Specific Cellular Processes Research Articles

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Ageing research: rethinking primary prevention of skin cancer.

Ageing research: rethinking primary prevention of skin cancer.

Integration and gene co-expression network analysis of scRNA-seq transcriptomes reveal heterogeneity and key functional genes in human spermatogenesis

Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis’s main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

Clinical Importance of Wnt5a in the Pathogenesis of Colorectal Cancer.

Wnt5a is one of the potent signaling molecules that initiates responses involved in cancer through activation of both canonical and noncanonical signaling cascades. Wnt5a both directly and indirectly triggers cancer-associated signaling pathways based on the cancer type. In colorectal cancer (CRC), altering Wnt5a expression can influence several cellular processes of tumor cells, including proliferation, differentiation, migration, invasion, and metastasis. This review summarizes the molecular mechanisms and clinical importance of Wnt5a in the pathogenesis of CRC for better understanding the pathogenesis and its potential role as a prognostic marker and as an appropriate therapeutic target in the treatment of this disease in the future.

MP55-03 CLINICAL ANALYSIS OF THE ROLE OF THYROID FUNCTION IN THE DIFFERENTIATION PROCESS OF SPERMATOGONIAL STEM CELLS IN CRYPTORCHID TESTES

You have accessJournal of UrologyPediatric Urology V (MP55)1 Sep 2021MP55-03 CLINICAL ANALYSIS OF THE ROLE OF THYROID FUNCTION IN THE DIFFERENTIATION PROCESS OF SPERMATOGONIAL STEM CELLS IN CRYPTORCHID TESTES Kentaro Mizuno, Hidenori Nishio, Daisuke Matsumoto, Taiki Kato, Akihiro Nakane, Tetsuji Maruyama, Takahiro Yasui, and Yutaro Hayashi Kentaro MizunoKentaro Mizuno More articles by this author , Hidenori NishioHidenori Nishio More articles by this author , Daisuke MatsumotoDaisuke Matsumoto More articles by this author , Taiki KatoTaiki Kato More articles by this author , Akihiro NakaneAkihiro Nakane More articles by this author , Tetsuji MaruyamaTetsuji Maruyama More articles by this author , Takahiro YasuiTakahiro Yasui More articles by this author , and Yutaro HayashiYutaro Hayashi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002085.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Boys with cryptorchidism may have a high risk for future fertility. The pool of spermatogonial stem cells (SSCs) in prepubertal testes has been reported to be disrupted in these cases. Spermatogenesis is a complex process in which SSCs undergo differentiation and morphogenesis. A previous study has revealed that SSCs differentiate from gonocytes until 3 months after birth during a period colloquially called “mini-puberty.” However, the factors involved in this cellular differentiation process remain unclear. In this study, we investigated the role of thyroid hormones in SSC differentiation. METHODS: After obtaining consent from the parents, we performed testicular biopsies in 138 boys with cryptorchidism who underwent orchidopexy at Nagoya City University Hospital between January 2008 and February 2021. We investigated the number of SSCs per seminiferous tubule using anti-DDX4 antibody immunohistochemistry in testicular tissues. We detected congenital hypothyroidism (CH) in 12 boys, who were compared with 126 boys without CH. This study was approved by the institutional review board of our hospital (no. 60180016). RESULTS: The mean age of patients with (Hypo-T group) and without (Normal-T group) CH at surgery was 23.3±14.5 and 34.8±30.4 months, respectively (p=0.45). The mean testicular volume was 0.54±0.27 cm3 (Normal-T group) and 0.62±0.36 cm3 (Hypo-T group) (p=0.30). The number of SSCs per tubule was 0.62±0.59 in the Normal-T group and 2.14±0.69 in the Hypo-T group (p<0.01). CONCLUSIONS: In patients with CH, the number of SSCs per tubule was significantly increased, and differentiation to SSCs occurred at a greater degree than in those without CH. We suspected that this was caused by the high thyroid-stimulating hormone (TSH) levels during mini-puberty. TSH is similar in structure to follicle-stimulating hormone, and their respective receptors are located in Sertoli cells. A previous study revealed that TSH promotes the proliferation of Sertoli cells, suggesting that Sertoli cells are involved in the SSC differentiation process. It provides a novel therapeutic method for preserving fertility potential in cryptorchid boys through stimulation of Sertoli cell function via thyroid hormones. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e958-e959 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kentaro Mizuno More articles by this author Hidenori Nishio More articles by this author Daisuke Matsumoto More articles by this author Taiki Kato More articles by this author Akihiro Nakane More articles by this author Tetsuji Maruyama More articles by this author Takahiro Yasui More articles by this author Yutaro Hayashi More articles by this author Expand All Advertisement Loading ...

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules.

Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here, we used a combination of targeted and unbiased approaches to look for other cases of “chemical rescue” by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from “off-target” effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued ∼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.

Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway

Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.

The Effects of Environmental Adversities on Human Neocortical Neurogenesis Modeled in Brain Organoids.

Over the past decades, a growing body of evidence has demonstrated the impact of prenatal environmental adversity on the development of the human embryonic and fetal brain. Prenatal environmental adversity includes infectious agents, medication, and substances of use as well as inherently maternal factors, such as diabetes and stress. These adversities may cause long-lasting effects if occurring in sensitive time windows and, therefore, have high clinical relevance. However, our knowledge of their influence on specific cellular and molecular processes of in utero brain development remains scarce. This gap of knowledge can be partially explained by the restricted experimental access to the human embryonic and fetal brain and limited recapitulation of human-specific neurodevelopmental events in model organisms. In the past years, novel 3D human stem cell-based in vitro modeling systems, so-called brain organoids, have proven their applicability for modeling early events of human brain development in health and disease. Since their emergence, brain organoids have been successfully employed to study molecular mechanisms of Zika and Herpes simplex virus-associated microcephaly, as well as more subtle events happening upon maternal alcohol and nicotine consumption. These studies converge on pathological mechanisms targeting neural stem cells. In this review, we discuss how brain organoids have recently revealed commonalities and differences in the effects of environmental adversities on human neurogenesis. We highlight both the breakthroughs in understanding the molecular consequences of environmental exposures achieved using organoids as well as the on-going challenges in the field related to variability in protocols and a lack of benchmarking, which make cross-study comparisons difficult.

The role of lncRNA Meg3 in the proliferation of all-trans retinoic acid-treated mouse embryonic palate mesenchymal cells involves the Smad pathway

Mesenchymal cell proliferation is critical for the growth of the palate shelf. All-trans retinoic acid (atRA), as well as pathways associated with TGF-β/Smad signaling, play crucial roles in the proliferation of mouse embryonic palate mesenchymal (MEPM) cells. We have found that MEPM-cell proliferation was regulated by atRA and exogenous TGF-β3 could significantly antagonize the atRA-mediated suppression of MEPM cell proliferation, which is closely associated with the regulation of TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been reported to activate TGF-β/Smad signaling, thereby regulating cellular proliferation, differentiation, and related processes. Here, we found that Meg3 expression increased significantly in atRA-treated MEPM cells while TGF-β3 treatment markedly inhibited Meg3 expression and antagonized the effect of atRA on Meg3. Moreover, Smad2 was found to interact directly with Meg3, and atRA treatment significantly enriched Meg3 in Smad2-immunoprecipitated samples. After Meg3 deletion, the effects of atRA on the proliferation of MEPM cells and TGF-β3-dependent protein expression were lost. Hence, we speculate that Meg3 has a role in the RA-induced suppression of MEPM cell proliferation by targeting Smad2 and thereby mediating TGF-β/Smad signaling inhibition.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

A Role of Alkbh5 at the Early Stage of Cell Differentiation in C2C12 Cells

ObjectivesN6-methyladenosine(m6A) is one of the most abundant mRNA modifications, controlling mRNA metabolism including splicing, stabilization, and translation efficiency. Although it has been reported that m6A was involved in cellular differentiation process, its underlying molecular mechanism is not fully understood. The object of this study was to discover ALKBH5, a m6A demethylase, as a key regulator in early myogenesis and adipogenesis using C2C12 model. MethodsTo identify the epigenetic modifier, we compared publicly available GEO data sets from two independent adipogenic and myogenic differentiation models. M6A levels of mRNAs were measured by dot blot assays upon differentiation. C2C12 cells were maintained under myogenesis or adipogenesis media(±rosiglitazone) for 5–8 days. Alkbh5 knockdown using siRNA and overexpression using GFP-tagged cDNA plasmid were transfected into cells 2 days before confluence. Gene expression analysis was done by qRT-PCR, following RNA isolation and conversion into cDNA. Oil-red-O staining was conducted to detect lipid droplets. Myotube length and width were measured by giemsa staining. ResultsWe found Alkbh5 as a common epigenetic modifier, expression of which was significantly different in both early myogenic and adipogenic cells, compared to undifferentiated cells. Consistently, m6A levels, measured by dot blots, decreased during myogenesis(day 2), while increased during adipogenesis(24h). To examine if modulation of Alkbh5 expression affected cell differentiation, we conducted both Alkbh5 knockdown and overexpression in C2C12 myoblasts, followed by differentiating stimulation into myotube and adipocyte by appropriate media. Knockdown of Alkbh5 facilitates lipid accumulation, while overexpression of Alkbh5 represents significant increase in myotube length and width. Expression of myogenesis and adipogenesis markers were consistent with the phenotypes. ConclusionsThese results suggested that Alkbh5 had an important role in determination of cell fates towards myogenesis over adipogenesis at the early point of cell differentiation. Funding SourcesThis study was supported by the National Research Foundation of Korea the Korean National Cancer Center. JYC was supported by Brain Korea Four Project(Education Research Center for 4IR-Based Health Care).

Nanomaterial Shape Influence on Cell Behavior.

Nanomaterials are proven to affect the biological activity of mammalian and microbial cells profoundly. Despite this fact, only surface chemistry, charge, and area are often linked to these phenomena. Moreover, most attention in this field is directed exclusively at nanomaterial cytotoxicity. At the same time, there is a large body of studies showing the influence of nanomaterials on cellular metabolism, proliferation, differentiation, reprogramming, gene transfer, and many other processes. Furthermore, it has been revealed that in all these cases, the shape of the nanomaterial plays a crucial role. In this paper, the mechanisms of nanomaterials shape control, approaches toward its synthesis, and the influence of nanomaterial shape on various biological activities of mammalian and microbial cells, such as proliferation, differentiation, and metabolism, as well as the prospects of this emerging field, are reviewed.

Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology

MicroRNAs (miRNAs) are small noncoding RNAs that play key roles in post- transcriptional gene regulation. Being involved in regulating virtually all cellular processes, from proliferation and differentiation to migration and apoptosis, they have emerged as important epigenetic players. While most interest has gone into which miRNAs are involved in specific cellular processes or pathologies, the dosage-dependent effects of miRNAs remain vastly unexplored. Different doses of miRNAs can cause selective downregulation of target genes, in turn determining what signaling pathways and cellular responses are triggered. To explore this behavior, the effects of incremental miRNA dosage need to be studied; however, current delivery methods for miRNAs are unable to control how much miRNA enters a cell. Herein, an approach is presented based on a nanostrawelectroporation delivery platform that decouples the delivery from biological mechanisms (e.g., endocytosis) to enable precise control over the amount of miRNA delivered, along with demonstrating ratiometric intracellular delivery into primary dermal fibroblasts for miR-181a and miR-27a. In addition, it is shown that the nanostraw delivery platform allows efficient delivery of miRNAs into primary keratinocytes, opening new opportunities for successful miRNA delivery into this hard-to-transfect cell type.

138 Expression profiling of the ventral colon, pelvic flexure, and dorsal colon epithelium by RNA-seq

The equine large intestine is essential for nutrient absorption and as a location for microbial digestion. The pelvic flexure is an important anatomical landmark that divides distinct microbial communities in the equine hindgut. Previous work from our group showed that several taxonomic groups were restricted to the cecum and ventral colon. Since no physical barrier separates these compartments from the more distal areas, we became curious about alternative mechanisms that support the segregation of different microbial groups. Physiological differences in each compartment likely result in different requirements from the microbiota at each location. This study evaluated gene expression in the mucosal and submucosal layers of the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). Samples were collected from 3 4-year-old American Quarter Horses, and total RNA was isolated. Single-end libraries were prepared using the NEBNextⓇ Ultra II Directional RNA library kit and sequenced on an IlluminaⓇ NextSeq 550 platform. The sequencing generated an average of 22.5 million reads per sample (range 14.5 to 30.9). Gene expression analysis was performed using the Galaxy platform by aligning quality trimmed sequence reads to EquCab3 using the HISAT2, assembling transcripts with StringTie, and quantifying transcript abundance with Salmon. DESeq2 was used to assess differential expression between the intestinal compartments. Expression (present in 2 of 3 biological replicates) was detected in at least one intestinal compartment for 21,987 transcripts and ranging from 14,766 to 15,134 transcripts per sample. A total of 13,944 transcripts were detected in all 5 areas, and 739 transcripts demonstrated a tissue-restricted expression pattern (detected above threshold in only on anatomical location). Gene ontology was used to identify the functional roles of transcripts expressed in all or only one hindgut compartment. Transcripts expressed in all tissues were associated with internal cell metabolic processes, which are common despite cell type. In contrast, transcripts with tissue-restricted expression patterns were associated with cellular differentiation and secretion processes. Matrix metalloproteinases, transcription factors, and integrin proteins were identified as differentially expressed (P < 0.05) in the PF compared with the surrounding VC and DC compartments. Continued investigation of these transcripts in the equine hindgut will enhance our understanding of digestive physiology and support future studies of interactions between host and microbiota.

Breast Cancer Cell Lines, HER2/Neu Phenotype, and a Higher Propensity to Reactive Oxygen Species Production

Background: The reactive oxygen species (ROS) generated in the biological systems play an important role in pathological conditions and specific normal cellular processes, such as signaling pathways and drug sensitivity. Methods: Intracellular ROS was determined using (2'-7'dichlorofluorescin diacetate) DCFH-DA fluorimetric probe, malondialdehyde as lipid peroxidation index detected by the Thiobarbituric acid reactive substances method. Cells' protein carbonyl contents were assessed with 2,4-Dinitrophenylhydrazine (DNPH) derivatization spectrophotometrically at 360-385 nm. Results: The oxidative stress induces ROS (p?0.05), MDA (Malondialdehyde) (p?0.05), and protein carbonylation (p?0.01) was significantly higher in HER2-positive BT-474, SK-BR-3, and MDA-MB-453 compared to the HER2-negative MDA-MB-231 and MCF-7 cell lines. Conclusion: We hypothesized that increased oxidative stress in HER2-positive cell lines is due to the oncogenic function of the HER2 and PI3K/Akt signaling activation, resulting in glycolysis induction. It is assumed that HER2-positive cell lines with high ROS levels are more vulnerable to further damage by increased ROS levels induced by pro-oxidant anticancer agents.

The molecular functions of Biodentine and mineral trioxide aggregate in lipopolysaccharide-induced inflamed dental pulp cells.

To explore the proliferation, adhesion and differentiation response and the underlying mechanisms that occur in lipopolysaccharide (LPS)-induced inflamed dental pulp cells (DPCs) in contact with Biodentine and mineral trioxide aggregate (MTA). The DPCs were isolated from three healthy donors and named DPC-H1 to DPC-H3. The DPCs were pre-cultured with 2 or 5μgmL-1 LPS for 24h to induce inflammation. The expression of inflammation marker miR-146a was detected by q-PCR. The normal and LPS-induced DPCs were further treated with 0.14mgmL-1 Biodentine or 0.13mgmL-1 MTA for 24h. MTT assay and adhesion assay were used to analyse the changes of cell phenotypes. DSPP, AKT and ERK expressions were detected by Western blotting. The data were analysed by Mann-Whitney test or two-way anova. Differences were considered statistically significant when P<0.05. In LPS-induced DPCs, Biodentine and MTA treatment neither induced nor aggravated LPS-induced inflammation, but their presence did increase the expression of the odontogenic differentiation marker DSPP. Under 2 or 5μgmL-1 LPS-induced inflammation, Biodentine and MTA promoted the proliferation of DPC cells, and significantly in DPC-H2 (P<0.0001 for both reagents). With the treatment of 2μgmL-1 LPS, the cell adhesion of DPCs on the fibronectin-coated culture plates was increased significantly by Biodentine (P=0.0413) and MTA (P<0.0001). Biodentine and MTA regulated cell adhesion on the fibronectin-coated culture plates (P<0.0001 for both reagents) and proliferation (P<0.0001 for both reagents) via the AKT pathway. However, the AKT pathway was not involved in the expression of DSPP induced by Biodentine and MTA. Biodentine and MTA enhanced the proliferation, adhesion and differentiation of LPS-induced DPCs. The proliferation and adhesion process induced by Biodentine and MTA was via the AKT pathway. However, the cellular differentiation process might not use the same pathway, and this needs to be explored in future studies.

Aromatic Rings as Molecular Determinants for the Molecular Recognition of Protein Kinase Inhibitors.

Protein kinases are key enzymes in many signal transduction pathways, and play a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. However, aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Over the past three decades, thousands of protein kinase inhibitors (PKIs) with varying molecular frames have been developed. Large-scale data mining of the Protein Data Bank resulted in a database of 2139 non-redundant high-resolution X-ray crystal structures of PKIs bound to protein kinases. This provided us with a unique opportunity to study molecular determinants for the molecular recognition of PKIs. A chemoinformatic analysis of 2139 PKIs resulted in findings that PKIs are “flat” molecules with high aromatic ring counts and low fractions of sp3 carbon. All but one PKI possessed one or more aromatic rings. More importantly, it was found that the average weighted hydrogen bond count is inversely proportional to the number of aromatic rings. Based on this linear relationship, we put forward the exchange rule of hydrogen bonding interactions and non-bonded π-interactions. Specifically, a loss of binding affinity caused by a decrease in hydrogen bonding interactions is compensated by a gain in binding affinity acquired by an increase in aromatic ring-originated non-bonded interactions (i.e., π–π stacking interactions, CH–π interactions, cation–π interactions, etc.), and vice versa. The very existence of this inverse relationship strongly suggests that both hydrogen bonding and aromatic ring-originated non-bonded interactions are responsible for the molecular recognition of PKIs. As an illustration, two representative PKI–kinase complexes were employed to examine the relative importance of different modes of non-bonded interactions for the molecular recognition of PKIs. For this purpose, two FDA-approved PKI drugs, ibrutinib and lenvatinib, were chosen. The binding pockets of both PKIs were thoroughly examined to identify all non-bonded intermolecular interactions. Subsequently, the strengths of interaction energies between ibrutinib and its interacting residues in tyrosine kinase BTK were quantified by means of the double hybrid DFT method B2PLYP. The resulting energetics for the binding of ibrutinib in tyrosine kinase BTK showed that CH–π interactions and π–π stacking interactions between aromatic rings of the drug and hydrophobic residues in its binding pocket dominate the binding interactions. Thus, this work establishes that, in addition to hydrogen bonding, aromatic rings function as important molecular determinants for the molecular recognition of PKIs. In conclusion, our findings support the following pharmacophore model for ATP-competitive kinase inhibitors: a small molecule features a scaffold of one or more aromatic rings which is linked with one or more hydrophilic functional groups. The former has the structural role of acting as a scaffold and the functional role of participating in aromatic ring-originated non-bonded interactions with multiple hydrophobic regions in the ATP binding pocket of kinases. The latter ensure water solubility and form hydrogen bonds with the hinge region and other hydrophilic residues of the ATP binding pocket.

Physcomitrium patens: A Single Model to Study Oriented Cell Divisions in 1D to 3D Patterning.

Development in multicellular organisms relies on cell proliferation and specialization. In plants, both these processes critically depend on the spatial organization of cells within a tissue. Owing to an absence of significant cellular migration, the relative position of plant cells is virtually made permanent at the moment of division. Therefore, in numerous plant developmental contexts, the (divergent) developmental trajectories of daughter cells are dependent on division plane positioning in the parental cell. Prior to and throughout division, specific cellular processes inform, establish and execute division plane control. For studying these facets of division plane control, the moss Physcomitrium (Physcomitrella) patens has emerged as a suitable model system. Developmental progression in this organism starts out simple and transitions towards a body plan with a three-dimensional structure. The transition is accompanied by a series of divisions where cell fate transitions and division plane positioning go hand in hand. These divisions are experimentally highly tractable and accessible. In this review, we will highlight recently uncovered mechanisms, including polarity protein complexes and cytoskeletal structures, and transcriptional regulators, that are required for 1D to 3D body plan formation.

DTFLOW: Inference and Visualization of Single-cell Pseudotime Trajectory Using Diffusion Propagation

One of the major challenges in single-cell data analysis is the determination of cellular developmental trajectories using single-cell data. Although substantial studies have been conducted in recent years, more effective methods are still strongly needed to infer the developmental processes accurately. This work devises a new method, named DTFLOW, for determining the pseudo-temporal trajectories with multiple branches. DTFLOW consists of two major steps: a new method called Bhattacharyya kernel feature decomposition (BKFD) to reduce the data dimensions, and a novel approach named Reverse Searching on k-nearest neighbor graph (RSKG) to identify the multi-branching processes of cellular differentiation. In BKFD, we first establish a stationary distribution for each cell to represent the transition of cellular developmental states based on the random walk with restart algorithm, and then propose a new distance metric for calculating pseudotime of single cells by introducing the Bhattacharyya kernel matrix. The effectiveness of DTFLOW is rigorously examined by using four single-cell datasets. We compare the efficiency of DTFLOW with the published state-of-the-art methods. Simulation results suggest that DTFLOW has superior accuracy and strong robustness properties for constructing pseudotime trajectories. The Python source code of DTFLOW can be freely accessed at https://github.com/statway/DTFLOW.