Liver Metastasis in Pancreatic Cancer Reveals PCSK6 as a Crucial Mediator

Liver metastasis occurs frequently in patients with pancreatic cancer. We analyzed the molecular profiling in liver metastatic lesions aiming to uncover novel genes responsible for tumor progression. Bioinformatics analysis was applied to identify genes directing liver metastasis. CRISPR/Cas9 technology was used to knock out the candidate gene. Proliferation assays, colony formation assays, cell cycle analysis, migration assays, wound healing assays, Immunofluorescence analysis, and the tumor xenograft model of intrasplenic injection were adopted to evaluate the effects of PCSK6 inactivation on cell growth, migration and liver metastasis. GSEA and Western blot were used to investigate the corresponding signaling pathway. PCSK6 was one of the obtained liver-metastasis-related genes in pancreatic cancer. PCSK6 inactivation inhibited cell growth and cell migration, due to G0/G1 cell cycle arrest and the remodeling of cell-cell junctions or the cell skeleton, respectively. PCSK6 inactivation led to fewer counts and lower outgrowth rates of liver metastatic niches in vivo. The Raf-MEK1/2-ERK1/2 axis was repressed by PCSK6 inactivation. Accordingly, we found PCSK6 inactivation could inhibit cell growth, cell migration, and liver metastasis, and explored the role of the Raf-MEK1/2-ERK1/2 axis in PCSK6 inactivation. PCSK6-targeted therapy might represent a novel approach for combatting liver metastasis in pancreatic cancer.

P-237 Non-invasive metabolic live cell imaging of early embryo development using adapted confocal microscopy; a safety study

Abstract Study question Is it safe to use metabolic imaging to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence during embryo development using adapted confocal microscopy? Summary answer Non-invasive metabolic imaging is safe as no differences were observed between controls and illuminated embryos in terms of embryo development, blastocyst formation and implantation potential. What is known already Developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to cell death or abnormal embryo development and low implantation potential. This could potentially be predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging in embryos has been investigated through complex methodologies, however, scientific evidence for its utility during embryo development using simple technology remains unexplored. Measurements of metabolic activity could be a useful tool as the auto-fluorescence of molecules such as NADH is a straightforward representation of mitochondrial function. Study design, size, duration Super-ovulated female mice (n = 30) were subjected to mating with 10 males. In-vivo produced embryos collected at the 2-cell stage were divided in control group (n = 151), sham control group (n = 151) and illuminated group (n = 152). Illuminated embryos were assessed for NADH levels during embryo development every 3 hours using arbitrary units of autofluorescence (AU). Produced blastocysts were assessed for total cell and inner-cell-mass (ICM) number (Oct4 immuno-staining) and implantation potential through outgrowth assays in separate experiments. Participants/materials, setting, methods F1 (CBA/C57Bl6) mouse strain was used. NADH auto-fluorescence levels were measured during embryo development using adapted confocal microcopy (Olympus FV1200). A confocal Z-stacking function was used to record 15 focal planes using a 20x/0.95NA air objective of entire embryos, opening the confocal pinhole system completely. Then, images were collected and analysed using FIJI software (version: 2.0.0-rc-69/1.52n;ImageJ). Blastocyst cell number, formation rates and outgrowth rates for 4 days post blastocyst formation were compared between study groups. Main results and the role of chance Embryo culture experiments showed no significant differences in blastocyst formation rates between study groups (Control: 71.7%; Sham: 64.9%; Illuminated 71.7%; p &amp;gt; 0.05). Similarly, the total number of cells (Control: 82.9±5.6; Sham: 76.5±3.3; Illuminated: 77.1±4.2; ± Standard error of mean [SEM]) and ICM cells (Control: 10.8±1.3; Sham: 9.4±0.7; Illuminated: 11.9±0.8; ± SEM) did not differ between groups (p &amp;gt; 0.05). Outgrowth assays presented similar outgrowth areas during day5 to day8 post-blastocyst development between study groups (p &amp;gt; 0.05). Illuminated embryos presented significantly different NADH activity levels during embryo development, particularly between the 2-cell stage (987.1±36.2AU), morulae stage (1226±31.5AU) and blastocyst stage (649±42.9AU; ± SEM; p &amp;lt; 0.05). Embryos that did not reach the blastocyst stage presented a significantly different NADH activity profile during embryo development compared to those that did(p &amp;lt; 0.05). Additionally, abnormal embryos also presented significantly decreased NADH activity levels at the 2-cell stage (Normal: 987.1±36.2; abnormal: 726.9±121.7AU; p &amp;lt; 0.05) to the morulae stage (Normal: 1226±31.5; Abnormal:893.3±189AU; p &amp;lt; 0.05). Our study indicates that measuring NADH activity levels during early embryo development present no negative effects in embryo developmental rates, blastocyst formation and implantation potential. Thus, non-invasive measurements of NADH could be applied to determine embryo metabolic activity during embryo development using simple technology and imaging techniques. Limitations, reasons for caution The study was conducted using a mouse model focused in early embryo development and implantation potential. Thus, studies on live birth are required to fully assess safety to further validate potential wider applications. Validation in ageing models is also required to assess potential applications for embryo selection. Wider implications of the findings Non-invasive measurements of metabolic activity could be applied to determine embryo metabolic activity using simple and safe technology. Further applications could link the use of simple non-invasive metabolic imaging with the latest time-lapse technology and artificial intelligence applications. Trial registration number N/A

Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro

To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells.

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

The retinal vascular growth rate in babies with retinopathy of prematurity could indicate treatment need.

Purpose:To analyze the weekly rate of retinal vascular growth in treatment-naïve babies with various stages of retinopathy of prematurity (ROP) and validate if this could be a predictor of treatment need.Methods:Retrospective review of medical charts and retinal images of babies with various stages of ROP. The images were enhanced using red-green image enhancement software. Using the length of the horizontal disc diameter (DD) of each eye, the vessel growth was measured from the disc margin up to the vessel tip in fixed quadrants. The rate of vessel growth was the ratio of vessel length to the number of weeks it took to reach this length. The babies were divided into treatment warranting ROP (group 1), low-risk pre-threshold (type II) ROP (group 2,), and no-ROP (group 3) for analysis. The “no-ROP” group acted as normal control. Group 1 was further subdivided into 1A (threshold ROP), IB (aggressive posterior ROP), 1C (hybrid ROP), and ID (high-risk pre-threshold ROP).Results:Out of 436 eyes, groups 1, 2, and 3 had 238, 108, and 90 eyes, respectively. The mean rate of vascular outgrowth along with 95% confidence interval (CI) was 0.490 [0.487,0.520], 0.612 [0.599, 0.638], and 0.719 [0.703, 0.740] DD/week, respectively, for babies with “treatment warranting,” “low risk pre-threshold” and “no ROP” groups, respectively. In our estimate, more than 80% of eyes with a vessel growth rate of 0.54 DD/week or less required treatmentConclusion:A rate of retinal vascular growth less than 0.54 DD/week can be used to determine treatment requirements in babies with ROP.

Three plant growth-promoting rhizobacteria alter morphological development, physiology, and flower yield of Cannabis sativa L.

The beneficial phytomicrobiome is a sustainable approach with the potential to enhance plant growth; it has been evaluated for a number of crop species, but not for Cannabis sativa. The legalization of cannabis and awareness of its end-use applications has resulted in the renewed consumer demand. An important challenge is achievement of high yield with minimum input for indoor production. This study evaluated three individual plant growth-promoting rhizobacteria (Bacillus sp., Mucilaginibacter sp. and Pseudomonas sp.) on the root development and subsequent plant growth of cannabis (cv. CBD Kush) cuttings. The hypothesis tested was that the application of plant growth-promoting rhizobacteria would improve rooting speed of cuttings, yield attributes, and physiological variables. When compared with control plants (mock inoculation with MgSO4), plants inoculated with plant growth-promoting rhizobacteria increased root length at the vegetative stage. At harvest, flower fresh weight was increased by 5.13%, 6.94% and 11.45%, compared to the control, for plants inoculated with Bacillus sp., Mucilaginibacter sp. and Pseudomonas sp. respectively. However, the plant height, node number, branch number and leaf area of plants treated with plant growth-promoting rhizobacteria were rarely different from the control group. Inoculation with Pseudomonas sp. resulted in the greatest increase in photosynthetic rate during the vegetative and reproductive growth stages, and harvest index, while Bacillus sp., and Mucilaginibacter sp. increased flower number and axillary bud outgrowth rate. Because cannabinoids composition is the one of most important attributes of cannabis flowers, future research should investigate the effects of plant growth-promoting rhizobacteria on flower secondary metabolite profile at harvest.

Engineering Fiber-Based Nervous Tissue Constructs for Axon Regeneration

Biomaterial-based scaffolds used in nerve conduits including channels for confining regenerating axons and 3-dimensional (3D) gels as substrates for growth have made improvements in models of nerve repair. Many biomaterial strategies, however, continue to fall short of autologous nerve grafts, which remain the current gold standard in repairing severe nerve lesions (<20 mm). Intraluminal nerve conduit fibers have also shown considerable promise in directing regenerating axons in vitro and in vivo and have gained increasing interest for nerve repair. It is unknown, however, how growing axons respond to a fiber when encountered in a 3D environment. In this study, we considered a construct consisting of a compliant collagen hydrogel matrix and a fiber component to assess contact-guided axon growth. We investigated preferential axon outgrowth on synthetic and natural polymer fibers by utilizing small-diameter microfibers of poly-L-lactic acid and type I collagen representing 2 different fiber stiffnesses. We found that axons growing freely in a 3D hydrogel culture preferentially attach, turn and follow fibers with outgrowth rates and distances that far exceed outgrowth in a hydrogel alone. Wet-spun type I collagen from rat tail tendon performed the best, associated with highly aligned and accelerated outgrowth. This study also evaluated the response of dorsal root ganglion neurons from adult rats to provide data more relevant to axon regenerative potential in nerve repair. We found that ECM treatments on fibers enhanced the regeneration of adult axons indicating that both the physical and biochemical presentation of the fibers are essential for enhancing axon guidance and growth.

Prevalence of allergic disorders and risk factors associated with food allergy in Turkish preschoolers.

The prevalence of allergic disorders is on the rise, affecting about 10% of the population. In this retrospective cohort, we investigated prevalence of allergic disorders, associated risk factors, and the outcome of food allergies. We analyzed data from birth cohorts of two university hospitals' well-child outpatient clinics. Factors related to onset and type of allergic diseases were assessed from demographic, socioeconomic, and clinical data. Analyses were performed on 949 (431F/518M) infants at a mean current age of 28±6 months. Any allergic disease was established among 177 cases (22%); atopic dermatitis in 123 (12.8%), respiratory allergies in 55 (5.7%), and food allergy in 41 (4.3%). The risk for allergic disorders was found to be significantly increased for male gender (OR: 2.31, 95% CI; 1.54-3.46), and positive parental atopy (OR: 1.94, 95% CI; 1.31-2.86). The risk of food allergies was significantly higher in the male gender (OR: 2.47, 95% CI; 1.21-5.02), who consumed egg-white between 6 and 12 months (OR: 2.34, 95% CI; 1.22-4.48), and who were formula-fed before 6 months (OR: 2.16, 95% CI; 1.14-4.10). We found no significant association between the rate of food allergy outgrowth or food induced-anaphylaxis with regards to the timing of introducing egg-white into the diet. Although the introduction of egg-white into infant diet at 6-12 months of life appeared as an independent risk for any food allergy, none of the patients developed anaphylaxis. Age at symptom onset and outgrowing food allergy were similar compared to those introduced egg-white after 12 months. We recommend promoting exclusive breastfeeding during the first 6 months of life, and avoidance of prolonged restrictive diets for children with food allergy.

A constant pool of Lgr5+ intestinal stem cells is required for intestinal homeostasis

Lgr5+ crypt base columnar cells, the operational intestinal stem cells (ISCs), are thought to be dispensable for small intestinal (SI) homeostasis. Using a Lgr5-2A-DTR (diphtheria toxin receptor) model, which ablates Lgr5+ cells with near-complete efficiency and retains endogenous levels of Lgr5 expression, we show that persistent depletion of Lgr5+ ISCs in fact compromises SI epithelial integrity and reduces epithelial turnover invivo. Invitro, Lgr5-2A-DTR SI organoids are unable to establish or survive when Lgr5+ ISCs are continuously eliminated by adding DT to the media. However, transient exposure to DT at the start of culture allows organoids to form, and the rate of outgrowth reduces with the increasing length of DT presence. Our results indicate that intestinal homeostasis requires a constant pool of Lgr5+ ISCs, which is supplied by rapidly reprogrammed non-Lgr5+ crypt populations when preexisting Lgr5+ ISCs are ablated.