Improved Cell Growth Research Articles

Plant Hormone Signals Mediate Melatonin Synthesis to Enhance Osmotic Stress Tolerance in Watermelon Cells

Melatonin, a multifunctional signaling molecule, has been shown to play a significant role in response to abiotic stress. Several species have been reported to unveil melatonin’s effect on osmotic stress; however, the signal transduction mechanism of phytohormone-mediated melatonin biosynthesis in plant species remains unclear. In this study, although plants can biosynthesize melatonin, the exogenous application of melatonin to watermelon cells can improve cell growth in response to osmotic stress by regulating the antioxidant machinery of cells. Regarding the melatonin synthesis pathway, ClOMT (ClASMT and ClCOMT) is a multi-gene family, and ClSNAT has two members. Both ClOMTs and ClSNATs harbor the cis-elements in their promoter regions responding to various hormones. Among abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), ABA treatment observably upregulated the expression of ClOMTs and ClSNATs, and the accumulation of melatonin with ABA treatment reached a level comparable to that with osmotic stress by mannitol treatment. Furthermore, when hormone biosynthesis inhibitors were added to cells before osmotic stress, the expression of ClOMTs and ClSNATs, as well as melatonin accumulation, were significantly suppressed with the ABA biosynthesis inhibitor. This study demonstrated the crucial role of melatonin biosynthesis in response to osmotic stress via plant hormone signal transduction. It showed that ABA signaling plays a dominant role in melatonin synthesis under osmotic stress.

Electrospun Polyvinylpyrrolidone/Soy protein nanofibers enriched with curcumin-loaded ZIF-8 nanoparticles: Synthesis and evaluation for wound healing application

In skin tissue engineering, biocompatible polymeric nanofibers (NFs) with high antimicrobial capability are used as wound dressings for the rapid healing of skin wounds. In this research, a novel NFs is produced by a combination of Polyvinylpyrrolidone (PVP) and soy protein (Soy) incorporated with Zeolite imidazolate framework-8 loaded with Curcumin (ZIF8 @Cur) nanoparticles (NPs) via electrospinning method. PVP/Soy NF with a ratio of 70:30 (P70S30) presented acceptable properties as a parent scaffold. ZIF8 @Cur NPs with concentrations of 10 % and 20 % wt. were loaded in P70S30 mat which coded as the PSZ10 and PSZ20, respectively. NFs were investigated in terms of chemical and physical structure such as FTIR and FESEM analyses, swelling, degradation, moisture retention and wettability. In addition, this study investigates cellular behavior, hemocompatibility and antioxidant properties of NFs. The produced scaffolds were not toxic in the cell media and ZIF8 @Cur NPs improved cell growth and proliferation. The results of the antibacterial test showed that the presence of ZIF8 @Cur NPs increases the antibacterial ability against pathogenic agents due to the release of Zn2+ and Cur. Among the NFs, the PSZ20 mat was chosen as the optimal scaffold due to its favorable characteristics. Therefore, the obtained results confirm the potential of NFs as a wound dressing.

A tri-layer decellularized, dehydrated human amniotic membrane scaffold supports the cellular functions of human tenocytes in vitro.

Differences in scaffold design have the potential to influence cell-scaffold interactions. This study sought to determine whether a tri-layer design influences the cellular function of human tenocytes in vitro. The single-layer decellularized, dehydrated human amniotic membrane (DDHAM) and the tri-layer DDHAM (DDHAM-3L) similarly supported tenocyte function as evidenced by improved cell growth and migration, reduced dedifferentiation, and an attenuated inflammatory response. The tri-layer design provides a mechanically more robust scaffold without altering biological activity.

Formulation and Evaluation of Chitosan-PLGA Biocomposite Scaffolds Incorporated with Quercetin Liposomes Made by QbD Approach for Improved Healing of Oral Lesions.

The current research aims to develop and evaluate chitosan-PLGA biocomposite scaffolds in combination with quercetin liposomes to accomplish the desired impact in oral lesions where pharmacotherapeutic agent treatment through circulation could only reach the low content at the target. Optimization of quercetin-loaded liposomes was carried out using 32 factorial design. The preparation of porous scaffolds comprising produced quercetin-loaded liposomes by thin-film method was carried out in the current study using a unique strategy combining solvent casting and gas foaming procedures. The prepared scaffolds were tested for physicochemical properties, in vitro quercetin release study, ex vivo drug permeation and retention research using goat mucosa, antibacterial activity, and cell migration studies on fibroblast L929 cell lines. Improved cell growth and migration were seen in the order control < liposomes < proposed system. The proposed system has been examined for its biological and physicochemical features, and it has the potential to be utilized as an efficient therapy for oral lesions.

Chitosan and cellulose-based composite hydrogels with embedded titanium dioxide nanoparticles as candidates for biomedical applications

Hydrogel based matrices and titanium dioxide (TiO2) nanoparticles (NPs) are well established materials in bone tissue engineering. Nevertheless, there is still a challenge to design appropriate composites with enhanced mechanical properties and improved cell growth. Progressing in this direction, we synthesized nanocomposite hydrogels by impregnating TiO2 NPs in a chitosan and cellulose-based hydrogel matrix containing polyvinyl alcohol (PVA), to enhance the mechanical stability and swelling capacity. Although, TiO2 has been incorporated into single and double component matrix systems, it has rarely been combined with a tri-component hydrogel matrix system. The doping of NPs was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy and small- and wide-angle X-ray scattering. Our results showed that incorporation of TiO2 NPs improved the tensile properties of the hydrogels significantly. Furthermore, we performed biological evaluation of scaffolds, swelling degree, bioactivity assessment, and hemolytic tests to prove that all types of hydrogels were safe for use in the human body. The culturing of human osteoblast-like cells MG-63 on hydrogels showed better adhesion of cells in the presence of TiO2 and showed increasing proliferation with increasing amount of TiO2. Our results showed that the sample with the highest TiO2 concentration, CS/MC/PVA/TiO2 (1 %) had the best biological properties.

Design of a novel tannic acid enriched hemostatic wound dressing based on electrospun polyamide-6/hydroxyethyl cellulose nanofibers

Recently, the use of scaffolds as wound dressings in skin tissue engineering with the aim of rapid healing and prevention of bleeding is growing quickly, materials and structures similar to the target tissue are used to improve its quality. In this research, polyamide6/hydroxyethyl cellulose (PA6/HEC) incorporated with TA (tannic acid) in different concentration (5, 10, 25%) has been used to produce nanofibers (NFs) by electrospinning method. Electrospun NFs were examined in terms of chemical and physical structure, swelling, degradation, moisture retention, thermal stability and mechanical properties. In addition, this study investigates cellular behavior, antibacterial, hemocompatibility and antioxidant properties. Among the NFs, PA/HEC/25%TA mat showed more favorable results. The produced scaffolds were non-toxic in the cell media, and increasing TA content improved cell growth and proliferation. The results of the hemostatic test showed that the presence of TA causes rapid blood coagulation, and TA has increased the antibacterial ability against pathogenic agents and antioxidant virtues. Therefore, the obtained results confirm the potential of NFs as a wound dressing.

BIO25: Toward a Biocompatible 3D Printing Resin for Microfluidic Organ Models

Background: Microfluidics is a desirable platform to create artificial organ models, such as organs-on-a-chip, to study disease and develop therapeutics in a 3D microenvironment. There is a gap in understanding and reproducing the microenvironment due to a lack of suitable models and means to fabricate these micron-scale architectures. 3D printed microfluidics offer a reliable fabrication approach with geometric control for replicating the tissue microenvironment at a relevant size-scale. However, gas-permeable, biocompatible materials for high-resolution microfluidic 3D printing do not exist. Here, we report a biocompatible, polydimethylsiloxane (PDMS) resin for facile 3D printing of microfluidics for tissue-based devices. Methods: 3D-printed parts were made using the Asiga MAX X UV27 digital light processing (DLP) 3D printer and our custom, high-resolution PDMS resin. Unreacted monomers and leachable components were extracted from 3D-printed well plates by soaking in ethanol for 72h and compared against untreated (no ethanol extraction) 3D-printed plates and standard cell culture plates. Absorbance of extraction media was measured to quantify the removal of leachables. Gas permeability of thin, 3D-printed films was measured for O2 and CO2 by Labthink International, Inc. according to ASTM D1434-82(R09)e1. Biocompatibility of the novel gas-permeable, high-resolution PDMS resin was tested by BioAssay Systems via a cell viability study with HepG2 cells in vitro. Results: Figure 1A shows a diminishing absorbance signal of the extractable compounds in the ethanol solution after each 24h soak of the cured resin. Reduction in the absorbance signal indicates removal of these unwanted constituents as confirmed by the lighter hue of extracted 3D-printed cell culture plate vs unextracted controls (Figure 1B). O2 and CO2 permeability are comparable to Sylgard 184 (Figure 1C). Cell viability studies performed with HepG2 cells (Figure 1D) grown on untreated 3D-printed plates, extracted 3D-printed plates, and control cell culture plates show that removal of unreacted groups and photoabsorbing compounds have a significant effect (p<0.05), improving cell growth and viability relative to the untreated and control groups. Conclusion: We have demonstrated the potential of this PDMS 3D printing resin as a biocompatible, gas-permeable material for cell-based microfluidic devices. Applications range from µALs as respiratory support devices to tissue-engineered, microfluidic organ models to study disease.Figure 1. (A) Absorbance signal of extraction media after soaking the 3D printed part in ethanol. (B) 3D printed well plates, untreated (no ethanol extraction) and extracted in ethanol (72h soak). (C) Gas permeability measurements of 100µm film made from PDMS resin. Sylgard 184 data were taken from literature. (D) Cell viability of HepG2 cells grown on control plates (standard 12-well), untreated 3D printed plates, and extracted 3D printed plates (n=3). Analyses of unpaired multiple t-tests between treatment groups (* indicates p-value<0.05) and 2-way ANOVA with Tukey’s multiple comparison test determined there was a significant interaction (p-value<0.0001) between the effects of treatment (control, untreated, and extracted) and time (24h and 72h) on cell viability (absorbance).

Understanding the impact of bioactive coating materials for human mesenchymal stromal cells and implications for manufacturing

Bioactive materials interact with cells and modulate their characteristics which enable the generation of cell-based products with desired specifications. However, their evaluation and impact are often overlooked when establishing a cell therapy manufacturing process. In this study, we investigated the role of different surfaces for tissue culture including, untreated polystyrene surface, uncoated Cyclic Olefin Polymer (COP) and COP coated with collagen and recombinant fibronectin. It was observed that human mesenchymal stromal cells (hMSCs) expanded on COP-coated plates with different bioactive materials resulted in improved cell growth kinetics compared to traditional polystyrene plates and non-coated COP plates. The doubling time obtained was 2.78 and 3.02 days for hMSC seeded in COP plates coated with collagen type I and recombinant fibronectin respectively, and 4.64 days for cells plated in standard polystyrene treated plates. Metabolite analysis reinforced the findings of the growth kinetic studies, specifically that cells cultured on COP plates coated with collagen I and fibronectin exhibited improved growth as evidenced by a higher lactate production rate (9.38 × 105 and 9.67 × 105 pmol/cell/day, respectively) compared to cells from the polystyrene group (5.86 × 105 pmol/cell/day). This study demonstrated that COP is an effective alternative to polystyrene-treated plates when coated with bioactive materials such as collagen and fibronectin, however COP-treated plates without additional coatings were found not to be sufficient to support cell growth. These findings demonstrate the key role biomaterials play in the cell manufacturing process and the importance of optimising this selection.

Abstract 1996: Prostate cancer androgen receptor signaling inhibition via romidepsin encapsulated lipid-polymer hybrid nanoparticle with PSMA617 targeting

Abstract Purpose/Objective(s): Androgen deprivation therapy (ADT) has been a clinical standard of care for the treatment of castration sensitive prostate cancer (CSPC). However, it is not curative, and ADT has significant systemic effects including hot flashes, osteoporosis, anemia, etc. We sought to identify agents that would inhibit AR signaling without causing these systemic effects. Histone deacetylase inhibitors (HDACis) are attractive agents because they impede AR signaling and DNA damage repair in PCa and show epigenetic modifications in prostate cancer (PCa) development and progression. This study demonstrated romidepsin as an alternative to ADT and the development of a romidepsin encapsulated CSPC PCa targeted nanoparticle for PCa cell killing and growth inhibition without the toxicity associated with free romidepsin or ADT. Materials/Methods: Three PCa cell lines, LNCaP, 22Rv1 and PC3 that express different androgen receptor (AR) levels were used for this study. Cell viability was tested by real-time cell analysis (RTCA), protein expression by western blot analysis, and quantification of γ-H2AX double stranded DNA (dsDNA) breaks by immunofluorescent microscopy. We developed a poly (D,L-lactide-co-glycolide) (PLGA) and 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG) polymer-lipid nanoparticle coated with vipivotide tetraxetan(PSMA-617) for prostate specific membrane antigen (PSMA) targeting. Nanoparticles were characterized by transmission electron microscope (TEM), dynamic light scattering (DLS) analysis, and nanoparticle tracking analysis (NTA). Romidepsin encapsulation efficiency was quantified by high-performance liquid chromatography(HPLC). Results: RTCA showed free romidepsin inhibited cell growth in all three PCa cell lines in a dose dependent manner. The addition of ADT exhibited an additive effect with low romidepsin doses, but did not significantly improve cell growth inhibition when combined with high doses of romidepsin. Western blot and γ-H2AX foci staining confirmed that romidepsin inhibited AR signaling and increased dsDNA damage, respectively. HPLC showed nanoparticle encapsulated romidepsin of 0.7%wt and TEM/DLS/NTA showed a size of 130 ±20nm. Furthermore, PSMA-617 coated nanoparticles exhibited enhanced uptake in LNCaP cells compared to non-coated nanoparticles. Conclusion: Our study showed that the romidepsin has a dual effect on inhibiting AR signaling as well as downregulating DNA damage repair genes. Additionally, we developed a PCa targeted nanoparticle for specific PCa delivery of romidepsin. PSMA-targeted, romidepsin-encapsulated nanoparticles would reduce the systemic effects of free romidepsin and a potential replacement for ADT. Citation Format: Wendi Ma, Daiki Hara, Yu-Ping Yang, Olivia Yolande Bosquet, Wei Zhang, Emre Dikici, Huayang Feng, Tim Horton, Wensi Tao, Jessi Hersh, Junwei Shi, Sylvia Daunert, Sapna Deo, Alan Pollack. Prostate cancer androgen receptor signaling inhibition via romidepsin encapsulated lipid-polymer hybrid nanoparticle with PSMA617 targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1996.

Microwell-based flow culture increases viability and restores drug response in prostate cancer spheroids.

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and down-regulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions. This article is protected by copyright. All rights reserved.

Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine.

L-threonine is an essential amino acid used widely in food, cosmetics, animal feed and medicine. The thrABC operon plays an important role in regulating the biosynthesis of L-theronine. In this work, we systematically analyzed the effects of separating thrAB and thrC in different proportions on strain growth and L-threonine production in Escherichia coli firstly. The results showed that higher expression of thrC than thrAB enhanced cell growth and L-threonine production; however, L-threonine production decreased when the thrC proportion was too high. The highest L-threonine production was achieved when the expression intensity ratio of thrAB to thrC was 3:5. Secondly, a stationary phase promoter was also used to dynamically regulate the expression of engineered thrABC. This strategy improved cell growth and shortened the fermentation period from 36h to 24h. Finally, the acetate metabolic overflow was reduced by deleting the ptsG gene, leading to a further increase in L-threonine production. With these efforts, the final strain P 2.1 -2901ΔptsG reached 40.06g/L at 60h fermentation, which was 96.85% higher than the initial control strain TH and the highest reported titer in shake flasks. The maximum L-threonine yield and productivity was obtained in reported fed-batch fermentation, and L-threonine production is close to the highest titer (127.30g/L). In this work, the expression ratio of genes in the thrABC operon in E. coli was studied systematically, which provided a new approach to improve L-threonine production and its downstream products.

In-situ ionic crosslinking of 3D bioprinted cell-hydrogel constructs for mechanical reinforcement and improved cell growth

Hydrogels are commonly used in 3D bioprinting technology owing to their ability to encapsulate living cells. However, their inherent delicate properties limit their applicability in the fabrication of mechanically reliable tissue engineering constructs. Herein, we propose a novel reinvented layering integration method for the functional enhancement of 3D cell-hydrogel bioprinting. This was implemented by inserting electrospun microfiber sheets with a crosslinker between the 3D bioprinted layers. When surface-modified microfiber sheets were combined with Ca2+ ionic crosslinkers, the as-printed cell-hydrogel strand was immediately crosslinked when it contacted the sheet surface. The in-situ crosslinking in the bioprinting process not only improved the overall structural stability, but also reinforced the compressive strength and elastic modulus. The enhanced structural stability guaranteed the shape fidelity of the 3D architecture, which included the internal channel network, resulting in improved perfusion conditions for cell growth. The growth of NIH3T3 fibroblasts in 3D bioconstructs with in-situ crosslinking increased by up to five times compared to that of normally bioprinted constructs. The strengthened structural integrity was distinctly sustainable during the cell culture period owing to the sustained release of Ca2+ ions from the embedded microfiber sheets. The synergistic effect of the reinforced mechanical properties with enhanced cell growth is expected to extend the applicability of the proposed hydrogel-based bioprinting technique for soft tissue engineering.

An efficient approach for the production of polyhydroxybutyrate from lignin by alkali-halophile Halomonas alkalicola M2

Lignin bioconversion into polyhydroxybutyrate (PHB) has emerged as a promising valorization, however, it is suffering from inadequate conversion efficiency. Herein, an efficient strategy for addressing this challenge by employing an alkali-halophilic strain Halomonas alkalicola M2 is developed. Up to 58.5% of lignin is degraded when incubates in the non-sterilized lignin (NSL) medium under a glucose concentration of 1.055 g/L, peptone concentration of 0.596 g/L, and incubation time of 6.89 d, demonstrating enhanced proficiency compared to thermally sterilized lignin (TSL) method due to the improved cell growth, lignin-degrading enzyme activities, and secreted protein contents. Consequently, PHB production demonstrates a 50% increase using NSL method compared to TSL method. A separate route of enzymatic hydrolysis and fermentation (SHF) is proposed to further improve the PHB production, resulting in a record yield of PHB which is 1.89 g/L. This work creatively develops a non-sterilization strategy to improve PHB production from lignin, showing great potential for promoting biological lignin valorization.

Enzyme-constrained metabolic model and in silico metabolic engineering of Clostridium ljungdahlii for the development of sustainable production processes

Constraint-based genome-scale models (GEMs) of microorganisms provide a powerful tool for predicting and analyzing microbial phenotypes as well as for understanding how these are affected by genetic and environmental perturbations. Recently, MATLAB and Python-based tools have been developed to incorporate enzymatic constraints into GEMs. These constraints enhance phenotype predictions by accounting for the enzyme cost of catalyzed model´s reactions, thereby reducing the space of possible metabolic flux distributions. In this study, enzymatic constraints were added to an existing GEM of Clostridium ljungdahlii, a model acetogenic bacterium, by including its enzyme turnover numbers (kcats) and molecular masses, using the Python-based AutoPACMEN approach. When compared to the metabolic model iHN637, the enzyme cost-constrained model (ec_iHN637) obtained in our study showed an improved predictive ability of growth rate and product profile. The model ec_iHN637 was then employed to perform in silico metabolic engineering of C. ljungdahlii, by using the OptKnock computational framework to identify knockouts to enhance the production of desired fermentation products. The in silico metabolic engineering was geared towards increasing the production of fermentation products by C. ljungdahlii, with a focus on the utilization of synthesis gas and CO2. This resulted in different engineering strategies for overproduction of valuable metabolites under different feeding conditions, without redundant knockouts for different products. Importantly, the results of the in silico engineering results indicated that the mixotrophic growth of C. ljungdahlii is a promising approach to coupling improved cell growth and acetate and ethanol productivity with net CO2 fixation.

Computational modeling of multiple myeloma interactions with resident bone marrow cells

The interaction of multiple myeloma with bone marrow resident cells plays a key role in tumor progression and the development of drug resistance. The tumor cell response involves contact-mediated and paracrine interactions. The heterogeneity of myeloma cells and bone marrow cells makes it difficult to reproduce this environment in in-vitro experiments. The use of in-silico established tools can help to understand these complex problems.In this article, we present a computational model based on the finite element method to define the interactions of multiple myeloma cells with resident bone marrow cells. This model includes cell migration, which is controlled by stress–strain equilibrium, and cell processes such as proliferation, differentiation, and apoptosis.A series of computational experiments were performed to validate the proposed model. Cell proliferation by the growth factor IGF-1 is studied for different concentrations ranging from 0–10 ng/mL.Cell motility is studied for different concentrations of VEGF and fibronectin in the range of 0–100 ng/mL. Finally, cells were simulated under a combination of IGF-1 and VEGF stimuli whose concentrations are considered to be dependent on the cancer-associated fibroblasts in the extracellular matrix.Results show a good agreement with previous in-vitro results. Multiple myeloma growth and migration are shown to correlate linearly to the IGF-1 stimuli. These stimuli are coupled with the mechanical environment, which also improves cell growth. Moreover, cell migration depends on the fiber and VEGF concentration in the extracellular matrix. Finally, our computational model shows myeloma cells trigger mesenchymal stem cells to differentiate into cancer-associated fibroblasts, in a dose-dependent manner.

Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli

BackgroundMembrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential—as therapeutic drug targets or in microbial cell factory optimization—many challenges remain for efficient and functional expression in a host such as Escherichia coli.ResultsA dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general.ConclusionsA dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selectionmay serve as a powerful tool for rRNA engineering.

Biosynthesis and biodegradation of poly(3-hydroxybutyrate) from Priestia flexa; A promising mangrove halophyte towards the development of sustainable eco-friendly bioplastics

The protracted persistence of petrochemical plastics in the environment and their non-biodegradability impede the survival of living creatures. Recently, biopolymers are being thoroughly researched as a potential replacement for conventional plastics. This present study sought to locate Poly(3-hydroxybutyrate) synthesizing bacterial species prevalent in the mangrove ecosystem. Six halophilic bacterial isolates were obtained from the mangrove habitat, four isolates displayed superior cell dry weight as well as PHB accumulation. Isolate PMPHB5 showed the highest cell dry weight (4.92 ± 0.02 g/L), while the maximum PHA yield (80%) was found with PMPHB7. Hence, PMPHB7 was chosen for further optimization of carbon source wherein glucose demonstrated improved cell growth as well as PHB production. The characterization of the PHB granules was performed by FT-IR spectroscopy and FE-SEM EDX. The presence of characteristic elements in the sample was confirmed using EDX. Isolate PMPHB7 was further identified as Priestia flexa through 16S rRNA gene sequencing (GenBank accession number: ON362236) and a phylogenetic tree was constructed to reveal the molecular relationships of this organism with others. The solvent-cast biopolymer film was made to check the biodegradability of the extracted PHB. When buried in soil, it was found that the biopolymer film exhibited approximately 73% biodegradation after 21 days. Thus, the present study sheds light on the potential of mangrove-associated halophytes to efficiently produce PHB that is readily biodegradable in soil.

PRDM1 Deletion and STAT3 Mutations Cooperatively Promote CD8+ T-Cell and NK-Cell Growth in Vitro

PRDM1 loss and STAT3 activating mutations commonly co-occur in several non-Hodgkin lymphomas, including ALK negative Anaplastic Large Cell Lymphoma (ALCL) and NK-cell lymphoma (NKCL). PRDM1 is a tumor suppressor gene in NKCL, and also plays important roles in the homeostasis and differentiation of CD8+ T-cells. STAT3 encodes a critical transcription factor involved in specific cytokine-mediated JAK/STAT pathway activation in lymphocytes, controlling proliferation, apoptosis, and other cellular functions. However, it remains unclear whether these two abnormalities cooperate during lymphomagenesis. Here, we introduced PRDM1 deletion and STAT3 mutants into primary human T- or NK-cells isolated from PBMCs of healthy donors and performed functional studies on the genetically modified cells. When either of the two common STAT3 mutants, STAT3Y640F or STAT3D661Y, was transduced into T- or NK-cells, the transduced cells showed short term growth advantage but could not persist, indicating that STAT3 activating mutations may provide stimulatory signals to T- and NK-cells initially, but inhibitory pathways could be activated that prevent long-term sustainable growth. When we knocked out PRDM1 first and then transduced the cells with the STAT3 mutants, the double modified T- or NK-cells showed sustained competitive growth advantage during in vitro culture. The effects were specific to STAT3 mutants as transduction of wild-type (WT) STAT3 or the STAT5BN642H mutant did not improve cell growth. These findings suggest that PRDM1 loss and STAT3 mutations synergistically promote growth in primary T- and NK-cells. We further showed that PRDM1-null, STAT3Y640F-transduced NK-cells displayed better survival and growth compared to PRDM1-deleted or WT cells with feeder cell stimulation. More importantly, the double mutant NK-cells were more independent of feeder cell stimulation, and they were able to expand when supplemented with a single cytokine (IL-15 or IL-2). This feature may be relevant to lymphoma development in vivo where stimulatory cytokines may be limiting. In addition, these double mutant NK-cells had reduced expression of exhaustion markers Tim-3 and Tigit. Similarly, genetically modified CD8+ T cells with PRDM1 knockout and STAT3-mutant were found to express much lower levels of PD-1, Tim-3, and Tigit, an important feature that may promote their growth in vivo where ligands of these co-inhibitory molecules may be expressed in the microenvironment. These double mutant CD8+ T cells exhibited greater stem memory (Tscm) phenotype, marked by CD45RA+, CD62L+, and CCR7+, suggesting the switch to a memory rather than an effector program. Taken together, our in vitro experiments using primary human cells have provided evidences for the functional cooperation between PRDM1 loss and STAT3 activating mutation in promoting CD8+ T- and NK-cell growth. Some features shown by the double mutant cells may facilitate their survival and growth more significantly in vivo. We are currently investigating the adoptive transfer of modified mouse or human cells into littermates or NSG-Tg(huIL15) mice respectively to observe the in vivo development and evolution of the transferred cells. The detailed mechanistic interactions between these two genetic abnormalities in lymphoma development will be further investigated.

Mitochondrial expression of metabolic enzymes for improving carotenoid production in Saccharomyces cerevisiae

The utilization of renewable feedstocks for producing bio-based chemicals by Saccharomyces cerevisiae is a viable option. Carotenoids are a class of pigments with important biological functions and are synthesized via the derived mevalonate pathway. Among the various approaches, the accumulation of intermediates is an important strategy to enhance carotenoid production in yeast. Acetyl-CoA is an important intermediate synthesized mainly in the cytosol and mitochondria. Here, we focused on the utilization of accumulated intermediates, including acetyl-CoA, in mitochondria by compartmentalization of enzymes. Hence, we attempted localizing enzymes related to carotenoid production in the mitochondria to improve carotenoid production in S. cerevisiae. The strain that expressed carotenoid-producing enzymes in the mitochondria (YPH-mtβ) showed improved production of carotenoid compared to that shown by the strain expressing carotenoid-producing enzymes in the cytosol (YPH-cyβ). In addition, the increase in glucose concentration resulted in improved cell growth and carotenoid production in YPH-mtβ, but not in YPH-cyβ (0.354 or 0.023 mg/L culture/OD600 with 120 g/L glucose after 48 h of cultivation with YPH-mtβ or YPH-cyβ, respectively). These results show that the mitochondrial compartmentalization of enzymes can provide a new strategy for the improvement of carotenoid production by S. cerevisiae.