A rare case of primary ovarian mesonephric adenocarcinoma and a review of the literature.

OBJECTIVE: Are reported in the cervix in the female genital tract, has been reported in very few studies in the literature. In this report, we aimed to present a case with mesonephric carcinoma, which was detected in the ovary and is very rarely seen. CASE REPORT: In a case since the frozen section results of the left adnexal mass were reported as malignant. CONCLUSION: Ovarian mesonephric carcinoma is very rare and exhibits very different morphological patterns. Therefore, immunohistochemical and morphological findings should be evaluated together. If the pathological picture does not fit the common carcinomas of ovarian origin and this entity must be brought to mind, because, if these tumors with different molecular developmental pathways are diagnosed correctly, treatment schemes will change and targeted therapies will be developed too. KEY WORDS: Mesonephric carcinoma, Mesonephric like carcinoma, Ovarian carcinoma.

Probing the interactions between air bubbles and (bio)interfaces at the nanoscale using FluidFM technology.

Understanding the molecular mechanisms underlying bubble-(bio)surfaces interactions is currently a challenge that if overcame, would allow to understand and control the various processes in which they are involved. Atomic force microscopy is a useful technique to measure such interactions, but it is limited by the large size and instability of the bubbles that it can use, attached either on cantilevers or on surfaces. We here present new developments where microsized and stable bubbles are produced using FluidFM technology, which combines AFM and microfluidics. The air bubbles produced were used to probe the interactions with hydrophobic samples, showing that bubbles in water behave like hydrophobic surfaces. They thus could be used to measure the hydrophobic properties of microorganisms' surfaces, but in this case the interactions are also influenced by electrostatic forces. Finally a strategy was developed to functionalize their surface, thereby modulating their interactions with microorganism interfaces. This new method provides a valuable tool to understand bubble-(bio)surfaces interactions but also to engineer them.

Towards a better understanding of microalgae natural flocculation mechanisms to enhance flotation harvesting efficiency.

In microalgae harvesting, flocculation is usually a compulsory preliminary step to further separation by sedimentation or flotation. For some microalgae species, and under certain growth conditions, flocculation can occur naturally. Natural flocculation presents many advantages as it does not require the addition of any flocculants to the culture medium and shows high efficiency rate. But because natural flocculation is so specific to the species and conditions, and thanks to the knowledge accumulated over the last years on flocculation mechanisms, researchers have developed strategies to induce this natural harvesting. In this review, we first decipher at the molecular scale the underlying mechanisms of natural flocculation and illustrate them by selected studies from the literature. Then we describe the developed strategies to induce natural flocculation that include the use of biopolymers, chemically modified or not, or involve mixed species cultures. But all these strategies need the addition of external compounds or microorganism which can present some issues. Thus alternative directions to completely eliminate the need for an external molecule, through genetic engineering of microalgae strains, are presented and discussed in the third part of this review.

Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media.

Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants PADH2-Cat8-L2 and PmAOX1 and with the naturally occurring promoter PGAP to enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under PADH2-Cat8-L2 and PmAOX1 or PGAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, PmAOX1, and PGAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations of cis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidentical cis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents PADH2-Cat8-L2 and PmAOX1, the bifunctional DPES4 enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts. KEY POINTS: • Design method with the constraint and parameters for the construction of the DPESs is presented. • Hybrid-architectured de novo DPESs are designed to enhance and fine-tune gene expression. • Bifunctional DPESs demonstrate enhanced transcription and expression. • Twin DPESs linearly increase cis-acting DNA sites and consequently increase the demand for the same TFs. • Bifunctional DPESs enable good coupling of supply and demand to bind with TFs. • Ethanol-controlled Snf1 pathway and crosstalk enable fine-tuned transcription and enhanced expression.

Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor.

Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (PAOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with PAOX1 on methanol, the expression on ethanol is increased with i) PAOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) PAOX1/Adr1-L3/Cat8-L3 (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) PeAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations.

Nanoscale Evidence Unravels Microalgae Flocculation Mechanism Induced by Chitosan.

Microalgae are a promising resource for biofuel production, although their industrial use is limited by the lack of effective harvesting techniques. Flocculation consists in the aggregation and adhesion of cells into flocs that can be more easily removed from water than individual cells. Although it is an efficient harvesting technique, contamination is a major issue as chemical flocculants are often used. An alternative is to use natural biopolymers flocculants such as chitosan. Chitosan is a biobased nontoxic polymer that has been effectively used to harvest Chlorella vulgaris cells at a pH lower than its pKa (6.5). While the reported flocculation mechanism is said to rely on electrostatic interactions between chitosan and the negative cell surface, no molecular evidence has yet confirmed this mechanism. In this study, we performed force spectroscopy atomic force microscopy (AFM) experiments to probe the interactions between C. vulgaris cells and chitosan at the molecular scale to decipher its flocculation mechanism. Our results showed that at pH 6, chitosan interacts with C. vulgaris cell wall through biological interactions rather than electrostatic interactions. These observations were confirmed by comparing the data with cationically modified cellulose nanocrystals, for which the flocculation mechanism, relying on an electrostatic patch mechanism, has already been described for C. vulgaris. Further AFM experiments also showed that a different mechanism was at play at higher pH, based on chitosan precipitation. Thus, this AFM-based approach highlights the complexity of chitosan-induced flocculation mechanisms for C. vulgaris.

Isolation of High-Quality RNA from Pichia pastoris.

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science) Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen).