Deep learning-based classification of microalgae using light and scanning electron microscopy images.

Microalgae possess diverse applications, such as food production, animal feed, cosmetics, plastics manufacturing, and renewable energy sources. However, uncontrolled proliferation, known as algal bloom, can detrimentally impact ecosystems. Therefore, the accurate detection, monitoring, identification, and tracking of algae are imperative, albeit demanding considerable time, effort, and expertise, as well as financial resources. Deep learning, employing image pattern recognition, emerges as a practical and promising approach for rapid and precise microalgae cell counting and identification. In this study, we processed light microscopy (LM) and scanning electron microscopy (SEM) images of two Cyanobacteria species and three Chlorophyta species to classify them, utilizing state-of-the-art Convolutional Neural Network (CNN) models, including VGG16, MobileNet V2, Xception, NasnetMobile, and EfficientNetV2. In contrast to prior deep learning based identification studies limited to LM images, we, for the first time, incorporated SEM images of microalgae in our analysis. Both LM and SEM microalgae images achieved an exceptional classification accuracy of 99%, representing the highest accuracy attained by the VGG16 and EfficientNetV2 models to date. While NasnetMobile exhibited the lowest accuracy of 87% with SEM images, the remaining models achieved classification accuracies surpassing 93%. Notably, the VGG16 and EfficientNetV2 models achieved the highest accuracy of 99%. Intriguingly, our findings indicate that algal identification using optical microscopes, which are more cost-effective, outperformed electron microscopy techniques.

Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties.

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

This study is the first report on the separation and reusability of ApoUnaG protein, indicating excellent fluorescence response with high affinity and specificity toward unconjugated bilirubin (UC-BR) molecules, from the UnaG-UC-BR complex structure. The fluorescence properties of the UnaG-UC-BR complex (holo-UnaG) are studied by addition of different metal ions to perform possible interactions with holo-UnaG through absorbance and emission spectra. After addition of metal ions, some changes with respect to the type of metal ions are observed in fluorescence intensity of the holo-UnaG. When compared to metal ions, an excellent quenching response is sighted in the presence of Cu2+ ions by binding with UC-BR in the UnaG-UC-BR complex structure. Obtained non-fluorescence holo-UnaG-Cu2+ complex mixture is passed through Ni-NTA agarose to remove the ingredients such as Cu2+, UC-BR and Cu2+-UC-BR coordination complex from holo-UnaG. From the obtained experiments, it is concluded that Cu2+ ion can be used as an agent for the recovery of ApoUnaG protein via binding with UC-BR molecules. Graphical Abstract Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

Sequential VEGF and BMP-2 releasing PLA-PEG-PLA scaffolds for bone tissue engineering: I. Design and in vitro tests.

Biodegradable PLA-PEG-PLA block copolymers were synthesized with desired backbone structures and molecular weights using PEG20000. Rectangular scaffolds were prepared by freeze drying with or without using NaCl particles. Bone morphogenetic protein (BMP)-2 was loaded to the matrix after the scaffold formation for sustained release while vascular endothelial growth factor (VEGF) was loaded within the pores with gelatin solution. VEGF release was quite fast and almost 60% of it was released in 2 d. However, sequential - sustained released was observed for BMP-2 in the following few months. Corporation of VEGF/BMP-2 couple into the scaffolds increased the cell adhesion and proliferation. Neither significant cytotoxicity nor apoptosis/necrosis were observed.