Low Cell Density Research Articles

Functional and structural changes in the neuroretina are accompanied by mitochondrial dysfunction in a type 2 diabetic mouse model

BackgroundDiabetic retinopathy (DR), one of the leading causes of blindness and vision impairment, is suggested to exhibit functional and structural changes in retinal neurons as the earliest manifestation, which could be used to predict the progression of related angiopathy. While neural function and survival rely on proper mitochondrial function, and a growing body of literature has supported the role of mitochondrial dysfunction in the development of DR, how diabetes affects mitochondrial function in retinal tissue remains elusive. This study primarily aimed to investigate mitochondrial functional changes in a diabetic rodent model. We also characterized the early DR phenotype, in particular, neurodegeneration.MethodsC57BLKsJ-db/db (db/db) mice (a type 2 diabetic mouse model) were used with their normoglycemic heterozygous littermates (db/+) serving as controls. Longitudinal changes in retinal function and morphology were assessed with electroretinography (ERG) and optical coherence tomography (OCT), respectively, at 9, 13, 17, and 25 weeks of age. At 25 weeks, the retinas were harvested for immunohistochemistry and ex vivo mitochondrial bioenergetics.ResultsDecreased ERG responses were observed in db/db mice as early as 13 weeks of age. OCT revealed that db/db mice had significantly thinner retinas than the controls. Immunohistochemistry showed that the retinas of the db/db mice at 25 weeks were thinner at the outer and inner nuclear layers, with lower photoreceptor and cone cell densities compared with the db/+ mice. The number of rod-bipolar cell dendritic boutons and axon terminals was significantly reduced in db/db mice relative to the db/+ mice, suggesting that diabetes may lead to compromised synaptic connectivity. More importantly, the retinas of db/db mice had weaker mitochondrial functions than the controls.ConclusionsOur longitudinal data suggest that diabetes-induced functional deterioration and morphological changes were accompanied by reduced mitochondrial function in the retina of db/db mice. These findings suggest that mitochondrial dysfunction may be a contributing factor triggering the development of DR. While the underlying mechanistic cause remains elusive, the db/db mice could be a useful animal model for testing potential treatment regimens targeting neurodegeneration in DR.

Physiology of the widespread pulsating soft coral Xenia umbellata is affected by food sources, but not by water flow.

Coral energy and nutrient acquisition strategies are complex and sensitive to environmental conditions such as water flow. While high water flow can enhance feeding in hard corals, knowledge about the effects of water flow on the feeding of soft corals, particularly those pulsating, is still limited. In this study, we thus investigated the effects of feeding and water flow on the physiology of the pulsating soft coral Xenia umbellata. We crossed three feeding treatments: (i) no feeding, (ii) particulate organic matter (POM) in the form of phytoplanktonand (iii) dissolved organic carbon (DOC) in the form of glucose, with four water volume exchange rates (200, 350, 500 and 650 L h-1) over 15 days. Various ecophysiological parameters were assessed including pulsation rate, growth rate, isotopic and elemental ratios of carbon (C) and nitrogen (N) as well as photo-physiological parameters of the Symbiodiniaceae (cell density, chlorophyll-a and mitotic index). Water flow had no significant effect but feeding had a substantial impact on the physiology of the X. umbellata holobiont. In the absence of food, corals exhibited significantly lower pulsation rates, lower Symbiodiniaceae cell density and lower mitotic indices compared to the fed treatments, yet significantly higher chlorophyll-a per cell and total N content. Differences were also observed between the two feeding treatments, with significantly higher pulsation rates and lower chlorophyll-a per cell in the DOC treatment, but higher C and N content in the POM treatment. Our findings suggest that the X. umbellata holobiont can be viable under different trophic strategies, though favouring mixotrophy. Additionally, the physiology of the X. umbellata may be regulated through its own pulsating behaviour without any positive or negative effects from different water flow. Therefore, this study contributes to our understanding of soft coral ecology, particularly regarding the competitive success and widespread distribution of X. umbellata.

A modified isooctane-based DNA extraction method from crude oil.

Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 101-103 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.

μDACS platform: A hybrid microfluidic platform using magnetic levitation technique and integrating magnetic, gravitational, and drag forces for density-based rare cancer cell sorting

Circulating tumor cells (CTCs) are crucial indicators of cancer metastasis. However, their rarity in the bloodstream and the heterogeneity of their surface biomarkers present challenges for their isolation. Here, we developed a hybrid microfluidic platform (microfluidic-based density-associated cell sorting (µDACS) platform) that utilizes density as a biophysical marker to sort cancer cells from the population of white blood cells (WBCs). The platform utilizes the magnetic levitation technique on a microfluidic chip to sort cells based on their specific density ranges, operating under a continuous flow condition. By harnessing magnetic, gravitational, and drag forces, the platform efficiently separates cells. This approach involves a microfluidic chip equipped with a microseparator, which directs cells into top and bottom outlets depending on their levitation heights, which are inversely proportional to their densities. Hence, low-density cancer cells are collected from the top outlet, while high-density WBCs are collected from the bottom outlet. We optimized the sorting efficiency by varying the flow rates, and concentrations of the sorting medium's paramagnetic properties using standard densities of polymeric microspheres. To demonstrate the platform's applicability, we performed hybrid microfluidic sorting on MDA-MB-231 human breast cancer cells and U-937 human monocytes. The results showed efficient sorting of rare cancer cells (≥100 cells/mL) from serum samples, achieving a sorting efficiency of ∼70% at a fast-processing speed of 1 mL h−1. This label-free approach holds promise for rapid and cost-effective CTC sorting, facilitating in-vitro diagnosis and prognosis of cancer.

Dynamic regulation using a quorum-sensing circuit enhances the production of tyrosol by Escherichia coli

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.

Digital pathology in pediatric nodular lymphocyte-predominant Hodgkin lymphoma: correlation with treatment response

Early-stage pediatric nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) can be treated effectively with low-intensity chemotherapy, most frequently cyclophosphamide in combination with vinblastine and prednisone (CVP). Descriptive histological risk factors based on the disease-defining lymphocyte predominant cells (LP cells) as used within the Fan classification, are less predictive in early-stage patients. We used deep learning-based cell detection and spatial analysis on digitized biopsy slides from 53 early-stage pediatric NLPHL patients to quantitatively assess LP cell histomorphometry. We found that poor responding patients had significantly fewer LP cells per cluster and lower LP cell density than good responding patients. In our exploratory analysis, we found no correlation between Fan classes or B cell pattern variables and therapy response. We hypothesize that the relationship between poor treatment response and decreased LP cell density may be explained by differences in LP cell proliferation.

Retraction Note: SNHG6 modulates oxidized low-density lipoprotein-induced endothelial cells injury through miR-135a-5p/ROCK in atherosclerosis

Retraction Note: SNHG6 modulates oxidized low-density lipoprotein-induced endothelial cells injury through miR-135a-5p/ROCK in atherosclerosis

Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction

ObjectiveStem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction.MethodsRPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining.ResultsThe rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF.ConclusionRPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.

Inkjet-printed morphogenesis of tumor-stroma interface using bi-cellular bioinks of collagen-poly(N-isopropyl acrylamide-co-methyl methacrylate) mixture

Recent advances in biomaterials and 3D printing/culture methods enable various tissue-engineered tumor models. However, it is still challenging to achieve native tumor-like characteristics due to lower cell density than native tissues and prolonged culture duration for maturation. Here, we report a new method to create tumoroids with a mechanically active tumor-stroma interface at extremely high cell density. This method, named “inkjet-printed morphogenesis” (iPM) of the tumor-stroma interface, is based on a hypothesis that cellular contractile force can significantly remodel the cell-laden polymer matrix to form densely-packed tissue-like constructs. Thus, differential cell-derived compaction of tumor cells and cancer-associated fibroblasts (CAFs) can be used to build a mechanically active tumor-stroma interface. In this methods, two kinds of bioinks are prepared, in which tumor cells and CAFs are suspended respectively in the mixture of collagen and poly (N-isopropyl acrylamide-co-methyl methacrylate) solution. These two cellular inks are inkjet-printed in multi-line or multi-layer patterns. As a result of cell-derived compaction, the resulting structure forms tumoroids with mechanically active tumor-stroma interface at extremely high cell density. We further test our working hypothesis that the morphogenesis can be controlled by manipulating the force balance between cellular contractile force and matrix stiffness. Furthermore, this new concept of “morphogenetic printing” is demonstrated to create more complex structures beyond current 3D bioprinting techniques.

Clinical observation of phacoemulsification under the low perfusion pattern and low negative pressure in patients with low corneal endothelial cell density

BackgroundTo observe the safety and effect of phacoemulsification combined with intraocular lens (IOL) implantation in patients with low corneal endothelial cell density (CD) under the low perfusion pattern with low negative pressure.MethodsIn this retrospective case series study, a total of 16 patients (17 eyes) were studied. They had all been diagnosed with low corneal endothelial (CD) and cataracts in the First Affiliated Hospital of Fujian Medical University from December 2019 to October 2021. They underwent phacoemulsification combined with IOL implantation under the low perfusion pattern with low negative pressure. The variations of corneal endothelial( CD), coefficient of variation (CV) of the cell area, central corneal thickness (CCT), visual acuity, and intraocular pressure before and after the operation were observed and assessed. Then a paired t-test, repeated measures analysis of variance, and Pearson correlation analysis were adopted for data analysis.ResultsThe mean intraocular pressure of the 17 eyes was 16.88 ± 6.47 mmHg before the operation and 14.41 ± 3.10 mmHg after the operation, showing a statistically significant difference of t = 2.222, and p = 0.041. Before the operation, the mean visual acuity was 0.16 ± 0.09, and after the operation, it was 0.45 ± 0.16, displaying a statistically significant difference of t = -9.917, p < 0.001. Before and after the operation, four of the 17 eyes had no detectable CD. The mean CD of the other 13 eyes at one month after the operation (644.308 ± 106.24 cells/mm2) was lower than that before the operation (709.62 ± 119.19 cells/mm2), and the differences were statistically significant (F = 20.044, p < 0.001). However, no statistically significant differences were found in the mean CV before the operation (31.23 ± 4.21), and at one month after the operation (32.62 ± 3.80; F = 2.130, p = 0.157). Moreover, the mean CCT of 14 eyes at one month after the operation (562.72 ± 27.82 μm) was larger than that before the operation (534.79 ± 24.69 μm).ConclusionsThe low perfusion pattern with low negative pressure is safe and effective for corneal endothelial dysfunction patients complicated with cataracts.

Natural silencing of quorum-sensing activity protects Vibrio parahaemolyticus from lysis by an autoinducer-detecting phage.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.

LIM Homeobox 2 Increases Adhesion-Regulating Molecule 1 Transcription to Facilitate the Pathological Progression of Oxidized Low-Density Lipoprotein-Stimulated Atherosclerotic Cell Models.

Endothelial-mesenchymal transition (EndMT) and endothelial cell apoptosis have been documented to have a role in atherosclerosis (AS) progression. To deepen knowledge in this aspect, our study investigated the effect of LIM homeobox 2 (LHX2) and adhesion-regulating molecule 1 (ADRM1) on EndMT and endothelial cell apoptosis in the oxidized low-density lipoprotein (ox-LDL) -stimulated AS cell model.Ox-LDL was utilized to treat human umbilical vein endothelial cells (HUVECs) for constructing an AS model in vitro, followed by measurement of LHX2 and ADRM1 expressions. Afterward, gain- and loss-of-function assays were performed in HUVECs, followed by detection of cell viability, invasion, migration, and apoptosis and the expression of inflammatory factors [tumor necrosis factor (TNF) -α, interleukin (IL) -1β, and IL-6], EndMT-related proteins [CD31, vascular epithelium (VE) -cadherin, vimentin, α-smooth muscle actin (SMA), Snai1, Snai2, and Twist1], and the apoptotic protein cleaved caspase-3. Interactions between LHX2 and ADRM1 were analyzed with dual-luciferase reporter gene and chromatin immunoprecipitation assays.High levels of LHX2 and ADRM1 were observed in ox-LDL-induced HUVECs. In ox-LDL-treated HUVECs, LHX2, or ADRM1 knockdown promoted CD31 and VE-cadherin levels, viability, invasion, and migration and reduced apoptosis and the expressions of TNF-α, IL-1β, IL-6, vimentin, α-SMA, Snai1, Snai2, Twist1, and cleaved caspase-3. Mechanistically, LHX2 bound to the ADRM1 promoter to promote ADRM1 transcription. Overexpression of ADRM1 annulled the aforementioned effects of LHX2 knockdown on ox-LDL-induced HUVECs.LHX2 facilitates the pathological progression of ox-LDL-stimulated AS cell models by increasing ADRM1 transcription.

Tumor Control Probability and Small-Scale Monte Carlo Dosimetry: Effects of Heterogenous Intratumoral Activity Distribution in Radiopharmaceutical Therapy.

In radiopharmaceutical therapy, intratumoral uptake of radioactivity usually leads to heterogeneous absorbed dose distribution. The likelihood of treatment success can be estimated with the tumor control probability (TCP), which requires accurate dosimetry, estimating the absorbed dose rate per unit activity to individual tumor cells. Methods: Xenograft cryosections of the prostate cancer cell line LNCaP treated with [177Lu]Lu-PSMA-617 were evaluated with digital autoradiography and stained with hematoxylin and eosin. The digital autoradiography images were used to define the source in a Monte Carlo simulation of the absorbed dose, and the stained sections were used to detect the position of cell nuclei to relate the intratumoral absorbed dose heterogeneity to the cell density. Simulations were performed for 225Ac, 177Lu, and 90Y. TCP was calculated to estimate the mean necessary injected activity for a high TCP. A hypothetical case of activity mainly taken up on the tumor borders was generated and used to simulate the absorbed dose. Results: The absorbed dose per decay to tumor cells was calculated from the staining and simulation results to avoid underestimating the tumor response from low absorbed doses in tumor regions with low cell density. The mean of necessary injected activity to reach a 90% TCP for 225Ac, 177Lu, and 90Y was found to be 18.3 kBq (range, 18-22 kBq), 24.3 MBq (range, 20-29 MBq), and 5.6 MBq (range, 5-6 MBq), respectively. Conclusion: To account for the heterogeneous absorbed dose generated from nonuniform intratumoral activity uptake, dosimetry models can estimate the mean necessary activity to reach a sufficient TCP for treatment response. This approach is necessary to accurately evaluate the efficacy of suggested radiopharmaceuticals for therapy.

Immunological and Histological Studies of Different Concentrations of Rosmarinus officinalis and Thymus vulgaris Extracts on Thymus Gland of Chick Embryos.

Humanity has an ancient history of consuming medicinal plants for prophylaxis. Within hours, and even months, embryonic cells undergo several processes to form an organism. This study aimed to prove the positive or negative effects of using rosemary and thyme extract on the thymus gland and level of IL-10, IgM, and IgG in serum of chick embryos. The immunological effect was measured by histological and immunological studies. A total of 160 fertilized eggs were randomly distributed into 8 groups; on the 0 and 8th day of incubation, all treated groups received a dose of 0.1 mL/egg. On the 14th and 20th days of incubation, the embryos were sacrificed and the samples were collected (serum and thymus gland). The data were analyzed using ANOVA. Simple damage in thymic tissue with a low cell density in the embryos was treated with high concentrations of rosemary and thyme extracts, as well as in the mixed group. A significant decrease in IgM levels in the group treated by a high concentration of thyme. A decrease in IgG levels was found in the group treated with a high concentration of rosemary and in the mixed group, while the group treated with a high concentration of thyme and the mixed group showed decreases on the 14th day. A significant decrease in IL-10 levels was found on the 14th day, followed by an increase on the 20th day. Despite the benefits of rosemary and thyme, inflammation signs appeared on embryos treated with these herbs.

Knockdown of circ_0113656 assuages oxidized low-density lipoprotein-induced vascular smooth muscle cell injury through the miR-188-3p/IGF2 pathway.

Circular RNA (circRNA) is involved in the pathogenesis of atherosclerosis (AS). The present work analyzed the RNA expression of circ_0113656, microRNA-188-3p (miR-188-3p), and insulin-like growth factor 2 (IGF2) by quantitative real-time polymerase chain reaction. The protein expression of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), and IGF2 was detected by Western blotting. Cell viability, proliferation, invasion, and migration were analyzed using the cell counting kit-8, 5-ethynyl-2'-deoxyuridine, transwell invasion, and wound-healing assays, respectively. The interactions among circ_0113656, miR-188-3p, and IGF2 were identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The results showed that circ_0113656 and IGF2 expression were significantly upregulated, while miR-188-3p was downregulated in the blood of AS patients and oxidized low-density lipoprotein (ox-LDL)-treated HVSMCs in comparison with controls. The ox-LDL treatment induced HVSMC proliferation, migration, and invasion accompanied by increases in PCNA and MMP2 expression; however, these effects were attenuated after circ_0113656 knockdown. Circ_0113656 acted as a miR-188-3p sponge and it regulated ox-LDL-induced HVSMC disorders by binding to miR-188-3p. Besides, the regulation of miR-188-3p in ox-LDL-induced HVSMC injury involved IGF2. Further, the depletion of circ_0113656 inhibited IGF2 expression by interacting with miR-188-3p. Thus, the circ_0113656/miR-188-3p/IGF2 axis may mediate ox-LDL-induced HVSMC disorders in AS, providing a new therapeutic strategy for AS.

Production of adeno-associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities.

In recent years, the use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells/mL) resulting in titers above 1010 VG/mL. No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells/mL) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing. This article is protected by copyright. All rights reserved.

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia.

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O2, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O2 permeability. High flow rates of 520μLmin-1 prevented biofilm sloughing in 1m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O2 concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8gm-2 and, thus, a successful CBR scale-up by a factor of 25. Practical application: Cyanobacteria use light energy to upgrade CO2, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O2 accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.

Circ_0033596 depletion ameliorates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell damage.

Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2. The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637. Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.

Lipid kinase PIP5Kα contributes to Hippo pathway activation via interaction with Merlin and by mediating plasma membrane targeting of LATS1

BackgroundThe Hippo pathway plays a critical role in controlled cell proliferation. The tumor suppressor Merlin and large tumor suppressor kinase 1 (LATS1) mediate activation of Hippo pathway, consequently inhibiting the primary effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid present in the plasma membrane (PM), binds to and activates Merlin. Phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) is an enzyme responsible for PIP2 production. However, the functional role of PIP5Kα in regulation of Merlin and LATS1 under Hippo signaling conditions remains unclear.MethodsPIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed.ResultsWe found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased.ConclusionsCollectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity.11kBwjrDVBy7xy7exwWkZdVideo

Diverse diterpenoids from Callicarpa rubella Lindl. as natural inhibitors of macrophage foam cell formation

Ten undescribed diterpenoids namely rubellawus E-N of structural types pimarane (1, 3-4), nor-abietane (2), nor-pimarane (5–6), isopimarane (7–9), and nor-isopimarane (10), along with eleven known compounds, were isolated and identified from the aerial parts of Callicarpa rubella Lindl. The structures of the isolated compounds were confirmed by comprehensive spectroscopic analyses and quantum chemical computations. Pharmacologically, almost all the compounds exhibited a potential inhibitory effect on oxidized low-density lipoprotein-induced macrophage foam cell formation, which suggests that these compounds may be promising candidates in the treatment of atherosclerosis.