Pretreatment Culture Research Articles

An added value of Azithromycin: mitigation of Doxorubicin associated oxidative damage and genotoxicity in normal human bronchial epithelium cells.

Doxorubicin, a well-known and widely used antineoplastic agent with direct ROS-accumulating activity, has proven effective in treating various cancer types. However, its non-specific cytotoxicity towards non-cancerous cells prompts concerns regarding potential adverse effects. Azithromycin is an antibiotic for treating bacterial infections and an anti-inflammatory agent, particularly beneficial in managing respiratory conditions like bronchitis and sinusitis. Despite azithromycin's well-documented antibacterial properties, its potential cellular/genomic protective effects remain unexplored. As an in vitro model, BEAS-2B cells (normal human bronchial epithelium cells) were employed in the present study to assess whether azithromycin possesses any protective properties against doxorubicin-induced cellular toxicity. Cells in pre-treatment culture were treated to various amounts of azithromycin (3.125, 6.25, 12.5, 25, and 50 μg/mL) in combination with doxorubicin at IC50 (0.08 μg/mL). Doxorubicin at 0.08 μg/mL highlighted cytotoxicity, oxidative stress, and genotoxicity. Azithromycin at 25 and 50 μg/mL markedly modulated oxidative stress and genomic damage by decreasing the ROS and LPO amounts, and suppressing DNA fragmentation in the comet assay parameters. Consequently, azithromycin may be regarded as a cytomodulating, antigenotoxic, and antioxidant agent.

Construction of an Efficient Genetic Transformation System for Watercress (Nasturtium officinale W. T. Aiton).

Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the distribution details of auxin in the growth process of watercress. Our findings provide an effective method for gene function research and lay the foundation for innovative utilization of germplasm resources of watercress.

Cytomodulatory characteristics of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) against cypermethrin on skin fibroblast cells (HFF-1)

The hematopoietic factor granulocyte macrophage-colony stimulating factor (GM-CSF) has been identified via its capacity to promote bone marrow progenitors' development and differentiation into granulocytes and macrophages. Extensive pre-clinical research has established its promise as a critical therapeutic target in an assortment of inflammatory and autoimmune disorders. Despite the broad literature on GM-CSF as hematopoietic of stem cells, the cyto/geno protective aspects remain unknown. This study aimed to assess the cyto/geno protective possessions of GM-CSF on cypermethrin-induced cellular toxicity on HFF-1 cells as an in vitro model. In pre-treatment culture, cells were exposed to various GM-CSF concentrations (5, 10, 20, and 40 ng/mL) with cypermethrin at IC50 (5.13 ng/mL). Cytotoxicity, apoptotic rates, and genotoxicity were measured using the MTT, Annexin V-FITC/PI staining via flow-cytometry, and the comet assay. Cypermethrin at 5.13 ng/mL revealed cytotoxicity, apoptosis, oxidative stress, and genotoxicity while highlighting GM-CSF's protective properties on HFF-1. GM-CSF markedly attenuated cypermethrin-induced apoptotic cell death (early and late apoptotic rates). GM-CSF considerably regulated oxidative stress and genotoxicity by reducing the ROS and LPO levels, maintaining the status of GSH and activity of SOD, and suppressing genotoxicity in the comet assay parameters. Therefore, GM-CSF could be promising as an antioxidant, anti-apoptotic, genoprotective and cytomodulating agent.

Modulatory effect of amifostine (WR-1065) against genotoxicity and oxidative stress induced by methotrexate in human umbilical vein endothelial cells (HUVECs)

Amifostine is used in chemotherapy and radiotherapy as a cytoprotective adjuvant alongside DNA-binding chemotherapeutic agents. It functions by reducing free radicals and detoxifying harmful metabolites. Methotrexate, as an antimetabolite drug has been considered for treating various cancers and autoimmune diseases. However, the cytotoxic effects of methotrexate extend beyond tumor cells to crucial organs, including the heart. This study applied the HUVEC cell line as a reference in vitro model for researching the characteristics of vascular endothelium and cardiotoxicity. The current study aimed to assess amifostine’s potential cytoprotective properties against methotrexate-induced cellular damage. Cytotoxicity was measured using the MTT assay. Apoptotic rates were evaluated by Annexin V-FITC/PI staining via flow cytometry. The genoprotective effect of amifostine was determined using the comet assay. Cells were exposed to various amifostine doses (10–200 μg/mL) and methotrexate (2.5 μM) in pretreatment culture condition. Methotrexate at 2.5 μM revealed cytotoxicity, apoptosis, oxidative stress and genotoxicity while highlighting amifostine’s cyto/geno protective properties on HUVECs. Amifostine significantly decreased the levels of ROS and LPO while preserving the status of GSH and SOD activity. Furthermore, it inhibited genotoxicity (tail length, %DNA in tail, and tail moment) in the comet assay. Amifostine markedly attenuated methotrexate-induced apoptotic cell death (early and late apoptotic rates). These findings convey that amifostine can operate as a cytoprotectant agent.

Regulatory Effects of Three-Dimensional Cultured Lipopolysaccharide-Pretreated Periodontal Ligament Stem Cell-Derived Secretome on Macrophages.

Phenotypic transformation of macrophages plays important immune response roles in the occurrence, development and regression of periodontitis. Under inflammation or other environmental stimulation, mesenchymal stem cells (MSCs) exert immunomodulatory effects through their secretome. It has been found that secretome derived from lipopolysaccharide (LPS)-pretreated or three-dimensional (3D)-cultured MSCs significantly reduced inflammatory responses in inflammatory diseases, including periodontitis, by inducing M2 macrophage polarization. In this study, periodontal ligament stem cells (PDLSCs) pretreated with LPS were 3D cultured in hydrogel (termed SupraGel) for a certain period of time and the secretome was collected to explore its regulatory effects on macrophages. Expression changes of immune cytokines in the secretome were also examined to speculate on the regulatory mechanisms in macrophages. The results indicated that PDLSCs showed good viability in SupraGel and could be separated from the gel by adding PBS and centrifuging. The secretome derived from LPS-pretreated and/or 3D-cultured PDLSCs all inhibited the polarization of M1 macrophages, while the secretome derived from LPS-pretreated PDLSCs (regardless of 3D culture) had the ability to promote the polarization of M1 to M2 macrophages and the migration of macrophages. Cytokines involved in the production, migration and polarization of macrophages, as well as multiple growth factors, increased in the PDLSC-derived secretome after LPS pretreatment and/or 3D culture, which suggested that the secretome had the potential to regulate macrophages and promote tissue regeneration, and that it could be used in the treatment of inflammation-related diseases such as periodontitis in the future.

Selection bias in multidrug-resistant tuberculosis cohort studies assessing sputum culture conversion.

Conversion of sputum culture from positive to negative for M. tuberculosis is a key indicator of treatment response. An initial positive culture is a pre-requisite to observe conversion. Consequently, patients with a missing or negative initial culture are excluded from analyses of conversion outcomes. To identify the initial, or "baseline" culture, researchers must define a sample collection interval. An interval extending past treatment initiation can increase sample size but may introduce selection bias because patients without a positive pre-treatment culture must survive and remain in care to have a culture in the post-treatment interval. We used simulated data and data from the endTB observational cohort to investigate the potential for bias when extending baseline culture intervals past treatment initiation. We evaluated bias in the proportion with six-month conversion. In simulation studies, the potential for bias depended on the proportion of patients missing a pre-treatment culture, proportion with conversion, proportion culture positive at treatment initiation, and proportion of patients missing a pre-treatment culture who would have been observed to be culture positive, had they had a culture. In observational data, the maximum potential for bias when reporting the proportion with conversion reached five percentage points in some sites. Extending the allowable baseline interval past treatment initiation may introduce selection bias. If investigators choose to extend the baseline collection interval past treatment initiation, the proportion missing a pre-treatment culture and the number of deaths and losses to follow up during the post-treatment allowable interval should be clearly enumerated.

Fenton-reaction-aid selective delignification of lignocellulose by Inonotus obliquus to improve enzymatic saccharification

Aiming at improving the efficiency of recovering sugars from wheat straw by the white rot fungus Inonotus obliquus in solid-state fermentation, two Fenton-reaction-aid systems, i.e., one system containing Fenton reaction pretreatment (FRP) and subsequent Inonotus obliquus culture, one system composed of fungal combined treatment with Fe3O4 nanomaterials (NMs), were developed. FRP and NMs improved the fungal growth and degradation efficiency of wheat straw (WS) by regulating ligninolytic enzyme secretion with manganese peroxidase (MnP) (471.0, 359.8 IU g−1), lignin peroxidase (LiP) (108.0, 48.6 IU g−1), and laccase (Lac) (18.0, 13.4 IU g−1) in the FRP group and NMs group within 15 days. Hemicellulose removal on Day 5 and delignification on Day 10 in FRP group (51.3 % and 20.93 %) and NMs group (40.7 % and 12.60 %) were more severe than the control group (19.2 %, 7.37 %). Sugar yield and conversion rate of 10-day-cultured FRP-WS were 228.4 g kg−1 and 37.2 %, 2.1 and 2.6 times higher than that of 10-day-cultured raw WS. Synergism of Fenton reaction and white-rot fungus quickened fermentation period, promoted degradation selectivity, and then subsequent enzymatic hydrolysis. The Fenton-like system established by NMs and I. obliquus made the synergistic system simpler and represented biocompatible chemistry for developing novel microbial cell factories.

Human Amnion-Derived Mesenchymal Stromal/Stem Cells Pre-Conditioning Inhibits Inflammation and Apoptosis of Immune and Parenchymal Cells in an In Vitro Model of Liver Ischemia/Reperfusion.

Ischemia/reperfusion injury (IRI) represents one of the leading causes of primary non-function acute liver transplantation failure. IRI, generated by an interruption of organ blood flow and the subsequent restoration upon transplant, i.e., reperfusion, generates the activation of an inflammatory cascade from the resident Kupffer cells, leading first to neutrophils recruitment and second to apoptosis of the parenchyma. Recently, human mesenchymal stromal/stem cells (hMSCs) and derivatives have been implemented for reducing the damage induced by IRI. Interestingly, sparse data in the literature have described the use of human amnion-derived MSCs (hAMSCs) and, more importantly, no evidence regarding hMSCs priming on liver IRI have been described yet. Thus, our study focused on the definition of an in vitro model of liver IRI to test the effect of primed hAMSCs to reduce IRI damage on immune and hepatic cells. We found that the IFNγ pre-treatment and 3D culture of hAMSCs strongly reduced inflammation induced by M1-differentiated macrophages. Furthermore, primed hAMSCs significantly inhibited parenchymal apoptosis at early timepoints of reperfusion by blocking the activation of caspase 3/7. All together, these data demonstrate that hAMSCs priming significantly overcomes IRI effects in vitro by engaging the possibility of defining the molecular pathways involved in this process.

Desflurane pretreatment can reduce sepsis-evoked lung injury in rats via inhibiting STAT3 pathway.

The purpose of this study was to investigate the effect of desflurane (Des) pretreatment on sepsisevoked lung injury in rats and its mechanism. The rat model of sepsis-evoked lung injury was prepared using lipopolysaccharide (LPS), while rat lung mesenchymal cell (MSC) model was cultured in vitro, followed by Des pretreatment or inhibitor S31-201 culture. The degree of lung tissue injury was analyzed by Hematoxylin-eosin (HE) staining. The expression levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α in the serum of rats were detected by enzyme-linked immunosorbent assay (ELISA). One-step terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was utilized to determine the apoptosis levels of lung tissues and MSCs cultured in vitro. The expressions of the signal transducer and activator of transcription 3 (STAT3) pathway in rat lung tissues and MSCs were detected by Western blotting. After modeling, LPS induced the lung injury in rats, the expression levels of IL-6, IL-1β and TNF-α were up-regulated (P<0.05), the apoptosis rate was increased (P<0.05), and phosphorylated-Janus kinase 2 (p-JAK2) and phosphorylated-STAT3 (p-STAT3) protein expressions were up-regulated (P<0.05). Des pretreatment can alleviate LPS-induced lung injury, down-regulate IL-6, IL-1β and TNF-α expression levels (P<0.05), reduce apoptosis (P<0.05), and downregulate p-JAK2 and p-STAT3 protein levels (P<0.05). LPS induced an increase in apoptosis rate of MSCs (P<0.05) and the up-regulation of p-STAT3 protein expression (P<0.05). Both Des pretreatment and S31-201 inhibitor culture could reduce the apoptosis rate (P<0.05) and down-regulate p-STAT3 protein level (P<0.05). Des pretreatment can reduce sepsis-evoked lung injury in rats, which may be related to the inhibition of protein expressions of STAT3 pathway.

Development of bacterial resistance during treatment with topical gentamicin for chronic rhinosinusitis in patients with cystic fibrosis and primary ciliary dyskinesis. Retrospective case series.

The management of chronic rhinosinusitis (CRS) in patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is still a challenge. At our institution we have used gentamycin nasal spray, extemporaneously produced, for prophylactic treatment of moderate-to-severe CRS. The aim of this study was to investigate the gentamycin susceptibility of bacteria in sputum samples in CF and PCD patients treated for CRS. Patients with CF and PCD who were prescribed gentamycin nasal spray for CRS and had sputum bacterial cultures taken pre-treatment and followed-up at least once after ≥6 months were retrospectively included. Microbiological data were descriptively analysed in terms of bacterial species and resistance to gentamycin. A case series of 17 CF and 12 PCD patients passed the inclusion criteria. Of those cases, three (18%) CF patients and one (8%) PCD patient developed resistance to gentamycin during treatment with gentamycin nasal spray. In all four cases, the resistant bacterial isolates were &lt;i&gt;P. aeruginosa&lt;/i&gt;. Additionally, two CF patients already had &lt;i&gt;P. aeruginosa &lt;/i&gt; isolates resistant to gentamycin in the pre-treatment culture. In further two CF patients, the multi-resistant &lt;i&gt;Burgdorferi cepacia &lt;/i&gt;complex, including gentamycin resistance, was identified. &lt;i&gt;P. aeruginosa &lt;/i&gt; and &lt;i&gt;S. aureus &lt;/i&gt; in CF and &lt;i&gt;P. aeruginosa&lt;/i&gt; and &lt;i&gt;H. influenza &lt;/i&gt; in PCD were the predominant bacterial species. The study showed that there was moderate incidence of gentamycin resistance in CF and PCD patients at our institution. However, further prospective studies are needed to confirm the outcomes.

Effects on Pre-treatment and Different Tissue Culture Media for Androgenesis in Stevia rebaudiana Bertoni

Haploids and doubled haploids are very important tools which shorten the breeding periods and 100% homozygous lines. Especially in cross-pollinated species, it is known that it takes a long time to obtain homozygosity, and these breeding periods can be reduced by haploids considerably. In this study, appropriate bud stage and the effects of different growth regulators in Murashige and Skoog’s nutrient medium (MS) and pre-treatments on in vitro androgenesis were investigated in Stevia rebaudiana Bertoni which is known as sweet herb. In the first year, callus, bar-shaped embryo, and embryogenic calli were obtained. Embryos were obtained from MS 2.0 mg l−1 α-naphthaleneacetic acid (NAA) + 0.5 mg l−1 6-benzylaminopurine (BAP) medium, MS 1.0 mg l−1 NAA + 0.5 mg l−1 BAP, and MS 0.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) + 0.5 mg l−1 BAP callus ratios are 13.33%, 10%, and 3.33%, respectively. No differentiation was observed from the obtained callus and embryo. In the second year, high-temperature (+ 35 °C) and cold (+ 4 °C) pre-treatments were applied to the anthers, and no difference was observed between the applications. MS 2.0 mg l−1 NAA + 0.5 mg l−1 Kinetin high-temperature application was observed as the most successful medium with 37.5% embryo ratio obtained from two calli. This medium was followed by MS 2.0 mg l−1 NAA + 0.5 mg l−1 BAP and cold application (MS 1.0 mg l−1 NAA + 0.5 mg l−1 BAP) with 7.5% callus formation rates. Only cold application resulted in organogenesis through the callus, but the plants obtained were in diploid structure.

Ex vivo Pretreatment of Islets with Mitomycin C

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

More than just an Anti-Infective Agent: A Prospective Pilot Clinical Trial to Determine the Effectiveness of IV Ertapenem in Severe Hidradenitis Suppurativa

Background Hidradenitis suppurativa (HS) is a chronic debilitating cutaneous inflammatory condition caused by follicular occlusion/rupture with resultant inflammatory response. No durable control of HS is seen with current management strategies including topical and/or oral antibiotics, immunomodulatory drugs and local surgery. A recent French study using IV ertapenem for ≥4 weeks showed significant control of severe HS in patients (patients) who failed alternate medical strategies. We performed a prospective trial to study the effectiveness of IV ertapenem in severe HS.Methods A prospective pilot clinical study was done at Henry Ford Hospital, Detroit from May 2013 to December 2015. All patients with severe HS (Hurley Stage III) and who failed medical management by Dermatology were referred to Infectious Diseases for daily IV ertapenem through a PICC line. Patients were followed up for at least 6 months after completion of therapy with weekly monitoring. Data including demographics, clinical response/adverse effects to ertapenem and any PICC line-related complications were recorded. Response to the treatment was based on clinical improvement in pain score/drainage and Hurley Staging of HS.ResultsTwenty-three patients with severe HS consented to participate with a median age of 40 years (range 23–78 years) and 56% were female. Median treatment duration was 8 weeks (range 6–12). A 100% response rate was observed within 2 weeks of initiation and near complete resolution of active inflammation/drainage and improvement of pain score was observed in all patients at the end of treatment. Durable response was variable after completion of ertapenem (median 4 weeks; range 2–12); all subjects relapsed with varying degree of severity. In three patients who relapsed back to Hurley Stage III, ertapenem was restarted after 3 months with successful response. Adverse events include diarrhea (four patients), Clostridium difficile infection (one patient), and PICC line-related DVT (three patients). Clinical benefit despite ertapenem non-responsive bacteria in pretreatment culture of cutaneous drainage suggests an unproven immune-modulatory activity. Role of gut/skin microbial alteration needs further study.Conclusion In selected patients with severe HS, IV ertapenem is highly effective in the control of inflammation and can be useful in combination with local surgery.Disclosures All authors: No reported disclosures.

Long-term operation of microbial electrosynthesis cell reducing CO2 to multi-carbon chemicals with a mixed culture avoiding methanogenesis

In microbial electrosynthesis (MES), CO2 can be reduced preferably to multi-carbon chemicals by a biocathode-based process which uses electrochemically active bacteria as catalysts. A mixed anaerobic consortium from biological origin typically produces methane from CO2 reduction which circumvents production of multi-carbon compounds. This study aimed to develop a stable and robust CO2 reducing biocathode from a mixed culture inoculum avoiding the methane generation. An effective approach was demonstrated based on (i) an enrichment procedure involving inoculum pre-treatment and several culture transfers in H2:CO2 media, (ii) a transfer from heterotrophic to autotrophic growth and (iii) a sequential batch operation. Biomass growth and gradual acclimation to CO2 electro-reduction accomplished a maximum acetate production rate of 400mgLcatholyte−1d−1 at −1V (vs. Ag/AgCl). Methane was never detected in more than 300days of operation. Accumulation of acetate up to 7–10gL−1 was repeatedly attained by supplying (80:20) CO2:N2 mixture at −0.9 to −1V (vs. Ag/AgCl). In addition, ethanol and butyrate were also produced from CO2 reduction. Thus, a robust CO2 reducing biocathode can be developed from a mixed culture avoiding methane generation by adopting the specific culture enrichment and operation procedures without the direct addition of chemical inhibitor.

Anakinra and Tocilizumab Enhance Survival and Function of Human Islets during Culture: Implications for Clinical Islet Transplantation

Pretreatment culture before islet transplantation represents a window of opportunity to ameliorate the proinflammatory profile expressed by human β-cells in duress. Anakinra (IL-1 receptor antagonist) and tocilizumab (monoclonal IL-6 receptor antibody) are two known anti-inflammatory agents successfully used in the treatment of inflammatory states like rheumatoid arthritis. Both compounds have also been shown to reduce blood glucose and glycosylated hemoglobin in diabetic patients. We therefore sought to evaluate the impact of anakinra and tocilizumab on human β-cells. The islets were precultured with or without anakinra or tocilizumab and then transplanted in a marginal mass model using human islets in immunodeficient mice. Islet viability was evaluated in an in vitro model. The pretreatment culture led to a significantly improved engraftment in treated islets compared to the vehicle. Anakinra and tocilizumab are not toxic to human islets and significantly reduce markers of inflammation and cell death. These results strongly support a pretreatment culture with anakinra and tocilizumab prior to human islet transplantation.

The influence of culture conditions on the identification ofMycobacteriumspecies by MALDI-TOF MS profiling

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints. However, cell-wall characteristics of mycobacterial species, and their well known stability, complicate MALDI-TOF MS profiling analysis. In this study, we tested two recently published protocols for inactivation and disruption of mycobacteria, and we also examined the influence of different culture conditions (four culture media and five cultivation times) on mass spectral quality and the discriminatory power of the method. We found a significant influence of sample pretreatment method and culture medium on species identification and differentiation for a total of 10 strains belonging to Mycobacterium phlei and Mycobacteriumsmegmatis. Optimum culture conditions yielding the highest identification success rate against the BioTyper database (Bruker Daltonics) and permitting the possibility of automatic acquisition of mass spectra were found to be distinct for the two mycobacterial species examined. Similarly, individual changes in growth conditions had diverse effects on the two species. For these reasons, thorough control over cultivation conditions should always be employed to maximize the performance and discriminatory power of MALDI-TOF MS profiling, and cultivation conditions must be optimized separately for individual groups of mycobacterial species/strains.

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.

Relative effect of different inorganic acids on selective enrichment of acidogenic biocatalyst for fermentative biohydrogen production from wastewater

The effect of different inorganic acids viz., HNO3, HCl, H2SO4 and H3PO4 on inoculum pretreatment to selectively enrich hydrogen (H2) producing acidogenic bacteria was evaluated in anaerobic sequencing batch bioreactors. Relative positive efficiency of HNO3 pretreated consortia in enhancing H2 production (11.85 mol H2/kg CODR) was noticed compared to other acids (HCl, 5.64 mol H2/kg CODR; H2SO4, 7.65 mol H2/kg CODR; H3PO4, 6.90 mol H2/kg CODR) and untreated-parent consortia (control, 6.80 mol H2/kg CODR). On the contrary, substrate degradation (COD removal) was higher with the control operation (ξCOD, 66.3%; substrate degradation rate (SDR), 1.42 kg CODR/m(3)-day) compared to pre-treated culture. HNO3 pre-treatment resulted in a shift in the fermentation pathway towards more acetic acid production, while other acid pretreatment and untreated culture showed mixed type fermentation (acetic, butyric, propionic acids). The bio-electrochemical analysis and dehydrogenase activity supported the biocatalyst performance after HNO3 pretreatment with specific enrichment of Firmicutes and Bacillus.

Protective effects of astragaloside IV on high-glucose-induced damage of retinal ganglion cells

To explore the protective effects of astragaloside IV on retinal ganglion cells (RGC) incubated under high glucose. Cultured RGC-5 were divided into 3 groups: control, high glucose and high glucose+astragaloside IV. The survival rate of cell was measured by CCK-8 kit and flow cytometry. The changes of mitochondrial membrane potential were monitored by confocal laser scanning microscopy while those of RGC-5 mitochondria observed by electron microscopy. When the cell survival rate was 100% in the control group, the survival rate improved after 1 h 5, 10, 50, 100, 200 µg/ml astragaloside IV pretreatment and high-glucose culture. The cell survival rates for 100 mg/L, 200 mg/L of astragaloside IV were 81.6%, 80.0%, the difference from the high glucose group (66%) was significant (P < 0.05) and 100 mg/L of astragaloside IV was the best protection concentration. The apoptotic rate for 100 mg/L astragaloside IV pretreated group of RGC-5 cell was 6.1%. And it could partially inhibit the RGC-5 cell apoptosis caused by high glucose (8.2%) (P < 0.05). In the high-glucose group, the cells showed marked increases in mitochondrial swelling, especially for cristae. Numerous normal mitochondria were observed in 100 mg/L astragaloside IV pretreated group. Astragaloside IV could elevate mitochondrial membrane potential and alleviate mitochondrial swelling. Astragaloside IV can protect RGC from high-glucose induced damage and may benefit the patients with diabetic retinopathy.

Effectiveness of 1.25 % povidone–iodine combined with topical levofloxacin against conjunctival flora in intravitreal injection

To compare the effect of a reduced concentration (1.25 %) of povidone-iodine (PI) eye drops, combined with 0.5 % topical levofloxacin (LVFX), with that of the standard of care (5 % PI) on conjunctival flora before intravitreal injections (IVT). A prospective, randomized, single-blind clinical trial. One hundred eyes from 100 patients who underwent IVT were included. Eyes were randomly assigned to two groups and underwent different preparatory procedures before therapeutic IVT. LVFX drops were instilled three times every 5 min and then either 1.25 % PI drops were instilled three times every few minutes in group A or 5 % PI drops were instilled three times every few minutes in group B, the control. Conjunctival flora and the injection needles were collected for culture both before and after preparation. The number of positive cultures, the species isolated, and the minimum inhibitory concentrations (MIC) of the antibiotics were assessed. The pretreatment culture was positive for 45 eyes (86.5 %) in group A (n = 52) and for 37 eyes (77.1 %) in group B (n = 48). After the preparatory procedure, the cultures were positive for 25 eyes (48.1 %) in group A and for 27 eyes (56.3 %) in group B (P = 0.43). After preparation, the number of positive cultures decreased by 46.7 % in group A and by 33.4 % in group B (P = 0.48). No needle samples were contaminated in either group. The most common isolate in both groups after preparation for surgery was Propionibacterium acnes. Among the different antibiotics, vancomycin and oxacillin had the lowest MIC >90 for overall isolates. 1.25 % PI with LVFX is as effective as 5 % PI. P. acnes was the most common conjunctival flora detected. Vancomycin has been confirmed as the best choice for treating infectious endophthalmitis after IVT.