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The aim of this study was to explore the concentrations of PAEs (Phthalate esters) in milk in Türkiye. For this purpose, a methodology was developed to quantify eight different PAEs in milk samples using a combination of dispersive solid-phase extraction (dSPE) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). Employing this methodology, the concentrations of PAEs were evaluated in 34 milk samples. Results indicated the presence of PAEs in the milk samples; however, all tested compounds remained within the specific migration limits established by the EU. Among the analyzed PAEs, BBP (Benzyl butyl phthalate) was not detected in any samples, while DMP (di-methyl phthalate) (ND-5.51 µg/L) and DBP (di-butyl phthalate) (ND-7.91 µg/L) exhibited the lowest concentrations. DEHP (bis(2-ethylhexyl)) was identified as the most prevalent plasticizer with a maximum concentration of 41.31 µg/L. In conclusion, this study successfully investigated PAE concentrations in Turkish milk samples using a developed methodology. The results indicated the presence of PAEs within EU-established limits, with DEHP being the predominant plasticizer. Further research and monitoring efforts are crucial to ensure ongoing safety in packaged milk products.

Different species of microalgae are highly efficient in removing nutrients from wastewater streams and are able to grow using flue gas as a CO2 source. These features indicate that application of microalgae has a promising outlook in wastewater treatment. However, practical aspects and process of integration of algae cultivation into an existing wastewater treatment line have not been investigated. The Climate-KIC co-funded Microalgae Biorefinery 2.0 project developed and demonstrated this integration process through a case study. The purpose of this paper is to introduce this process by phases and protocols, as well as report on the challenges and bottlenecks identified in the case study. These standardized technical protocols detailed in the paper help to assess different aspects of integration including biological aspects such as strain selection, as well as economic and environmental impacts. This process is necessary to guide wastewater treatment plants through the integration of algae cultivation, as unfavourable parameters of the different wastewater related feedstock streams need specific attention and management. In order to obtain compelling designs, more emphasis needs to be put on the engineering aspects of integration. Well-designed integration can lead to operational cost saving and proper feedstock treatment enabling algae growth.

Recently, much focus has been given to a new type of chemical production plant, with the aim of a much faster time-to-market (“50% idea”) and better cash-flow revenue. The main enabling technology is to have the plants pre-manufactured and assembled by a modular construction and to use innovative, smart-scale processing and apparatus technology, in order to achieve a compact overall plant footprint. Focal points in such technology are on the one hand, flow processing, with micro process technology as a cutting-edge cornerstone, and on the other hand, the container framework. Yet, other process-intensified technologies are suitable as well such as milli-flow or upgraded batch technologies. Finally, process robustness and short-time applicability make the decision. In this paper, for the first time, a CAPEX and OPEX analysis of the new plant technology is done, at the example of the Evotrainer production platform. This platform is pre-manufactured in serial and provides all the utilities needed around the reactor and e.g., separator to be tailored and inserted. The unit-operational modularization, with respective definition of interfaces, was developed further to a so-called functional modularization, where different cabinets with pre-defined functions and equipment are proposed. Three virtual microreactor applications were used and stand as model-based scenarios for market applications in bulk chemistry, fine chemistry and pharmacy. It is shown, in many facets, that the Evotrainer infrastructure based plants indeed have a faster payback and higher earnings as compared to conventional technology, particularly when serving high-priced markets such as pharmaceutical applications. Further, the combination with novel chemical routes or novel processing (Novel Process Windows) is advantageous. Micro process technology is one of the key enablers and was taken here, since the dataset of such technology was readily available to the authors due to past research efforts and there is some general belief in the combination to the so-called “Future Factories”. Yet, it stands also for any other process intensification technology which can achieve the same performance level and which is able to satisfy the needs of a producing industry.

Background: Blood components should be gamma-irradiated (γ-IR) in order to prevent transfusion-associated graft-versus-host disease. The aim of this study is to determine the effect of γ-IR and storage time on the exosomes released from apheresis platelet concentrates (aPC) and to investigate their impact on the maximum platelet aggregation (MPA) and hemostasis., Materials and Methods: Eight units of aPC were included in this study. These were divided into four equal portions. Two portions were irradiated before storage while the other two were not. Thus, irradiated and non-irradiated aPC samples for storage Days 0 (D0) and 5 (D5) were obtained. Exosomes were isolated from these samples using a commercial kit and were evaluated to ascertain their parent cells by flow cytometry. For the following steps, exosomes were pooled according to their features. Pooled exosomes were then used for aggregometry and thromboelastography., Results: Platelet-derived exosome (PD-EX) levels decreased in D5 compared to D0 in NI-aPC, whereas granulocyte-derived exosome (GD-EX) levels increased. Exosome pools had no effect on MPA compared to saline groups. Exosome pools decreased the time to initial fibrin formation (R), whereas they increased the rate of clot formation (α-angle) and coagulation index (CI) compared to saline groups., Discussion: Storage time and γ-IR each have almost the opposite effects on PD-EX and GD-EX. Exosomes have no impact on MPA, but enhance the clot strength. The impact of exosomes on aPC quality and effectiveness can be ignored or considered as a positive effect.

In the last decade, lignin has received much attention as a feedstock to produce bio-based products. This study investigates the potential benefits of using lignin to mitigate the environmental impact of the road construction sector. An environmental life cycle assessment (LCA) of various top-layer bio-based asphalts using kraft lignin was conducted. From a cradle-to-grave perspective, lignin-based asphalts were compared with conventional asphalts. The results of the LCA revealed that the climate change impact of lignin-based asphalts could be 30-75% lower than conventional asphalts. For the other ten impact categories, trade-offs were observed. Overall, two key factors to make the environmental impact of lignin-based asphalts lower than conventional asphalts are 1) increasing the amount of bitumen-substituted and 2) using low-grade biomass fuels for process steam in the pulp mill. The substitution of weak filler with lignin was beneficial only for climate change and could lead to a worse overall environmental performance than conventional asphalts. Similarly, higher environmental impacts for lignin-based asphalts could be obtained if the pulp mill consumed natural gas to complete the energy balance to replace the part of the black liquor from which lignin is extracted. This study also includes an in-depth discussion on methodological choices such as the allocation methods for lignin, functional units, and asphalt layers considered. We believe that such a methodological discussion could be helpful to support future Product Category Rules for asphalt mixtures., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tobias Strating is employed by an industrial partner (Roelofsgroep) that might have financial interests in the technology investigated by this article. Richard Gosselink is the inventor of a patent regarding lignin-based bio-asphalt. The other authors do not have any known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the 'rdar' (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the last decade, the use of lignin as a bio-based alternative for fossil-based products has attracted significant attention, and the first LCAs of lignin and derived products have been conducted. Assessing side-stream products like lignin and potential benefits compared to their fossil counterparts presents complex methodological issues. This article provides a critical review of forty-two peer-reviewed LCAs regarding lignin and derived products. Methodological issues and their influence on the LCA results include the choice of the modeling approach and system boundaries, functional unit definition, impact categories considered, type of data used, handling multifunctionality and biogenic carbon modeling. The review focused on climate change impacts, as this is also the main impact category considered in most studies. Other impact categories in the comparison between lignin-based products and counterparts were also discussed with examples from the studies. Based on ten lessons learned, recommendations were provided for LCA practitioners to increase future consistency of environmental claims made about lignin and lignin-based products. The finding suggest that the environmental performance of lignin-based products is significantly affected by both 1) LCA methodological problems such as allocation practices and biogenic carbon modeling and 2) technical aspects such as the percentage of lignin in the composition of products and the selection of the fuel to replace lignin in internal energy uses. Beyond this, the reviewed LCAs showed that often lignin-based products offer better environmental performances than fossil-based products, especially for climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Discovery of novel and broad-acting immunomodulators is of critical importance for the prevention and treatment of disorders occurring due to overexuberant immune responseincluding SARS-CoV-2 triggered cytokine storm leading to lung pathology and mortality during the ongoing viral pandemic. Mesenchymal stem/stromal cells (MSCs), highly regarded for their regenerative capacities, also possessesremarkable immunoregulatory functions affecting all types of innate and adaptive immune cells. Owing to that, MSCs have been heavily investigated in clinic for the treatment of autoimmune and inflammatory diseases along with transplant rejection. Extensive research in the last decaderevealed that MSCs carry out most of their functions through paracrine factors which are soluble mediators and extracellular vesicles (EVs). EVs, including exosomes and microvesicles, are an efficient way of intercellular communication due to their unique ability to carry biological messages such as transcription factors, growth factors, cytokines, mRNAs and miRNAs over long distances. EVs originate through direct budding of the cell membrane or the endosomal secretion pathway and they consist of the cytosolic and membrane components of their parent cell. Therefore, they are able to mimic the characteristics of the parent cell, affecting the target cells upon binding or internalization. EVs secreted by MSCs are emerging as a cell-free alternative to MSC-based therapies. MSC EVs are being tested in preclinical and clinical settings where they exhibit exceptional immunosuppressivecapacity. They regulate the migration, proliferation, activation and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory response. Being as effective immunomodulators as their parent cells, MSC EVs are also preferable over MSC-based therapies due to their lower risk of immunogenicity, tumorigenicity and overall superior safety. In this review, we present the outcomes of preclinical and clinical studies utilizing MSC EVs as therapeutic agents for the treatment of a wide variety of immunological disorders., Competing Interests: CONFLICT OF INTEREST: none declared, (Copyright © 2020 The Author(s).)

Pasteurella multocida serotype A:3 is a Gram-negative bacterial pathogen, one of the causative agents of shipping fever of cattle. In this study, outer membrane protein H (ompH) and Pasteurella lipoprotein E (plpE) genes were cloned and plpEC-ompH fusion was constructed and expressed in Escherichia coli. Recombinant PlpE, OmpH and PlpEC-OmpH fusion proteins were purified and formulated with oil-based and oil-based CpG ODN adjuvants. Antibody responses in mice vaccinated with recombinant PlpE and PlpEC-OmpH proteins formulated with both adjuvants were significantly (p<0.05) increased. However, a significant (p<0.05) increment in serum IFN-γ level was only observed upon immunization with oil-based CpG formulations. Protectivity of the vaccines were evaluated via intraperitoneal challenge of mice with 10 LD50 of P. multocida A:3. The recombinant proteins PlpE and PlpEC-OmpH fusion conferred 100% protection when formulated with oil-based CpG ODN while the protectivity was found to be 80% and 60%, respectively when only oil-based adjuvant was used in respective formulations. These findings indicated that the recombinant PlpE or PlpEC-OmpH fusion proteins formulated with oil-based CpG ODN adjuvant are possible acellular vaccine candidates against shipping fever., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Studies regarding the physiological role of acidic mammalian chitinase (AMCase) and the effects of its genetic variants on asthma have produced conflicting results., Objectives: We aimed to determine the genetic variants in the AMCase gene that could regulate the gene expression and thus influence disease severity., Methods: Genetic variants of the AMCase gene were determined by sequencing of asthmatics and healthy controls in up to -1 kb in the promoter region and exon 1 and 2. In an association study, a population of asthmatic (n = 504) and healthy Turkish children (n = 188) were genotyped for the observed SNPs. A replication study was performed in a North American adult population of patients with mild (n = 317) and severe (n = 145) asthma. The functional properties of the insertion were determined by promoter reporter assay, electromobility shift assay and transcription factor ELISA experiments., Results: Of the identified SNPs, only a ten base pair insertion (CAATCTAGGC) in the 5'UTR region of exon 2 was significantly associated with lower FEV(1) (β = -14.63 SE = 6.241, P = 0.019) in Turkish children with asthma. However, in the adult population, the same insertion showed a trend toward higher FEV(1). The insertion was shown to have enhancer activity and the mutant probe possessing the insertion had higher binding affinity for the nuclear extracts., Conclusion: Our study shows that a ten base pair insertion in the 5'UTR region of AMCase gene may modify gene expression and thus may affect the severity of asthma. However, its effects appear to be different in different populations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The immunogenicity of a vaccine formulation is closely related to the effective internalization by the innate immune cells that provide prolonged and simultaneous delivery of antigen and adjuvant to relevant antigen presenting cells. Endosome associated TLR9 recognizes microbial unmethylated CpG DNA. Clinical applications of TLR9 ligands are significantly hampered due to their pre-mature in vivo digestion and rapid clearance. Liposome encapsulation is a powerful tool to increase in vivo stability as well as enhancing internalization of its cargo to relevant immune cells. The present study established that encapsulating CpG motifs in different liposomes having different physicochemical properties altered not only encapsulation efficiency, but also the release and delivery rates that ultimately impacted in vitro and ex-vivo cytokine production rates and types. Moreover, different liposomes encapsulating CpG ODN significantly increased Th1-biased cytokines and chemokines gene transcripts Additional studies demonstrated that co-stimulatory and surface marker molecules significantly upregulated upon liposome/CpG injection. Finally, co-encapsulating model antigen ovalbumin with CpG ODN adjuvant in nanoliposomes profoundly augmented Th1 and cell mediated anti-Ova specific immune response. Collectively, this work established an unappreciated immunoregulatory property of nanoliposomes mediating immunity against protein antigen and could be harnessed to design more effective therapeutic vaccines or stand alone immunoprotective agents targeting infectious diseases, as well as cancer or allergy., (2010 Elsevier Ltd. All rights reserved.)

Synthetic oligodeoxynucleotides (ODN) expressing "CpG motifs" show promise as immune adjuvants, antiallergens, anticancer, and immunoprotective agents. Two structurally distinct classes of CpG ODN have been identified that stimulate human PBMC. This work establishes that both types of ODN bind to and are internalized by the same individual B cells, NK cells, and monocytes. However, the intracellular localization of "D" and "K" ODN differs, as does their functional activity: "K" type ODN trigger monocytes and B cells to proliferate and secrete IL-6 and IgM, whereas "D" type ODN induce NK cells to produce IFN-gamma and monocytes to differentiate into CD83(+)/CD86(+) dendritic cells. In monocytes, these two types of ODN (which differ in backbone composition and CpG motif) cross-inhibit one another's activity. Thus, different types of CpG ODN have distinct and in some cases incompatible effects on the same cells, a finding with important implications for the therapeutic use of these agents.

Polyhydroxyethyl methacrylate (pHEMA) membranes coated on one side with chondroitin sulfate (CS) were used to block adhesion physically and to reduce friction between healing flexor tendons and the surrounding tissue in rabbit forepaws after surgical repair. Digits with pHEMA-only, standard tendon sheath repair, and with no sheath repair were the controls. Over 12 weeks the CS-coated membranes were evaluated for joint flexion, adhesion limitation, and tendon healing progress. The membranes initially allowed for better flexion (ie, for 6 weeks), but their relative superior effectiveness faded afterward. Histology showed that adhesions were less severe and healing was better in the CS-pHEMA membranes at 3 and 6 weeks. If further studies determine precise amounts or thicknesses of CS coats that will maximize its healing properties, CS-pHEMA should prove useful in clinical settings in which restoration of tendon sheath integrity with a minimum of adhesions is not possible.

In this study, the major goal was to evaluate in vitro and in vivo findings by macroscopy, radiology, and histology to determine the effectiveness of therapy of experimental implant-related osteomyelitis with antibiotic carrier rods constructed of microbial polyesters. The polymers used were poly(3-hydroxybutyrate-co-4-hydroxyvalerate) [P(3-HB-co-4-HB)] and poly(3-hydroxybutyrate-co-3-hydroxy- valerate) [P(3-HB-co-3-HV)]. Both the Sulperazone and the Duocid-P(3-HB-co-4-HB) rods with a drug to polymer ratio of 1:1 (w/w) were effective in treating the bone infection that was experimentally initiated by inoculation of a hemolytic strain of Staphylococcus aureus (coagulase positive; phage type 52/52b) together with metal implants into the medullary area of rabbit tibia. Macroscopical data revealed that the effectiveness of therapy was apparent at week 6 for all categories tested. Radiological findings with Duocid- and Sulperazone-loaded P(3-HB-co-4-HB) rods improved significantly when judged by changes in periosteal elevation, widening of bone shaft, new bone formation, and soft-tissue deformation after 6 weeks of implantation. Histologically the signs of infection were found to subside by weeks 3 and 6. Inflammatory cells were replaced with bone-forming cells upon treatment with Sulperazone-P(3-HB-co-4-HB) and Duocid-P(3-HB-co-4-HB). Osteoblastic activity was prominent. Intramedullary inflammation, although still present, started to be replaced by fibrous or bony tissue. Histological findings presented the subsidence of infection. In summary, the antibiotic-loaded biopolymeric rods appeared to have potential as a new controlled-release system for the treatment of implant related osteomyelitis and chronic osteomyelitis., (Copyright 2001 John Wiley & Sons, Inc.)

In this study the construction and in vivo testing of antibiotic-loaded polyhydroxyalkanoate rods were planned for use in the treatment of implant-related osteomyelitis. The rods were constructed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), carrying 50% (w/w) Sulperazone or Duocid. They were implanted in rabbit tibia in which implant-related osteomyelitis (IRO) had been induced with Staphylococcus aureus. The effectiveness of the antibiotics in the treatment of IRO was determined. The establishment of IRO with bacterial inoculation was complete after 3 weeks with 100% infection rate in all groups. There was no contamination or super-infection. Both antibiotics were found to be highly effective against the bacteria. Following the application of Sulperazone-P(3-HB-co-4-HB) rods, no infective agents could be isolated from the infection site within the 6-week test period, indicating complete treatment of the infection. Macroscopical evaluation at follow-up revealed no drainage, minimal swelling and increase in local warmth, most probably due to the surgery rather than to a reaction towards the implant. The overall scores for radiological findings by the end of 6 weeks were 0.8/5 for the antibiotic-loaded rod implanted in the right limb, and 1.1/5 for the antibiotic-free rod implanted in the left limb. There was no statistical difference between the antibiotic-loaded and antibiotic-free polymeric rods. In vivo drug release was almost complete within the first week. One interesting observation, however, was that the therapy was still very effective even when the release rate was very high. In the SEM of in vitro tested rods, the polymeric component was unchanged in 2 weeks while the drug leached out, leaving voids behind. In vivo, however, the morphology of the implant was significantly modified within 6 weeks post-implantation. Since a substantial degree of the in vivo drug release was complete within 1 week, we believe that dissolution of the drug must be the predominant mechanism through which the drug release is controlled.

In this study, a novel antibiotic carrier system for use in the treatment of implant-related and chronic osteomyelitis was developed. Sulbactam-cefoperazone was introduced to rods of polyhydroxybutyrate-co-hydroxyvalerate (22 mol % HV, w/w), a member of a family of microbial-origin polymer that is biodegradable, biocompatible, and osteoconductive due to its piezoelectric property. The antibiotic-loaded carrier was implanted into the infection site that was induced by Staphylococcus aureus inoculation into the rabbit tibia. The effectiveness of this was assessed macroscopically, radiographically, bacteriologically, and histopathologically. Findings of infection subsided on day 15 and almost complete remission was observed on day 30. The control side that contained antibiotic-free rods, however, worsened. These findings prompted us to conclude that the novel biodegradable antibiotic carrier developed in the present study seems to be a promising candidate for use in the treatment of severe bone infection., (Copyright 1999 John Wiley & Sons, Inc.)

For the treatment of periodontal diseases, design of a controlled release system seemed very appropriate for an effective, long term result. In this study a novel, biodegradable microbial polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV of various valerate contents containing a well established antibiotic, tetracycline, known to be effective against many of the periodontal disease related microorganisms, was used in the construction of a controlled release system. Tetracycline was loaded in the PHBV microspheres and microcapsules both in its acidic (TC) and in neutral form (TCN). Microcapsules of PHBV were prepared under different conditions using w/o/w double emulsion and their properties such as encapsulation efficiency, loading, release characteristics, and morphological properties were investigated. It was found that concentration of emulsifiers polyvinyl alcohol (PVA) and gelatin (varied between 0-4%) influenced the encapsulation efficiency appreciably. In order to increase encapsulation efficiency (from the obtained range of 18.1-30.1%) and slow down the release of the highly soluble tetracycline.HCl, it was neutralized with NaOH. Encapsulation efficiency of neutralized tetracycline was much higher (51.9-65.3%) due to the insoluble form of the drug used during encapsulation. The release behaviour of neither of the drugs was found to be of zero order. Rather the trends fitted reasonably well to Higuchi's approach for release from spherical micropheres. Biodegradability was not an appreciable parameter in the release from microcapsules because release was complete before any signs of degradation were observed.