Bioscience Research Research Articles

Micro‐Lensed Fiber Laser Desorption Mass Spectrometry Imaging Reveals Subcellular Distribution of Drugs within Single Cells

AbstractThe visualization of temporal and spatial changes in the intracellular environment has great significance for chemistry and bioscience research. Mass spectrometry imaging (MSI) plays an important role because of its unique advantages, such as being label‐free and high throughput, yet it is a challenge for laser‐based techniques due to limited lateral resolution. Here, we develop a simple, reliable, and economic nanoscale MSI approach by introducing desorption laser with a micro‐lensed fiber. Using this integrated platform, we achieved 300 nm resolution MSI and successfully visualized the distribution of various small‐molecule drugs in subcellular locations. Exhaustive dynamic processes of anticancer drugs, including releasing from nanoparticle carriers entering nucleus of cells, can be readily acquired on an organelle scale. Considering the simplicity and universality of this nanoscale desorption device, it could be easily adapted to most of laser‐based mass spectrometry applications.

Micro‐Lensed Fiber Laser Desorption Mass Spectrometry Imaging Reveals Subcellular Distribution of Drugs within Single Cells

The visualization of temporal and spatial changes in the intracellular environment has great significance for chemistry and bioscience research. Mass spectrometry imaging (MSI) plays an important role because of its unique advantages, such as being label-free and high throughput, yet it is a challenge for laser-based techniques due to limited lateral resolution. Here, we develop a simple, reliable, and economic nanoscale MSI approach by introducing desorption laser with a micro-lensed fiber. Using this integrated platform, we achieved 300 nm resolution MSI and successfully visualized the distribution of various small-molecule drugs in subcellular locations. Exhaustive dynamic processes of anticancer drugs, including releasing from nanoparticle carriers entering nucleus of cells, can be readily acquired on an organelle scale. Considering the simplicity and universality of this nanoscale desorption device, it could be easily adapted to most of laser-based mass spectrometry applications.

Bioinspired Underwater Superoleophobic Microlens Array With Remarkable Oil-Repellent and Self-Cleaning Ability.

Underwater superoleophobic microlens array (MLA) has been emerging as a crucial device for its wide applications in ocean optical imaging and sensing, endoscopic surgery, microfluidics and optofluidics, and other biomedical applications. Fabrication of microlens arrays integrated with excellent optical performance as well as underwater superoleophobicity remains a great challenge. In this paper, we report an underwater super oil-repellent MLA on a transparent optical glass substrate via femtosecond laser-induced phase and structural modification and chemical isotropic etching. The fabricated sample simultaneously possesses microlens structures with a smooth surface to enable optical imaging function, and grid-patterned biomimetic micro/nano hierarchical surface structures to produce underwater oil-resistance with a contact angle of 160.0° and a sliding angle of 1.5°. The resultant oil-repellent MLA exhibits underwater superoleophobicity and self-cleaning abilities in water. Meanwhile, it was demonstrated to have impressive imaging capability even after oil contamination. We believe that this novel resultant anti-oil MLA will be helpful for underwater detection and bioscience research, especially in oil polluted underwater workspaces.

A Comparative Evaluation and Utilization of Entrapped Bacillus sp. (Gen Bank MN 243657) and Pseudomonas aeruginosa SU-1 in Poultry Waste Degradation

Bacillus sp. (Genbank MN243657) was obtained from the Center for Bioscience and Nanoscience Research, and P. aeruginosa SU-1 were used in this study. Initially, biomass was optimized for the incubation period, pH, and temperature, following biomass optimization with RSM. The obtained organisms were able to produce keratinase, which was found to be a 64 KDa protein and 56KDa. Now the biomass produced using the optimized condition was immobilized and checked for its ability to degrade keratinase, and efficiency was compared. Immobilized P. aeruginosa SU-1 was degrading keratin and feather better than Bacillus sp. (Genbank MN243657). It was found to reduce featherweight by 57.3% and 41%. Hence it was found that both the immobilized bacterium can be used for poultry waste treatment, and P. aeruginosa SU-1 was more efficient than Bacillus sp. (Genbank MN243657).

Optimization of Biomass of Keratinase Producing Bacillus sp CBNRBT2 to Utilize in Whole-Cell Immobilization for Feather Degradation

Bacillus sp CBNRBT2, obtained from Centre for Bioscience and Nanoscience Research, was used in this study. Initially, biomass was optimized for the incubation period, pH, and temperature, following biomass optimization with RSM. The obtained organism was able to produce keratinase, which was found to be a 64 KDa protein. Now the biomass produced using the optimized condition was immobilized and checked for its ability to degrade keratinase. Efficiency was compared with the whole organism. Immobilized organisms were degrading keratin and feather better. It was found to reduce featherweight by 41 %. Thus, this immobilized bacterium can be used for poultry waste treatment.

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

BackgroundGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques.ResultsIn the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated.ConclusionsThe optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

A practical criterion for focusing of unstained cell samples using a digital holographic microscope.

Digital holographic microscopy (DHM) is an important technique that may be used for quantitative phase imaging of unstained biological cell samples. Since the DHM technology is not commonly used in clinics or bioscience research labs, at present there is no well-accepted focusing criterion for unstained samples that users can follow while recording image plane digital holograms of cells. The usual sharpness metrics that are useful for auto-focusing of stained cells do not work well for unstained cells as there is no amplitude contrast. In this work, we report a practical method for estimating the best focus plane for unstained cells in the digital hologram domain. The method is based on an interesting observation that for the best focus plane the fringe pattern associated with individual unstained cells predominantly shows phase modulation effect in the form of bending of fringes and minimal amplitude modulation. This criterion when applied to unstained red blood cells shows that the central dip in the doughnut-like phase profile of cells is maximal in this plane. The proposed methodology is helpful for standardizing the usage of DHM technology across different users and application developmentefforts. LAY DESCRIPTION: Digital holographic microscopy (DHM) is slowly but steadily becoming an important microscopy modality and gaining acceptability for basic bio-science research as well as clinical usage. One of the important features of DHM is that it allows users to perform quantitative imaging of unstained transparent cells. Instead of using dyes or fluorescent labelling, DHM systems use quantitative phase as a contrast mechanism which depends on the natural refractive index variation within the cell samples. Since minimal wet lab processing is required in order to image cell samples with a DHM, cells can be imaged in their natural state. While DHM is gaining popularity among users, the imaging protocols across the labs or users need to be standardized in order to make sure that the same quantitative phase parameters are used for tasks such as quantitative phased based cellclassification. One of the important operational tasks for any microscopy work is to focus the sample under study. While focusing comes naturally to users of brightfield microscopes based on image contrast, the focusing is not straightforward when samples are unstained so that they do not offer any amplitude contrast. When performing quantitative phase imaging, defocus can actually change the phase profile of the cell due to near-zone (Fresnel) diffraction effects. So unless a standardized focusing methodology is used, it will be difficult for multiple DHM users (potentially at different sites) to agree on quantitative results out of their phase images. DHM literature has prior works which perform numerical focusing of recovered complex wave-field in the hologram plane to find the best focus plane. However such methods are not user friendly and do not allow user the same focusing experience as in a brightfield microscope. The numerical focusing is therefore a reasonably good method for an optics researcher but not necessarily so for a microscopy technician looking at cell samples with a DHM system in a clinicalsetting. The present work provides a simple focusing criterion for unstained samples that works directly in the hologram domain. The technique is based on an interesting observation that the when an unstained cell sample is in the best-focus plane, its corresponding hologram (or fringe pattern) predominantly shows phase modulation manifested by bending of fringes at the location of the cell. This criterion can be converted into a simple numerical method as we have used to find the best-focus plane using a stack of through focus holograms. We believe that the technique can be used manually by visually observing the holograms or can be converted to an auto-focus algorithm for a motorized DHMsystem.

Editorial and News

Editorial and News

Improving Specimen Labelling and Data Collection in Bio-science Research using Mobile and Web Applications

AbstractResearchers in bio-sciences are increasingly harnessing technology to improve processes that were traditionally pegged on pen-and-paper and highly manual. The pen-and-paper approach is used mainly to record and capture data from experiment sites. This method is typically slow and prone to errors. Also, bio-science research activities are often undertaken in remote and distributed locations. Timeliness and quality of data collected are essential. The manual method is slow to collect quality data and relay it in a timely manner. Capturing data manually and relaying it in real time is a daunting task. The data collected has to be associated to respective specimens (objects or plants). In this paper, we seek to improve specimen labelling and data collection guided by the following questions; (1) How can data collection in bio-science research be improved? (2) How can specimen labelling be improved in bio-science research activities? We present WebLog, an application that we prototyped to aid researchers generate specimen labels and collect data from experiment sites. We use the application to convert the object (specimen) identifiers into quick response (QR) codes and use them to label the specimens. Once a specimen label is successfully scanned, the application automatically invokes the data entry form. The collected data is immediately sent to the server in electronic form for analysis.

Changing Infrastructural Practices: Routine and Reproducibility in Automated Interdisciplinary Bioscience.

Proponents of engineering and design approaches to biology aim to make interdisciplinary bioscience research faster and more reproducible. This paper outlines and deploys a practice-based approach to analyses of infrastructure that focuses on the routine epistemic activities and charts how two such routines are unsettled and resettled in the background of epistemic culture. This paper describes attempts to bring about new research infrastructures in synthetic biology using robotics and software-enabled design. A focus on the skills of pipetting shows how established manual labor has to be reconfigured to fit with novel robotic automations. An analysis of curating frozen materials shows that automated design presents new problems for the established activities of storing and retrieving biological materials. These movements, while transient, have implications for organizing interdisciplinary collaboration, research productivity, and enabling greater reproducibility. This paper explores the idea of infrastructure as practice and shows how this has important implications for studies of research infrastructures. This article discusses the main contributions of this approach for analysts of infrastructure in terms of movements, temporalities, and ethics and offers suggestions for what the research implies for synthetic biology.

Salivary total Immunoglobulin G as a surrogate marker of oral immune activity in salivary bioscience research

The integration of salivary biomeasures in biobehavioral, psychophysiological, and clinical research has greatly expanded our ability to study the biopsychosocial processes underlying health. Much of this research, however, has failed to adequately assess and adjust for the impact of oral immune activity on salivary biomeasure concentrations and associations with serum levels. Aiming to improve the validity and reliability of salivary biomeasure data, we examine salivary total Immunoglobulin G (IgG) as a potential surrogate marker of oral inflammation and immune activity. During a single study visit in Baltimore, Maryland, healthy young adult participants provided matched blood and saliva samples (N=99; age 18–37 years, 42% female) and completed an oral health questionnaire. Biospecimens were assayed for total IgG and immune markers related to inflammation (cytokines), blood in saliva (transferrin), and tissue remodeling (matrix metalloproteinase-8). Total IgG (μg/mL) concentrations were higher in serum than saliva. Salivary total IgG was associated with some self-reported oral health measures, and strongly positively associated with all salivary immune markers. Controlling for salivary total IgG may be a feasible, affordable approach to adjusting salivary biomeasure findings for the influence of the oral immune environment when it is not possible or practical to obtain clinical oral health data.

Underwater superoleophobic and anti-oil microlens array prepared by combing femtosecond laser wet etching and direct writing techniques.

As an important micro-optical device, microlens array (MLA) also has broad applications in aqueous environment apart from atmosphere, such as bioscience research, ocean exploration, and microfluidic systems. However, the surface of the normal MLA is easily polluted by oil contaminations when the MLA is practically applied in a water medium, leading to the loss of its optical imaging ability. Herein, we fabricated a functional MLA with underwater anti-oil and self-cleaning abilities by combining the femtosecond laser wet etching (FLWE) and the femtosecond laser direct writing (FLDW) techniques. The as-prepared close-packed MLA is composed of 10000 single microlenses with the aperture diameter of 50 µm. The surface of each microlens is further textured with micro/nanoparticles. Clear and uniform images could be captured by using the resultant MLA in water, demonstrating great underwater imaging ability. The modulation transfer function value is larger than 0.6 at 55 lp/mm. In addition, the micro/nanostructures endow the as-fabricated MLA surface with underwater superoleophobicity and oil-repellent performance. Various oils can be repelled by the resultant MLA in water. Underwater 1,2-dichloroethane oil droplet on the textured MLA has a contact angle of 158.0 ± 0.5° and a sliding angle of 2.0 ± 0.2°. The underwater superoleophobic MLA also has good mechanical durability. The anti-oil and self-cleaning functions will broaden the applications of the MLA in ocean exploration, bioscience research, microfluidic system, and many underwater MLA-based systems.

Transitions to Excellence in Molecular and Cellular Biosciences Research (NSF)

Transitions to Excellence in Molecular and Cellular Biosciences Research (NSF)

Refining animal research: The Animal Study Registry.

The Animal Study Registry (ASR; www.animalstudyregistry.org) was launched in January 2019 for preregistration of animal studies in order to increase transparency and reproducibility of bioscience research and to promote animal welfare. The registry is free of charge and is designed for exploratory and confirmatory studies within applied science as well as basic and preclinical research. The registration form helps scientists plan their study thoroughly by asking detailed questions concerning study design, methods, and statistics. With registration, the study automatically receives a digital object identifier (DOI) that marks it as intellectual property of the researcher. To accommodate the researchers concerns about theft of ideas, users can restrict the visibility of their registered studies for up to 5 years. The full content of the study becomes publicly accessible at the end of the embargo period. Because the platform is embedded in the infrastructure of the German Federal Government, continuity and data security are provided. By registering a study in the ASR, researchers can show their commitment to transparency and data quality to reviewers and editors, to third-party donors, and to the general public.

A simple, real-time assay of horseradish peroxidase using biolayer interferometry.

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.

1422P - Molecular fingerprinting in breast cancer (BC) screening using Quantum Optics (QO) technology combined with an artificial intelligence (AI) approach applying the concept of “molecular profiles at n variables (MPnV)”: A prospective pilot study

BackgroundBC screening by mammography is associated with a significant reduction in mortality (19 % overall reduction of the relative risk), however with significant limitations and debatable cost-effectiveness. Screening individuals for cancer using liquid biopsies (LB) represents an unmet need. We report the first prospective “proof of concept” study using the QO technology combined with an AI approach using the concept of “MPnV” applied to BC detection. MethodsQO (femto/atto-second infrared laser spectroscopy) on LB is a simple, non-invasive and reproducible method allowing to identify individualized molecular spectra (MS). These highly detailed MS can be correlated to physiological or pathological changes allowing detection and translation of differences. Integrated into a super-computational approach using a non-hierarchical deep data mining strategy (MPnV concept), MS could discriminate individuals with and without BC. The plasma of 68 controls and 27 BC patients, accrued at the King Saud University BC screening program, Riyadh, Saudi Arabia (KSA), were studied by QO (Max Planck Institute of Quantum Optics /Ludwig Maximillian University Munich, Garching, Germany). The MS were analysed on the Shaheen II supercomputer at King Abdallah University for Science and Technology (KAUST), KSA, in order to generate comparative algorithms. ResultsThe use of special feature selection, followed by class analysis allowed to differentiate profiles between the two groups with a sensitivity of 97% and a specificity of 72% (variables n=1,100). A more in-depth analysis led to 99% sensitivity, but with a lower specificity of 64%. Further analysis of the series, using an age-matched approach led to 97% sensitivity with 98% specificity in differentiating women with or without BC. ConclusionsThese results warrant a large scale prospective validation BC screening trial (ongoing) and “proof of concept trials” in other frequent cancers, in particular those without existing screening programs. Editorial acknowledgementProf. Ferenc Krausz, Director, and Dr. Mihaela Zigman, Leader of the Broadband Infrared Diagnostics, Max Planck Institute of Quantum Optics (MPQ), Faculty of Physics at Ludwig-Maximilians Universität München (LMU), Garching, Germany. Legal entity responsible for the studyJean Marc Nabholtz, Oncology Centre, King Saud University, Riyadh, Saudi Arabia. Funding1. Oncology Centre, King Saud University Medical City, King Saud University, Saudi Arabia. 2. Max-Planck-Institut für Quantenoptik and Faculty of Physics at Ludwig-Maximilians Universität München, Garching, Germany. 3. Computational Bioscience Research Centre, King Abdallah University for Science and Technology (KAUST), Thuwal, Saudi Arabia. DisclosureM.R.K. Bahadoor: Travel / Accommodation / Expenses, ASCO Participation Funded: Pfizer; Advisory / Consultancy, Pancreas Expert Opinion Advisory: Baxter. All other authors have declared no conflicts of interest.

Metabolomic Profiling of Staphylococcus aureus.

Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.

Redox control of vascular biology.

Redox control is lost when the antioxidant defense system cannot remove abnormally high concentrations of signaling molecules, such as reactive oxygen species (ROS). Chronically elevated levels of ROS cause oxidative stress that may eventually lead to cancer and cardiovascular and neurodegenerative diseases. In this review, we focus on redox effects in the vascular system. We pay close attention to the subcompartments of the vascular system (endothelium, smooth muscle cell layer) and give an overview of how redox changes influence those different compartments. We also review the core aspects of redox biology, cardiovascular physiology, and pathophysiology. Moreover, the topic‐specific knowledgebase DES‐RedoxVasc was used to develop two case studies, one focused on endothelial cells and the other on the vascular smooth muscle cells, as a starting point to possibly extend our knowledge of redox control in vascular biology.

Uterine spiral artery muscle dedifferentiation.

Is vascular smooth muscle cell (VSMC) dedifferentiation a feature of uterine spiral artery (SpA) remodelling in early human pregnancy? Remodelling of human uterine SpAs is associated with dedifferentiation of VSMCs and can be induced in vitro by uterine natural killer (uNK) cells and extravillous trophoblast cells (EVTs). Uterine SpAs undergo profound morphological changes in normal pregnancy with replacement of the musculoelastic arterial wall structure by fibrinoid containing EVTs. The fate of VSMCs in SpA remodelling is unknown; in guinea pig uterine artery VSMCs dedifferentiate, remain in the vessel wall and differentiate after parturition to restore the arterial wall. There is increasing evidence that uNK cells play a role in SpA remodelling. We hypothesized that SpA remodelling in human pregnancy is associated with VSMC dedifferentiation, initiated by uNK cell-derived growth factors. Formalin fixed, paraffin embedded placental bed biopsies were immunostained for angiogenic growth factor (AGF) receptors and markers of VSMC differentiation. An in vitro model of SpA remodelling using chorionic plate arteries (CPAs) was used to test the effect of different cell types and AGFs on VSMC differentiation. Placental bed biopsies were immunostained for vascular endothelial growth factor receptors 1-3 (VEGF-R1, VEGF-R2, VEGF-R3), transforming growth factor beta 1 receptors I and II (TGF-βRI, TGF-βRII), interferon gamma receptors 1 and 2 (IFN-γR1, IFN-γR2), Tie2, α-smooth muscle actin (α-SMA), H-caldesmon (H-Cal), myosin heavy chain (MyHC), osteopontin and smoothelin. Staining intensity was assessed using a modified quickscore. Expression by VSMCs of the AGF receptors was confirmed by laser capture microdissection and real-time RT-PCR of non-remodelled SpAs, after laser removal of the endothelium. As an in vitro model, VSMC differentiation was assessed in CPAs by immunohistochemistry after culture in uNK cell-conditioned medium (CM), EVT-CM, uNK cell/EVT co-culture CM, Ang-1, Ang-2, IFN-γ, VEGF-A and VEGF-C, and after blocking of both Ang-1 and Ang-2 in uNK-CM. SpA VSMC expression of Tie-2 (P= 0.0007), VEGF-R2 (P= 0.005) and osteopontin (P= 0.0001) increased in partially remodelled SpAs compared with non-remodelled SpAs, while expression of contractile VSMC markers was reduced (α-SMA P< 0.0001, H-Cal P= 0.03, MyHC P= 0.03, smoothelin P= 0.0001). In the in vitro CPA model, supernatants from purified uNK cell (H-Cal P< 0.0001, MyHC P= 0.03, α-SMA P= 0.02, osteopontin P= 0.03), EVT (H-Cal P =0.0006, MyHC P= 0.02, osteopontin P= 0.01) and uNK cell/EVT co-cultures (H-Cal P =0.001, MyHC P= 0.05, osteopontin P= 0.02) at 12-14 weeks, but not 8-10 weeks, gestational age induced reduced expression of contractile VSMC markers and increased osteopontin expression. Addition of exogenous (10ng/ml) Ang-1 (P= 0.006) or Ang-2 (P= 0.009) also reduced H-Cal expression in the CPA model. Inhibition of Ang-1 (P= 0.0004) or Ang-2 (P= 0.004) in uNK cell supernatants blocked the ability of uNK cell supernatants to reduce H-Cal expression. This is an in vitro study and the role of uNK cells, Ang-1 and Ang-2 in SpA remodelling in vivo has not yet been shown. VSMC dedifferentiation is a feature of early SpA remodelling and uNK cells and EVT play key roles in this process by secretion of Ang-1 and Ang-2. This is one of the first studies to suggest a direct role for Ang-1 and Ang-2 in VSMC biology. This work was supported by a grant from British Biotechnology and Biosciences Research Council (BB/E016790/1). The authors have no competing interests to declare.

The International Seminar on Bioscience and Biogical Education

PREFACEThe rapid development of information and communication technology (ICT) in recent years has shifted us to digital era. The biggest challenge in our world of education is how to reform education system and integrate modern technology in teaching and learning. The development of science and technology has also supported the acceleration of research in this field, as well as giving influence to researchers to continuously improve themselves.In a highly open global information era, sharing creative ideas and cooperations in the field of biosciences and biological education need to be improved through various fora. International Seminar on Bioscience and Biological Education (ISBBE) facilitates to evoke creative and innovative ideas among educators and researchers in the field of bioscience and biological education. In accordance with the focus of the problem, this international seminar has a theme: “Research and Education Innovation on Bioscience for the 21st Century Challenges.”This seminar brought us to discuss recent development and innovation in biological and bioscience research. Several expert in the field of Bioscience and biological education were invited, including Prof. Dr. Mamoru Yamada (Yamaguchi University, Japan), Prof. David F. Treagust (Curtin University, Australia), Dr. Mahani Mokhtar (University of Technology Malaysia, Johor, Malaysia) and Dr. Kriengkrai Satapornvanit (Kasetsart University, Bangkok, Thailand). We are also invited special keynote speaker Dr. Ir. Paristiyanti Nurwardani, MP, Director of Learning Affair, General Directorate of Learning and Student Affairs, The Ministry of Research, Technology, and Higher Education.On behalf of the committee and the institution, I would like to thanks to the participants who had been participated in this seminar. I realy hope that we are going to meet again at the next ISBBE event. Thank you.Yogyakarta, Indonesia, October 2018ISBBE ChairmanDr. Suyitno Aloysius