Impact of Neospora caninum infection on the bioenergetics and transcriptome of cerebrovascular endothelial cells

In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm−1), amide I protein (1649 cm−1), amide II protein (1537 cm−1), nucleic acids and carbohydrates (1092 cm−1, 1047 cm−1, and 939 cm−1). By using quantitative synchrotron radiation X-ray fluorescence (μSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells

3D Bioprinting of Mature Bacterial Biofilms for Antimicrobial Resistance Drug Testing

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (>4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl 2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing

Surface enhanced spatially offset raman spectroscopy (SESORS) imaging of bacterial biofilms

This thesis was previously held under moratorium until 19th October 2022.Bacterial biofilm formation is crucial to establishing chronic infections including respiratory infection, orthopaedic infection and medical device infection et.al. Many antibiotics are unable to eradicate dense biofilms since extracellular polymeric substances (EPS) make up the matrix of the biofilm which retard the diffusion penetration of antibiotics. Current methods of bacteria detection rely upon laboratory-based techniques that are time-consuming and costly and require specialist trained users. Hence, there is an urgent need for in-situ methodologies to detect and prevent the formation of bacterial biofilms. Raman spectroscopy (RS) is based on the inelastic scattering of photons following monochromatic laser excitation. This powerful technique has the advantages of being non-destructive, non-invasive and label-free. However, the main disadvantage is that spontaneous Raman spectroscopy has low signal levels and long acquisition time. To address these issues, surface-enhanced Raman scattering (SERS) has been used to enhance the Raman signal up to 1013 - 1015 orders of magnitudes and can increase acquisition speeds as well as improving the accuracy of detection. Therefore, the focus of this research is to use specially designed bionanosensors (lectin and DNA aptamer) with resonant nanotag chalcogenpyrylium dyes and low-pH sensing probes PhagoGreen as optical imaging tools showing spectral change in response to the interaction with defined target molecules via enhanced SERRS signals to detect biofilm. This research focuses on developing new biomolecular sensing Raman-active nanotags as highly sensitive surface enhanced Raman probes. The specific nanosnesor was designed such that they will detect bacterial biofilms in vitro. This approach involves using galactophilic lectin PA-IL functionalised silver nanoparticles as a molecular recognition agent to detect the carbohydrates on the surface of bacteria using SERS. This research demonstrated this lectin biosensor is not only capable of detecting bacteria but also providing a rapid, sensitive discrimination between Gram-negative and Gram-positive bacteria, offering opportunities for future SERS biosensing in biomedical applications. None of current biofilm models can mimic the complexity of the 3D microenvironment and host defence mechanisms. In this study, clinically relevant bacterial species including Escherichia coli (E.coli), methicillinsensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. 3D biofilm constructs produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing. The advantage of using Raman rather than fluorescence as the optical imaging technique is the molecular specificity of the optical response, however more importantly in this case, is the combination of surface enhanced spectroscopy and spatially offset Raman (SESORS) which allows detection of Raman signals at depth. Herein, we have developed a novel approach for the detection of bacterial biofilms at depth using a 3D bioprinted biofilm model combined with gold nanoparticles functionalised with resonant Raman reporters and bacteria specific DNA aptamers. Detection was carried out using surface enhanced spatially offset resonant Raman spectroscopy (SESORRS) allowing detection of the bacterial biofilms to be achieved at penetration depths up to 2.1 cm through tissue for single bacteria and 1.5 cm for multiple bacteria. This work uses a low-pH sensing fluorescent probe, PhagoGreen, as a Raman reporter attached to a silver nanoparticle, to detect phagosome acidification in Gram-negative bacteria strain Escherichia coli activated macrophages by surface enhanced Raman spectroscopy (SERS). The SERS intensity of PhagoGreen conjugates at peak 759 cm-1 was shown to be highly responsive at a lower pH range (pH5-pH3).Bacterial biofilm formation is crucial to establishing chronic infections including respiratory infection, orthopaedic infection and medical device infection et.al. Many antibiotics are unable to eradicate dense biofilms since extracellular polymeric substances (EPS) make up the matrix of the biofilm which retard the diffusion penetration of antibiotics. Current methods of bacteria detection rely upon laboratory-based techniques that are time-consuming and costly and require specialist trained users. Hence, there is an urgent need for in-situ methodologies to detect and prevent the formation of bacterial biofilms. Raman spectroscopy (RS) is based on the inelastic scattering of photons following monochromatic laser excitation. This powerful technique has the advantages of being non-destructive, non-invasive and label-free. However, the main disadvantage is that spontaneous Raman spectroscopy has low signal levels and long acquisition time. To address these issues, surface-enhanced Raman scattering (SERS) has been used to enhance the Raman signal up to 1013 - 1015 orders of magnitudes and can increase acquisition speeds as well as improving the accuracy of detection. Therefore, the focus of this research is to use specially designed bionanosensors (lectin and DNA aptamer) with resonant nanotag chalcogenpyrylium dyes and low-pH sensing probes PhagoGreen as optical imaging tools showing spectral change in response to the interaction with defined target molecules via enhanced SERRS signals to detect biofilm. This research focuses on developing new biomolecular sensing Raman-active nanotags as highly sensitive surface enhanced Raman probes. The specific nanosnesor was designed such that they will detect bacterial biofilms in vitro. This approach involves using galactophilic lectin PA-IL functionalised silver nanoparticles as a molecular recognition agent to detect the carbohydrates on the surface of bacteria using SERS. This research demonstrated this lectin biosensor is not only capable of detecting bacteria but also providing a rapid, sensitive discrimination between Gram-negative and Gram-positive bacteria, offering opportunities for future SERS biosensing in biomedical applications. None of current biofilm models can mimic the complexity of the 3D microenvironment and host defence mechanisms. In this study, clinically relevant bacterial species including Escherichia coli (E.coli), methicillinsensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. 3D biofilm constructs produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing. The advantage of using Raman rather than fluorescence as the optical imaging technique is the molecular specificity of the optical response, however more importantly in this case, is the combination of surface enhanced spectroscopy and spatially offset Raman (SESORS) which allows detection of Raman signals at depth. Herein, we have developed a novel approach for the detection of bacterial biofilms at depth using a 3D bioprinted biofilm model combined with gold nanoparticles functionalised with resonant Raman reporters and bacteria specific DNA aptamers. Detection was carried out using surface enhanced spatially offset resonant Raman spectroscopy (SESORRS) allowing detection of the bacterial biofilms to be achieved at penetration depths up to 2.1 cm through tissue for single bacteria and 1.5 cm for multiple bacteria. This work uses a low-pH sensing fluorescent probe, PhagoGreen, as a Raman reporter attached to a silver nanoparticle, to detect phagosome acidification in Gram-negative bacteria strain Escherichia coli activated macrophages by surface enhanced Raman spectroscopy (SERS). The SERS intensity of PhagoGreen conjugates at peak 759 cm-1 was shown to be highly responsive at a lower pH range (pH5-pH3)

SOSIALISASI STRATEGI DALAM MEMBANGUN BRAND AWARNESS DI SEKOLAH ALAM TAHFIDZPRENEUR

Brand awareness adalah salah satu cara untuk meningkatkan kepekaan calon pelanggan terhadap produk yang kita punya,dengan melakukan brand awareness menjadi langkah besar untuk mengembangkan sebuah bisnis,salah satu cara yang bisa kita lakukan adalah dengan melalui sosial media,untuk pemula yang ingin atau baru belajar sebuah bisnis, sosial media adalah wadah yang cocok  karena gratis dan mempunyai efek yang sangat besar,namun tak hanya bagi pemula nyatanya masih banyak para perintis usaha yang belum menjajaki ranah sosial media dalam membangun sebuah kesadaran merek yang mereka punya,faktor utamanya adalah mereka masih mengira bahwa melakukan hal tahapan membangun kepekaan terhadap merek adalah hal yag sulit dan memakan banyak biaya, di wilayah Sekolah Alam Tahfidzpreneur, kami melihat banyak nya pengusaha kecil menengah yang sudah mempunyai sebuah bisnis namun belum memanfaatkan media social yang mereka bisa akses secara optimal, sudah semestinya social media yang mempunyai pengguna hingga miliaran orang menjadi lahan untuk meningkatkan pengetahuan bahkan penjualan sebuha produ

Serenade untuk sebuah kisah : selaksa cinta menyatu dengan sewindu waktu

Kumpulan cerita pendek ini memperlihatkan kisah-kisah dari tokoh utama yang berkelana dengan berbagai moda dan cara dalam menghargai Warisan Budaya Takbenda (WBTb) di berbagai daerah di Indonesia. Tampak juga reaksi dan pergulatan sikap para tokoh dalam menghargai berbagai adat istiadat masyarakat, ritus, dan perayaan-perayaan. Kehadiran buku ini diharapkan dapat menjadi pengayaan yang mendukung program merdeka belajar dan program literasi