Subsequent Culture Research Articles

Prevalence and Identification of Carbofuran Degrading Bacteria from Agricultural Fields and Associated Water Bodies

The extensive use of pesticides in agricultural areas is done to assure protection against insect invasion, to raise crop production, to improve fertility and soil quality, and therefore to efficiently contribute to an agriculture-based economy. However, the unfortunate reality is that the long-term presence of these pesticides in the environment as a result of their complex and xenobiotic chemical structures leads to bioaccumulation as well as bio-magnification, which in turn causes toxicity, chronic disabilities, hormonal imbalance, reproductive and endocrine dysfunctionalities, and many other health issues in humans as well as other vertebrates and causes ecosystem derangement. Similar results are produced by one of the most widely used N-methylcarbamate-based pesticides, carbofuran. There is no better solution to this issue than breakdown of leftover pesticide in the environment to harmless, native components through biological agents, ideally indigenous bacteria. Carbofuran, one of the most widely used N-methylcarbamate-based insecticides, has comparable effects. In light of this, a study was conducted to identify and characterize bacteria that have the innate ability to not only survive pesticide applications to rice and vegetable fields, but also to use the pesticides as a source of energy and break them down into harmless, environmentally friendly components. As a result, samples were taken from a Savar rice and tomato field shortly after a pesticide application, and Carbofuran 5G was the insecticide selected to check for degradability given to its widespread use there. Bushnell Haas broth and Bushnell Haas agar media were employed for enrichment, plating, and subsequent cultures to generate pure culture, with supplemented carbofuran of different concentrations serving as the only carbon source. The identities of the 17 isolates that could grow there were verified by sequencing after PCR and product purification. The evolutionary relatedness was determined using phylogenetic analysis with MEGAX. Acinetobacter spp., Rahnella spp., Chitinophaga spp., Serratia spp., Stenotrophomonas spp., Achromobacter spp., Pseudoxanthomonas spp., and Klebsiella spp. were the bacteria that were found.
 Bangladesh J Microbiol, Volume 39, Number 2, December 2022, pp 68-74

758 Correlation between initial and diagnostic bronchoalveolar lavage isolates among patients with inhalation injury and pneumonia

Abstract Introduction Inhalation injury is associated with increased risk of pneumonia. Bronchoscopy with bronchoalveolar lavage (BAL) is commonly used to diagnose and grade inhalation injury while obtaining a microbiological sample. It has been shown that a quarter of patients with inhalation injury have a single bacteria isolated from initial BAL within 12-48 hours of injury. The clinical significance of this and role of antibiotics in this setting are unknown. We investigated the incidence that bacteria isolated during a pneumonia episode were previously isolated on surveillance BAL among patients with inhalation injury. Methods Among patients with inhalation injury admitted to a large ABA-verified burn center from 2009-2022, microbiological isolates from initial BAL and subsequent culture obtained during a pneumonia episode (BAL, sputum), were extracted from medical records. Abbreviated injury severity score (AIS) was used to grade inhalation injury. Hospital-acquired pneumonia (HAP) was defined using CDC PNU1 criteria, and ventilator-associated pneumonia (VAP) was defined using PNU2 criteria with BAL cutoff >10,000 colony forming units (CFU). Results Two-hundred forty-eight patients with inhalation injury who underwent bronchoscopy for airway inspection and surveillance BAL within 48 hours of injury were included in the analysis. Of the 111 who had a bacterial isolate from initial BAL, 23 (21%) subsequently developed HAP or VAP during the hospitalization. Median (IQR) days between bronchoscopy and pneumonia diagnosis was six (2-8.5). In three of the 11 VAP cases (27%), the organisms identified at pneumonia diagnosis matched the organisms identified on initial BAL. Antibiotic use on admission was not associated with re-culture of initial BAL organism during a subsequent pneumonia episode (p=0.071). Conclusions Among patients with inhalation injury and bacteria isolated on surveillance BAL, 21% developed pneumonia during their hospital stay, usually with different bacteria than those isolated at initial bronchoscopy. There was no association between empiric antibiotic use and incidence of initial BAL organisms re-cultured during subsequent pneumonia episode. Applicability of Research to Practice Findings suggest initial BAL isolates cannot guide empiric coverage of pneumonia for patients with inhalation injury. This remains an important area of future research.

A rare cause of necrotizing pneumonia in an adult woman: p40phox (NCF4) deficient autosomal recessive Chronic Granulomatous Disease

IntroductionChronic Granulomatous Disease (CGD) is a rare inherited primary immunodeficiency typically diagnosed during childhood due to recurrent severe bacterial and fungal infections. Bi-allelic neutrophil cytoplasmic factor 4 (NCF4) mutations lead to P40phox deficient CGD which is a rare subtype of CGD. CaseA 37-year-old Pakistani woman with consanguineous parents presented with a left lower lobe necrotizing pneumonia. She reported tuberculosis as a child, but did not have a personal or family history of recurrent infections, colitis or other major health issues. Bronchoscopy with lung biopsy and bronchoalveolar lavage grew Burkholderia multivorans, and subsequent sputum culture grew Pseudomonas aeruginosa. Mycobacterium avium complex grew in 1 of 2 acid-fast bacillus cultures. Immune gene panel testing revealed homozygous pathologic mutations (c.118–1G > A-Splice acceptor) in NCF4 which encodes the p40phox subunit of the NADPH oxidase complex. Evaluation of her neutrophils by DHR oxidation revealed a slightly diminished response to PMA, but markedly reduced responses to multiple doses of opsonized, heat-killed S. aureus. PMN production of reactive oxygen species by luminol-enhanced chemiluminescence was also diminished after treatment with either opsonized zymosan or heat-killed S. aureus. Her neutrophils completely lacked p40phox by immunoblotting. Patient was treated with meropenem and levofloxacin. Interferon-gamma was added to her regimen following identification of her p40phox deficiency. After 10 weeks of therapy, computed tomography imaging showed improvement. Discussionp40phox is a regulatory component of the NADPH oxidase complex and impaired p40phox expression in phagocytes is associated with atypical presentations of CGD. Less than 30 cases have been described worldwide. NCF4-related CGD has fewer infections than other forms of CGD and is typically marked by granulomatous inflammation of the GI tract and skin. No adults previously identified have been asymptomatic past the second decade of life. All reported cases have had normal or only mildly impaired levels of DHR oxidation. This case report expands the clinical phenotype of NCF4-related CGD and illustrates the necessity of genetic testing for diagnosis, especially with near-normal DHR testing in the setting of opportunistic infection. Further studies are being pursued to identify why this patient presented so late in life with this infection and without colitis.

Pre-isolation Molecular Screening for High-throughput Sampling and Sequencing of Bacterial Microbes from the Environment.

Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic characterization of microorganisms to study their functional properties. For efficient isolation of low-prevalence organisms, enrichment followed by PCR screening is performed to identify positive samples for subsequent culture. Molecular characterization, strain-typing, and genotyping of isolated microorganisms is best comprehensively performed using whole-genome sequencing. This article outlines end-to-end protocols for screening, isolation, and sequencing of microbes from environmental samples. We provide systematic methods for environmental study design, enrichment, screening, and isolation of target microorganisms. Species identification is performed using qPCR or MALDI-TOF MS. Genomic DNA is extracted for whole-genome sequencing using the Oxford Nanopore platform. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Designing and conducting an environmental soil sampling study Basic Protocol 2: Enrichment of microbes from environmental soil samples Alternate Protocol 1: Collection and enrichment of microbes from environmental water samples Basic Protocol 3: Screening of enriched samples by direct qPCR Basic Protocol 4: Enumeration and isolation of enriched samples using selective medium Basic Protocol 5: Species confirmation using colony qPCR Alternate Protocol 2: Species identification using a MALDI-TOF MS Biotyper Alternate Protocol 3: Species identification of bacterial isolates using universal PCR primers and Sanger sequencing Basic Protocol 6: Cryostorage of bacterial isolates Basic Protocol 7: Extraction of genomic DNA Basic Protocol 8: Quality check of extracted genomic DNA Basic Protocol 9: Whole-genome sequencing using the Oxford Nanopore MinION Platform.

Guanidinylated/PEGylated chitosan in the bioink promotes the formation of multi-layered keratinocytes in a human skin equivalent

The biological differences of skin between rodent and human beings and the strong appeal to replace the experimental animals have led to the development of alternative models with structures similar to the real human skin. Keratinocytes cultured in vitro on conventional dermal scaffolds tend to form monolayer rather than multi-layer epithelial tissue architectures. How to construct human skin or epidermal equivalents with multi-layered keratinocytes similar to real human epidermis remains one of the greatest challenges. Herein, a human skin equivalent with multi-layered keratinocytes was constructed by 3D bioprinting fibroblasts and subsequent culturing epidermal keratinocytes. Biocompatible guanidinylated/PEGylated chitosan (GPCS) was used as the main component of bioink to 3D bioprint tissue-engineered dermis. The function of GPCS to promote HaCat cell proliferation and connection was confirmed at the genetic, cellular, and histological levels. Compared with the skin tissues with mono-layered keratinocytes engineered with collagen and gelatin, adding GPCS in the bioink generated tissue-engineered human skin equivalents with multi-layered keratinocytes. Such human skin equivalents could be alternative models for biomedical, toxicological, and pharmaceutical research.

Salmonella Typhimurium-based inactivated vaccine containing a wide spectrum of bacterial antigens which mimics protein expression changes during different stages of an infection process

Salmonella infections are still considered a persistent problem in veterinary medicine. Vaccination is one of the tools for decreasing the burden of many pathogens on animals. However, the efficiency of available commercial or experimental vaccines against non-typhoid Salmonella strains is not yet sufficient. We followed the path of an inactivated vaccine that is safe and well accepted, but whose presented antigen spectrum is limited. We improved this issue by using diverse cultivation conditions mimicking bacterial protein expression during the natural infection process. The cultivation process was set up to simulate the host environment to enhance the expression of SPI-1 (Salmonella pathogenicity island) proteins, SPI-2 proteins, siderophore-related proteins, and flagellar proteins. Three different cultivation media were used and subsequent cultures were mixed together, inactivated, and used for the immunization of post-weaned piglets. A mixture of recombinant Salmonella proteins was also used as a recombinant vaccine for comparison. The clinical symptoms during the subsequent experimental infection, antibody response, and organ bacterial loads were examined. One day after the infection, we observed an increased rectal temperature in the group of unvaccinated animals and the animals vaccinated with the recombinant vaccine. The increase in the temperature of the pigs vaccinated with the inactivated Salmonella mixture was significantly lower. In the same group, we also found lower bacterial loads in the ileum content and the colon wall. The IgG response to several Salmonella antigens was enhanced in this group, but it did not reach the titers of the group vaccinated with the recombinant vaccine. To summarize, the pigs vaccinated with an inactivated mixture of Salmonella cultures mimicking protein expression changes during the natural infection exhibited less serious clinical symptoms and lower bacterial load in the body after the experimental infection compared to the unvaccinated pigs and the pigs vaccinated with a mixture of recombinant Salmonella proteins.

Isolation and characterization of halotolerant plant growth promoting rhizobacteria from mangrove region of Sundarbans, India for enhanced crop productivity.

Halotolerant plant growth promoting rhizobacteria (PGPR) are beneficial microorganisms utilized to mitigate the biotic and abiotic stresses in plants. The areas of Sundarban mangroves of West Bengal, India have been reported to be rich in halotolerant microflora, yet major area remains unexplored. The present study, therefore, aims to map down the region-specific native microbial community potent of salt tolerance, plant growth promoting (PGP) activity and antagonistic activity against fungal pathogens. Bacterial samples were isolated from the saline soil of the Sundarban mangroves. A total of 156 bacterial samples were isolated and 20 were screened for their salt tolerance potential. These isolates were characterised using morphological, biochemical, and molecular approaches. Based on 16s rRNA sequencing, they were classified into 4 different genera, including Arthrobacter sp. (01 isolate), Pseudomonas plecoglossicida (01 isolate), Kocuria rosea (01 isolate), and Bacillus (17 isolates). The halotolerant isolates which possessed plant growth promoting traits including phosphate, and zinc solubilization, indole acetic acid production, siderophore, and ammonia generation were selected. Further, the effect of two halotolerant isolates GN-5 and JR-12 which showed most prominent PGP activities was evaluated in pea plant under high salinity conditions. The isolates improved survival by promoting germination (36 to 43%) and root-shoot growth and weight of pea plant in comparison to non-inoculated control plants. In a subsequent dual culture confrontation experiment, both these halo-tolerant isolates showed antagonistic activities against the aggressive root rot disease-causing Macrophomina phaseolina (Tassi) Goid NAIMCC-F-02902. The identified isolates could be used as potential bioagents for saline soils, with potential antagonistic effect on root rot disease. However, further studies at the physiological and molecular level would help to delineate a detail mechanistic understanding of broad-spectrum defence against salinity and potential biotic pathogen.

Diagnosis and unsuccessful management of iatrogenic fungal septic arthritis caused byWickerhamomyces anomalous(formerlyCandida pelliculosa) and concurrent osteochondral lesion in Ireland

SummaryEquine septic fungal arthritis is poorly documented in the literature. There is an absence of efficacy and safety data for antifungal medications, highlighting the importance of documenting this case. A 15‐year‐old Irish sport horse gelding presented 4/5 (AAEP) left hindlimb lame 13 days after intra‐articular medication of the left tarsocrural joint with triamcinolone and hyaluronic acid. Radiography revealed an osteochondral fragment at the distal intermediate ridge of the tibia and shallow subchondral bone defect on the medial trochlear ridge. Synoviocentesis yielded a turbid sample with an elevated total nucleated cell count (49 × 109cells/L; total neutrophil count of 62.8%). Arthroscopic lavage of the tarsocrural joint (TCJ) was performed. Ceftriaxone was administered and systemic antimicrobials initiated. The fungusWickerhamomyces anomaluswas identified on culture. The horse was subsequently intensively treated with antimicrobial and antifungal medications, both systemically and intra‐articularly over the following 60 plus days. Multiple subsequent synovial cultures revealed the persistence of the fungal organism, until a negative culture on day 55. However, on day 113, a grade 5/5 lameness was present, and a further positive synovial fungal culture. Radiographs revealed a lytic lesion at the distal tibia with marked progression. The horse was euthanised on day 126.

IRISIN STIMULATES ANABOLISM AND MATRIX SYNTHESIS IN HUMAN NUCLEUS PULPOSUS CELLS IN VITRO: NEW INSIGHTS INTO A CROSS-TALK BETWEEN THE MUSCLE AND THE INTERVERTEBRAL DISC

This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.

A Quality Improvement Project to Reduce Antibiotic Exposure in Premature Neonates.

Overuse of antibiotics in NICUs is a problem worldwide. Unnecessary antibiotic exposure leads to resistance, changes in the microbiome, and increases the risk of bronchopulmonary dysplasia, retinopathy of prematurity, periventricular leukomalacia, necrotizing enterocolitis, late-onset sepsis (LOS), and mortality in neonates. We aimed to safely reduce the antibiotic usage rate (AUR) in our level IV unit by 10% by December 2018. A multidisciplinary quality improvement project took place as part of a Vermont Oxford Network initiative in 2018. Multiple interventions took place, including identification of variations in practices and subsequent standardization through the creation of early onset and LOS guidelines, mass education, improved visibility of the guidelines, and standardized documentation. The main outcome measure for this project was the AUR for infants born <35 weeks' gestation expressed as antibiotic doses per 1000 patient days. The AUR decreased from a mean of 524 to 394, for a decrease of 24.8%. Results have been sustained for 3 years. Main contributors that led to the sustained success include decreasing the overall use of antibiotics for early onset sepsis, as well as the duration when cultures are negative. The number of LOS courses also decreased slightly. We noted no cases of inadequately treated sepsis resulting in subsequent positive cultures. Creation of guidelines with mass education and ongoing feedback/monitoring can result in a safe reduction of AUR in the NICU.

From Monitors to Monitors: A Primitive History

As computers became multi-component systems in the 1950s, handling the speed differentials efficiently was identified as a major challenge. The desire for better understanding and control of ‘concurrency’ spread into hardware, software, and formalism. This paper examines the way in which the problem emerged and was handled across various computing cultures from 1955 to 1985. In the machinic culture of the late 1950s, system programs called ‘monitors’ were used for directly managing synchronisation. Attempts to reframe synchronisation in the subsequent algorithmic culture pushed the problem to a higher level of abstraction; Dijkstra’s semaphores were a reaction to the algorithms’ complexity. Towards the end of the 1960s, the culture of ‘structured programming’ created a milieu in which Dijkstra, Hoare, and Brinch Hansen (among others) aimed for a concurrency primitive which embodied the new view of programming. Via conditional critical regions and Dijkstra’s ‘secretaries’, the co-produced ‘monitor’ appeared to provide the desired encapsulation. The construct received embodiment in a few programming languages; this paper ends by considering Modula and Concurrent Pascal.

MP34-10 NATURAL HISTORY OF RECURRENT UTI: POOR RESOLUTION RATES WITHOUT PREVENTATIVE APPROACHES

You have accessJournal of UrologyCME1 Apr 2023MP34-10 NATURAL HISTORY OF RECURRENT UTI: POOR RESOLUTION RATES WITHOUT PREVENTATIVE APPROACHES Emma Dixon, Ashley Caron, and A. Lenore Ackerman Emma DixonEmma Dixon More articles by this author , Ashley CaronAshley Caron More articles by this author , and A. Lenore AckermanA. Lenore Ackerman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recurrent UTIs (rUTIs) have an enormous healthcare burden. Many patients continue to experience recurrence of symptoms despite appropriate antibiotic treatment. We aimed to understand the natural history and progression of rUTI in women to determine the influence of patient factors, laboratory findings, and management approaches on recurrence. METHODS: Women aged 18-95 diagnosed between 2013-2015 by urogynecologic specialists with rUTI after in-person evaluation were followed for up to three subsequent visits for symptomatic complaints over the year following initial presentation. Patients with complicated UTIs or any history of pyelonephritis were excluded. Patient demographics, comorbidities, laboratory findings, treatments administered, and symptom resolution for each episode was recorded. RESULTS: Of a total of 101 women with rUTI, 70% (70/101) experienced recurrent symptoms within 1 year; 90% of these recurrences included acute dysuria prompting reevaluation. On multivariable logistic regression, only prior pelvic surgery (OR 0.15 [95% CI 0.03-0.8], p=0.02) was associated with lower recurrence rates, while preventative medications (prophylactic or post-coital antibiotics, vaginal estrogen) was the only variable significantly associated with symptomatic resolution (OR 4.85 [95% CI 1.1-22], p=0.04). Post-menopausal women more commonly experienced symptomatic resolution than pre-menopausal women (OR 20.4 [95% CI 0.83-502], p=0.06), and a comorbid diagnosis of anxiety or depression was more common in patients whose symptoms did not resolve (OR 0.09 [95% CI 0.01-1.06], p=0.08), although these trends did not reach statistical significance. Across all symptomatic episodes, 60% of urine cultures did not have microbiological evidence of infection. Only 2/70 (3%) subjects had positive cultures during all symptomatic episodes. There was no correlation between initial culture results and rates of symptomatic resolution or subsequent culture positivity. CONCLUSIONS: Spontaneous resolution of symptoms is not the natural history of rUTIs once established. Use of intermittent antibiotics to treat rUTIs is inferior to preventative measures at achieving symptomatic resolution. As most symptomatic episodes displayed negative urine cultures despite symptoms consistent with UTI, future studies will need to determine whether this discrepancy is due to poor sensitivity of standard urine culture or a higher than previously appreciated rate of confounding conditions presenting with similar symptoms as UTI. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e462 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Emma Dixon More articles by this author Ashley Caron More articles by this author A. Lenore Ackerman More articles by this author Expand All Advertisement PDF downloadLoading ...

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Development of a novel ex vivo organ culture system to improve preservation methods of regenerative tissues

Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial–mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine.

Extended preconditioning on soft matrices directs human mesenchymal stem cell fate via YAP transcriptional activity and chromatin organization.

Dynamic extracellular matrix (ECM) mechanics plays a crucial role in tissue development and disease progression through regulation of stem cell behavior, differentiation, and fate determination. Periodontitis is a typical case characterized by decreased ECM stiffness within diseased periodontal tissues as well as with irreversible loss of osteogenesis capacity of periodontal tissue-derived human periodontal tissue-derived MSCs (hMSCs) even returning back to a physiological mechanical microenvironment. We hypothesized that the hMSCs extendedly residing in the soft ECM of diseased periodontal tissues may memorize the mechanical information and have further effect on ultimate cell fate besides the current mechanical microenvironment. Using a soft priming and subsequent stiff culture system based on collagen-modified polydimethylsiloxane substrates, we were able to discover that extended preconditioning on soft matrices (e.g., 7 days of exposure) led to approximately one-third decrease in cell spreading, two-third decrease in osteogenic markers (e.g., RUNX2 and OPN) of hMSCs, and one-thirteenth decrease in the production of mineralized nodules. The significant loss of osteogenic ability may attribute to the long-term residing of hMSCs in diseased periodontal tissue featured with reduced stiffness. This is associated with the regulation of transcriptional activity through alterations of subcellular localization of yes-associated protein and nuclear feature-mediated chromatin organization. Collectively, we reconstructed phenomena of irreversible loss of hMSC osteogenesis capacity in diseased periodontal tissues in our system and revealed the critical effect of preconditioning duration on soft matrices as well as the potential mechanisms in determining ultimate hMSC fate.

Micropropagation of Strawberry Crop (Fragaria ananassa): A Review

The objective of this review paper is to summarize on the history, advantages, disadvantages of in vitro micropropagation of strawberry crop. Since the traditional method of propagation, that is, the use of runners as propagating material, is not that efficient because it leads to the spread of viral diseases. We have to go for in vitro micropropagation, the crops also yield more when compared to traditional methods of propagation. The paper also reviews a study done to develop a protocol for large-scale propagation of strawberry cv. ‘Sweet Charlie’ and ‘Winter Dawn’ using the micropropagation technique. Shoot cultures were obtained from shoot tips on Murashige and Skoog (MS) medium with 4% table sugar, 0.75% agar, 5 mg L-1 6-benzyladenine and 0.01 mg L-1 kinetin. These shoots were multiplied and maintained on MS medium with 1 mg L-1 6-benzyladenine and 0.1 mg L-1 kinetin. Rooting of in vitro raised shoots was successfully conducted by pulse treatment with 500 mg L-1 indole-3-butyric acid for 30 s and subsequent culturing in MS medium with 1 mg L-1 indole-3-butyric acid, 0.1 g L-1 activated charcoal and 6% table sugar. Plants were successfully acclimatized and survived in field conditions.

A Three-Dimensional Xeno-Free Culture Condition for Wharton's Jelly-Mesenchymal Stem Cells: The Pros and Cons.

Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton's Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.

Capture and release of circulating tumor cells stimulated by pH and NIR irradiation of magnetic Fe3O4@ZIF-8 nanoparticles

Detecting and analyzing circulating tumor cells(CTCs) is significant for early diagnosis, management, and personalized treatment of tumors. Herein, a smart magnetic aptamer modified Fe3O4@ZIF-8 core/shell structured nanoparticle (NPs) was successfully constructed using for capture and simultaneous pH- and NIR-irradiation responsive release of CTCs. Taking MCF-7 as model CTCs, it could be captured ca. 60 % in low-concentration artificial blood by aptamer (SYL3C) on the surface of Fe3O4@ZIF-8 NPs. After magnetic separation, the ZIF-8 shell in aptamer-modified Fe3O4@ZIF-8 NPs carrying captured CTCs would disintegrate within 20 min under the synergistic effect of an acidic environment (pH=6.0) and NIR irradiation leading to the release of CTCs with high cell viability, which was benefited for the subsequent culture and analysis. This magnetic and core/shell structured device integrated high-efficiency capture, quick isolation and perfect release into one system, which showed great potentials for the detection of CTCs in the clinic.

Electrochemical and biological characterization of Ti–Nb–Zr–Si alloy for orthopedic applications

The performance of current biomedical titanium alloys is limited by inflammatory and severe inflammatory conditions after implantation. In this study, a novel Ti–Nb–Zr–Si (TNZS) alloy was developed and compared with commercially pure titanium, and Ti–6Al–4V alloy. Electrochemical parameters of specimens were monitored during 1 h and 12 h immersion in phosphate buffered saline (PBS) as a normal, PBS/hydrogen peroxide (H2O2) as an inflammatory, and PBS/H2O2/albumin/lactate as a severe inflammatory media. The results showed an effect of the H2O2 in inflammatory condition and the synergistic behavior of H2O2, albumin, and lactate in severe inflammatory condition towards decreasing the corrosion resistance of titanium biomaterials. Electrochemical tests revealed a superior corrosion resistance of the TNZS in all conditions due to the presence of silicide phases. The developed TNZS was tested for subsequent cell culture investigation to understand its biocompatibility nature. It exhibited favorable cell-materials interactions in vitro compared with Ti–6Al–4V. The results suggest that TNZS alloy might be a competitive biomaterial for orthopedic applications.

Evaluation of Moringa oleifera and corn starch as feed for seed production of the pearl oyster Pteria sterna (Gould,1851)

In the search for complementary diets to improve performance in bivalve farming, the use of terrestrial plants with nutritional and nutraceutical properties has been proposed as an alternative. The effectiveness of moringa leaf meal Moringa oleifera (Mo) was evaluated under controlled laboratory conditions (30 days), as a dietary supplement during the pre-growth stage of Pteria sterna seeds (7.2 ± 0.59 mm), as well as its combinations with microalgae and corn starch (Co), on growth and survival in the laboratory and its subsequent initial suspended culture in the sea. Diets were formulated with a mixture of the microalgae Tetraselmis suecica and Chaetoceros gracilis (M), diet M; M and 5% Mo (M + Mo); diet M and 5% corn starch (M + Co); 100% moringa leaf meal (Mo); 100% corn starch (Co), and diet M with 2.5% Mo and 2,5% Co (M + Mo + Co). The Mo diet did not provide pre-seed sustainability, resulting in 100% mortality at 30 days. From the rest of the diets, M obtained the lowest oyster survival, while M + Mo and M + Mo + Co showed the highest growth rates. At the end of the laboratory bioassay, the seeds were sown in a culture system in the open sea (50 days), where the highest growth occurred in the juveniles previously fed with M + Mo + Co. The results suggest that, in the nursery, P. sterna pre-seeds can be maintained with a diet of 100% corn starch, but not with 100% moringa flour, probably due to its poor digestibility. However, moringa used as an additive to the microalgae diet provided a higher yield in the oyster, which is reflected in a higher yield in the initial culture outdoors.