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W S Lai

Publications and source records attributed to W S Lai.

At least 19 recordsLinked to original sources
PubMed2001

Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway.

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.

3' Untranslated Regions↗
PubMed2001

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Alleles↗
PubMed2001

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail.

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.

Animals↗
PubMed2000

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA.

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.

3' Untranslated Regions↗
PubMed2000

Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability.

Deficiency of tristetraprolin (TTP), the prototype of the CCCH zinc finger proteins, results in a complex inflammatory syndrome in mice. Most aspects of the syndrome are secondary to excess circulating tumor necrosis factor (TNF)-alpha, a consequence of increased stability of TNF-alpha messenger RNA (mRNA) in TTP-deficient macrophages. TTP can bind directly to the AU-rich element in TNF-alpha mRNA, increasing its lability. Here we show that TTP deficiency also results in increased cellular production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and increased stability of its mRNA, apparently secondary to decreased deadenylation. Similar findings were observed in mice also lacking both types of TNF-alpha receptors, excluding excess TNF-alpha production as a cause of the increased GM-CSF mRNA levels and stability. TTP appears to be a physiological regulator of GM-CSF mRNA deadenylation and stability. (Blood. 2000;95:1891-1899)

Adenosine Monophosphate↗
PubMed1999

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression.

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

Amino Acid Sequence↗
PubMed1999

Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA.

Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Base Sequence↗
PubMed1998

Transgenic complementation of MARCKS deficiency with a nonmyristoylatable, pseudo-phosphorylated form of MARCKS: evidence for simultaneous positive and dominant-negative effects on central nervous system development.

MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure-function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, "pseudo-phosphorylated" form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.

Animals↗
PubMed1998

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin.

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.

3T3 Cells↗
PubMed1998

Promoter sequence, expression, and fine chromosomal mapping of the human gene (MLP) encoding the MARCKS-like protein: identification of neighboring and linked polymorphic loci for MLP and MACS and use in the evaluation of human neural tube defects.

The MARCKS-like protein (MLP), also known as F52, MacMARCKS, or MARCKS-related protein, is a widely distributed substrate for protein kinase C (PKC). Recent studies using gene disruption in vivo have demonstrated the importance of both MARCKS and MLP to the development of the central nervous system; specifically, mice lacking either protein exhibit a high frequency of neural tube defects. We isolated a genomic clone for human MLP and discovered a directly linked polymorphism (MLP1) useful for genetic linkage analysis. The MLP promoter was 71% identical over 433 bp to that of the corresponding mouse gene, Mlp, with conservation of many putative transcription factor-binding sites; it was only 36% identical over 433 bp to the promoter of the human gene, MACS, which encodes the MLP homologue MARCKS. This 433-bp fragment drove expression of an MLP-beta-galactosidase transgene in a tissue-specific and developmental expression pattern that was similar to that observed for the endogenous gene, as shown by in situ hybridization histochemistry. In contrast to MACS, the MLP and Mlp promoters contain a TATA box approximately 40 bp 5' of the presumed transcription initiation site. MLP was localized to chromosome 1p34-->1pter by analysis of human-mouse somatic cell hybrid DNA and to 1p34 by fluorescence in situ hybridization. Radiation hybrid mapping of MLP placed it between genetic markers D1S511 (LOD > 3.0) and WI9232. MACS was localized to 6q21 between D6S266 (LOD > 3.0) and AFM268uh5 by the same technique. We tested the novel MLP1 polymorphism and the MACS flanking markers in a series of 43 Caucasian simplex families in which the affected child had a lumbosacral myelomeningocele. We found no evidence of linkage disequilibrium, suggesting that these loci were not major genes for spina bifida in these families. Nonetheless, the identification of linked and neighboring polymorphisms for MACS and MLP should permit similar genetic studies in other groups of patients with neural tube defects and other neurodevelopmental abnormalities.

Animals↗
PubMed1998

Characteristics of the intron involvement in the mitogen-induced expression of Zfp-36.

Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in fibroblasts by a variety of growth factors. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and autoimmunity, all apparently mediated by an excess of tumor necrosis factor alpha. We recently showed that full serum inducibility of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited serum-induced TTP expression. We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the splice sites, each resulted in the maintenance of approximately 45 and 19%, respectively, of full serum-induced expression. In addition, deletion of intron sequences base pairs 601-655 decreased the serum-induced expression of TTP by 65%. Sequence base pairs 618-626 bound specifically to the transcription factor Sp1; mutation of this binding motif decreased TTP expression by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its natural location also contributes significantly to the regulated expression of this gene.

3T3 Cells↗
PubMed1996

Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency.

The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.

Animals↗
PubMed1996

Cloning and characterization of two yeast genes encoding members of the CCCH class of zinc finger proteins: zinc finger-mediated impairment of cell growth.

Members of the CCCH zinc finger (Zf) protein family have in common two or more repeats of a novel Zf motif consisting of Cys and His residues in the form Cx8Cx5Cx3H [where x is a variable amino acid (aa)]. We used a degenerate polymerase chain reaction (PCR) strategy to clone members of this gene family from Saccharomyces cerevisiae. The deduced aa sequences encoded by these genes, designated CTH1 and CTH2, share 46% overall identity and 59% similarity, largely due to the two highly conserved Zf domains. We found readily detectable expression of a 1.4-kb mRNA encoding Cth1p. The 1.1-kb mRNA encoding Cth2p was barely detectable under normal growth conditions; however, disruption of CTH1 resulted in at least a threefold increase in CTH2 mRNA accumulation. No change in phenotype was detected following disruption of CTH1 and CTH2, either singly or together. In contrast, overexpression of the CTH genes or one of the related mammalian genes, tris-tetraprolin (TTP), caused delayed entry of cell cultures into exponential growth, and a decrease in final cell density. Removal of the Zf domain of Cth1p by truncation or deletion completely reversed this slow growth phenotype, indicating that it was mediated through this highly conserved structural motif.

Amino Acid Sequence↗
PubMed1996

A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.

Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.

Animals↗
PubMed1996

Mitogens stimulate the rapid nuclear to cytosolic translocation of tristetraprolin, a potential zinc-finger transcription factor.

Tristetraprolin (TTP) is the prototype of a group of potential transcription factors that contain two or more unusual CCCH zinc fingers. TTP is encoded by the immediate-early response gene Zfp-36, which is rapidly induced in fibroblasts in response to insulin and other growth factors. Indirect evidence suggests that TTP might function as an inhibitory transcription factor. The present studies evaluated the effect of mitogens on the subcellular localization of TTP using Western blotting of cellular nuclear and cytosolic fractions. In NIH/3T3 mouse fibroblasts that constitutively express TTP, 70% of the protein was located in the nucleus of quiescent, serum-deprived cells. Immunoreactive TTP began to increase in the cytosolic compartment within 1 min of serum stimulation of the cells; this increase in cytosolic protein was essentially complete within 5 min of serum stimulation (81% of total) and was accompanied by a commensurate decrease in nuclear TTP. This translocation was complete well before the increase in TTP synthesis that occurred after serum stimulation. Similar experiments in cells expressing a mutant TTP, in which the major mitogen-activated protein kinase site (serine 220) had been mutated to alanine, revealed normal nuclear to cytosolic translocation after serum stimulation, indicating that phosphorylation of this site is not necessary for this translocation to occur. These results suggest that TTP is rapidly modified in response to mitogens so that it is rapidly released from the nucleus to the cytosol, or that proteins retaining TTP in the nucleus are modified to release it into the cytosol. Thus, TTP's proposed function as a transcription factor, possibly an inhibitory one, may be regulated in cells in part by a novel mechanism, i.e. that of rapid, mitogen-stimulated translocation out of the cellular nucleus.

3T3 Cells↗
PubMed1995

Promoter analysis of Zfp-36, the mitogen-inducible gene encoding the zinc finger protein tristetraprolin.

The gene encoding the putative zinc finger protein tristetraprolin (TTP), Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show here that 77 base pairs 5' of the transcription start site are sufficient for full serum inducibility of the mouse Zfp-36 promoter. This region of the promoter includes consensus sequences for the binding of the transcription factors EGR-1, AP2, and Sp1. In addition, we have identified a previously undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence includes a palindrome and is identical in the human, bovine, and mouse genes. Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the serum induction of Zfp-36 and can confer serum-inducible expression on a heterologous minimal promoter. Gel mobility shift assays demonstrated the formation of complexes consisting of this region of the promoter and cellular nuclear proteins and demonstrated that the extent of complex formation could be altered by treatment of the cells with serum or insulin. These results suggest that the response of Zfp-36 to serum and other mitogens is mediated by a series of cis-acting elements acting in concert to confer full inducible transcription of this gene.

Animals↗
PubMed1995

Phosphorylation of tristetraprolin, a potential zinc finger transcription factor, by mitogen stimulation in intact cells and by mitogen-activated protein kinase in vitro.

Tristetraprolin (TTP) is a potential transcription factor that contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is encoded by the early response gene Zfp-36, which is highly expressed in response to growth factors and in several hematopoietic cell lines. In the present studies, we investigated the possibility that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were made to overexpress TTP constitutively, we found that the protein was phosphorylated on serine residues, and that this phosphorylation was rapidly (within 10 min) stimulated by several mitogens. In cell-free assays, recombinant mouse TTP was a substrate for the mitogen-activated protein (MAP) kinase. By a combination of protease digestion experiments and site-directed mutagenesis strategies, we found that serine 220 was phosphorylated by p42 MAP kinase in vitro. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function.

3T3 Cells↗