We described a silver(I)-mediated intramolecular oxidative C–H amination that enables the construction of assorted 1H-indazoles that are widely applicable in medicinal chemistry. The developed ...amination was found to be efficient for the synthesis of a variety of 3-substituted indazoles that are otherwise difficult to be synthesized by other means of C–H aminations. Preliminary mechanistic studies suggested that the current amination proceeds via single electron transfer (SET) mediated by Ag(I) oxidant.
•Rapid phenol toxicity tests (1h) were developed based on Chl a fluorescence and the movement parameters of Euglena agilis.•Phenol significantly reduced Fv/Fm of PS II and rETRmax with EC50 values of ...8.94 and 4.67mM, respectively.•Among the movement parameters tested, velocity was the most sensitive biomarker with an EC50 of 3.17mM.•The EC50 values for Fv/Fm, motility, and velocity appear to overlap the environmental permissible levels of phenol.
Phenol, a monosubstituted aromatic hydrocarbon with various commercial uses, is a major organic constituent in industrial wastewaters. The ecotoxic action of phenol for aquatic environment is well known. In this study, rapid phenol toxicity tests (1h) were developed based on chlorophyll a (Chl a) fluorescence and the movement parameters of the freshwater flagellate, Euglena agilis Carter. Phenol significantly reduced the maximum quantum yield (Fv/Fm) of photosystem II (PS II) and the maximum photosynthetic electron transport rate (rETRmax) with median effective concentration (EC50) values of 8.94 and 4.67mM, respectively. Phenol reduced the motility and triggered change in the swimming velocity of the test organism. Among the parameters tested, velocity was the most sensitive biomarker with an EC50 of 3.17mM. The EC50 values for Fv/Fm, motility, and velocity appear to overlap the permitted levels of phenol. In conclusion, the photosynthesis and movement of E. agilis can be fast and sensitive risk assessment parameters for the evaluation of phenol toxicity in municipal and industrial effluents.
Highly enantioselective lipase has been widely utilized in the preparation of versatile enantiopure chiral sec-alcohols through kinetic or dynamic kinetic resolution. Lipase is intrinsically ...(R)-selective, and it is difficult to obtain (S)-selective lipase. Recent crystal structures of a family VIII carboxylesterase have revealed that the spatial array of its catalytic triad is the mirror image of that of lipase but with a catalytic triad that is distinct from lipase. We, therefore, hypothesized that the family VIII carboxylesterase may exhibit (S)-enantioselectivity toward sec-alcohols similar to (S)-selective serine protease, whose catalytic triad is also spatially arrayed as its mirror image. In this study, a homologous enzyme (carboxylesterase from Proteobacteria bacterium SG_bin9, PBE) of a known family VIII carboxylesterase (pdb code: 4IVK) was prepared, which showed not only moderate (S)-selectivity toward sec-alcohols such as 3-butyn-2-ol and 1-phenylethyl alcohol but also (R)-selectivity toward particular sec-alcohols among the substrates explored. Furthermore, the (S)-selectivity of PBE has been significantly improved by rational redesign based on molecular modeling. Molecular modeling identified a binding pocket composed of Ser381, Ala383, and Arg408 for the methyl substituent of (R)-1-phenylethyl acetate and suggested that larger residues may increase the enantioselectivity by interfering with the binding of the slow-reacting enantiomer. As predicted, substituting Ser381with larger residues (Phe, Tyr, and Trp) significantly improved the (S)-selectivity of PBE toward all sec-alcohols explored, even the substrates toward which the wild-type PBE exhibits (R)-selectivity. For instance, the enantioselectivity toward 3-butyn-2-ol and 1-phenylethyl alcohol was improved from E = 5.5 and 36.1 to E = 2001 and 882, respectively, by single mutagenesis (S381F).
Processing of interleukin RNAs must be tightly controlled during the immune response. Here we report that a subnuclear body called the interleukin-6 and -10 splicing activating compartment (InSAC) is ...a nuclear site of cytokine RNA production and stability. Tat-activating regulatory DNA-binding protein-43 (TDP-43) acts as an InSAC scaffold that selectively associates with IL-6 and IL-10 RNAs in a sequence-specific manner. TDP-43 also recruits key spliceosomal components from Cajal bodies. LPS induces posttranslational modifications of TDP-43; in particular, TDP-43 ubiquitination provides a driving force for InSAC formation. As a consequence, in vivo depletion of TDP-43 leads to a dramatic reduction in the RNA processing and the protein levels of IL-6 in serum. Collectively, our findings highlight the importance of TDP-43-mediated InSAC biogenesis in immune regulation.
BackgroundChimeric antigen receptor (CAR) -T cell therapies have proven to be effective against various liquid tumors. However, the development of CAR-T against solid tumors has been challenging due ...to insufficient efficacy and potential on-target off-tumor toxicities caused by low expression of tumor antigens on normal tissues. Testing various affinities of CARs has demonstrated that lower affinity CARs maintain its anti-tumor effect while minimizing safety concerns (1). In order to develop a CAR-T against solid tumors expressing Mucin1, we have screened for Mucin1 binding antibodies and tested their anti-tumor effect in vitro and in vivo. The potential of on-target off-tumor toxicity was also measured in vitro.MethodsAnti-Mucin1 human single chain variable fragments (scFv) were obtained via screening against a scFv display library. Anti-Mucin1 scFvs were incorporated into CARs and in vitro, in vivo functions against various tumor cells expressing Mucin1 were tested. For in vivo studies, tumor bearing NOG mice (HCC1954 cells) received anti-Mucin1 CAR-T cells. Therapeutic efficacy was evaluated by measuring tumor volumes. Potential on-target off-tumor toxicity against Mucin1 on normal cells was tested by investigating the killing effect of anti-Mucin1 CAR-T against cancer cell line (HCC70) and non-tumorigenic breast epithelial cell line (MCF-10A) in co-culture systemsResultsIn vitro activity of anti-Mucin1 CAR-T cells that displayed a range of affinities for Mucin1 (27nM to 320nM) showed similar cytokine secretion levels and cytotoxicity against Mucin-1 expressing tumor cell lines (HCC70 and T47D). Robust anti-tumor activity was also demonstrated in vivo against large tumors (400~500 mm3) with relatively small numbers of CAR-T cells (0.5 x 106 CAR-T cells per mouse). In vivo expansion of CAR-T cells were observed in all scFv-CAR-T cases and accompanied by close to complete regression of tumors within 25 days post CAR-T cell injection. Of the 4 scFv CAR-Ts, 2H08 (with a Kd of 94nM) was tested for activity against normal breast epithelial cells. When 2H08-CAR-T was cocultured with a mixture of HCC70 and MCF-10A cells, they preferentially killed only the Mucin1 overexpressing HCC70 cells leaving MCF-10 cells intact.ConclusionsOur study demonstrates anti-tumor activity of a novel scFv-derived CAR-T recognizing Mucin1 and its effectiveness in large pre-established tumors in vivo. We also demonstrate that 2H08-CAR-T can distinguish between target overexpressing cancer cells and normal epithelial cells, which suggests that by toning down the affinity of CAR against antigen one can improve the safety profile of solid tumor antigen targeting CAR-T cell therapies.ReferenceCastellarin M, Sands C, Da T, Scholler J, Graham K, Buza E, Fraietta J, Zhao Y, June C. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight 2020; 5:e136012Ethics ApprovalAll experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) (Study#LGME21-011).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
•First cavitation-microstreaming-facilitated microfluidic chemical cell lysis and microbead-based DNA extraction.•Enhanced assay performance by agitation from bubbles oscillating at resonance ...frequency determined using the EMIS method.•Excellent performance of 77 % extraction efficiency and 1.85 purity on a par with that of a commercial kit.•Successful DNA extraction from as scarce as 18 cells and numbers of cells spanning 5 orders of magnitude.•Precise, rapid polycarbonate-chip fabrication (25 min) using optimized laser machining and solvent-assisted thermal bonding.
We for the first time present a microfluidic cavitation-microstreaming-based cell lysis and DNA extraction method. Chemical lysis and DNA extraction have been demonstrated in a microfluidic format but the performance is limited by ineffective mass transport due to low Reynolds number. Here we propose to employ cavitation microstreaming for enhancing chemical lysis and magnetic-bead-based dynamic solid phase extraction (dSPE) of DNA. Cavitation microstreaming condition is optimized by exciting a microfluidic chip at its flexural resonance frequency (fr) measured via electrical impedance spectroscopy. Strong circulatory flows around bubbles excited at fr yields vigorous agitation, allowing fast lysis, and DNA extraction and purification. The microfluidic device is rapidly fabricated using CO2-laser machining and solvent-assisted thermal bonding of polycarbonate (∼25 min). Laser cutting conditions are experimentally determined to achieve a clean sidewall for negligible nonspecific binding and minimal burrs for unobstructed bonding. Solvent exposure and thermal bonding conditions are also experimentally determined for a leakage-free device with excellent dimensional integrity. Our method, although not fully optimized, exhibits an excellent DNA extraction performance, compared to a commercial kit and previous microfluidic methods. High extraction efficiency (76.9 %) and purity (A260/A280 = 1.85) are achieved for a relatively short assay time (∼25 min). Notably, DNA from as few as 18 cells is successfully extracted even from a highly diluted cell sample (0.18 cells/μl). PCR and electrophoresis results confirm the excellent quality of the extracted DNA. Considering these notable performances, and straightforward fabrication and operation, we anticipate our DNA extraction method will be widely used in microfluidic nucleic-acid analysis devices.
Display omitted
It is known that Candida antarctica lipase B (CAL-B) exhibits low enantioselectivity towards sec-alcohols bearing small substituents such as (±)-but-3-yn-2-ol (E = 5.4) or ...(±)-butan-2-ol (E = 7), although it possesses high enantioselectivity towards various chiral sec-alcohols. In a previous study, we reported that the Ser47Asn variant, prepared based on the sequence of a homologous enzyme (lipase from Pseudozyma brasiliensis GHG001, PBL), exhibits high enantioselectivity towards (R)-but-3-yn-2-ol (E > 200). However, the molecular basis of high enantioselectivity was unclear because the residue is quite distant from the substituents, making it challenging to establish any interaction. In this study, we prepared a series of variants at the Ser47 residue to understand the molecular basis. We introduced Asp, Glu, Gln, and Leu at the position 47 and found that the Ser47Leu variant also exhibits high enantioselectivity (E > 200). We conducted a molecular dynamics study to elucidate its high enantioselectivity. The introduced leucine shifted the side chain of Trp104, which is a component for constructing the binding pocket for medium substituents, thereby interfering with the binding of the larger substituent of the slow enantiomer.
Abstract
The humoral immunity is mediated by antibodies produced by plasma cells, which are terminally differentiated effector cells of the B cell lineage. The antibody production should be tightly ...regulated to prevent unwanted responses against self-molecules and efficiently defend the host against foreign antigens. In this study, we aimed to examine the roles of PX domain-containing kinase in B cell-mediated immune responses. PX domain-containing kinase is a cytosolic protein composed of a PX domain, a pseudokinase domain, and a WH2 domain. It is expected that PX domain-containing kinase regulates intracellular trafficking of membrane proteins based on its structural features and previously reported functions. PX domain-containing kinase is most highly expressed in B cells both in humans and mice. Furthermore, several reports indicated that SNPs of the PX domain-containing kinase gene are linked to autoimmune diseases commonly characterized by excessive production of autoantibodies, such as systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis. However, the in vivo function of PX domain-containing kinase in immune responses has not been reported. In order to investigate the immunoregulatory functions of PX domain-containing kinase, we firstly generated PX domain-containing kinase-deficient mice. Although PX domain-containing kinase is not essential for the development of immune cells, we found that foreign antigen-induced antibody responses were affected by deletion of PX domain-containing kinase in mice. Currently, we are in the process of uncovering the underlying mechanisms. This study is expected to elucidate the functions of PXK as a novel modulator of antibody-mediated immune responses.
배추좀나방(Plutella xylostella)은 시설재배지를 중심으로 국내 자연 상태에서 월동한다. 안동지역에서 이른 봄부터 배추좀나방 성페로몬트 랩을 이용하여 배추좀나방의 성충 발생 시기를 주기적으로 조사한 결과 연중 4 회의 성충 발생 피크를 보였다. 월동 집단을 대상으로 서로 다른 지 역 집단 간 생물적 특성을 조사한 결과 내한성, 약제감수성 및 ...발육속도에서 뚜렷한 집단 특성을 나타냈다. 분자마커로 집단변이를 분석한 결과 월동세대의 높은 집단변이는 계절이 진행됨에 따라 낮아지는 양상을 나타냈다. 이 결과는 배추좀나방의 국내 월동 집단 사이의 생물적 특성 차이 를 나타냈고, 이들의 높은 유전적 분화는 계절이 진행됨에 따라 감소하여 이들 집단 사이의 개체들의 이동에 따른 유전적 교환이 이뤄졌다는 것을 제시했다.
The diamondback moth, Plutella xylostella, overwinters in some protected areas in Korea. Using a sex pheromone trap, the adults were monitored since the occurrence of the overwintering populations. In Andong, P. xylostella exhibited four adult peaks in a year. Biological characters, such as cold tolerance, insecticide susceptibility, and developmental rate, were analyzed and showed a significant variation among different local overwintering populations. Population genetic variation was assessed with molecular markers, in which the initial high genetic variation among the overwintering populations decreased with the progress of seasons. These results suggests that there may be a significant migration of P. xylostella to decrease the genetic variation among the different local populations that are different in biological characters.
PDF
