Microfluidics for adaptation of microorganisms to stress: design and application

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas

Microfluidic cultivation and analysis of productive biofilms

We here report the application of a machine-based microfluidic biofilm cultivation and analysis platform for studying the performance of biocatalytically active biofilms. By using robotic sampling, we succeeded in spatially resolving the productivity of three microfluidic reactors containing biocatalytically active biofilms that inducibly overexpress recombinant enzymes. Escherichia coli biofilms expressing two stereoselective oxidoreductases, the (R)-selective alcohol dehydrogenase LbADH and the (S)-selective ketoreductase Gre2p, as well as the phenolic acid decarboxylase EsPAD were used. The excellent reproducibility of the cultivation and analysis methods observed for all three systems underlines the usefulness of the new technical platform for the investigation of biofilms. In addition, we demonstrated that the analytical platform also opens up new opportunities to perform in-depth spatially resolved studies on the biomass growth in a reactor channel and its biochemical productivity. Since the platform not only offers the detailed biochemical characterization but also broad capabilities for the morphological study of living biofilms, we believe that our approach can also be performed on many other natural and artificial biofilms to systematically investigate a wide range of process parameters in a highly parallel manner using miniaturized model systems, thus advancing the harnessing of microbial communities for technical purposes

Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance

Microfluidic devices can mimic naturally occurring microenvironments and create microbial population heterogeneities ranging from planktonic cells to biofilm states. The exposure of such populations to spatially organized stress gradients can promote their adaptation into complex phenotypes, which are otherwise difficult to achieve with conventional experimental setups. Here a microfluidic chip that employs precise chemical gradients in consecutive microcompartments to perform microbial adaptive laboratory evolution (ALE), a key tool to study evolution in fundamental and applied contexts is described. In the chip developed here, microbial cells can be exposed to a defined profile of stressors such as antibiotics. By modulating this profile, stress adaptation in the chip through resistance or persistence can be specifically controlled. Importantly, chip‐based ALE leads to the discovery of previously unknown mutations in Escherichia coli that confer resistance to nalidixic acid. The microfluidic device presented here can enhance the occurrence of mutations employing defined micro‐environmental conditions to generate data to better understand the parameters that influence the mechanisms of antibiotic resistance

Intracellular Assembly of Interacting Enzymes Yields Highly‐Active Nanoparticles for Flow Biocatalysis

All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for >4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes

Enhanced Bioactivity of Tailor-Made Glycolipid Enriched Manuka Honey

Glycolipids can be synthetized in deep eutectic solvents (DESs) as they possess low water content allowing a reversed lipase activity and thus enables ester formation. Based on this principle, honey can also serve as a media for glycolipid synthesis. Indeed, this supersaturated sugar solution is comparable in terms of physicochemical properties to the sugar-based DESs. Honey-based products being commercially available for therapeutic applications, it appears interesting to enhance its bioactivity. In the current work, we investigate if enriching medical grade honey with in situ enzymatically-synthetized glycolipids can improve the antimicrobial property of the mixture. The tested mixtures are composed of Manuka honey that is enriched with octanoate, decanoate, laurate, and myristate sugar esters, respectively dubbed GOH, GDH, GLH, and GMH. To characterize the bioactivity of those mixtures, first a qualitative screening using an agar well diffusion assay has been performed with methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Candida bombicola, Escherichia coli, and Pseudomonas putida which confirmed considerably enhanced susceptibility of these micro-organisms to the different glycolipid enriched honey mixtures. Then, a designed biosensor E. coli strain that displays a stress reporter system consisting of three stress-specific inducible, red, green, and blue fluorescent proteins which respectively translate to physiological stress, genotoxicity, and cytotoxicity was used. Bioactivity was, therefore, characterized, and a six-fold enhancement of the physiological stress that was caused by GOH compared to regular Manuka honey at a 1.6% (v/v) concentration was observed. An antibacterial agar well diffusion assay with E. coli was performed as well and demonstrated an improved inhibitory potential with GOH upon 20% (v/v) concentration

Macroporous Silicone Chips for Decoding Microbial Dark Matter in Environmental Microbiomes

Natural evolution has produced an almost infinite variety of microorganisms that can colonize almost any conceivable habitat. Since the vast majority of these microbial consortia are still unknown, there is a great need to elucidate this “microbial dark matter” (MDM) to enable exploitation in biotechnology. We report the fabrication and application of a novel device that integrates a matrix of macroporous elastomeric silicone foam (MESIF) into an easily fabricated and scalable chip design that can be used for decoding MDM in environmental microbiomes. Technical validation, performed with the model organism Escherichia coli expressing a fluorescent protein, showed that this low-cost, bioinert, and widely modifiable chip is rapidly colonized by microorganisms. The biological potential of the chip was then illustrated through targeted sampling and enrichment of microbiomes in a variety of habitats ranging from wet, turbulent moving bed biofilters and wastewater treatment plants to dry air-based environments. Sequencing analyses consistently showed that MESIF chips are not only suitable for sampling with high robustness but also that the material can be used to detect a broad cross section of microorganisms present in the habitat in a short time span of a few days. For example, results from the biofilter habitat showed efficient enrichment of microorganisms belonging to the enigmatic Candidate Phyla Radiation, which comprise ∼70% of the MDM. From dry air, the MESIF chip was able to enrich a variety of members of Actinobacteriota, which is known to produce specific secondary metabolites. Targeted sampling from a wastewater treatment plant where the herbicide glyphosate was added to the chip’s reservoir resulted in enrichment of Cyanobacteria and Desulfobacteria, previously associated with glyphosate degradation. These initial case studies suggest that this chip is very well suited for the systematic study of MDM and opens opportunities for the cultivation of previously unculturable microorganisms

Gene expression of IQGAPs and Ras families in an experimental mouse model for hepatocellular carcinoma: a mechanistic study of cancer progression

IQGAPs genes play critical role in either induction or suppression of cancer and its progression, however the relationship between Ras genes and these genes are still unclear. In this study, we tried to understand the mechanistic action of IQGAPs genes and its correlation with Ras genes in mouse hepatic cancer model. The genetic expressions of IQGAP1, IQGAP2, IQGAP3, Hras, Kras, Nras, Mras, Caspase3, and BAX were followed in both hepatocellular carcinoma and normal liver cells of Balbc mice. Genotoxic agent diethylnitrosamine (DEN)-induced hepatic cancer model was induced in male mice and recorded the occurrence of hepatocellular carcinoma by morphological and histological changes in the liver. It was observed that mRNA expressions of IQGAP1, Hras, Kras, Nras, Mras, Caspase3, and BAX genes were highly elevated in hepatocellular carcinoma cells when compared with normal liver cells, additionally their expressions increased by concentrating the dose of DEN. While, the expressions of IQGAP2 and IQGAP3 were significantly decreased in hepatocellular carcinoma cells when compared with normal liver cells, as well as their expressions decreased more with increasing the dose of DEN. It was concluded from this study that IQGAP1 has a strong signaling relationship with Ras genes in induction of cancer and it is considered as a key gene for induction or suppression of the hepatocellular carcinoma

Transcriptome comparisons detect new genes associated with apoptosis of cattle and buffaloes preantral follicles

Abstract Background To develop new breeding technology to improve the breeding ability of bovine, it is the development trend to find the main reason for the occurrence of atresia in these organisms. Transcriptomes of small (100–120 μm) and large (200–220 μm) preantral follicles from cattle and buffalo ovaries were evaluated in vivo and in vitro to understand the transcriptional modulation in preantral follicles that leads to the phenomenon of atresia. Methods The preantral follicles were checked as dead, damage, or live follicles in vivo and in vitro by using trypan blue then bisbenzimide and propidium iodine. Transcriptomes of small (100–120 μm) and large (200–220 μm) preantral follicles of cattle and buffalo were evaluated in vivo and in vitro by microarray and RT-PCR. Healthy preantral follicles were selected based on staining results, and then RNA was extracted from them. Results The viability percentage of preantral follicles in cattle was higher (26.7% and 20%) than buffalo (10%) in vivo and in vitro, respectively. According to the microarray data analysis for cattle preantral follicles, only eleven genes were detected corresponding to five upregulated and six downregulated in large size (200–220 μm) compared to small (100–120 μm) size preantral follicles, while in buffalo, 171 genes were detected (92 upregulated and 79 downregulated) in large size compared to small preantral follicle size. The results of RT-PCR of the selected genes (FASTKD1, BAG2, RHOB, AGTR2, MEF2C, BCL10, G2E3, TM2D1, IGF-I, IGFBP3, PRDX3, and TRIAP1) validated the microarray results. In conclusion, the data of gene expression showed significant differences between small and large sizes in both buffalo and cattle preantral follicles. Conclusion Apoptotic genes were upregulated in the large preantral follicle compared with the small preantral follicles. Moreover, the expression level of these apoptotic genes was significantly upregulated in buffalo than in the cattle. Most of these genes were significantly upregulated in the large buffalo preantral follicle compared with the small size. However, anti-apoptotic genes were upregulated in large cattle preantral follicle and downregulated in large buffalo preantral follicle

Protective Effects of Naringin–Dextrin Nanoformula against Chemically Induced Hepatocellular Carcinoma in Wistar Rats: Roles of Oxidative Stress, Inflammation, Cell Apoptosis, and Proliferation

Nanotechnology holds great promise for the development of treatments for deadly human diseases, such as hepatocellular carcinoma (HCC). In the current study, we compared the hepatoprotective effects of naringin–dextrin nanoparticles (NDNPs) against HCC in male Wistar rats with those of pure naringin and investigated the underlying cellular and molecular mechanisms. HCC was induced by intraperitoneal injection of diethylnitrosamine (DEN, 150 mg/kg body weight (b.w.) per week) for two weeks, followed by oral administration of 2-acetylaminofluorene (2AAF, 20 mg/kg b.w.) four times per week for three weeks. DEN/2AAF-administered rats were divided into three groups that respectively received 1% carboxymethyl cellulose (as vehicle), 10 mg/kg b.w. naringin, or 10 mg/kg b.w. NDNP every other day by oral gavage for 24 weeks. Both naringin and NDNP significantly attenuated the harmful effects of DEN on liver function. Both compounds also suppressed tumorigenesis as indicated by the reduced serum concentrations of liver tumor markers, and this antitumor effect was confirmed by histopathological evaluation. Additionally, naringin and NDNP prevented DEN-induced changes in hepatic oxidative stress and antioxidant activities. In addition, naringin and NDNP suppressed inflammation induced by DEN. Moreover, naringin and NDNP significantly reduced the hepatic expression of Bcl-2 and increased Bax, p53, and PDCD5 expressions. Naringin and NDNP also reduced expression of IQGAP1, IQGAP3, Ras signaling, and Ki-67 while increasing expression of IQGAP2. Notably, NDNP more effectively mitigated oxidative stress and inflammatory signaling than free naringin and demonstrated improved antitumor efficacy, suggesting that this nanoformulation improves bioavailability within nascent tumor sites