Measuring and modeling investigation of the net photochemical ozone production rate via an improved dual-channel reaction chamber technique

Current process-based research mainly uses box models to evaluate photochemical ozone production and destruction rates, and it is unclear to what extent the photochemical reaction mechanisms are elucidated. Here, we modified and improved a net photochemical ozone production rate (NPOPR, P(O3)net) detection system based on the current dual-channel reaction chamber technique, which makes the instrument applicable to different ambient environments, and its various operating indicators were characterized, i.e., “airtightness”, light transmittance, wall losses of the reaction and reference chambers, conversion rate of O3 to NO2, air residence time, and performance of the reaction and reference chambers. The limits of detection of the NPOPR detection system were determined to be 0.07, 1.4, and 2.3 ppbv h−1 at sampling flow rates of 1.3, 3, and 5 L min−1, respectively. We further applied the NPOPR detection system to field observations at an urban site in the Pearl River Delta (China). During the observation period, the maximum value of P(O3)net was 34.1 ppbv h−1, which was ∼ 0 ppbv h−1 at night within the system detection error and peaked at approximately noon local time. The daytime (from 06:00–18:00 LT) average value of P(O3)net was 12.8 (± 5.5) ppbv h−1. We investigated the detailed photochemical O3 formation mechanism in the reaction and reference chambers of the NPOPR detection system using a zero-dimensional box model. We found that the photochemical reactions in the reaction chamber were very close to those in ambient air, but there was not zero chemistry in the reference chamber because the reaction related to the production and destruction of RO2 (= HO2 + RO2) continued in the reference chamber, which led to a small amount of P(O3)net. Therefore, the P(O3)net measured here can be regarded as the lower limit of the real P(O3)net in the atmosphere; however, the measured P(O3)net was still ∼ 7.5 to 9.3 ppbv h−1 higher than the modeled P(O3)net value depending on different modeling methods, which may be due to the inaccurate estimation of HO2 / RO2 radicals in the modeling study. Short-lived intermediate measurements coupled with direct P(O3)net measurements are needed in the future to better understand O3 photochemistry. Our results show that the NPOPR detection system can achieve high temporal resolution and continuous field observations, which helps us to better understand photochemical O3 formation and provides a key scientific basis for continuous improvement of air quality in China.</p

Calcium phosphate-hybridized tendon graft to enhance tendon-bone healing two years after ACL reconstruction in goats

Abstract Background We developed a novel technique to improve tendon-bone attachment by hybridizing calcium phosphate (CaP) with a tendon graft using an alternate soaking process. However, the long-term result with regard to the interface between the tendon graft and the bone is unclear. Methods We analyzed bone tunnel enlargement by computed tomography and histological observation of the interface and the tendon graft with and without the CaP hybridization 2 years after anterior cruciate ligament (ACL) reconstruction in goats using EndoButton and the postscrew technique (CaP, n = 4; control, n = 4). Results The tibial bone tunnel enlargement rates in the CaP group were lower than those in the control group (p p p Conclusions The CaP-hybridized tendon graft enhanced the tendon-bone healing 2 years after ACL reconstruction in goats. The use of CaP-hybridized tendon grafts can reduce the bone tunnel enlargement and gap area associated with the direct insertion-like formation in the interface near the joint.</p

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands

Improvement of Cardiac Function in Mouse Myocardial Infarction after Transplantation of Epigenetically-Modified Bone Marrow Progenitor Cells

OBJECTIVE: To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice. METHODS AND RESULTS: We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells. CONCLUSION: Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells

Lipid Modifications of Sonic Hedgehog Ligand Dictate Cellular Reception and Signal Response

Sonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.We analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.The findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues

Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration

A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR™) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming

The serologically defined colon cancer antigen-3 (SDCCAG3) is involved in the regulation of ciliogenesis

A primary cilium is present on most eukaryotic cells and represents a specialized organelle dedicated to signal transduction and mechanosensing. Defects in cilia function are the cause for several human diseases called ciliopathies. The serologically defined colon cancer antigen-3 (SDCCAG3) is a recently described novel endosomal protein mainly localized at early and recycling endosomes and interacting with several components of membrane trafficking pathways. Here we describe localization of SDCCAG3 to the basal body of primary cilia. Furthermore, we demonstrate that decreased expression levels of SDCCAG3 correlate with decreased ciliary length and a reduced percentage of ciliated cells. We show that SDCCAG3 interacts with the intraflagellar transport protein 88 (IFT88), a crucial component of ciliogenesis and intraciliary transport. Mapping experiments revealed that the N-terminus of SDCCAG3 mediates this interaction by binding to a region within IFT88 comprising several tetratricopeptide (TRP) repeats. Finally, we demonstrate that SDCCAG3 is important for ciliary localization of the membrane protein Polycystin-2, a protein playing an important role in the formation of polycystic kidney disease, but not for Rab8 another ciliary protein. Together these data suggest a novel role for SDCCAG3 in ciliogenesis and in localization of cargo to primary cilia