Time-series analyses of directional sequence changes in SARS-CoV-2 genomes and an efficient search method for candidates for advantageous mutations for growth in human cells

SummaryTo establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

Host range analysis of a chimeric simian virus 40 genome containing the BKV capsid genes

Mutations in the gene encoding the adenovirus (Ad) early region 1B 19-kDa protein (the 19K gene) result in multiple phenotypic effects upon infection of permissive human cells. It has been reported, for example, that Ad type 2 (Ad2) and Ad5 with mutations in the 19K gene (19K-defective mutants) have a marked growth advantage compared with wild-type virus in human diploid WI38 cells (E. White, B. Faha, and B. Stillman, Mol. Cell. Biol. 6:3763-3773, 1986), and it was proposed that this host range phenotype stems from the large increase in viral early gene expression reported to occur in the mutant-infected cells. These observations gave rise to the hypothesis that the 19-kDa protein (the 19K protein) normally functions as a negative regulator of Ad early gene expression and growth. We have tested this hypothesis and find that Ad5 and Ad12 wild-type viruses grow as efficiently as their respective 19K-defective mutants, in1 and dl337 and pm700 and in700, in WI38 and other human cell types. Neither the accumulation of E1A cytoplasmic mRNAs nor the synthesis of E1A and other viral early proteins in these cells is altered as a result of these mutations in the 19K gene, and we conclude that the 19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cells.

The attachment and growth of human endothelial cells and fibroblasts was studied on polymer surfaces fabricated by the polymerization of volatile amine and amide compounds in a low pressure gas plasma, and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen content of the surface layers. Infrared spectra showed the presence of amide groups, including those cases where the volatile compound ('monomer') did not contain oxygen. The performance of the surfaces in cell attachment correlated with the surface hydrophilicity and the nitrogen content, although for the latter a fair degree of scatter indicated that a more complex relationship applies. All these surfaces supported the attachment and growth of human cells. Generally, amide plasma polymers were best but the individual monomer and the plasma parameters also played a role. From comparisons of the various surfaces, it is suggested that the amide group is the main promoter of cell attachment in nitrogen-containing plasma surfaces.

The purpose of this study was to determine whether the growth of human melanoma cells in the brain parenchyma is selective and represents the growth of unique cells. Six human melanoma cell lines derived from cutaneous lymph node or brain metastases (from six different patients) and melanoma cells isolated from fresh surgical specimens of two primary cutaneous melanomas, two lymph node metastases and two brain metastases (each from a different patient) were injected into the subarachnoid space of nude mice. All melanomas produced growths in the leptomeninges, but only melanoma cells isolated from brain metastases infiltrated into and grew in the brain parenchyma of nude mice. The results from in vitro assays for cell motility or production of gelatinase activity did not correlate with in vivo growth pattern. However, the in vitro growth of human melanoma cells in the presence of TGF-beta 2 inversely correlated with potential for brain parenchyma metastasis, i.e. the growth of cells from brain metastases was least inhibited by TGF-beta 2. These data suggest that melanoma brain parenchyma metastases are produced by unique cells that may be resistant to the antiproliferative effects of TGF-beta 2.

WtSV40 and its variant EL-SV40 (contains two complementing defective genomes) fail to productively infect human embryonic kidney cells or human fibroblasts. However, early SV40 (E-SV40) genomes can propagate in human cells when complemented by a particular late RF virus (L-RFV) genome or the closely related wtBKV genome. The L-RFV genome (L-RFV clone H) contains a deleted early region, a complete set of BKV capsid genes, and a single SV40 regulatory region (acquired by recombination). In contrast, it was not possible to make the reciprocal genome cross in human cells; late SV40 genomes containing a deleted early region do not complement early RFV or early BKV DNAs. The L-RFV clone H genome was also shown to complement wtSV40 in human cells. However, wtSV40 DNA was rapidly lost and replaced by a defective SV40 genome. The SV40 defective (E-SV40 alpha) contained a deletion of the late region, an intact early region, and paired with L-RFV clone H DNA to form a new hybrid virus. In human cells wtSV40 was also complemented by wtBKV DNA, but after two serial passages SV40 DNA disappeared. These findings indicate that SV40 late or capsid gene sequences, but not SV40 early sequences, generate a block to SV40 growth in human cells. When the SV40 late region is replaced by a RFV or a BKV late region, E-SV40 DNA propagates efficiently in human cells and in some cases more rapidly than wtBKV. Northern blot hybridization indicates that SV40 DNA is poorly transcribed in human cells when the SV40 late region is present.

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/β and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.

Objective To investigate the effect and mechanism of sodium valproate on inhibiting the growth of human cholangiocarcinoma cells. Methods Human cholangiocarcinoma TFK-1 cells were respectively treated with 0.5, 1.0, 2.0, 4.0 and 8.0 mmol/L of sodium valproate for 1, 3 and 5 d. And the control and blank groups were also established. The inhibitory effect of sodium valproate on the growth of human cholangiocarcinoma cells was detected by cell counting kit (CCK)-8 assay. The effect of sodium valproate on the cell apoptosis rate and cell cycle was detected by flow cytometry. The nude mice subcutaneous implantation tumor models of TFK-1 cells was established and the experimental and control groups were assigned to observe the effect of sodium valproate on the growth of tumor. The experiment data in two groups were compared using t test, and the multi-group comparison was conducted using one way analysis of variance and LSD-t test. Results The inhibitory effect of sodium valproate on the growth of human cholangiocarcinoma TFK-1 cells was observed in a dose- and time-dependent manner. As the concentration of sodium valproate increased, the cell apoptosis rate and the percentage of cells in the G2/M phase significantly increased. Compared with (8.6±2.3)% in the control group, the cell apoptosis rate (57.5±6.2)% in the 8.0 mmol/L experimental group significantly increased at 3 d. Compared with (12.0±2.6)% in the control group, the percentage of cells in the G2/M phase (42.5±5.4)% in the 8.0 mmol/L experimental group significantly increased (LSD-t=17.557, 12.465; P<0.05). At 31 d after the first treatment, the tumor volume (338±11) mm3 in the 8.0 mmol/L experimental group significantly decresed, compared with (426±14) mm3 in the control group (t=-15.630, P<0.05). Conclusions Sodium valproate can significantly inhibit the growth of human cholangiocarcinoma cells, and its mechanisms include cell cycle arrest and cell apoptosis. Key words: Sodium valproate; Bile duct neoplasms; Cell cycle; Apoptosis; Neoplasm seeding; Mice

The muscadine grape (Vitis rotundifolia) is native to the Southeastern United States and muscadine grape seed products are marketed as dietary supplements, based upon their antioxidant properties. Extracts from muscadine grapes contain a number of antioxidants, including resveratrol and ellagic acid; however, the natural antioxidants found in grape seed extracts are predominantly procyanidins, while the grape skin extracts contain more anthocyanins. We investigated the effect of muscadine grape seed extracts (MSE) and muscadine grape skin extracts (MSKE) on the growth of human lung, colon, prostrate, skin, brain and breast cancers as well as human leukemias. Cells were incubated with increasing concentrations (from 0.5 to 50 μg/mL) of aqueous extracts of either MSE or MSKE and the total number of cells was quantified after 7 days. Both the MSE and MSKE inhibited the growth of A549 and SK-LU-1 human lung adenocarcinoma cancer cells (81.8% inhibition at the highest concentration) and HT29 and HCT116 human colon cancer cells (80.5% inhibition at the highest dose). Similarly, extracts from both muscadine grape seeds and skins inhibited the growth of LNCaP and PC3 human prostate cancer cells. The growth of U87 and U373 human glioblastoma cells and RPMI 7951 and SKMEL28 human skin cancer cells was dose-dependently reduced by treatment with the MSE or MSKE. Human leukemia cells were also incubated with increasing concentrations of the extracts; the growth of THP-1 human acute monocytic leukemia cells, HL-60 human acute promyelocytic cells, and K562 human chronic myelogenous leukemia cells was reduced by both muscadine grape extracts (average of 74.2% inhibition at the highest dose). Both the MSE and MSKE inhibited the growth of estrogen receptor-dependent ZR-75-1, HER2 over-expressing SKBR3, and triple negative MDA-MB-231 human breast cancer cells. The inhibition of growth by either extract was highest in the triple negative breast cancer cells (92.6% inhibition at the highest concentration). The reduction in human breast cancer cell growth was accompanied by a significant decrease in the phosphorylation and activation of the mitogen-activated protein kinases ERK1 and ERK2; MAP kinase activities were reduced 91% by MSE and 80% by MSKE in ZR-75-1 estrogen receptor-dependent breast cancer cells and 73% by MSE and 66% by MSKE in MDA-MB-231 triple negative breast cancer cells. There were no significant differences between the effects of the grape seed extract compared to the grape skin extract with any of the cell lines. These results demonstrate that extracts from muscadine grape seeds and muscadine grape skins inhibit the growth of human lung, colon, prostate, breast, skin, brain and leukemia cells in vitro, suggesting that further studies are warranted to investigate their potential use in the prevention or treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4220. doi:10.1158/1538-7445.AM2011-4220

The composition of the vaginal microbiome is closely tied to host health. Bacterial vaginosis (BV), caused by the overgrowth of specific anaerobes (e.g., Gardnerella vaginalis), is associated with negative health outcomes. A vaginal microbiome dominated by Lactobacillus species is thought to protect against BV. However, the role of Lactobacillus iners is controversial, with evidence suggesting that some strains may not protect against BV while others do. To better characterize L. iners strains, their interactions with vaginal bacteria and human cells need to be investigated in vitro, but this has been impeded by the lack of liquid media that supports rapid L. iners growth. We have developed three liquid media formulations for L. iners growth: Serrador's Lactobacillus-adapted Iscove's medium (SLIM), which supports robust L. iners growth; a vaginally adapted version of SLIM (SLIM-V); and a chemically defined version (SLIM-CD). SLIM and SLIM-V improve L. iners growth compared to previously published formulations and also support the growth of other vaginal bacteria, including Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Gardnerella vaginalis. SLIM-CD leads to slower growth but may be useful for characterizing L. iners nutrient requirements or metabolite production. Importantly, SLIM and SLIM-V also support the growth of human vaginal epithelial cells, providing a foundation for future co-culture studies. Here, we present the formulations of SLIM, SLIM-V, and SLIM-CD and compare the growth of bacterial strains and human cells in these media.IMPORTANCELactobacillus iners is one of the most prevalent members of the vaginal microbiome, but whether it promotes health or leads to bacterial vaginosis is not well understood. We have developed media formulations that lead to improved L. iners growth and support growth of other vaginal bacteria and human vaginal cells. This will allow for investigation of how L. iners interacts with vaginal bacteria and the host, improving our understanding of its role in the vaginal microbiome.

Prostate cancer is the second leading cause of cancer death in men in the United States. Polymethoxyflavonoids (PMFs) such as tangeretin which exclusively exist in citrus fruit peels have been studied to have many bioactivities. In this study, we investigated the inhibitory effect of the metabolites of tangeretin in human prostate cancer LNCaP and PC3 cells. The results showed that PMF1 is a synthetic tangereitin metabolite can significantly inhibits the growth of LNCaP and PC-3 cells (IC50 14.0 and 13.5 µM, respectively) while PMF1 at 15 µM cannot affect the growth of human normal prostate epithelial RWPE-1 cells. The results of the anchorage-independent growth analysis indicated that PMF1 can suppress the colony formation in LNCaP and PC-3 cells as well as LNCaP and PC-3 stem-like cells which were isolated when the parent cells were cultured in the serum-free medium on the ultra-low attached dishes. Furthermore, PMF1 can induce apoptosis in LNCaP cells in which PMF1 increases protein expressions of Bax and Bad and decreases protein expressions of procaspases-3 and Bcl-2. We also found that PMF1 also decreases the protein levels of DNA Methyltransferase 1 and histone deacetylases 1, 2, and 4/5/9. These results suggested that PMF1 might effectively inhibit the growth of human prostate cancer cells as well as prostate cancer stem cells, which provides new insights in the prevention and treatment of prostate cancer. Citation Format: Yen-Hsiang Chao, Zheng-Yuan Su. A novel metabolite of citrus tangeretin epigenetically inhibits the growth of human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2017-LB-167

Cell sheet engineering is a novel and effective means of forming tissue substitutes in regenerative medicine. This technology allows for the manufacturing of cell sheets for either direct transplantation or the assembly of three-dimensionally organized tissue-mimicking structures. In the last decade, cell sheet engineering emerged as a promising alternative to injected cell delivery and biomaterial vehicles. Cell sheets manufactured on thermosensitive polymer coatings are already successfully employed in clinical applications such as human corneal reconstruction and treatment of human myocardial dysfunction. The main goal of my thesis was to develop an alternative, electrochemically responsive surface coating for the site-specific release of cell sheets by applying of micro-electrical currents. In context of my work, polyelectrolyte multilayer (PEM)-based platforms should be used for the synthesis of cell sheets composed of human stem / progenitor and end-differentiated cells. In the first part of my thesis, I determined the critical parameters for the isolation and long-term cultivation of human term placenta-derived mesenchymal stem cells (PD-MSCs). My results revealed that the isolated cells were of maternal origin and possessed very high proliferation and very low mortality rates up to passage twenty. The phenotypic profile of PD-MSC-isolates fulfilled the recently defined minimal criteria for the determination of mesenchymal stem cells (MSCs) and remained constant throughout continuous sub-confluent culture. The plasticity of PD-MSCs allowed their differentiation into adipogenic, chondrogenic, and osteogenic lineages as well as endothelium. In the second part of my thesis, I designed and optimized a PEM-based platform for the growth and intact peeling of cell sheets generated from different human cell types. Therefore, I established PEM substrates of a constant thickness with variations in rigidity. As a preliminary experiment, I used the assembly composed from nine layer-pairs of positively-charged poly-L-lysine (PLL) and negatively-charged hyaluronic acid (HA). The use of such polymers allowed for the construction of soft coatings and has been previously described as an attractive system for culturing different cell types. Moreover, the coating stiffness was adjusted by modulating the heterobifunctional crosslinkers present. For the assembly of stiff coatings, I used a film comprised of positively-charged poly-(allylamine)-hydrochloride (PAH) and negatively-charged poly-(styrene)-sulfonate (PSS). The potential of such coatings in cell sheet formation was previously demonstrated by endothelial cell cultivation. I demonstrated that the stiffness of the PEM substrate plays a critical role in the adhesion, spreading, and growth of human cells. Although the soft substrate supported the adhesion of the control murine muscle cell line C2C12, it only provided a minimal support for the adhesion of human cell types such as placenta-derived MSCs (PD-MSCs), adipose tissue-derived MSCs (AT-MSCs), human muscle cells (HMCs), human umbilical cord endothelial cells (HUVECs), and human late outgrowth endothelial cells (OECs). In contrast, the semi-stiff and stiff substrates were deemed suitable in culturing all of the tested human cell types and allowed for the complete mesodermal differentiation of stem cell sheets generated from AT-MSCs and PD-MSCs. In the final part of my thesis, I established the optimal conditions for the generation and detachment of PD-MSC sheets. In order to find a PEM architecture that minimally affected the cell sheet growth, I decided to utilize stiff (PAH/PSS) coatings. Such coatings supported the successful formation of PD-MSC sheets without any chemical modification. They also allowed for cell sheet detachment mediated either electrochemically by applying the micro-electrical current about 1.8 mV or by decreasing the environmental pH to 4. The viable PD-MSC sheets that detached from the (PAH/PSS) platform were able to adhere on the fresh TCPS substrates and could be successfully differentiated towards adipogenesis and osteogenesis. In summary, my thesis explored and implemented a PEM-based platform as substrates for cell sheet engineering and differentiation. Comparable with costly classical thermosensitive platforms, economically priced PEM based platforms allowed formation of cell sheets from different human stem / progenitor and adult cell types during a very short time period. Moreover, charge and pH-mediated peeling of cell sheets supported by PEM platforms are not only faster, but easier to automatize than thermosensitive platforms as well. My results lead me to believe that PEM based platforms indeed have potential as substrates for cell sheet engineering. They can be used for manufacturing single stem cell sheets geared for direct transplantation or for the generation of tissue-like structures destined for drug screening applications.

This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells. Optimal conditions for stimulating clonal growth were determined by testing the efficacy of horse serum (HS), fetal bovine serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors, UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated dishes and DME/F12 medium with 10% FBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal human adrenocortical cells without fibroblast overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal and nonclonal early passage human adrenocortical cells had similar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland fibroblasts, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts. In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of human adrenocortical cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and increased the cloning efficiency of cultured bovine adrenocortical cells.

This study evaluated the effects of sodium chloride reduction and its substitution with potassium chloride on Akawi cheese during storage for 30 d at 4 °C. Survival of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum) and starter bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), angiotensin-converting enzyme-inhibitory and antioxidant activities, and concentrations of standard amino acids as affected by storage in different brine solutions (10% NaCl, 7.5% NaCl, 7.5% NaCl+KCl [1:1], 5% NaCl, and 5% NaCl+KCl [1:1]) were investigated. Furthermore, viability of human colon cells and human colon cancer cells as affected by the extract showing improved peptide profiles, highest release of amino acids and antioxidant activity (that is, from cheese brined in 7.5% NaCl+KCl) was evaluated. Significant increase was observed in survival of probiotic bacteria in cheeses with low salt after 30 d. Calcium content decreased slightly during storage in all cheeses brined in various solutions. Further, no significant changes were observed in ACE-inhibitory activity and antioxidant activity of cheeses during storage. Interestingly, concentrations of 4 essential amino acids (phenylalanine, tryptophan, valine, and leucine) increased significantly during storage in brine solutions containing 7.5% total salt. Low concentration of cheese extract (100 μg/mL) significantly improved the growth of normal human colon cells, and reduced the growth of human colon cancer cells. Overall, the study revealed that cheese extracts from reduced-NaCl brine improved the growth of human colon cells, and the release of essential amino acids, but did not affect the activities of potential bioactive peptides.

The effects of antiepidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) 528 and 225 and a 528-ricin A conjugate on the growth of normal and malignant human cells were tested in vitro. Malignant human cell lines with EGF receptor numbers ranging from 0 to 4 X 10(5) receptors/cell, human fetal fibroblasts, and normal marrow granulocyte/macrophage progenitors (CFU-gm) showed no effect when grown with 10(-12) M to 10(-7) M MAb 225 or 528. MAbs 225 and 528 and EGF also had no effect on the ability of marrow stromal cells to maintain CFU-gm viability in long-term marrow cultures. Reversible growth inhibition of A431 epidermoid and MDA-468 breast carcinoma cells with 2 and 3 X 10(6) EGF receptors/cell, respectively, was observed with both antibodies and with 10(-8) M EGF. In contrast, an immunoconjugate prepared with MAb 528 and recombinant ricin A chain (528-rRA) showed dose-dependent killing over a concentration range of 10(-12) M to 10(-8) M against cells with greater than or equal to 1.2 X 10(5) EGF receptors/cell [concentration that causes 50% inhibition of growth (IC50) values, approximately 10(-12) M to 10(-10) M]. Human fetal fibroblasts (5.6 X 10(4) EGF receptors/cell), melanoma cells without detectable EGF receptors, and human CFU-gm showed IC50 values of greater than 10(-8) M. Killing of KB epidermoid carcinoma cells and 547 ovarian carcinoma cells with 4 and 1.2 X 10(5) EGF receptors/cell by 10(-10) or 10(-11) M 528-rRA was time dependent, but cytotoxicity to 547 cells was not complete even with 48 hours of immunotoxin treatment. Cytotoxicity of 528-rRA was not enhanced by chloroquine or verapamil. In vitro, anti-EGF receptor MAbs cause reversible antiproliferative effects only against malignant cell lines with amplified EGF receptor expression. In contrast, 528-rRA shows potent, specific toxicity to cells with greater than 50,000 EGF receptors/cell. However, kinetics of cell killing with 528-rRA are protracted, suggesting that prolonged exposure may be required for in vivo antitumor effects.