A Guide to Biodetection in Droplets.

Droplet-based methods for optical biodetection enable unprecedented high-throughput experimental parameters. The methods, however, remain underused due to the accompanying multidisciplinary and complicated experimental workflows. Here, we provide a tutorial for droplet-based optical biodetection workflows with a focus on the key aspect of label selection. By discussing and guiding readers through recent state-of-the-art studies, we aim to make droplet-based approaches more accessible to the general scientific public.

Microfluidic production, stability and loading of synthetic giant unilamellar vesicles.

In advanced drug delivery, versatile liposomal formulations are commonly employed for safer and more accurate therapies. Here we report a method that allows a straightforward production of synthetic monodisperse (~ 100 µm) giant unilamellar vesicles (GUVs) using a microfluidic system. The stability analysis based on the microscopy imaging showed that at ambient conditions the produced GUVs had a half-life of 61 ± 2 h. However, it was observed that ~ 90% of the calcein dye that was loaded into GUVs was transported into a surrounding medium in 24 h, thus indicating that the GUVs may release these small dye molecules without distinguishable membrane disruption. We further demonstrated the feasibility of our method by loading GUVs with larger and very different cargo objects; small soluble fluorescent proteins and larger magnetic microparticles in a suspension. Compared to previously reported microfluidics-based production techniques, the obtained results indicate that our simplified method could be equally harnessed in creating GUVs with less cost, effort and time, which could further benefit studying closed membrane systems.

Replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt.

The incorporation of lipid droplets and further characterization of matrices within dairy products may be possible using such adjacent particles as protein complexes/lipids. Among the range of varied emulsions and their functionalities, great attention has recently focused on the fabrication of high internal phase types. Feasibly, stable alternatives structured with health-beneficial lipids like those derived from plants could replace saturated fatty acids. As a fat replacement strategy, the fate of incorporated HIPE would require some adjustments either with storage stability and/or structural feat for the food matrix. Therefore, the replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt was investigated. This involved 25%, 50% and 75% rapeseed oil respectively assigned as low (LIPE), medium (MIPE), and high internal phase emulsion (HIPE). Specifically, emulsions were examined by droplet size, encapsulation, pH, zeta potential, phase separation, and rheology. The fat free yogurt supplemented by HIPE were examined by droplet size, zeta potential, pH, color, sensory, texture and microbiological aspects against positive (regular milk fat) and negative (fat free) yogurt controls. Results showed increasing rapeseed oil contents would form smaller droplet-like emulsions. Within the yogurt matrix however, incorporating HIPE would seemingly reduce oil droplet size without much compromise to bacterial viability, sensory, or texture. Overall, this simple method of lipid alternation shows promise in dairy products.

User-friendly analysis of droplet array images.

Water-in-oil droplets allow performing massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, analyzing such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits accessibility for a wider user base in the fields of chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet experiments. We built pipelines that combine various open-source image-analysis software with a custom-developed data processing tool called "EasyFlow". Our pipelines are applicable to the typical experimental scenarios that users encounter when working with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence).

A COST Action on microbial responses to low pH: Developing links and sharing resources across the academic-industrial divide.

We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-organisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Microbial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.

Investigation of Different Free Image Analysis Software for High-Throughput Droplet Detection.

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software's modules. We test and evaluate the software's (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses.

The snapping shrimp dactyl plunger: a thermomechanical damage-tolerant sandwich composite.

The dactyl plunger of Alpheus sp. was found to be a layered composite, with mineral-rich outer and inner layers and a chitin-rich middle layer of high porosity. The chitin-rich middle layer is itself composed of several porous chitin laminae. Modelling heat conduction through the plunger cross-section revealed that the chitin-rich layer is able to insulate heat and retard its progress through the material. Heat accumulates in the plunger after a series of successive snaps and as such, its thermally resistant design can be considered most useful under the conditions of successive snapping. The plunger has a concurrent mechanical damage-tolerant design with biogenic mineral layers, viscous (chitin-mineral) interfaces, energy-dissipating porous chitin, and sidewalls composed of ordered, layered aragonite. The snapping shrimp plunger has a design that may protect it and internal soft tissues from thermomechanical damage during plunger-socket compression prior to cavitation bubble release.

α-globin Alteration in α-thalassemia Disorder: Prediction and Interaction Defect.

BACKGROUND AND OBJECTIVE: The α-thalassemia is an inherited blood disorder affecting quality and quantity of hemoglobin. It caused mostly by deletion of one or two α-globin genes and characterized by deficient production of α-globin chain in hemoglobin leading from mild anemia to lethal. The α-globin gene with partial deletion could reduce chain production or produce abnormal chain. Its effect depends on mechanism of chain production affected. This study aimed to analyze the effect of partial deletion in α-globin gene influencing the mechanisms to produce functional α-globin chain in α-thalassemia cases. MATERIALS AND METHOD: The three mutant genes from genebank were selected and processed. The analysis performed in deleted sequences determination, mRNA sequences, protein structures and protein chains interaction to form hemoglobin by SWISS MODEL, CHIMERA and SABLE Polyview 2D. RESULTS: The result showed 76 amino acids deleted in one mutant α-globin gene (V00516.1). The mutation gave effect in every mechanism of the α-globin chain conformation and production. It affected protein conformation by losing over half the helical chains. It reduced the function completely, in which, disturb hemoglobin A (HbA) production with emergence of ß-sheets conformation. CONCLUSION: The analysis concluded that the protein produced by the α-globin gene with partial deletion lost its function and unable to form hemoglobin.

The effects of diatom pore-size on the structures and extensibilities of single mucilage molecules.

Diatoms secrete extracellular polymeric substances (EPS), or mucilage, around the cell wall that may serve to aid in motility and form a discrete layer that may help maintain thicker layers of EPS that have a greater role in adhesion. Mucilage molecules adhere to the diatom frustules, which are biosilica skeletons that develop from the diatom cell walls. Here, molecular dynamics methods were used to determine the characteristics of mucilage molecules as a function of pore size; notably 1,4-α-D-galacturonic acid, 1,4-ß-glucuronic acid and 1,4-ß-D-mannuronic acid. These uronic acids differ from each other in structure and extensibility as a function of their folding characteristics. Here, we find that when overlain upon a pore, mucilage molecules try to return to their native folded states but are restrained by their interactions with the silica surfaces. Furthermore, the extensibility of mucilage molecules over pore spaces affects the extent of mechanical energy required to straighten them. As such, different EPS molecules will affect sliding, friction and adhesion to subsequent layers of EPS in different ways. We conclude that higher EPS extensibility is homonymous with higher adhesive or frictive resistance since the molecules will be able to strain more before they reach the most extended (and thus rigid) conformation. The research herein is applicable to modern engineering as it yields insight into the biomimetic design of molecules and surfaces for improved adhesion or motility.

Carapace surface architecture facilitates camouflage of the decorator crab Tiarinia cornigera.

UNLABELLED: This paper elucidates the unique setal morphology of the decorator crab Tiarinia cornigera, and further presents evidence to that setal morphology promotes micro-organism nucleation and adhesion. The carapace of this crab is covered by clusters of setae, each comprising a hollow acicular stem that is enveloped by a haystack-like structure. Using computational fluid dynamics, we find that these setae are responsible for manipulating water flow over the carapace surface. Micro-organisms in the sea water, nest in areas of flow stagnation and as a result, nucleate to and biofoul the setae by means of chemical adhesion. Attached micro-organisms secrete extracellular polymeric substances, which we deduce must also provide an additional element of chemical adhesion to mechanically interlocked mesoscopic and macroscopic biomatter. By coupling physical and chemical methods for adhesion, T. cornigera is able to hierarchically decorate its carapace. STATEMENT OF SIGNIFICANCE: Our paper brings to light the unique decorator crab carapace morphology of T. cornigera; and furthermore evidences its function in micro-organism nucleation and adhesion. We show how this special carapace morphology directs and guides water flow to form nesting regions of water stagnation where micro-organisms can nucleate and adhere. In the literature, decorator crab carapaces are presumed to be able to mechanically interlock biomatter as camouflage using hook-like setal outgrowths. T. cornigera contrarily exhibits clusters of hay-stack like structures. By encouraging micro-organism adhesion to the carapace setae, T. cornigera is able to effectively attach biomatter using both chemical and physical principles of adhesion. T. cornigera essentially has a super-biofouling carapace surface, for at least micro-organisms. Our work will have an impact on researchers interested in biofouling, adhesion, biomedical and purification filter systems, and in the development of novel biomimetic surfaces with tailored properties.