VDACs are pore-forming proteins, coating the mitochondrial outer membrane, and playing the role of main regulators for metabolites exchange between cytosol and mitochondria. In mammals, three isoforms have evolutionary originated, VDAC1, VDAC2, and VDAC3. Despite similarity in sequence and structure, evidence suggests different biological roles in normal and pathological conditions for each isoform. We compared Homo sapiens and Mus musculus VDAC genes and their regulatory elements. RNA-seq transcriptome analysis shows that VDAC isoforms are expressed in human and mouse tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3, with the exception of reproductive system. Numerous transcript variants for each isoform suggest specific context-dependent regulatory mechanisms. Analysis of VDAC core promoters has highlighted that, both in a human and a mouse, VDAC genes show features of TATA-less ones. The level of CG methylation of the human VDAC genes revealed that VDAC1 promoter is less methylated than other two isoforms. We found that expression of VDAC genes is mainly regulated by transcription factors involved in controlling cell growth, proliferation and differentiation, apoptosis, and bioenergetic metabolism. A non-canonical initiation site termed “the TCT/TOP motif,” the target for translation regulation by the mTOR pathway, was identified in human VDAC2 and VDAC3 and in every murine VDACs promoter. In addition, specific TFBSs have been identified in each VDAC promoter, supporting the hypothesis that there is a partial functional divergence. These data corroborate our experimental results and reinforce the idea that gene regulation could be the key to understanding the evolutionary specialization of VDAC isoforms.

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

In this work, a 3D printed biocompatible micro-optofluidic (MoF) device for two-phase flow monitoring is presented. Both an air–water bi-phase flow and a two-phase mixture composed of micrometric cells suspended on a liquid solution were successfully controlled and monitored through its use. To manufacture the MoF device, a highly innovative microprecision 3D printing technique was used named Projection Microstereolithography (P$\mu$SL) in combination with the use of a novel 3D printable photocurable resin suitable for biological and biomedical applications. The concentration monitoring of biological fluids relies on the absorption phenomenon. More precisely, the nature of the transmission of the light strictly depends on the cell concentration: the higher the cell concentration, the lower the optical acquired signal. To achieve this, the microfluidic T-junction device was designed with two micrometric slots for the optical fibers’ insertion, needed to acquire the light signal. In fact, both the micro-optical and the microfluidic components were integrated within the developed device. To assess the suitability of the selected biocompatible transparent resin for optical detection relying on the selected working principle (absorption phenomenon), a comparison between a two-phase flow process detected inside a previously fully characterized micro-optofluidic device made of a nonbiocompatible high-performance resin (HTL resin) and the same made of the biocompatible one (BIO resin) was carried out. In this way, it was possible to highlight the main differences between the two different resin grades, which were further justified with proper chemical analysis of the used resins and their hydrophilic/hydrophobic nature via static water contact angle measurements. A wide experimental campaign was performed for the biocompatible device manufactured through the P$\mu$SL technique in different operative conditions, i.e., different concentrations of eukaryotic yeast cells of Saccharomyces cerevisiae (with a diameter of 5 $\mathsf{\mu }$m) suspended on a PBS (phosphate-buffered saline) solution. The performed analyses revealed that the selected photocurable transparent biocompatible resin for the manufactured device can be used for cell concentration monitoring by using ad hoc 3D printed micro-optofluidic devices. In fact, by means of an optical detection system and using the optimized operating conditions, i.e., the optimal values of the flow rate $FR=0.1$ mL/min and laser input power $P\in \{1,3\}$ mW, we were able to discriminate between biological fluids with different concentrations of suspended cells with a robust working ability $R^2=0.9874$ and $R_{adj}^2=0.9811$.

Background VDACs (Voltage-Dependent Anion-selective Channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to their presence. In higher eukaryotes three isoforms raised during the evolution: they have the same exon-intron organization and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. Results VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3. However, RNA-seq CAGE approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription Factor Binding Sites (TFBSs) distribution in the promoters was investigated. The main regulators common to the three VDAC genes were identified as E2FF, NRF1, KLFS, EBOX transcription factors family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each isoform gene were identified, supporting the results in the literature, indicating a general role of VDAC1, as actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. Conclusions For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoforms genes.

In this paper, the combination of two algorithms, a cell counting algorithm and a velocity algorithm based on a Digital Particle Image Velocimetry (DPIV) method, is presented to study the collective behavior of micro-particles in response to hydrodynamic stimuli. A wide experimental campaign was conducted using micro-particles of different natures and diameters (from 5 to 16μm), such as living cells and silica beads. The biological fluids were injected at the inlet of a micro-channel with an external oscillating flow, and the process was monitored in an investigated area, simultaneously, through a CCD camera and a photo-detector. The proposed data analysis procedure is based on the DPIV-based algorithm to extrapolate the micro-particles velocities and a custom counting algorithm to obtain the instantaneous micro-particles number. The counting algorithm was easily integrated with the DPIV-based algorithm, to automatically run the analysis to different videos and to post-process the results in time and frequency domain. The performed experiments highlight the difference in the micro-particles hydrodynamic responses to external stimuli and the possibility to associate them with the micro-particles physical properties. Furthermore, in order to overcome the hardware and software requirements for the development of a real-time approach, it was also investigated the possibility to detect the flows by photo-detector signals as an alternative to camera acquisition. The photo-detector signals were compared with the velocity trends as a proof of concept for further simplification and speed-up of the data acquisition and analysis. The algorithm flexibility underlines the potential of the proposed methodology to be suitable for real-time detection in embedded systems.