Cell Strains Research Articles

Two-way strategy for enhanced pectinase production: Random mutagenesis and utilization of a halophytic biomass

Strain improvement through mutagenesis has remained an effective strategy for the cost-effective production of industrially important enzymes. Here, we focused on random mutagenesis of a pectinolytic yeast, Geotrichum candidum AA15, and on utilization of halophytic biomass along with apple waste as substrate for pectinase production to make the process cost effective. G. candidum AA15 was treated with a physical (UV) and a chemical (Ethidium bromide) mutagen separately and in a sequential way. After mutagenic treatments, a total of eight mutants (AHU0 to AHU5, AHC1 and AHUC) were obtained and assessed for pectinase production but a mutant strain (AHC1) yielded the highest pectinase production of 12.03 IU mL−1 which was >2 folds higher than the wildtype strain. The reason for positive effect of mutagenesis on pectinase production was investigated, however, the gene encoding polygalacturonase in the mutant strains was found similar to that of the wildtype strain. The purified pectinase from the mutant strains also shared molecular mass of 60–66 kDa with the wildtype. The mutant strain's cell density was higher than the wildtype and it can be linked with the higher productivity of pectinase reported in this study. The mutant strain, AHC1, yielded 8.32 IU mL−1 and 6.35 IU mL−1 of pectinase after the fermentation of apple peels and biomass from a halophytic plant, Cressa cretica, respectively. The utilization of the substrate was observed through Scanning Electron Microscopy. Thus, the study established a path to proceed for cost effective pectinase production through strain improvement and utilization of crude substrates.

A new hyperelastic lookup table for RT-DC.

Real-time deformability cytometry (RT-DC) is an established method that quantifies features like size, shape, and stiffness for whole cell populations on a single-cell level in real-time. A lookup table (LUT) disentangles the experimentally derived steady-state cell deformation and the projected area to extract the cell stiffness in the form of the Young's modulus. So far, two lookup tables exist but are limited to simple linear material models and cylindrical channel geometries. Here, we present two new lookup tables for RT-DC based on a neo-Hookean hyperelastic material numerically derived by simulations based on the finite element method in square and cylindrical channel geometries. At the same time, we quantify the influence of the shear-thinning behavior of the surrounding medium on the stationary deformation of cells in RT-DC and discuss the applicability and impact of the proposed LUTs regarding past and future RT-DC data analysis. Additionally, we provide insights about the cell strain and stresses, as well as the influence resulting from the rotational symmetric assumption on the cell deformation and volume estimation. The new lookup tables and the numerical cell shapes are made freely available.

Cold-adapted SARS-CoV-2 variants with different temperature sensitivity exhibit an attenuated phenotype and confer protective immunity.

As novel SARS-CoV-2 Variants of Concern emerge, the efficacy of existing vaccines against COVID-19 is declining. A possible solution to this problem lies in the development of a live attenuated vaccine potentially able of providing cross-protective activity against a wide range of SARS-CoV-2 antigenic variants. Cold-adapted (ca) SARS-CoV-2 variants, Dubrovka-ca-B4 (D-B4) and Dubrovka-ca-D2 (D-D2), were obtained after long-term passaging of the Dubrovka (D) strain in Vero cells at reduced temperatures. Virulence, immunogenicity, and protective activity of SARS-CoV-2 variants were evaluated in experiments on intranasal infection of Syrian golden hamsters (Mesocricetus auratus). In animal model infecting with ca variants, the absence of body weight loss, the significantly lower viral titer and viral RNA concentration in animal tissues, the less pronounced inflammatory lesions in animal lungs as compared with the D strain indicated the reduced virulence of the virus variant. Single intranasal immunization with D-B4 and D-D2 variants induced the production of neutralizing antibodies in hamsters and protected them from infection with the D strain and the development of severe pneumonia. It was shown that for ca SARS-CoV-2 variants, the temperature-sensitive (ts) phenotype was not obligate for virulence reduction. Indeed, the D-B4 variant, which did not possess the ts phenotype but had lost the ability to infect human lung cells Calu-3, exhibited reduced virulence in hamsters. Consequently, the potential phenotypic markers of attenuation of ca SARS-CoV-2 variants are the ca phenotype, the ts phenotype, and the change in species specificity of the virus. This study demonstrates the great potential of SARS-CoV-2 cold adaptation as a strategy to develop a live attenuated COVID-19 vaccine.

Effect and Mechanism of Dexmedetomidine on Inhibiting Proliferation, Migration and Invasion of Ovarian Cancer Cell Strain SKOV3

To investigate the effect and mechanism of dexmedetomidine on SKOV3 proliferation, migration and invasion in ovarian cancer cell strains and the effect on high mobility group protein A2 expression. Western blot was used to detect the protein expression of high mobility group protein A2 in SKOV3 ovarian cancer cells; control group (without any treatment), (dexmedetomidine group 100 ng/ml was treated for 24 h), si-control group (transfection with si-control), si-high mobility group protein A2 group (transfection with si-high mobility group protein A2), dexmedetomidine+plasmid cloning deoxyribonucleic acid group (after transfection with plasmid cloning deoxyribonucleic acid group followed by dexmedetomidine 100 ng/ml treated for 24 h), dexmedetomidine+plasmid cloning deoxyribonucleic acid group-high mobility group protein A2 group (after transfection with plasmid cloning deoxyribonucleic acid-high mobility group protein A2 followed by dexmedetomidine 100 ng/ml treated for 24 h), were all transfected into SKOV3 cells using Lipofectamine; the proliferation of cells was detected by methyl thiazolyl tetrazolium assay; cell migration and invasion were detected by transwell assay; high mobility group protein A2 expression was measured by quantitative reverse transcription-polymerase chain reaction. The proliferation, migration and invasion of SKOV3 cells treated with dexmedetomidine were significantly down-regulated and high mobility group protein A2 expression was significantly down-regulated compared with the control group (p<0.05); knockdown of high mobility group protein A2 significantly inhibited SKOV3 cell proliferation, migration and invasion and overexpression of high mobility group protein A2 reversed the inhibitory effects of dexmedetomidine on ovarian cancer cell proliferation, migration and invasion. Dexmedetomidine can inhibit the proliferation, migration and invasion of ovarian cancer cells SKOV3 and the mechanism may be related to the direct down-regulation of high mobility group protein A2, which will provide a theoretical basis for dexmedetomidine treatment of ovarian cancer.

ASCs-EVs Inhibit Apoptosis and Promote Myocardial Function in the Infarcted Heart via miR-221.

Extracellular vehicles (EVs) secreted from adipose-derived stem cells (ASCs) (ASCs-EVs) have the potential to treat myocardial infarction (MI), although the underlying mechanism remains unclear. The current study explored the ability of ASCs-EVs to inhibit apoptosis and promote myocardial function in the infarcted heart via microRNAs (miRNAs)-221. In hypoxia-induced H9C2 cells, a cardiac cell strain derived from the SD Rat left ventricle, we measured the cell viability and apoptosis-related protein expression after transfection with the ASCs-EVs-NC (negative control for EVs-miR-221) or ASCs-EVs-miR-221 mimics. We then verified the cardioprotective effects of miR-221-overexpressing ASCs-EVs by investigating myocardial cell apoptosis and cardiac function in a MI rat model treated with ASCs-EVs from miR-221-overexpressing ASCs by comparing control with ASC treatment. The in vitro experiment results showed that the proliferation of H9C2 cells and the anti-apoptotic protein expression were significantly enhanced by the ASCs-EVs-miR-221 mimic. The in vivo experiment results found that ASCs-EVs from miR-221-overexpressing ASCs have cardioprotective effects, as demonstrated by lower serum troponin levels and left ventricular end-systolic volume, and a lower number of apoptotic myocardial cells than those in control and ASC-treated rats. ASCs-EVs have therapeutic effects on MI by inhibiting cardiomyocyte apoptosis via miR-221.

The Cold-Adapted, Temperature-Sensitive SARS-CoV-2 Strain TS11 Is Attenuated in Syrian Hamsters and a Candidate Attenuated Vaccine.

Live attenuated vaccines (LAVs) replicate in the respiratory/oral mucosa, mimic natural infection, and can induce mucosal and systemic immune responses to the full repertoire of SARS-CoV-2 structural/nonstructural proteins. Generally, LAVs produce broader and more durable protection than current COVID-19 vaccines. We generated a temperature-sensitive (TS) SARS-CoV-2 mutant TS11 via cold-adaptation of the WA1 strain in Vero E6 cells. TS11 replicated at >4 Log10-higher titers at 32 °C than at 39 °C. TS11 has multiple mutations, including those in nsp3, a 12-amino acid-deletion spanning the furin cleavage site of the S protein and a 371-nucleotide-deletion spanning the ORF7b-ORF8 genes. We tested the pathogenicity and protective efficacy of TS11 against challenge with a heterologous virulent SARS-CoV-2 D614G strain 14B in Syrian hamsters. Hamsters were randomly assigned to mock immunization-challenge (Mock-C) and TS11 immunization-challenge (TS11-C) groups. Like the mock group, TS11-vaccinated hamsters did not show any clinical signs and continuously gained body weight. TS11 replicated well in the nasal cavity but poorly in the lungs and caused only mild lesions in the lungs. After challenge, hamsters in the Mock-C group lost weight. In contrast, the animals in the TS11-C group continued gaining weight. The virus titers in the nasal turbinates and lungs of the TS11-C group were significantly lower than those in the Mock-C group, confirming the protective effects of TS11 immunization of hamsters. Histopathological examination demonstrated that animals in the Mock-C group had severe pulmonary lesions and large amounts of viral antigens in the lungs post-challenge; however, the TS11-C group had minimal pathological changes and few viral antigen-positive cells. In summary, the TS11 mutant was attenuated and induced protection against disease after a heterologous SARS-CoV-2 challenge in Syrian hamsters.

Isolation and characterization of a SARS-CoV-2 variant with a Q677H mutation in the spike protein

We isolated 20 SARS-CoV-2 strains from positive clinical samples collected in Columbus, Ohio, and investigated the replication of one pair of isolates: a clade 20G strain and a variant of this strain carrying a Q677H mutation in the spike protein and six other amino acid mutations. The OSU.20G variant replicated to a higher peak infectious titer than the 20G base strain in Vero-E6 cells, but the titers were similar when both strains were grown in Calu-3 cells. These results suggest that the OSU.20G variant has increased replication fitness compared to the 20G base strain. This may have contributed to its emergence in December 2020-January 2021.

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

Iron–sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe–S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe–4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron–sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered “SufFeScient” derivative of BL21(DE3). Our results show that co-expression of a Fe–S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe–S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe–S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe–S cluster maturation and points out the pitfalls of chemical reconstitution.Graphical abstract

Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML.

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 109 and 2.61 × 109IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.

Biological properties of domestic strain vRub-Ant of rubella virus

Introduction. Rubella is a mild infectious disease affecting mainly children and is caused by the rubella virus, part of the Matonoviridae family, genus Rubivirus. Rubella causes congenital rubella syndrome (CRS) and is the main cause of developmental abnormalities, especially blindness and deafness.&#x0D; There is no specific treatment for rubella and CRS. In order to avoid possible complications from rubella infection, a live attenuated rubella vaccine based on the foreign strain of Wistar RA 27/3 rubella virus is used. However, the actual, more effective and preferred vaccine strain the rubella virus for the Russian Federation is considered to be a viral strain of rubella circulating on its territory.&#x0D; The aim of the study was to study the biological properties of the developed domestic cold-adapted strain vRub-Ant circulating in the territory of the Russian Federation.&#x0D; Materials and methods. Following cell cultures were used in the study human embryo lung diploid cell strain LECH-3, transferable cell line from embryonic kidney cells of green monkeys Vero CCL-81 and Vero ECC, human mesenchymal stem cells, human peripheral blood mononuclear cells (PBMC). Cell cultures were grown on a DMEM/F12 nutrient medium with the addition of 5% fetal bovine serum. Swabs from the pharynx and nasal passages from a child with rubella were used as clinical virus-containing material. Monoclonal anti-idiotypic antibodies m(anti-ID)Ab were used to assess the expression level of alpha/beta and gamma interferon receptors (/ and IFN-R)Ab, imitating the biological effects of alpha/beta and gamma interferons (/ and IFN) of humans. The cultural, virological, immunochemical and serological research methods were applied in the study.&#x0D; Results. Attenuation of the vRub-Ant clinical isolate of rubella virus was carried out for 20 consecutive passages on LECH-3 diploid cells at a reduced temperature of 30C. The main biological markers of attenuation were determined to be ts and ca phenotypes. The avirulence of the attenuated viral strain (att-phenotype) was assessed by the level of expression of / and IFN-R. A lower level of / and IFN-R expression was found on the membranes of human PBMC induced by the vaccine strain vRub-Ant in comparison with the parent wild variant of the rubella virus. This trait,the att phenotype, is characteristic of attenuated viral strains. It has been shown that the vaccine strain vRub-Ant has lost neurotropism and was unable to bind to the membrane receptors of the brain (MRB) of guinea pig embryos, unlike its parent rubella virus strain. The high immunogenicity of the domestic cold-adapted strain vRub-Ant was confirmed by high titers of neutralizing rubella antibodies observed in guinea pigs immunized subcutaneously with one vaccination dose of the virus.&#x0D; Conclusion. A domestic attenuated vaccine strain vRub-Ant of the rubella virus that has the main biological markers of attenuation (ts-ca and att phenotypes) has been developed. The vaccine strain vRub-Ant induces a high levels of neutralizing antibodies in guinea pigs following the immunization with a single vaccination dose of the vaccine. The viral strain vRub-Ant has lost its tropism to the MRB of guinea pig embryos, unlike its parent variant.

In vitro study of glyphosate effects on thyroid cells

Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro.Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1–0.25- 0.5-1.0-2.0–10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR.Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model.The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells.

Hortaea werneckii isolates exhibit different pathogenic potential in the invertebrate infection model Galleria mellonella.

Hortaea werneckii is a black yeast with a remarkable tolerance to salt. Most studies have been dedicated to understanding how H. werneckii adapts to hypersaline environments. H. werneckii has an unconventional cell cycle in which it alternates between fission and budding, which is modulated by cell density. Additionally, H. werneckii can cause superficial mycosis of the palm and sole of humans. Here, we determine the impact of salt concentration on the EXF-2000 strain's cell division pattern and morphology by performing timelapse microscopy at different salt concentrations. At low density and no salt, EXF-2000 primarily grows as pseudohyphae dividing mainly by septation. When grown in the presence of salt at a similar concentration to saltwater or hypersaline environments, we observe it grows first by undergoing fission followed by budding at the poles. Then, we examined a collection of 16 isolates in the presence of 0.6M NaCl, including isolates from marine and hypersaline environments and isolates from patients. These isolates exhibit a wide diversity in colony shape and cellular morphology. The isolates grew as yeast, pseudohyphae, and true hyphae, indicating that isolates can exhibit various cell morphologies under similar environmental conditions. We used the insect larvae Galleria mellonella to determine the pathogenic potential of our isolates. We observe that only a subset of isolates can cause death in our model, and there was no correlation between H. werneckii morphology and capacity to cause disease. Taken together, H. werneckii genomic and phenotypic diversity can serve as a model to better understand how phenotypes and pathogenic potential evolve in environmental fungi.

Multifunctionality of AlBaFe12O19 /CoZnFe2O4 hybrid nanocomposite: Promising structural, elastic, morphological, compositional, optical, and magnetic properties

A detailed report on the influence of Aluminum doped Barium Hexaferrite (ABH) on structure, elasticity, morphology, composition, and optical and magnetic behavior of hybrid ABH-CZF (AlBaFe12O19)1-x/(CoZnFe2O4) x with X = 0.1, 0.2, 0.3, 0.4 nanocomposites is presented. A new study on the role of ABH in the composite made by the physical mixing method. The composites were characterized by XRD, SEM, EDAX, FTIR, UV, PL, and VSM analysis. XRD confirms the formation of hexagonal and spinel structures in the composite along with their cell volume, lattice parameters, stress, strain, and other structural parameters. Elastic parameters and Debye temperature were measured using FTIR. The composite AC 60–40 is found best suited for high-density optical storage devices and as lead-free radiation shielding materials. From the UV analysis, Both the direct and indirect band gap energies are found to be increasing with increasing ABH concentration. The overall structural and optical properties also prove that the material can be used in tunable photonic applications. The refractive index of the composite is between 3.0 and 3.2, which can be used for photo-electrochemical cells, optical detectors, or reflectors. Magnetic properties examined using VSM showed a decrease in Ms, Mr, and Hc values along with ABH concentration which may be suitable for data storage applications such as rewritable storage hard drives. The maximum values for Ms (7.85 emu/g), Mr (20.44 emu/g), and Hc (2.01 kOe) are found for AC90-10 nanocomposite material. By plotting loop width (ΔH) versus magnetization (M), the magnetic interaction between the composite is studied.

Obtaining Callus Culture of Sage Medicinal (&lt;i&gt;Salvia officinalis&lt;/i&gt; L.) and its Characteristics

Introduction. Cultivation of biomass of plant cells as a method of obtaining raw materials has existed for quite a long time. Plant cells cultivated in vitro act as a source of valuable secondary metabolites such as phenols, alkaloids, phytosteroids, glycosides, etc. It is important to create conditions under which the accumulation of valuable biologically active substances will be observed in the strains. Cultivation involves the use of complex multicomponent nutrient media containing a certain set of macro-, microelements, vitamins, growth stimulants. Salvia officinalis has a wide spectrum of pharmacological action. Due to the limited growing area of medicinal sage, as well as the deterioration of the ecological situation in the growing regions, the use of a phytobiotechnological method for obtaining raw materials is relevant.Aim. The aim of the study is to obtain a viable callus culture of salvia officinalis (Salvia officinalis L.).Materials and methods. Leaves of an intact plant sage medicinal, of the Lamiaceae family (Salvia officinalis, Lamiaceae) were used as explants. The explants were pre-sterilized with 6 % sodium hypochlorite solution for 20 minutes and 70 % ethanol for 1 minute. It was cultivated on a nutrient medium according to the Murasig – Skoog recipe. Determination of cell viability using vital dyes was assessed using microscopy (digital microscope Bresser LCD 50x-2000x, Germany). High performance thin layer chromatography was performed using a HPTLC PRO SYSTEM (CAMAG AG, Switzerland).Results and discussion. After two weeks of cultivation, the formation of primary callus was observed on the surface of the explants. Visually, it was a thin layer of intensely dividing undifferentiated light yellow cells. During cultivation, the biomass of the resulting callus increased, it became looser and acquired a darker shade, and the nutrient medium also began to darken. The detected cells during microscopy can be divided into two types: the first type is cells of the meristematic type, the second type is cells of the parenchymal type. Microscopy showed that more than 95 % of all visualized cells are alive. In the following passages, no significant changes in the morphotype of the culture were noted. In the eleventh passage, a study of the growth activity of the strain was carried out. The maximum specific growth rate of 0.42 day-1 is observed on the 14–18th day of growth, while the biomass doubling time is the smallest and corresponds to the value of 1.66 days. During one cultivation cycle, the amount of biomass increases by 7.73 times. The results of a qualitative analysis by the method of high-performance thin-layer chromatography (HTPLC) show that the qualitative composition of the biomass of medicinal sage is generally close to that of intact plants.Conclusion. A viable stable strain of plant cells of salvia officinalis was obtained on a nutrient medium according to the Murasig – Skoog recipe with a half content of micro- and macrosols and phytohormones 2,4-D (6 mg/ml) and kinetin (1 mg/ml). For the following passages, it is recommended to use a nutrient medium according to the Murasig – Skoog recipe with a full content of micro and macrosols and phytohormones naphthylacetic acid (1 mg/ml) and kinetin (1 mg/ml). The bulk of the obtained heterogeneous callus is made up of cells of the meristematic and parenchymal type. The qualitative composition of BAS biomass of medicinal sage is generally close to that of intact plants.

Anti-influenza properties of tiliroside isolated from Hibiscus mutabilis L.

Ethnopharmacological relevanceFu Rong Ye (FRY), the leaf of Hibiscus mutabilis L., is a Chinese medicinal herb used to treat coughs and respiratory diseases. FRY is the major herbal component of the patent medicine Fupo Ganmao Granules for treating common cold. However, its anti-influenza active components and mechanism were not identified. AimHere, we aim to a) isolate the anti-influenza phytochemicals from FRY extract and b) explore its anti-flu mechanism. Material and methodsBioassay guided isolation was performed to get anti-influenza virus components. Influenza virus infected cells and mouse model were employed for efficacy evaluation. ResultsUsing bioassay-guided isolation, the flavonoid tiliroside was obtained, which inhibited four IAV strains in MDCK cells with EC50 ranging from 3.87 to 27.61 μM by suppressing the viral ribonucleoprotein activity. Tiliroside also significantly downregulated the expression of cytokines/chemokines in A549 cells, and protected 50% of PR8-infected BALB/c mice from death and at 800 mg/kg/day, improved lung edema conditions. ConclusionTiliroside is effective for influenza virus infection treatment and promising for further drug development. This study is the first to demonstrate that tiliroside in FRY acts against influenza virus.

Retracted: Reversal of Radiotherapy Resistance of Ovarian Cancer Cell Strain CAOV3/R by Targeting lncRNA CRNDE.

[This retracts the article DOI: 10.1155/2021/8556965.].

The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression.

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.

Feedforward growth rate control mitigates gene activation burden

Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that actuates growth rate upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme (SpoTH) with sole hydrolysis activity, which lowers ppGpp level and thus increases growth rate. An inducible RelA+ expression cassette further allows to precisely set the basal level of ppGpp, and thus nominal growth rate, in any bacterial strain. Without the controller, activation of the GOI decreased growth rate by more than 50%. With the controller, we could activate the GOI to the same level without growth rate defect. A cell strain armed with the controller in co-culture enabled persistent population-level activation of a GOI, which could not be achieved by a strain devoid of the controller. The feedforward controller is a tunable, modular, and portable tool that allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications.

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Mycobacterium tuberculosis sRNA MTS2823 regulates the growth of the multidrug-resistant strain in macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a serious contagious disease. MTB-encoded small regulatory RNA (sRNA) MTS2823 was reported to be upregulated in the plasma of TB patients. Nevertheless, whether MTS2823 is implicated in MTB drug resistance is unclear. Human macrophage cell line THP-1 was infected with the drug-susceptible strain H37Rv or the multidrug-resistant (MDR) strain 8462. Colony-forming unit assay was implemented for evaluating intracellular growth of the MTB strains. Enzyme-linked immunosorbent assay was used for measurement of inflammatory cytokines. Real-time quantitative polymerase chain reaction was utilized to assess MTS2823 and recombinase A (recA) expression in strains 8462 and H37Rv. Nitric oxide (NO) production in the MDR strain-infected THP-1 cells was measured. In this study, MTS2823 was found to display a low level in the MDR strain. Overexpressing MTS2823 promoted intracellular growth of the MDR strain and inhibited inflammatory cytokine and NO production in infected THP-1 cells. RecA might be a target of MTS2823 in the MDR strain. Overall, MTB-encoded sRNA MTS2823 displays a low level and regulates the growth of the MDR strain in THP-1 cells by modulating recA.