Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant.

In this study, an in silico screening approach was employed to mine potential bacteriocin clusters in genome-sequenced isolates of Lacticaseibacillus zeae UD 2202 and Lacticaseibacillus casei UD 1001. Two putative undescribed bacteriocin gene clusters (Cas1 and Cas2) closely related to genes encoding class IIa bacteriocins were identified. No bacteriocin activity was recorded when cell-free supernatants of strains UD 2202 and UD 1001 were tested against Listeria monocytogenes. Genes encoding caseicin A1 (casA1) and caseicin A2 (casA2) were heterologously expressed in Escherichia coli BL21 (DE3) using the nisin leader peptide cloned in-frame to the C-terminal of the green fluorescent gene (mgfp5). Nisin protease (NisP) was used to cleave caseicin A1 (casA1) and caseicin A2 (casA2) from GFP-Nisin leader fusion proteins. Both heterologously expressed peptides (casA1 and casA2) inhibited the growth of L. monocytogenes, suggesting that casA1 and casA2 are either silent in the wild-type strains or are not secreted in an active form. The minimum inhibitory concentration (MIC) of casA1 and casA2, determined using HPLC-purified peptides, ranged from < 0.2 µg/mL to 12.5 µg/mL when tested against Listeria ivanovii, Listeria monocytogenes, and Listeria innocua, respectively. A higher MIC value (25 µg/mL) was recorded for casA1 and casA2 when Enterococcus faecium HKLHS was used as the target. The molecular weight of heterologously expressed casA1 and casA2 is 5.1 and 5.2 kDa, respectively, as determined with tricine-SDS-PAGE. Further research is required to determine if genes within Cas1 and Cas2 render immunity to other class IIa bacteriocins.

Komagataella phaffii (previously described as Pichia pastoris) is a yeast that produces high-level heterologous proteins with a wide range of applications in medicine and industry. The methanol-induced alcohol oxidase I promoter (PAOX1) is frequently used for protein expression in this yeast. However, limitations on the use of methanol have been observed in large-scale production, including its flammability, toxicity, and need for special handling. Here, we propose to develop a system using recombinant cells constitutively expressing pectinmethyl esterase for expression of two reporter proteins, GFP and azurin, under the control of PAOX1 using pectin in production medium. So, this system is coherent with yeast culture medium containing pectin and heterologous gene inserted downstream of PAOX1 can be successfully expressed without the addition of methanol. Therefore, this novel Self-inducibLe heterologous protein EXpression (SILEX) system, which does not require the addition of methanol, can be used for the production of any protein. It can also be adapted for large-scale production.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-024-04039-x.

Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is critical for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on Lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged, and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope (NE) ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit Emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others may explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

OBJECTIVE: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis.
METHODS: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer.
RESULTS: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer.
CONCLUSIONS: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its\' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.

The endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+-binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca2+ Basic Protocol 3: Monitoring ER- and cytosolic-Ca2+ Support Protocol: Generation of a stable cell line expressing erGAP3.

Various bacterial species are associated with plant roots. However, symbiotic and free-living plant growth-promoting bacteria (PGPB) can only help plants to grow and develop under normal and stressful conditions. Several biochemical and in vitro assays were previously designed to differentiate between the PGPB and other plant-associated bacterial strains. This chapter describes and summarizes some of these assays and proposes a strategy to screen for PGPB. To determine the involvement of the PGPB in abiotic stress tolerance, assays for the ability to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, ammonium, gibberellic acid (GA), indole acetic acid (IAA), and microbial volatile organic compounds (mVOCs) are described in this chapter. Additionally, assays to show the capacity to solubilize micronutrients such as potassium, phosphorus, and zinc by bacteria were also summarized in this chapter. To determine the contribution of the PGPB in biotic stress tolerance in plants, Fe-siderophore, hydrogen cyanide, and antibiotic and antifungal metabolites production assays were described. Moreover, assays to investigate the growth-promotion activities of a bacterium strain on plants, using the gnotobiotic root elongation, in vitro, and pots assays, were explained. Finally, an assay for the localization of endophytic bacterium in plant tissues was also presented in this chapter. Although the assays described in this chapter can give evidence of the nature of the mechanism behind the PGPB actions, other unknown growth-promoting means are yet to decipher, and until then, new methodologies will be developed.