Days Of Culture Research Articles

First Report of Fusarium Yellows and Rhizome Rot Caused by Fusarium oxysporum f. sp. zingiberi on Ginger in the Continental United States.

Ginger (Zingiber officinale Roscoe) is one of the most widely consumed medicinal herb in the world, and the U.S. imports of ginger have risen in recent years because of its health benefits. Seed rhizome and soilborne diseases are serious concerns of ginger worldwide (Stirling 2004; Moreira et al. 2013), including the recent observations of Fusarium yellows and rhizome rot in the Commonwealth of Virginia. In October 2018 and 2019, ginger plants with yellowing of leaf margins and stunted growth were uprooted from a 9.1 m × 14.6 m high tunnel (HT) and from an outdoor raised bed at Virginia State University's Randolph farm. Disease incidence in the HT and the raised bed was estimated between 5-70%. Small pieces (2-5 mm) of symptomatic rhizomes were disinfected with 0.6% sodium hypochlorite solution and placed on potato dextrose agar (PDA) Petri plates to recover fungal isolates. Hyphal tips from these isolates were transferred to fresh PDA to obtain pure cultures. The fungal colonies were pinkish-white initially, and turned purplish-pink after 5-7 days of incubation at 25 °C. The microconidia were aseptate, oval or elliptical, hyaline, and measured 5 to 12 × 4 to 6 µm in size. Macroconidia were with 3 to 5 septations, curved like a sickle towards the ventral side, hyaline, smooth and thin-walled, and 15 to 40 × 3 to 6 µm in size. Fungal genomic DNA of one isolate (Gf-VA-3) was extracted from a 7-days old culture using PrepMan®Ultra (Thermo Fischer Scientific, Cheshire UK). Four conserved regions of the isolated pathogen, internal transcribed spacer (ITS), translation elongation factor (EF), β-tubulin (Bt), and calmodulin (cal) gene regions were amplified using ITS1 and ITS4 (White et al. 1990), ef1α and ef2α (O'Donnell et al. 1998), Bt2a and Bt2b (Glass and Donaldson 1995), and calA1 and calQ1 (Carbone and Kohn 1999), respectively. PCR products were sequenced, and amplicons deposited in GenBank with accession numbers MT337417 for ITS, MT436712 for Bt, MT802441 for cal and MW816632 for EF. A 99-100% identity with Fusarium oxysporum was matched with accession nos. MW776326 for ITS, MN646766 for the β-tubulin, MT010904 for the calmodulin and MN258350 for the translation elongation factor genes. For pathogenicity test, six 6-week-old healthy ginger plants grown on sterilized potting mix in the greenhouse were inoculated by injecting 3-ml of a 1 × 108 micro- and macro-conidia suspension per ml at the crown area transcending to the rhizome. Another set of six plants were injected with distilled and autoclaved water in the same way. After four weeks, leaves withered, plants exhibited yellowing and wilt followed by stunted growth and eventually complete collapse of the six inoculated plants, however control plants showed none of the symptoms. The same pathogen was re-isolated from the inoculated plants. The pathogenicity test was repeated, and the same results were observed. Fusarium yellows and rhizome rot has been reported from Hawaii in the U.S. (Trujillo 1963), Brazil (Moreira et al. 2013), Australia (Stirling 2004), China (Li et al. 2014), and India (Shanmugam et al. 2013). To our knowledge, this is the first report of Fusarium yellows and rhizome rot on ginger in the Continental U.S. The disease is seed rhizome and soilborne leading to poor establishment and hence economic loss in ginger production.

Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology.

Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a 'bacteriophage-like' behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.

Biphasic Calcium Phosphate/Chitosan/Polyacrylonitrile/Polylactic Acid-Glycolic Acid (PLGA) Nanocomposite Stent for Repair and Osteogenesis of Oral Alveolar Bone Defect

ABSTRACTThe ability of sol-gel micro-nano biphasic calcium stent to repair oral alveolar bone defects was investigated in this study, and its osteogenesis performance was also analyzed. Biphasic calcium phosphate (BCP) was synthesized by wet method, which was combined with chitosan (CS), polyacrylonitrile (PAN), and polylactic acid-glycolic acid (PLGA). Then, the BCP/CS/PAN/PLGA nanocomposite stent was prepared by vacuum freeze-drying technology. The micro-nano composite stent was combined with the bone morphogenetic protein-2 (BMP-2) gene, so as to obtain the sol-gel micro-nano biphasic calcium BMP-2/BCP/CS/PAN/PLGA composite stent. Besides, the composite stent should be measured in terms of compressive strength, porosity, structure, and morphological features. The healthy female rhesus monkeys were taken as the research animals, and the iliac bone marrow was extracted by puncture. The mesenchymal stem cells (MSCs) were obtained by density gradient centrifugation, and their osteogenic differentiation ability was observed. The MSCs were culturedin vitrowith BMP-2/BCP/CS/PAN/PLGA composite stent, methylthiazolyldiphenyl-tetrazolium bromide (MTT) was applied to detect cell adhesion and proliferation, and the alkaline phosphatase (ALP) activity was employed to analyze its osteogenic properties on stent materials. In addition, the expression of BMP-2 was detected by Western blot. The alveolar bone defect models were established and divided into group A (MSCs + BMP-2/BCP/CS/PAN/PLGA), group B (BMP-2/BCP/CS/PAN/PLGA), group C (BCP/CS/PAN/PLG), and group D (control group, reposition of gingival flap and suture) according to different implant materials. The changes of bone defect area in different groups were detected by gross examinations and X-ray, so that the new bone density was analyzed. The results showed that the BCP/CS/PAN/PLGA composite stent exhibited a porous structure combining multiple pores/small pores, with an average pore diameter (PD) of 400–500µm, maximum compressive strength of 6.02 Mpa, and porosity of 86.82%. MSCs differentiated into osteoblasts under osteogenic induction conditioned medium, and the optical density (OD) of CS + MSCs/BMP-2/BCP/CS/PAN/PLGA cells was greater in contrast to that of MSCs/BMP-2/BCP/CS/PAN/PLGA cells on the 1st and 7th day of culture, showing a statistical difference(P< 0.05). The gross examination and X-ray of bone defect area in group A showed that its bone structure and density were very close to those of normal bone (all materials were absorbed, and newly formed bone cells were active); the CT value of alveolar bone in groups A, B, C, and D was 1,092.45± 15.87 g/cm3, 932.26± 16.75 g/cm3, 859.51 ±17.86 g/cm3, and 787.96± 16.54 g/cm3, respectively. There was no marked difference in CT values between group A and normal alveolar bone (P> 0.05), while the CT value of alveolar bone in group A was higher obviously than the value of groups C and D(P< 0.05). It indicated that the composite stent based on sol–gel micro-nano biphasic calcium BMP-2/BCP/CS/PAN/PLGA could promote the repair of oral alveolar bone defect and its osteogenesis, thereby providing a reference for the oral clinical treatment of periodontal bone defects.

Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology

Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a ‘bacteriophage-like’ behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.

Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology

Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a ‘bacteriophage-like’ behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.

Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology

Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a 'bacteriophage-like' behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.

Successful Use of Telemedicine Infectious Diseases Consultation With an Antimicrobial Stewardship-Led Staphylococcus aureus Bacteremia Care Bundle.

BackgroundTelemedicine (TM) programs can be implemented to deliver specialty care through virtual platforms and overcome geographic/resource constraints. Few data exist to describe outcomes associated with TM-based infectious diseases (ID) management. The purpose of this study was to compare outcomes associated with TM and onsite standard-of-care (SOC) ID consultation after implementation of an antimicrobial stewardship (AMS)-led Staphylococcus aureus bacteremia (SAB) bundle.MethodsA retrospective cohort study was conducted on the effects of a SAB bundle comparing ID consult delivery (SOC or TM) at 10 US hospitals within Atrium Health in adult patients admitted from September 2016 through December 2017. The type of ID consult provided was based on the admitting hospital; no hospital had both modalities. Bundle components included the following: (1) ID consult, (2) appropriate antibiotics, (3) repeat blood cultures until clearance, (4) echocardiogram obtainment, and (5) appropriate antibiotic duration. The AMS facilitated bundle initiation and compliance. The primary outcome was bundle adherence between groups. Differences in clinical outcomes were also assessed.ResultsWe evaluated 738 patients with SAB (576 with SOC, 162 with TM ID). No differences were observed in overall bundle adherence (SOC 86% vs TM 89%, P = .33). In addition, no significant differences resulted between groups for hospital mortality, 30-day SAB-related readmission, persistent bacteremia, and culture clearance. Groups did not differ in 30-day mortality when controlling for demographics, bacteremia source, and physiological measures with multivariable logistic regression.ConclusionsOur findings provide evidence to support effective use of TM ID consultation and AMS-led care bundles for SAB management in resource-limited settings.

Nursery and grow-out culture of the abalone Haliotis asinina on a reef flat: A comparison of growth and survival using different culture containers

The study was conducted to compare four different culture containers [cage, recycled oil container, tray (control), and tube] in the nursery and grow-out culture of the abalone Haliotis asinina on a reef flat. For nursery culture, early juvenile abalone with mean shell length (SL) of 1.30 ± 0.006 cm and mean body weight (BW) of 0.45 ± 0.007 g were stocked in the containers at 100 ind m−2 and reared until they attained the 3 cm SL size required for grow-out culture in three of the four culture containers. For grow-out culture, juvenile abalone harvested from nursery culture with mean SL of 3.23 ± 0.018 cm and mean BW of 7.33 ± 0.131 g were stocked in the containers at 50 ind m−2 and reared until they were harvestable at 5 cm SL cocktail size in three of the four culture containers. The 3 cm SL grow-out size was attained at 90 days of culture (DOC) in recycled containers, trays, and tubes, while the 5 cm SL cocktail size at 180 DOC in the same containers. Abalone reared in tubes had the highest mean SL after 90 DOC in nursery culture (3.21 cm), while those in trays after 180 DOC in grow-out culture (5.30 cm). However, at the end of the nursery and grow-out culture, there were no significant differences in mean SL among the abalone reared in these three culture containers. In both culture phases, abalone reared in cages had the lowest mean SL, 2.88 and 4.44 cm, respectively. Survival was highest in trays (98.64%) during nursery culture and in tubes (96.57%) during grow-out culture. With comparable results in recycled containers, trays, and tubes for both growth and survival at the nursery and grow-out phases, tubes are recommended for use when culture is to be done on reef flats. Tubes are the most stable and durable among the four culture containers tested. They can withstand strong winds and waves and may be reused for several culture runs.

Morphoanatomical aspects of the starting material for the improvement of pineapple cryopreservation by the droplet-vitrification technique.

Cryopreservation of pineapple shoot tips has been established from various protocols, including droplet vitrification. Thus, this work aimed to evaluate the morphoanatomical conditions of the starting material over different times (30, 45 and 60 days) of culture before freezing and its correlation with the survival percentage of the cryopreserved shoot tips. Four accessions, Ananas comosus var. comosus (BGA-009); var. bracteatus (BGA-119); var. parguazensis (BGA-376), var. erectifolius (BGA-750) from the Pineapple Active Germplasm Bank (BGA Pineapple) and two hybrids from the Embrapa Genetic Breeding Program, FIB-ROX1 (var. bracteatus X var. erectifolius) and FIB-ROX2 ( var. erectifolius X var. bracteatus), recently introduced in the field from in vitro storage, were used. Histological sections before freezing and the percentages of survival after freezing were obtained taking into account the different times of cultivation of the donor plants. The results showed a significative interaction between genotypes (accessions and hybrids) and the culture period. The accessions BGA-009 and BGA-119 showed the highest survival rates, with 95% and 90% respectively for the 30-day culture time. Different results were obtained for each genotype, showing the need for improvements in the standardization of starting material, which would allow better repeatability of the protocol.

Antifungal Activity of Bioactive Metabolites Produced by Trichoderma asperellum and Trichoderma atroviride in Liquid Medium.

Trichoderma spp. are known as biocontrol agents of fungal plant pathogens and have been recognized as a potential source of bioactive metabolites. The production of antimicrobial substances from strains T. atroviride (TS) and T. asperellum (IMI 393899) was investigated. The bioactivity of 10- and 30-day culture filtrate extracted with ethyl acetate was assessed against a set of pathogenic fungi and oomycetes. The 30-day extracts of both strains had significant cytotoxic effects against the tested pathogens, with values of minimum fungicidal concentration (MFC) ranging between 0.19 and 6.25 mg/mL. Dual culture assay (direct contact and nondirect contact) and the percentage inhibition of radial growth (PIRG) was calculated. The highest PIRG values were 76% and 81% (direct contact) with IMI 393899 and TS, respectively. Nondirect contact does not show inhibition on any of pathogens tested, indicating that the inhibition is not due to the secretion of volatile substances. Culture filtrates were analyzed by GC-MS and HPLC-Q-TOF-MS for the identification of volatile organic compounds (VOCs) and nonvolatile organic compounds (nVOCs), respectively. Seven classes of VOCs and 12 molecules of nVOCs were identified. These results indicate that these strains of Trichoderma had antimicrobial activities and they are potential natural sources of compounds with biological activity.

Metronomic Celecoxib Therapy in Clinically Available Dosage Ablates Hepatocellular Carcinoma via Suppressing Cell Invasion, Growth, and Stemness in Pre-Clinical Models

ObjectiveTo investigate the anti-carcinogenic effect of metronomic Celecoxib (i.e., frequent administration in clinically available doses) against hepatocellular carcinoma (HCC) in the perspective of metastasis, spontaneous hepatocarcinogenesis, cancer invasion, proliferation, and stemness in vivo and in vitro.BackgroundCelecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is known to cause anti-carcinogenic effects for HCC in suprapharmacological doses. However, the effects of metronomic Celecoxib treatment on HCC cells remain unclear.MethodsThe in vivo chemopreventive effect of metronomic Celecoxib (10mg/kg/d) was investigated by the syngeneic HCC implantation model and spontaneous hepatocarcinogenesis in HBV-transgenic(HBVtg) mice individually. HCC cell lines were treated by either suprapharmacological (100 μM) or metronomic (4 μM) Celecoxib therapy. Anti-carcinogenic effects were evaluated using cell invasion, cancer proliferation, angiogenesis, and phenotype of cancer stem/progenitor cells (CSPC). The molecular mechanism of metronomic Celecoxib on HCC was dissected using Luciferase assay.ResultsIn vivo metronomic Celecoxib exerted its chemopreventive effect by significantly reducing tumor growth of implanted syngeneic HCC and spontaneous hepatocarcinogenesis in HBVtg mice. Unlike suprapharmacological dose, metronomic Celecoxib can only inhibit HCC cell invasion after a 7-day course of treatment via NF-κB/MMP9 dependent, COX2/PGE2 independent pathway. Metronomic Celecoxib also significantly suppressed HCC cell proliferation after a 7-day or 30-day culture. Besides, metronomic Celecoxib reduced CSPC phenotype by diminishing sphere formation, percentage of CD90+ population in sphere cells, and expression of CSPC markers.ConclusionsMetronomic Celecoxib should be investigated clinically as a chemopreventive agent for selected high-risk HCC patients (e.g., HCC patients after curative treatments).

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

MP02-16 CONTEMPORARY ANTIBIOTIC PRACTICE PATTERNS AND RATES OF INFECTIOUS COMPLICATIONS IN THE REAL WORLD SETTING

You have accessJournal of UrologyGeneral & Epidemiological Trends & Socioeconomics: Practice Patterns, Quality of Life and Shared Decision Making I (MP02)1 Apr 2020MP02-16 CONTEMPORARY ANTIBIOTIC PRACTICE PATTERNS AND RATES OF INFECTIOUS COMPLICATIONS IN THE REAL WORLD SETTING Kelly Pekala*, Anup Shah, Olutiwa Akomolede, Hermoon Worku, Devin Rogers, Jathin Bandari, Benjamin Davies, and Bruce Jacobs Kelly Pekala*Kelly Pekala* More articles by this author , Anup ShahAnup Shah More articles by this author , Olutiwa AkomoledeOlutiwa Akomolede More articles by this author , Hermoon WorkuHermoon Worku More articles by this author , Devin RogersDevin Rogers More articles by this author , Jathin BandariJathin Bandari More articles by this author , Benjamin DaviesBenjamin Davies More articles by this author , and Bruce JacobsBruce Jacobs More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000816.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: There is increasing awareness surrounding the unintended deleterious downstream effects of antibiotics such as antibiotic resistance and cancer-specific survival. The American Urological Association recently released guidelines in June of 2019 consistent with more judicious antibiotic use and tailored antibiotic prophylaxis. In anticipation of the changes in antibiotic practice patterns, our objective was to review prior antibiotic practice patterns and infectious rates of three of the most common urologic procedures—cystoscopy, prostate biopsy, and post-prostatectomy catheter removal. METHODS: We retrospectively reviewed the Urology clinic schedule for three months (November 2018-January 2019) for cystoscopy and prostate biopsy and for five months (August 2018-January 2019) for post-prostatectomy catheter removal and identified patients who underwent the procedures of interest. We then recorded antibiotics given for prophylaxis at time of procedure, individual patient risk factors for infection, 30-day infection outcomes, and urine and blood culture data. RESULTS: We identified 550 cystoscopies, 113 prostate biopsies, and 157 post-prostatectomy catheter removals. Antibiotic prophylaxis practices varied widely. (Figure 1). Infection rates for cystoscopy, prostate biopsy, and post-prostatectomy catheter removal were 3.6%, 5.3%, and 7.0%, respectively. Among those who had an infection following cystoscopy, we fitted a multilinear regression analysis and found that indication for cystoscopy (eg. bladder cancer surveillance) (p<0.05), clean intermittent catheterization (p<0.05), indwelling foley catheter (p<0.05) were statistically significant. For post-prostatectomy catheter removal, we performed a logistic regression analysis, and a patient that did not receive antibiotics was 4.3 times more likely to have a urinary tract infection to patients receiving a single dose (p = 0.03). CONCLUSIONS: We found tremendous variation in antibiotic practices within one academic institution. In the future, we will be evaluating the effect of the new guideline changes on antibiotic procedural prophylaxis patterns and infection rates. Source of Funding: Shadyside Hospital Foundation © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e18-e18 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kelly Pekala* More articles by this author Anup Shah More articles by this author Olutiwa Akomolede More articles by this author Hermoon Worku More articles by this author Devin Rogers More articles by this author Jathin Bandari More articles by this author Benjamin Davies More articles by this author Bruce Jacobs More articles by this author Expand All Advertisement PDF downloadLoading ...

Advanced gelatin-based vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents

Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.

Effects of dendritic epidermal T cells on proliferation and apoptosis of epidermal cells in wound margin of mice

Effects of dendritic epidermal T cells on proliferation and apoptosis of epidermal cells in wound margin of mice

Change of hepatopancreas conditions in intensive shrimp aquaculture (Litopenaeus vannamei) at Mayangan Village, Legon Kulon District West Java

The study of hepatopancreas purpose to monitor changes in the health condition of shrimp, which is hepatopancreas is a vital organ of metabolism and digestive system. This study was conducted in intensive shrimp ponds in Mayangan Village, Legon Kulon for 82 days. Sampling was carried out every 10 days. Hepatopancreas preparation was conducted by taking 30-50 mg with adding 0.1-0.2 ml of immersion liquid and observing used a microscope at 40x magnification. The parameters of hepatopancreas observed such as microphili, lipids, wrinkles, and necrosis. After monitoring the microphili in the Day of Culture (DOC) 1 - 40, the average score range shows that the culture shrimp is 90-100% healthy. in DOC 43 until the last sampling has decreased by 40-0% which is assumed to be the effect of a White Feces Disease (WFD). The lipid and wrinkle in DOC 1-40 sampling showed that the average range shrimp culture was 90-100% healthy. While in DOC-43 sampling until the last of lipid and wrinkle sampling decreased by 60-0%. Hepatopancreas necrosis in DOC 1-31 sampling does not exceed from 20%, while DOC-40 sampling until the last of necrosis sampling continues to increase to 100% assumed by WFD and WSSV (White Spot Syndrome Virus).

CD244 Marks Non-Functional Hematopoietic Stem Cells with a Mast Cell Signature after Induction of Endoplasmic Reticulum Stress

Hematopoietic stem cells (HSCs) are capable of replenishing the entire blood system when needed and transplantation of HSCs remains as one of the most effective, curative treatments for patients with genetic diseases and hematopoietic malignancies. In vitro culture is an essential process for ex vivo expansion and modification of HSCs, however engraftment levels of cultured HSCs cannot be accurately estimated. This is mainly due to lack of reliable cell surface markers representing functionality of HSCs after culture, which also limits the resolution of molecular analyses. We have previously shown that HSCs are vulnerable to endoplasmic reticulum (ER) stress responses fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). Importance of ER stress suppression is also evident in vivo, as proliferative FL-HSCs fail to expand upon ER stress induction when natural molecular chaperone, bile acid, is reduced (Sigurdsson et al., Cell Stem Cell. 2016). Thus, ER stress elevation severely impairs the potential of HSCs, however usual marker profile is no longer representative of their functionality. In this study we aimed to discover the key signature and novel markers that represent functional retardation of HSC under activation and stress induction. Initially we compared gene expression profiles of fresh and 14-days cultured Lineage-Sca-1+c-kit+(LSK) CD48- (CD48-LSK) cells from mouse bone marrow using microarray analysis, since CD48 has been reported to enrich functional HSCs after in vitro culture (Noda et al., Stem Cells, 2008). We discovered abnormal up-regulations of genes frequently associated with mast cells (MC) in cultured CD48-LSK cells, and identified Cd244 as one of the top upregulated genes. CD244 is a member of the slam family of genes but is considered to be redundant with other slam markers in isolating HSCs from untreated mice. Indeed, freshly isolated CD150+CD48-LSK cells are negative for CD244. However, after 14-days in vitro culture with stem cell factor (SCF) and thrombopoietin (TPO), majority of CD150+CD48-LSK cells were positive for CD244. After shorter (7-days) culture, we found that CD48-LSK cells could be subdivided to CD244+ and CD244- populations (CD244-HSC and CD244+HSC). CD244-HSCs expressed high levels of HSC-related genes such as Fgd5, Hlf, Fhl1 and thrombopoietin receptor Mpl, In contrast, CD244+HSCs expressed MC-related genes, e.g. Cpa3, Gzmb and Mcpt8. In transplantation settings, CD244+HSCs showed no engraftment while CD244-HSCs showed long-term engraftment revealing them as functional stem cells. Since our and other groups have demonstrated that induction of ER stress impairs potential of mouse and human HSCs, we asked if ER stress induction would lead to the elevation of MC signature. Using an ER stress inducing chemical, thapsigargin, we could see increased ratio of CD244+HSCs within CD48-LSK cells. Conversely, the addition of TUDCA, a bile acid known to suppress ER stress, resulted in decreased frequency of CD244+HSCs. These findings strongly indicate that ER stress could be influencing the number of non-functional HSCs. To further substantiate the connection to ER stress and MC signature we analyzed a knock out mouse model of the ER stress modulator Trib3 (Trib3-/-) that is known to show an abnormal differentiation towards mast cells. Trib3-/- HSCs expressed MC genes including Cpa3 already at the steady-state condition. The number of CD244-HSCs after 7-days culture was significantly lower than control mice, and showed poor long-term engraftment potential in transplantation settings. To further elucidate the key molecular changes that impair HSCs, we compared gene expression profiles between fresh HSCs and CD244+/CD244-HSCs after 7-days culture. Gene expression comparison between CD244+ and CD244-HSCs independently confirmed the enrichment of MC cell related genes including Granzyme B (Gzmb), known to have negative impact on HSC potential (Carnevali et al., J Exp Med. 2014). Moreover, the Rel-A pathway was significantly lower in CD244-HSCs compared to fresh HSCs, suggesting a potential implication of NF-kB signal in the first alterations in HSCs during in vitro culture. We conclude that the induction of a MC cell signature fueled by ER stress is critical for normal HSC potential, and CD244 is a novel marker predicting the functionality of activated HSCs and allowing more detailed molecular analysis of activated HSCs. Disclosures No relevant conflicts of interest to declare.

Considerations for Maintaining Family Diversity in Commercially Mass-Spawned Penaeid Shrimp: A Case Study on Penaeus monodon.

Skewed family distributions are common in aquaculture species that are highly fecund, communally (mass) spawned, and/or communally reared. The magnitude of skews pose challenges for maintaining family-specific genetic diversity, as increased resources are required to detect individuals from underrepresented families, or reliably determine relative survival as a measure of family performance. There is limited understanding of family skews or changes in family proportion of communally reared shrimp under commercial rearing conditions and particularly how this may affect genotyping strategies to recover family performance data in breeding programs. In this study, three separate batches of shrimp, Penaeus monodon, were communally spawned and reared, and then sampled as larvae when ponds were stocked at 30 days of culture (DOC) and as juveniles from commercial ponds during harvest at 150 DOC. A total of 199 broodstock contributed to the 5,734 progeny that were genotyped with a custom multiplex single nucleotide polymorphism (SNP) panel, and family assignments were cross-referenced using two parentage assignment methods, CERVUS and COLONY. A total of 121 families were detected, with some families contributing up to 11% of progeny at 30 DOC and up to 18% of progeny at harvest. Significant changes were detected for 20% of families from 30 to 150 DOC, with up to a 9% change in relative contribution. Family skew data was applied in several models to determine the optimal sample size to detect families, along with the ability to detect changes in relative family contribution over time. Results showed that an order of magnitude increase in sampling was required to capture the lowest represented 25% of families, as well as significantly improve the accuracy to determine changes in family proportion from 30 to 150 DOC. Practical measures may be implemented at the hatchery to reduce family skews; a cost-effective measure may be to address the initial magnitude differences in viable progeny produced among families, by pooling equal quantities of hatched larvae from each family. This study demonstrates the relationships between skews in families under commercial conditions, the ability to accurately detect families, and the balance of sampling effort and genotyping cost in highly fecund species such as shrimp.

Cymothoa indica Schioedte and Meinert, 1884 (Crustacea: Isopoda: Cymothoidae) infestation in cage-cultured Asian seabass Lates calcarifer (Bloch, 1790) from the south-west coast of India

Infestation of the isopod parasite Cymothoa indica in Asian seabass Lates calcarifer (Bloch, 1790) fingerlings stocked in low volume cages is reported here. Hatchery produced seabass fry, reared to fingerling size (average size, 7±1 g and 63±12 mm) were used for stocking cages in Ashtamudi Lake, Kerala, south-west India. Instances of mortality were first observed on the 25th day of culture (DOC). By the 35th DOC, cumulative mortality of 84.6% was recorded. Intensity of parasitic infestation was found to be one parasite per fish. Parasites were observed to be latched to the tongue of the host fish. Reddening of buccal cavity, dark pigmentation, tongue degeneration/replacement were observed in the affected fish. On the 35th DOC, surviving fish in the cages were examined and the prevalence of C. indica was 53.08% in the remaining live fish. Though experimental infestation of Asian seabass by C. indica has been reported previously under laboratory conditions, this forms the first report on infestation of C. indica in cage-cultured Asian seabass.

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.