Using ANOVA Models To Compare and Optimize Extraction Protocols of P3HBHV from Cupriavidus necator

Polyhydroxyalkanoates (PHAs), produced by microorganisms, have attracted considerable attention due to their biodegradability. However, the cost for the production of PHA is still too high to be competitive against petro-based polymers, greatly caused by the expensive downstream processing (DSP). The DSP, moreover, is often ecologically unfriendly due to usage of large amounts of highly volatile organic solvents. To overcome these limitations, we systematically compared for the first time seven different extraction protocols reported previously using the same starting biomass of Cupriavidus necator. Design of experiments (DoE) and analysis of variance (ANOVA) models were applied to further improve two of the most promising protocols. Finally, we developed a method where a combination of mechanical disruption of cells via bead milling with an addition of sodium lauryl sulfate (SDS) was used. This method was optimized with a response surface methodology and allowed a fast PHA extraction within approximately 2 h with a PHA recovery of almost 100% and PHA purity of 94%

Beneficial Oral Biofilms as Smart Bioactive Interfaces

Periodontitis is a very common health problem caused by formation of pathogenic bacterial biofilm that triggers inflammation resulting in either reversible gingivitis or irreversible periodontal hard and soft tissue damages, leading to loss of teeth when left untreated. Commensal bacteria play an important role in oral health in many aspects. Mainly by colonizing oral tissues, they (i) contribute to maturation of immune response, and (ii) foreclose attachment of pathobiont and, therefore, prevent from infection. The main goal of the study was to investigate if blocking of receptors on a commensal biofilm can prevent or reduce the attachment of pathogenic strains. To do so, biofilm produced by commensal Streptococcus sanguinis was treated with whole cell lysate of pathobionts Fusobacterium nucleatum or Porphyromonas gingivalis, followed by incubation with respective strain(s). The study revealed significant reduction in pathobiont adhesion to lysate-treated commensal biofilm. Therefore, adhesion of pathobionts onto the lysate-blocked biofilm was hindered; however, not completely eliminated supporting the idea that such approach in the oral cavity would benefit the production of a well-balanced and healthy bioactive interface

The Pneumococcal Cell Envelope Stress-Sensing System LiaFSR Is Activated by Murein Hydrolases and Lipid II-Interacting Antibiotics ▿ §

In the Firmicutes, two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system in Streptococcus pneumoniae displays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transfer in vitro and is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon, spr0810 and PcpC (spr0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis

Slowing down of dynamics and orientational order preceding crystallisation in hard-sphere systems

Despite intensive studies in the past decades, the local structure of disordered matter remains widely unknown. We show the results of a coherent x-ray scattering study revealing higher-order correlations in dense colloidal hard-sphere systems in the vicinity of their crystallization and glass transition. With increasing volume fraction, we observe a strong increase in correlations at both medium-range and next-neighbor distances in the supercooled state, both invisible to conventional scattering techniques. Next-neighbor correlations are indicative of ordered precursor clusters preceding crystallization. Furthermore, the increase in such correlations is accompanied by a marked slowing down of the dynamics, proving experimentally a direct relation between orientational order and sample dynamics in a soft matter system. In contrast, correlations continuously increase for nonequilibrated, glassy samples, suggesting that orientational order is reached before the sample slows down to reach (quasi-)equilibrium

Enhanced Antimicrobial Activity and Structural Transitions of a Nanofibrillated Cellulose–Nisin Biocomposite Suspension

Resistance to antibiotics has posed a high demand for novel strategies to fight bacterial infections. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. However, their poor solubility in water and sensitivity to degradation has limited their application. Here, we report the design of a smart, pH-responsive antimicrobial nanobiocomposite material based on the AMP nisin and 2,2,6,6-tetramethyl-1-piperidinyloxyl-oxidized nanofibrillated cellulose (TONFC). Morphological transformations of the nanoscale structure of nisin functionalized-TONFC fibrils were discovered at pH values between 5.8 and 8.0 using small-angle X-ray scattering. Complementary ζ potential measurements indicate that electrostatic attractions between the negatively charged TONFC surface and the positively charged nisin molecules are responsible for the integration of nisin. Modification of the pH level or increasing the ionic strength reduces the nisin binding capacity of TONFC. Biological evaluation studies using a bioluminescence-based reporter strain of Bacillus subtilis and a clinically relevant strain of Staphylococcus aureus indicated a significantly higher antimicrobial activity of the TONFC–nisin biocomposite compared to the pure nisin against both strains under physiological pH and ionic strength conditions. The in-depth characterization of this new class of antimicrobial biocomposite material based on nanocellulose and nisin may guide the rational design of sustainable antimicrobial materials

Core–Shell Silver Nanoparticles in Endodontic Disinfection Solutions Enable Long-Term Antimicrobial Effect on Oral Biofilms

To achieve effective long-term disinfection of the root canals, we synthesized core–shell silver nanoparticles (AgNPs@SiO₂) and used them to develop two irrigation solutions containing sodium phytate (SP) and ethylene glycol-bis­(β-aminoethyl ether)<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetraacetic acid (EGTA), respectively. <i>Ex vivo</i> studies with instrumented root canals revealed that the developed irrigation solutions can effectively remove the smear layer from the dentinal surfaces. Further <i>in vitro</i> experiments with single- and multispecies biofilms demonstrated for the first time that AgNPs@SiO₂-based irrigation solutions possess excellent antimicrobial activities for at least 7 days, whereas the bare AgNPs lose the activity almost immediately and do not show any antibacterial activity after 2 days. The long-term antimicrobial activity exhibited by AgNPs@SiO₂ solutions can be attributed to the sustainable availability of soluble silver, even after 7 days. Both solutions showed lower cytotoxicity toward human gingival fibroblasts compared to the conventionally used solution (3% NaOCl and 17% EDTA). Irrigation solutions containing AgNP@SiO₂ may therefore be highly promising for applications needing a long-term antimicrobial effect

Six Hydrophobins Are Involved in Hydrophobin Rodlet Formation in <i>Aspergillus nidulans</i> and Contribute to Hydrophobicity of the Spore Surface

<div><p>Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In <i>Aspergillus nidulans</i> two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in <i>A. nidulans</i> and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of <i>rodA</i> caused loss of the rodlet structure. Analysis of the rodlets in the <i>dewB-E</i> deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the <i>rodA</i> promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking <i>rodA</i>-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.</p></div

Localization of mRFP-tagged hydrophobins.

<p>Constructs (as indicated) were transformed into GR5 or TN02A3. Strains: SCOS170-SCOS175, SAGR19a, and STT08. Scale bar, 2 μm in <b>A</b> and 5 μm in <b>B</b>.</p