Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Design and Construction of His-tagged Protein Purification Workflow in Aquarium

Thesis (Master's)--University of Washington, 2019Protein purification is a long process starting from cloning, protein induction, extraction, isolation to protein verification. Carrying out this experiment can be very time-consuming and pricey, especially when some high-end instruments are required. Proper laboratory experiences and background knowledge of protein properties are also crucial for people who are conducting protein purification. As a result, the aforementioned considerations lead to the motivation of designing and building a robust protein purification workflow in Aquarium. The laboratory operating system, Aquarium, will compute required information such as protein concentration, sample amount, and buffer volume to yield step-by-step instructions for laboratory technicians for generating reproducible outcomes. The protein purification using poly-histidine-tag (His-tag) is implemented to provide an inexpensive purifying method. With Aquarium, the protocols are represented as executable code with negative controls. Moreover, the system automates multiple purifications in batches, which not only significantly saves time and budget, but also elevates the success rate. The success rate is verified by 10 identical purifications which are performed in 2 batches; each batch includes 5 purifications in parallel. This demonstrates that this workflow is capable of processing multiple batches of purification. The results of the test run also demonstrate this workflow is robust. The isopropyl-β-D-thio-galacto-pyranoside (IPTG) induced samples have a significant enrichment on the protein expression by comparing with uninduced samples. The high purity of purified proteins is verified by gel images of SDS-PAGE analysis as well as abundant protein yield is determined by NanoDrop. This workflow in Aquarium provides a robust, straightforward, economic and highly applicable tool to perform protein purification for further biomedical research

Radiation Recall Pneumonitis: A Rare Syndrome That Should Be Recognized

Radiation recall pneumonitis (RRP) is a rare but severe condition which has been mainly detected in the previously irradiated lung of patients with cancer after administering inciting agents, most commonly antineoplastic regimens including chemotherapy, targeted therapy, or immunotherapy. More recently, coronavirus disease vaccines were found to induce RRP. In addition to typical radiation pneumonitis (RP) or drug-induced interstitial lung disease, the management of RRP requires withholding inciting agents and steroid therapy. Thus, the occurrence of RRP could significantly impact cancer treatment, given that inciting agents are withheld temporarily and even discontinued permanently. In the present review, we discuss the current understanding and evidence on RRP and provide additional insights into this rare but severe disease

The Cytokine CXCL12 Promotes Basket Interneuron Inhibitory Synapses in the Medial Prefrontal Cortex

Prenatally, the cytokine CXCL12 regulates cortical interneuron migration, whereas its postnatal functions are poorly understood. Here, we report that CXCL12 is expressed postnatally in layer V pyramidal neurons and localizes on their cell bodies in the medial prefrontal cortex (mPFC), while its receptors CXCR4/CXCR7 localize to the axon terminals of parvalbumin (PV) interneurons. Conditionally eliminating CXCL12 in neonatal layer V pyramidal neurons led to decreased axon targeting and reduced inhibitory perisomatic synapses from PV+ basket interneurons onto layer V pyramidal neurons. Consequently, the mPFC of Cxcl12 conditional mutants displayed attenuated inhibitory postsynaptic currents onto layer V pyramidal neurons. Thus, postnatal CXCL12 signaling promotes a specific interneuron circuit that inhibits mPFC activity

Traditional Chinese Medicine Enhances Survival in Patients with Gastric Cancer after Surgery and Adjuvant Chemotherapy in Taiwan: A Nationwide Matched Cohort Study

Background. Gastric cancer remains a major cancer globally. More than half of patients with gastric cancer undergo surgery in Taiwan; however, few large nationwide studies have investigated the effects of traditional Chinese medicine (TCM) on gastric cancer management after surgery. This study aimed to evaluate the effect of TCM on patients with gastric cancer following surgery and adjuvant chemotherapy in Taiwan and its prescription trends. Methods and Materials. The cohort sampling data set was obtained from the Registry of Catastrophic Illness Patient Database, a research database of patients with severe illnesses from the National Health Insurance Research Database, Taiwan. Patients who had received a new diagnosis of gastric cancer and had undergone surgery were enrolled. We matched TCM users and nonusers at a ratio of 1 : 3 based on the propensity score, and TCM users were also grouped into short-term and long-term users. Results. The number of TCM users and nonusers was 1701 and 5103 after applying the propensity score at a ratio of 1 : 3. Short-term users and long-term TCM users were independently associated with a decreased risk of death with HRs of 0.59 (95% confidence interval (CI), 0.55–0.65) and 0.41 (95% CI, 0.36–0.47), respectively, compared with TCM nonusers. We also obtained similar results when we adjusted for covariates in the main model, as well as each of the additional listed covariates. We also observed similar HR trends in short-term users and long-term TCM users among men and women aged <65 years and ≥65 years. The most commonly prescribed single herb and herbal formula in our cohort were Hwang-Chyi (Radix Hedysari; 11.8%) and Xiang-Sha-Liu-Jun-Zi-Tang (15.5%), respectively. Conclusion. TCM use was associated with higher survival in patients with gastric cancer after surgery and adjuvant chemotherapy. TCM could be used as a complementary and alternative therapy in patients with gastric cancer after surgery and adjuvant chemotherapy

Arsenic phytotoxicity and accumulation in rice seedlings grown in arsenic-contaminated soils as influenced by the characteristics of organic matter amendments and soils

Organic matter (OM) application into soils is a common agricultural practice. Previous studies have shown that in arsenic (As)-contaminated paddy soils, OM has the potential to alter the behavior of As and affects the growth and As accumulation of rice plants. In this study, pot experiments were conducted to investigate the differences in the amounts of As released into soil solutions, its toxicity, and accumulation in rice seedlings caused by application of three different OM amendments [soybean meal (SB), sugarcane dreg compost (SC), and cattle-dung compost (CD)]. These OM amendments were each applied to three As-contaminated soils, Guandu (Gd), Pinchen (Pc), and Chengchung (Cf), which have different characteristics. The results indicate that after addition of two easily decomposable OMs (SB and SC), the As toxicity and concentrations increased in rice plants, especially in As-spiked Cf soils which had low As retention capacity. This was the result of elevated As concentration in soil solutions due to a decrease in soil redox potential and competition between dissolved organic carbon (DOC) and As for sorption sites, as well as the formation of As-DOC complexes. However, there were no significant effects on plant growth and As accumulation in rice seedlings after treatments with CD (not easily decomposable OM). Another important finding was that the amount of iron plaque on the surface of rice roots increased with OM amendments in the Gd soils rich in iron oxides and hydroxides, thus reducing the As uptake by rice plants. These results suggest that the characteristics of OM and soils should be considered when OM amendments are applied to As-contaminated soils

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies.

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo