Better under stress: Improving bacterial cellulose production by Komagataeibacter xylinus K2G30 (UMCC 2756) using adaptive laboratory evolution

Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1-25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production

Assessment of alumina nano fluid as a coolant in double pipe gas cooler for trans-critical CO2 refrigeration cycle

In this study, the performance of an alumina nanofluid cooled double pipe gas cooler fortrans-critical C02 refrigeration cycle is theoretically compared to that of water cooled gas cooler. Equal pumping power comparison criterion is adopted besides conventional equal Reynolds number comparison base. Nanofluid is loaded with 0.5%, 1.5% and 2.5% of particle volume fraction under turbulent flow conditions. Drastic variation of thermal and transport properties of CO2 in the vicinity of pseudo criticai temperature is taken care of by employing an appropriate discretization technique. Effect of gas cooler pressure, Reynolds number, pumping power and nanoparticle volume fraction on COP of refrigeration system, gas cooler overall conductance, effectiveness and its capacity has been studied. Results indicate that at equal Reynolds number comparison, performance for alumina nanofluid cooled system is better than that of water cooled system. On the other hand, at equal pumping power comparison basis, the performance of water cooled system is superior. Even at equal mass flow rate comparison criterion, the performance of nanofluid cooled system degrades with increase in particle volume fraction. This study is expected to help to assess the nano fluid as a coolant before expensive experimentation

Use of an Online Ultrasound Simulator to Teach Basic Psychomotor Skills to Medical Students During the Initial COVID-19 Lockdown: Quality Control Study.

Teaching medical ultrasound has increased in popularity in medical schools with hands-on workshops as an essential part of teaching. However, the lockdown due to COVID-19 kept medical schools from conducting these workshops. The aim of this paper is to describe an alternative method used by our medical school to allow our students to acquire the essential psychomotor skills to produce ultrasound images. Our students took online ultrasound courses. Consequently, they had to practice ultrasound exercises on a virtual simulator, using the mouse of their computer to control a simulated transducer. Our team measured the precision reached at the completion of simulation exercises. Before and after completion of the courses and simulator's exercises, students had to complete a questionnaire dedicated to psychomotor skills. A general evaluation questionnaire was also submitted. A total of 193 students returned the precourse questionnaire. A total of 184 performed all the simulator exercises and 181 answered the postcourse questionnaire. Of the 180 general evaluation questionnaires that were sent out, 136 (76%) were returned. The average precourse score was 4.23 (SD 2.14). After exercising, the average postcourse score was 6.36 (SD 1.82), with a significant improvement (P<.001). The postcourse score was related to the accuracy with which the simulator exercises were performed (Spearman rho 0.2664; P<.001). Nearly two-thirds (n=84, 62.6%) of the students said they enjoyed working on the simulator. A total of 79 (58.0%) students felt that they had achieved the course's objective of reproducing ultrasound images. Inadequate connection speed had been a problem for 40.2% (n=54) of students. The integration of an online simulator for the practical learning of ultrasound in remote learning situations has allowed for substantial acquisitions in the psychomotor field of ultrasound diagnosis. Despite the absence of workshops, the students were able to learn and practice how to handle an ultrasound probe to reproduce standard images. This study enhances the value of online programs in medical education, even for practical skills

Effect of Re-acidification on Buffalo Grass Rhizosphere and Bulk Microbial Communities During Phytostabilization of Metalliferous Mine Tailings

Phytostabilized highly acidic, pyritic mine tailings are susceptible to re-acidification over time despite initial addition of neutralizing amendments. Studies examining plant-associated microbial dynamics during re-acidification of phytostabilized regions are sparse. To address this, we characterized the rhizosphere and bulk bacterial communities of buffalo grass used in the phytostabilization of metalliferous, pyritic mine tailings undergoing re-acidification at the Iron King Mine and Humboldt Smelter Superfund Site in Dewey-Humboldt, AZ. Plant-associated substrates representing a broad pH range (2.35-7.76) were sampled to (1) compare the microbial diversity and community composition of rhizosphere and bulk compartments across a pH gradient, and (2) characterize how re-acidification affects the abundance and activity of the most abundant plant growth-promoting bacteria (PGPB; including N2-fixing) versus acid-generating bacteria (AGB; including Fe-cycling/S-oxidizing). Results indicated that a shift in microbial diversity and community composition occurred at around pH 4. At higher pH (>4) the species richness and community composition of the rhizosphere and bulk compartments were similar, and PGPB, such as Pseudomonas, Arthrobacter, Devosia, Phyllobacterium, Sinorhizobium, and Hyphomicrobium, were present and active in both compartments with minimal presence of AGB. In comparison, at lower pH (<4) the rhizosphere had a significantly higher number of species than the bulk (p < 0.05) and the compartments had significantly different community composition (unweighted UniFrac; PERMANOVA, p < 0.05). Whereas some PGPB persisted in the rhizosphere at lower pH, including Arthrobacter and Devosia, they were absent from the bulk. Meanwhile, AGB dominated in both compartments; the most abundant were the Fe-oxidizer Leptospirillum and Fe-reducers Acidibacter and Acidiphilium, and the most active was the Fe-reducer Aciditerrimonas. This predominance of AGB at lower pH, and even their minimal presence at higher pH, contributes to acidifying conditions and poses a significant threat to sustainable plant establishment. These findings have implications for phytostabilization field site management and suggest re-application of compost or an alternate buffering material may be required in regions susceptible to re-acidification to maintain a beneficial bacterial community conducive to long-term plant establishment.National Institute of Environmental and Health Sciences (NIEHS) Superfund Research Program (SRP) [P42 ES004940]; National Science Foundation Graduate Research Fellowhip Program (NSF GRFP) [DGE-1143953]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

10.1186/1476-8518-6-2Journal of Immune Based Therapies and Vaccines6

Use of Ultraviolet Light Irradiated Multiple Myeloma Cells as Immunogens to generate Tumor Specific Cytolytic T Lymphocytes

Background: As the eradication of tumor cells in vivo is most efficiently performed by cytolytic Tlymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described. Methods: Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity. Results: By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p \u3c 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells. Conclusion: Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens

Assessing effectiveness of Komagataeibacter strains for producing surface-microstructured cellulose via guided assembly-based biolithography.

In this study, a medical device made of surface microstructured bacterial cellulose was produced using cellulose‑producing acetic acid bacteria wild‑type strains in combination with guided assembly‑based biolithography. The medical device aims at interfering with the cell’s focal adhesion establishment and maturation around implantable devices placed in soft tissues by the symmetrical array on its surface. A total of 25 Komagataeibacter strains was evaluated over a three‑step selection. In the first step, the ability of strains to produce a suitable bacterial cellulose layer with high production yield was examined, then nine strains, with a uniform and smooth layer of bacterial cellulose, were cultured in a custom‑made silicone bioreactor and finally the characteristics of the symmetrical array of topographic features on the surface were analysed. Selected strains showed high inter and intra species variability in bacterial cellulose production. The devices obtained by K2G30, K1G4, DSM 46590 (Komagataeibacter xylinus), K2A8 (Komagataeibacter sp.) and DSM 15973T (Komagataeibacter sucrofermentas) strains were pouched‑formed with hexagonal surface pattern required for reducing the formation of fibrotic tissue around devices, once they are implanted in soft tissues. Our findings revealed the effectiveness of the selected Komagataeibacter wild‑type strains in producing surface microstructured bacterial cellulose pouches for making biomedical devices

Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation

Acetic acid bacteria (AAB) are obligately aerobic bacteria within the family Acetobacteraceae, widespread in sugary, acidic and alcoholic niches. They are known for their ability to partially oxidise a variety of carbohydrates and to release the corresponding metabolites (aldehydes, ketones and organic acids) into the media. Since a long time they are used to perform specific oxidation reactions through processes called “oxidative fermentations”, especially in vinegar production. In the last decades physiology of AAB have been widely studied because of their role in food production, where they act as beneficial or spoiling organisms, and in biotechnological industry, where their oxidation machinery is exploited to produce a number of compounds such as l-ascorbic acid, dihydroxyacetone, gluconic acid and cellulose. The present review aims to provide an overview of AAB physiology focusing carbon sources oxidation and main products of their metabolism

Phase 1 study of fianlimab, a human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, plus cemiplimab in advanced melanoma

Background: Concurrent LAG-3 blockade may enhance efficacy of anti-program cell death-1 (PD-1) therapies such as cemiplimab. We present updated safety and clinical activity data from patients with advanced melanoma treated concurrently with cemiplimab and fianlimab (NCT03005782). Methods: Patients were included with unresectable or metastatic melanoma (excluding uveal melanoma) who were anti-PD-ligand (L) 1 treatment naive (expansion cohort [EC] 6) or anti-PD-(L)1 experienced within 3 months of screening (EC7). Patients received fianlimab 1600 mg + cemiplimab 350 mg intravenously every 3 weeks for 12 months (optional extra 12 months if clinically indicated). Tumours were measured every 6 weeks for 24 weeks, then every 9 weeks. In EC6 (n = 40) and EC7 (n = 15), respectively (data cutoff 9th February 2022), median age was 69.5 and 59.0 years, and median treatment duration was 37.1 and 9.0 weeks. Results: In EC6 and EC7, respectively, incidence of Grade ≥3 treatment-emergent adverse events (TEAEs) were 38% and 47%, incidence of serious TEAEs was 33% and 33%, and 18% and 13% of patients discontinued treatment due to a TEAE. Adrenal insufficiency rate was 13% and 7% in EC6 and EC7, respectively; no instances led to treatment discontinuation. Investigator-assessed objective response rate was63%(six complete responses; 19 partial responses) in EC6 and 13% (two partial responses) in EC7. Kaplan-Meier estimate of median progression-free survival was 14.2 (95% CI: 5.6-not estimated) months in EC6 and 1.4 (95% CI: 1.3-7.7) months in EC7. Median duration of response was not reached in EC6 or EC7. Conclusion: Fianlimab plus cemiplimab in advanced melanoma had a similar safety profile to anti-PD-1 monotherapies. Clinical activity in anti-PD-(L)1-naive patients appeared higher than previously reported for anti-PD-1monotherapy or anti-LAG-3 plus anti-PD-1. A Phase 3 trial (NCT05352672) investigating fianlimab plus cemiplimab in advanced melanoma is ongoing