Incubator Chamber Research Articles

ОПРЕДЕЛЕНИЕ ИНТЕНСИВНОСТИ ВЕНТИЛЯЦИИ ИНКУБАТОРА ПО ДИНАМИКЕ РАСХОДА ВОДЫ В УВЛАЖНИТЕЛЕ

It is known that the air composition in the incubator chamber is critically important for the development of bird embryos, since the embryo consumes oxygen and produces carbon dioxide during development. High embryonic mortality is observed with insufficient ventilation of the incubator chamber, however, excessive ventilation leads to unreasonable costs of electricity and water in the humidification system. Gas analyzers that allow precise regulation of gas exchange between the incubator chamber and the surrounding air are not used in tabletop incubators, and the problem of determining the optimal ventilation parameters for tabletop incubators remains relevant at present. The possibility of interpreting data on water consumption by humidification systems of tabletop incubators in order to determine the characteristics of oxygen and carbon dioxide gas exchange between the air in the incubator chamber and the environment was studied. Grumbach tabletop incubators, a carbon dioxide detector based on the ZGm053UK IR sensor, and an AT-8100 electrochemical oxygen detector were used. It was established that the dynamics of water consumption of the incubator humidification system correlates with the volume of gas exchange between the incubator and the environment. The danger of stopping the respiratory activity of embryos occurs in sealed incubators, while the marker of the absence of gas exchange is a constant water level in the humidification system. With the rate of water evaporation by the evaporator of at least 3 cm3 / day per 1 chicken egg, the difference in relative humidity in the incubator room and the incubator chamber of at least 10% and the difference in temperatures in the incubator and the incubator room of at least 10 ° C, the gas exchange of the incubator with the room provides a carbon dioxide level of no more than 3000 ppm, and an oxygen level of at least 20.6%. The air composition in the incubator room has a direct effect on the air composition in the incubator, however, the required ventilation level of the incubator room, providing satisfactory air quality, can be determined organoleptically.

Drug pair of Cornus officinalis and Radix achyranthis bidentatae improves renal injury of hypertension by regulating metabolic reprogramming mediated by eNOS

ObjectiveTo explore the effects and possible mechanisms of the drug pair Cornus officinalis and Radix achyranthis bidentatae (SYR-NX) on improving hypertensive kidney damage. MethodSYR-NX, a formulation of Cornus officinalis and Radix Achyranthis Bidentatae with a dose ratio 1:2.5, was used in this experiment. We investigated the effects of SYR-NX on spontaneously hypertensive rats (SHR) fed with a high-salt diet and Human Kidney-2 (HK2) cells exposed to hypoxia. After 8 weeks of treatment with SYR-NX, blood pressure was tested, and β 2-Microglobulin(β2-MG), blood creatinine (S-cr), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH), M2 pyruvate kinase (PKM2), adenosine triphosphate (ATP), pyruvate, lactate, connective tissue growth factor (CTGF) and tumor necrosis factor-α (TNF-α)were measured. HK2 cells pre-treated with SYR-NX were cultured in a three-gas hypoxic incubator chamber (5 % CO2, 1 % O2, 94 % N2) for 12 h, and then eNOS, PKM2, NADPH, ATP, pyruvate, lactate, CTGF and TNF-α were assessed. ResultsSYR-NX significantly reduced SBP, DBP, β2-MG, S-cr, PKM2, pyruvate, lactate, CTGF and TNF-α, and increased eNOS, NADPH, and ATP. ConclusionSYR-NX can regulate metabolic reprogramming through eNOS and improves hypertensive kidney injury.

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells.

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs.

Design of Incu Analyzer for IOT based Baby Incubator Calibration (Chamber Temperature)

Incubator Analyzer is a calibration tool used to measure temperature, mattress temperature, humidity, airflow, and sound level in baby incubators. The purpose of this research is to make a tool "Design of Incu Analyzer for IOT based Baby Incubator Calibration (Chamber Temperature)” with LCD display and Thingspeak. The design of this calibration tool consists of baby incubator chamber temperature parameters consisting of 5 points, namely T1, T2, T3 , T4, T5 using the DS18B20 sensor and using the ESP32 microcontroller to take advantage of the IOT system. Utilization of IOT on ESP32 is using Wi-Fi. The ESP32 microcontroller processes data from the DS18B20 temperature sensor which is then displayed on the LCD display and Thingspeak. Testing of this tool is done by comparing incubator analyzer module with a standard measuring instrument, INCU II. In this study, the temperature parameter obtained the smallest error value of -0,059293% at T5 with a setting temperature of 36°C and the largest error value of -0,0254188% at T2 with a setting temperature of 35°C. Overall the conclusions obtained after carrying out the literature study process, and planning, it can be concluded that the tool "Incu Analyzer Design for IOT based Baby Incubator Calibration" can be used according to the plan, namely Incubator Analyzer with IOT based.

Exploring the Role of Drug Repurposing in Bridging the Hypoxia-Depression Connection.

High levels of oxidative stress are implicated in hypoxia, a physiological response to low levels of oxygen. Evidence supports a connection between this response and depression. Previous studies indicate that tryptophan hydroxylase can be negatively affected in hypoxia, impairing serotonin synthesis and downstream pathways. Some studies also hypothesize that increasing hypoxia-inducible factor-1 (HIF-1) levels may be a new therapeutic modality for depression. Hence, this study delved into the influence of hypoxia on the cellular response to drugs designed to act in depression. By the induction of hypoxia in SH-SY5Y cells through a hypoxia incubator chamber or Cobalt Chloride treatment, the effect of Mirtazapine, an antidepressant, and other drugs that interact with serotonin receptors (TCB-2, Dextromethorphan, Ketamine, Quetiapine, Scopolamine, Celecoxib, and Lamotrigine) on SH-SY5Y cellular viability and morphology was explored. The selection of drugs was initially conducted by literature search, focusing on compounds with established potential for employment in depression therapy. Subsequently, we employed in silico approaches to forecast their ability to traverse the blood-brain barrier (BBB). This step was particularly pertinent as we aimed to assess their viability for inducing potential antidepressant effects. The effect of these drugs in hypoxia under the inhibition of HIF-1 by Echinomycin was also tested. Our results revealed that all the potential repurposed drugs promoted cell viability, especially when hypoxia was chemically induced. When combined with Echinomycin, all drugs decreased cellular viability, possibly by the inability to interact with HIF-1.

Monitoring Baby Incubator Central through Internet of Things (IoT) based on Raspberry Pi Zero W with Computer Monitoring)

Premature infants, with a gestational age of 37 weeks or less and weighing less than 2500 grams, face challenges in adapting to the outside world due to the underdeveloped state of their organ systems. Special care is essential to support their growth and development, including maintaining optimal temperature, humidity, and oxygen levels similar to those experienced in the mother's womb. Baby incubators play a crucial role in providing these conditions. This study aims to develop a convenient monitoring tool for midwives and healthcare workers responsible for multiple baby incubators. The tool operates independently and is compatible with various brands of baby incubators. Leveraging an IoT system, it utilizes ESP32 and Raspberry Pi Zero W modules for data transmission. The tool incorporates a DS18B20 sensor for monitoring skin temperature and a DHT22 sensor for monitoring the temperature inside the incubator chamber. Using a pre-experimental design with an after-only research design, the tool's measurements are compared against an incubator analyzer. The study reveals that the developed module still exhibits some measurement errors. However, despite these limitations, the research is expected to greatly assist medical personnel in simultaneously monitoring multiple baby incubators.
 Keywords : Premature infants, Baby incubators, IoT, Temperature, ESP32, Raspberry Pi Zero W

P-363 Ovarian endometriosis deteriorates embryo development in ART cycles: An embryonic time-lapse study

Abstract Study question Does ovarian endometriosis deteriorate embryo quality in terms of morphokinetic parameters determined by time-lapse images? Summary answer Embryos from ovarian endometriosis patients accelerate their early developmental events but decelerate after t5. Completion of late developmental events were significantly delayed in endometriosis group. What is known already Endometriosis may affect the oocyte quality and consequently affect the embryo quality in terms of embryo morphology and implantation ability. Morphokinetic evaluation of embryos provided more developmental details and might be utilised as a tool to distinguish the embryo developmental ability. Only scarce studies with limited case numbers have evaluated the association between endometriosis and developments of embryo in terms morphokinetic observation. Hardly any consensus has ever been drawn. Study design, size, duration The morphokinetic parameters of embryos were compared between patients with or without ovarian endometriosis. A total of 52 ovarian endometriosis cases and 208 controls without endometriosis, receiving their assisted reproductive techniques from April 2020 through June 2022, were included for analysis. Propensity Score Matching with age, BMI and AMH was performed in a ratio of 1:4 in order to eliminate the confounding factors. Participants/materials, setting, methods We analyzed a total of 737 top quality embryos obtained from 348 oocyte retrieval cycles performed at Taipei Fertility Center. 127 embryos were obtained from women affected by ovarian endometriosis and 617 were obtained from unaffected controls. All embryos were cultured in a time-lapse incubator chamber and completed blastulation. Main results and the role of chance Morphokinetic data showed that embryo development is distorted in embryos obtained from women with endometriosis. The timing of early developmental events, including tPNf (22.7 +/- 2.9 hr vs 23.5 +/- 4.1 hr, p = 0.03) and t2 (25.2 +/- 3.1 hr vs 26.1 +/- 4.4 hr, p = 0.03) were significantly shortened in the endometriosis group. However, the timing of late developmental events, including tM (89.7 +/- 9.8 hr vs 86.6 +/- 11.4 hr, p = 0.006) and tB (111.0 +/- 11.0 hr vs 108.3 +/- 12.5 hr, p = 0.03) were significantly prolonged in the endometriosis group. The timing of t5 by morphokinetic observation was found to be equal both in ovarian endometriosis group and control group (50.4 +/- 8.7 hr vs 50.4 +/- 8.4 hr, p = 0.99). The average of KID score of embryos from ovarian endometriosis group was lower than that from normal control, but it did not reached statistical significance (4.07 +/- 2.11 vs 4.49 +/- 2.70, p = 0.27). The initial serum hCG levels in pregnant women from both endometriosis and non-endometriosis participants showed that hCG levels were significantly higher in non-endometriosis cases (628.8 +/- 967.7 mIU/ml vs 1581.5 +/- 1885.1 mIU/ml, p = 0.01). Limitations, reasons for caution This is a retrospective observational study with propensity score matching method to eliminate confounding factors such as age, BMI and AMH. Wider implications of the findings The effects of prolonged morphokinetic parameters on the embryo quality need further study to clarify; however, they may serve as markers in selecting embryos with implantation potential. Trial registration number Not applicable

Abstract 3960: Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is one of the most aggressive human cancers with poor prognosis, and the overall 5-year survival rate is less than 20 percent. Prognosis for EAC remains poor even with modern combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches for EAC treatment improvements are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to cancer aggressiveness and poor clinical prognosis. Overexpression of hypoxia-inducible factor 1-alpha (HIF-1 alpha) and immunosuppressive CD73, an ecto-5’-nucleotidase enzyme in cancer can give rise to tumor progression with drug resistance. CD73 has never been proposed as a therapeutic target in EAC and its relationship with hypoxia or HIF-1 alpha has not also been investigated in EAC. In this study, we therefore investigated the therapeutic targeting of HIF-1 alpha and CD73 by acriflavine in experimental EAC. Methods: Hypoxia in EAC cells were induced by 3D culture and hypoxic exposure. NanoCulture® plates and dishes were used for 3D cultures. For hypoxic exposure, cells were placed in a sealed modular incubator chamber flushed with a gas mixture containing 1% O2, 5% CO2 and 94% N2. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Nanoparticle albumin-bound paclitaxel (NPT) was used as chemotherapeutic agent, whereas acriflavine was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 and Cell Titer-Glo (CTG) luminescent assays, in vivo tumor growth was detected by measuring subcutaneous xenografts, apoptosis was detected by cleaved caspase 3/PARP expressions and hypoxia-targeting was detected by HIF-1 alpha/CD73 expressions. Results: We observed overexpression of both HIF-1 alpha and CD73 in 3D culture and hypoxic exposure of EAC cells. Interestingly acriflavine treatment drastically inhibited both HIF-1 alpha and CD73 expression in EAC 3D culture and hypoxic exposure. 3D culture was more resistant to antiproliferative effect of chemotherapeutic agent NPT over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavine showed stronger antiproliferative effects in 3D culture than in 2D culture. We also observed hypoxia inside the 3D culture spheroids. In addition, acriflavine showed significant in vivo antitumor efficacy both as monotherapy and in combination with NPT. In subcutaneous xenografts using OE19 EAC cells, acriflavine monotherapy exhibited a significant decrease in relative tumor volume to 55.02% compared to control (p=0.04) and addition of NPT with acriflavine also showed a significant enhancement effect of tumor regression as tumor size decreased to 32.70% compared to control (p=0.002). Conclusion: These results support the potential of acriflavine as HIF-1 alpha and CD73 targeting and its combination with chemotherapy NPT as an effective option for EAC therapy. Citation Format: Md Sazzad Hassan, Aktar Ali, Saisantosh Ponna, Dimitri Scofield, Niranjan Awasthi, Mark Jantz, Urs von Holzen. Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3960.

Prototype of a Baby Incubator Physical Parameter Measurement Tool: Temperature, Humidity, Airflow and Noise Level

The Incubator Analyzer is equipment to measure the physical parameters contained in the baby incubator to verify the output value of the device and ensure the safety of the equipment for patient care. Currently, there are several products of baby incubator analyzer in Indonesia, but the availability of goods is still imported with relatively expensive product prices, so in this article, we report the development of a prototype baby incubator physical parameter measurement equipment to support the domestic product independence program. The method that has been carried out in this research is experimental in designing and developing a baby incubator physical parameter by maximizing the availability of components in the local market. Design of the prototype refers to SNI IEC 60601-2-19-2014 which is a standard of special requirements for basic safety and essential performance of infant incubators, with the measured parameters consisting of measurements of air temperature in the incubator room, mattress temperature, humidity, airflow, and noise level. The temperature measurement design uses a resistive temperature detector PT 100, SHT 11 as a humidity sensor, GFS131 as an airflow sensor, MAX4466 as a microphone amplifier as a sound sensor, and a 3.5” nextion TFT LCD as a display. This prototype is also equipped with a rechargeable battery system as a portable power supply. The testing is carried out by comparing the results read by a device designed with the Incu Analyzer II Fluke Biomedical. The results show that the prototype of the baby incubator parameter measuring instrument is able to read the values of temperature, humidity, airflow, and noise level in the baby incubator chamber. The accuracy of temperature parameter measurement reaches more than 98%, so this prototype has the potential to be tested by authorized institutions.

Development and Evaluation of a Locally Designed Solar Integrated Egg Incubator

There is need for subsistence poultry farmers to maximize chick production to supply the increasing demand for poultry meat. This research developed and evaluated an egg incubator powered by a renewable energy source generated from solar modules. The incubator was incorporated with temperature and humidity sensors which measured and regulated the conditions suitable for the egg incubation. The direct current (DC) heater, sensor, microcontroller and DC fan provided suitable temperature and humidity for the eggs. The developed incubator was evaluated by monitoring the temperature and Relative Humidity (RH) readings over a range of time and the hatchability rate was also evaluated. The temperature within the incubator chamber ranged 37-38°C with an error margin of +0.1oC while the RH ranged between of 30-35% with an error margin of +5%. An average hatchability rate of 92% was obtained from the evaluation of the incubator. The evaluation gave a standardized temperature and relative humidity range of values necessary for incubation of eggs. The study has shown that solar energy could be used to successfully power incubator systems with good hatchability for the whole incubation period.

TRAP-induced PAR1 expression with its mechanism during AMI in a rat model

Background and objectiveProtease-activated receptor 1 (PAR1) is crucial in individuals with acute myocardial infarction (AMI). The continuous and prompt PAR1 activation mainly dependent on PAR1 trafficking is essential for the role of PAR1 during AMI in which cardiomyocytes are in hypoxia. However, the PAR1 trafficking in cardiomyocytes specially during the hypoxia is still unclear.Methods and resultA rat AMI model was created. PAR1 activation with thrombin-receptor activated peptide (TRAP) had a transient effect on cardiac function in normal rats but persistent improvement in rats with AMI. Cardiomyocytes from neonatal rats were cultured in a normal CO2 incubator and a hypoxic modular incubator chamber. The cells were then subjected to western blot for the total protein expression and staining with fluorescent reagent and antibody for PAR1 localization. No change in total PAR1 expression following TRAP stimulation was observed; however, it led to increased PAR1 expression in the early endosomes in normoxic cells and decreased expression in the early endosomes in hypoxic cells. Under hypoxic conditions, TRAP restored the PAR1 expression on both cell and endosomal surfaces within an hour by decreasing Rab11A (8.5-fold; 179.93 ± 9.82% of the normoxic control group, n = 5) and increasing Rab11B (15.5-fold) expression after 4 h of hypoxia. Similarly, Rab11A knockdown upregulated PAR1 expression under normoxia, and Rab11B knockdown downregulated PAR1 expression under both normoxic and hypoxic conditions. Cardiomyocytes knocked out of both Rab11A, and Rad11B lost the TRAP-induced PAR1 expression but still exhibited the early endosomal TRAP-induced PAR1 expression under hypoxia.ConclusionsTRAP-mediated activation of PAR1 in cardiomyocytes did not alter the total PAR1 expression under normoxic conditions. Instead, it triggers a redistribution of PAR1 levels under normoxic and hypoxic conditions. TRAP reverses the hypoxia-inhibited PAR1 expression in cardiomyocytes by downregulating Rab11A expression and upregulating Rab11B expression.

First description of conspecific hyperparasitism in Amblyomma sculptum

The present study reports a case of conspecific hyperparasitism for Amblyomma sculptum. Two partially engorged females were collected from two naturally infested dairy cows and placed in a single petri dish to be transported to the laboratory, where they were held in a biological oxygen demand (BOD) incubator chamber under controlled conditions of 27 ± 1 °C and 85 ± 5% RH. After one day, it was observed that the smaller female (female 1) was attached to the dorsal surface of the larger female (female 2). The hypostome and chelicerae of the female 1 were entirely inserted into the cuticle of the female 2, characterizing tick attachment. Pictures were taken to illustrate the finding. After a few hours, the female 1 detached spontaneously and showed no visible signs of additional enlargement (engorgement). The females did not lay eggs and the survival period was not recorded. To our knowledge this is the first report of hyperparasitism for A. sculptum and the fourth for the genus Amblyomma.

Performance assessment of a solar powered egg incubator with a backup heater

This study presents the performance of a novel solar-powered incubator which uses water as thermal mass. Energy absorbed by the water is transferred to the incubator chamber by a fan powered with a 12 V battery. To enable adequate control of chamber temperature and humidity, a ProNem Mini sensor and controller (ESM-3723) were incorporated into the design. The thermal mass was sized adequately to supply energy for night operations as well as periods of inclement weather. The incubator was tested with and without load. Field measurements were obtained using UT330A USB datalogger. Tests under no load conditions gave an average temperature range of 36 to 42.9°C and relative humidity of 53 % to 70.5 %. Incubation tests showed average chamber temperature and relative humidity ranges of 35 ℃ to 41 ℃ and 45 % to 61 %. Candling test gave percentage fertility and hatchability of 60% and 56%. A cost-benefit analysis gave a capital cost of 617.32 USD, incubation cost of 69 cents per chick and simple payback period of 15 months. The performance indices obtained make the proposed design a suitable architecture to build upon in order to accelerate the promotion of livelihood empowerment through poultry farming in Ghana.

The Ocean Plastic Incubator Chamber (OPIC) system to monitor in situ plastic degradation at sea

Marine plastic pollution is a global and pervasive environmental issue. Knowledge on plastic degradation in natural settings is still very limited due to current technological limitations, hampering our understanding of plastic fate (including its breakdown into micro- and nanoplastics) and of its risk for marine ecosystems. Here we present the proof of concept of the Ocean Plastic Incubator Chamber (OPIC), a novel equipment to follow plastic degradation in situ at sea over time. OPIC consists of a frame containing a motorised rotating stage with transparent tubes sub-assemblies where reference plastic materials are incubated and exposed to natural weathering conditions for defined time multi-years period. OPIC has been designed, tested and adapted for deployment with mooring line platforms in the open ocean with potential future application in remote environments at different depths (from shallow waters to deep sea environments). This incubator will allow us to measure different markers of plastic aging in situ in the ocean for the first time, providing new insights into the multiple and locally driven dynamics regulating plastic transformations and fate at sea.

P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE

Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability. Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented. NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo, highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression. Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM. Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O2, 5% CO2 and 94%N2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05. Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival. Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment.

The impact of endometriosis on embryo morphokinetics: embryos from endometriosis patients exhibit delayed cell cycle milestones and decreased blastulation rates.

To compare morphokinetic parameters in embryos obtained from women with and without endometriosis. We evaluated a total of 3471 embryos resulting from 434 oocyte retrievals performed at a single academic center. One thousand seventy-eight embryos were obtained from women affected by endometriosis and 2393 came from unaffected controls. All embryos were cultured in a time-lapse incubator chamber for up to 6days. IVF cycle outcomes and morphokinetic parameters collected prospectively were retrospectively reviewed. Morphokinetic data suggest that embryo development is impaired in embryos obtained from women with endometriosis (EE). EE were slower to achieve the 2-8 cell stages compared to control embryos (CE) (p < 0.001); additionally, time to compaction was delayed compared to CE (p = 0.015). The timing of late developmental events, including morulation and blastulation was also delayed in the endometriosis cohort (p < 0.001). In addition to demonstrating delayed cell cycle milestones, EE were less likely than controls to progress to morula, blastocyst, and expanded blastocyst stages (p < 0.001). Furthermore, a smaller proportion of embryos in the endometriosis group fell into optimal kinetic ranges for cc2 (p = 0.003), t5 (p = 0.019), tSB (p < 0.001), and tEB (p = 0.007). There were no significant differences in clinical pregnancy or live birth rates between groups. Embryos from endometriosis patients demonstrate impairments in both early and late developmental events, and progress to the morula, blastocyst, and expanded blastocyst stages at lower rates than control embryos. Despite these differences, IVF outcomes are similar for patients with and without endometriosis.

First Report of Leaf Spot Disease Caused by Stemphylium eturmiunum on Garlic in Korea.

Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 - 22 × 16 - 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch's postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.

A Systematic Approach for the Evaluation, Validation, and Implementation of Automated Colony Counting Systems.

For several years, automated colony counting systems have been available with varying degrees of automation. Ever more sophisticated instruments are now increasingly used in microbiological laboratories of pharmaceutical quality control. In addition to the colony counting device, the instruments are now also equipped with robotic systems performing the entire handling of the petri dishes, e.g., automated internal transportation of petri dishes from the incubator chamber to the instrument's enumeration device and back. Moreover, the subjective evaluation of microbial enumeration tests by analysts is replaced with a more accurate and precise process. This leads to significant improvements to data integrity compliance. Automated colony counting systems also often enable cost reduction in the microbiological laboratory, e.g., by not requiring a contemporaneous verification by a second analyst. They also enable direct integration of count data into an existing laboratory information management system, reducing the hands-on time, costs per test and also preventing human errors caused by manual transcription. Altogether, these instruments will lead to improved monitoring and assurance of control of biopharmaceutical processes and manufacturing environments, as well as shortened cycle times in the supply chain. Regulators are encouraging the biopharmaceutical industry to adopt these innovative systems. For example, this year a BioPhorum member company received the first health authority approvals from EU, US, CH, Canada, Australia, and China for the use of automated colony counting systems for in-process bioburden testing and the release of drug substance lots, with an incubation time reduced by about 50%. Although these approvals are for release testing of drug substance lots, the instruments can also be used for environmental monitoring, testing of water samples, etc. This article describes a systematic 9-step approach to the evaluation, equipment qualification, and deployment of automated colony counting systems, which can be applied by biopharmaceutical companies wanting to take advantage of their numerous benefits.

Effective temperature for overcoming dormancy of ‘Fuyu’ persimmon tree buds

The objective of this study was to evaluate the effective temperature for overcoming the dormancy of ‘Fuyu’ persimmon tree buds. Stem samples were collected three times between 2013 and 2014. Stems were maintained in a climate incubator chamber at 3, 6, 9, and 12 °C. For each temperature, five numbers of additional chilling hours (CH) (0, 240, 384, 528, and 672 CH) were studied. The experimental design was completely randomized in a 5 × 4 factorial design (chilling hours × temperatures) with four replications with 10 cuttings. The maintenance of branches at cold temperatures from 3 to 12 °C intensified endodormancy of the buds when the plants were at the beginning of endodormancy. The most effective temperatures for overcome dormancy when the buds were in transition from paradormancy to endodormancy were from 3 to 6 °C. When the buds were already in endodormancy, temperatures of 3, 6, 9, and 12 °C were effective for the accumulation of cold and overcoming dormancy. The increase in the number of chilling hours from 3 to 12 °C induced budburst and the temperature of 12 °C was able to slowly induce and overcome bud dormancy.

Construction a CO2 Incubator for Cell Culture with Capability of Transmitting Microwave Radiation.

Background:The objective of this study was to design and construct a CO2 incubator with nonmetallic walls and to investigate the viability of the cells and microwave irradiance inside this incubator.Methods:Because the walls of conventional incubators are made of metal, this causes scattering, reflection, and absorption of electromagnetic waves. We decided to build a nonmetallic wall incubator to examine cells under microwave radiation. Incubator walls were made using polyvinyl chloride and Plexiglas and then temperature, CO2 pressure, and humidity sensors were placed in it. Atmel® ATmega1284, a low-power CMOS 8-bit microcontroller, collects and analyzes the sensor information, and if the values are less or more than the specified limits, the command to cut off or connect the electric current to the heater or CO2 solenoid valve is sent. Using a fan inside the incubator chamber, temperature and CO2 are uniforms. The temperature of the points where the cell culture plates are placed was measured, and the temperature difference was compared. Ovarian cancer cells (A2780) were cultured in the hand-made and commercial incubators at different times, and cell viability was compared by the MTT method. Microwave radiation in the incubator was also investigated using a spectrum analyzer. The survival of cells after microwave irradiation in the incubator was measured and compared with control cells.Results:The data showed that there was no significant difference in temperature of different points in hand-made incubator and also there was no significant difference between the viability of cells cultured in the hand-made and commercial incubators. The survival of irradiated cells in the incubator was reduced compared to control cells, but this reduction was not significant.Conclusion:This incubator has the ability to maintain cells and study the effects of electromagnetic radiations on the desired cells, which becomes possible by using this device.