Evolution of the therian face through complete loss of the premaxilla

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreER^T2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.     

Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factor-treated human microvascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the synthesis of interleukin-6 (IL-6) and collagenase in human omental microvascular endothelial (HOME) cell (Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J. V., Welgus, H. G., and Kuwano, M. (1989) J. Immunol. 143, 1619-1627). In the present study, we have examined whether the TNF-alpha-induced synthesis of IL-6 or collagenase in HOME cells is mediated by an inducible growth factor. Among several growth factors examined, we found that the expression of basic fibroblast growth factor (bFGF) mRNA was the one most prominently enhanced when HOME cells were treated with TNF-alpha. The increase of bFGF mRNA by TNF-alpha in HOME cells was observed in both a dose- and time-dependent manner when assayed by Northern blot analysis. The induction of bFGF mRNA was observed by 3 h after incubation with TNF-alpha, and the maximal increase of 5-fold was obtained after 12 h of incubation with 100 units/ml TNF-alpha. Western blot analysis confirmed the enhanced synthesis of bFGF by TNF-alpha. Metabolic labeling and immunoprecipitation assays of bFGF showed that exposure to TNF-alpha enhanced secretion of bFGF into culture medium and also that TNF-alpha stimulated the production of bFGF molecules with molecular masses of 18, 21, and 22.5 kDa in HOME cells. TNF-alpha induced the expression of collagenase mRNA and IL-6 mRNA in HOME cells as well, and the coadministration of neutralizing anti-bFGF antibody almost completely blocked the effects of TNF-alpha. The treatment of HOME cells with exogenous bFGF significantly stimulated the expression of collagenase mRNA and IL-6 mRNA in HOME cells. Therefore, the biological effects of TNF-alpha on HOME cells may be mediated, at least in part, by TNF-alpha-induced bFGF.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide

Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es). Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal. The signal intensity varied depending on the number of unpaired bases on the DNA duplex. From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased. These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in a peroxisome proliferator-activated receptor gamma-independent manner

Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy.     

A modified cornish pasty method for ex ovo culture of the chick embryo development

Chick embryos grown in ex ovo culture by the modified Cornish pasty method reported in Nagai, Lin and Sheng in this issue.     

Risk factors for bone loss in patients with rheumatoid arthritis treated with biologic disease-modifying anti-rheumatic drugs

Objective

Osteoporosis is a complication of rheumatoid arthritis. We examined the risk factors for bone loss in rheumatoid arthritis patients receiving biological disease-modifying anti-rheumatic drugs. Lumbar spine and femoral neck bone mineral density was measured at two time points in 153 patients with rheumatoid arthritis managed with biological disease-modifying anti-rheumatic drugs. We examined patients’ variables to identify risk factors for least significant reduction of bone mineral density.

Results

Least significant reduction of lumbar spine bone mineral density (≤ ? 2.4%) was seen in 13.1% of patients. Least significant reduction of femoral neck bone mineral density (≤ ? 1.9%) was seen in 34.0% of patients. Multiple logistic regression analysis showed that a risk factor for least significant reduction of the lumbar spine was high-dose methylprednisolone use. Multiple regression analysis showed that a risk factor for least significant reduction of the femoral neck was short disease duration. Our findings showed that a risk factor for femoral neck bone mineral density reduction was a short disease duration. These findings suggest that rheumatoid arthritis patients receiving treatment with biological disease-modifying anti-rheumatic drugs may benefit from earlier osteoporosis treatments to prevent femoral neck bone loss.