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Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Successful Plasmodium falciparum merozoite invasion requires the activation of red blood cell (RBC) signalling pathways. The binding of parasite ligand reticulocyte binding protein homologue 5 (RH5) to its host receptor Basigin is essential for merozoite invasion and triggers a Ca2+ influx in RBCs. Here we observed that RH5-bound RBCs form a multimeric protein complex containing Basigin, CD44 and β2-adrenergic receptor (β2AR), suggesting that RH5-Basigin interaction is functionally associated with the host cAMP signalling pathway. Interestingly, we detected a characteristic rise in cAMP levels in the RBC upon RH5-Basigin interaction, which can be blocked by G protein and cAMP-synthesising adenylyl cyclase (AC) inhibitors. Furthermore, we demonstrated that RBC L-type Ca2+ channel inhibitor and cAMP signalling inhibitors are able to block merozoite invasion. Checkerboard invasion inhibition assay containing different combinations of signalling inhibitors also exhibited a drastic amplification of inhibition levels, indicating that these signalling proteins are functioning in a common signalling cascade to activate the L-type Ca2+ channels. Taken together, this study provides new insights into the role of a host cAMP-Ca2+ signalling pathway during merozoite invasion and sheds new light on antimalarial therapeutic strategies to tackle the high infection rate and growing threat of drug resistant parasites.Key PointsA pre-existing Basigin-associated membrane protein complex undergoes increased protein assembly upon RH5 binding on the RBC surface.Plasmodium falciparum merozoite exploits host cAMP signalling to initiate Ca2+ influx in the RBC.

177Lu-PSMA-617 is a promising radiotheranostic agent for metastatic castration-resistant prostate cancer (mCRPC) patients that is composed of prostate-specific membrane antigen (PSMA) conjugated with 177Lu. PSMA-INER-56 is a molecule with PSMA-617 targeting activity and an albumin-binding motif and extends the blood retention time. PSMA-INER-56 is radiolabeled with indium-111 (111In) as a diagnostic agent and with lutetium-177 (177Lu) as a therapeutic agent. This study determines the efficacy of molecular targeted imaging and the preliminary therapeutic effect of 111In/177Lu-PSMA-INER-56 in PSMA expressing mice that bear a prostate tumor mice. The stability of 111In-PSMA-INER-56 and 177Lu-PSMA-INER-56 is evaluated in normal saline and human serum. Head-to-head comparisons between 177Lu-PSMA-INER-56 and 177Lu-PSMA-617 use NanoSPECT/CT molecular imaging to determine the distribution, targeting and anti-tumor efficacy in LNCaP tumor-bearing mice. The radiochemical purity of 111In-PSMA-INER-56 or 177Lu-PSMA-INER-56 is 99.09 ± 0.38 and 98.87 ± 0.73%, respectively. The in vitro stability for both radio-labeled peptides is more than 95% up to 96 h. The greatest uptake of 111In-PSMA-INER-56 or 177Lu-PSMA-INER-56 in the tumor occurs at 24 h. The uptake is 43.78 ± 6.55 and 22.08 ± 6.63%ID/g, respectively. The 177Lu-PSMA-INER-56 more effectively inhibits tumor growth (96.0%) than 177Lu-PSMA-617 (54.7%) 44 days post injection (p.i). PSMA-INER-56 that is labeled with 111In or 177Lu is demonstrated to be a potential radiotheranostic pair for PSMA-positive tumors. 177Lu-PSMA-INER-56 also exhibits better targeting than 177Lu-PSMA-617.

BackgroundTo explore the optimal treatment strategy for patients who harbor sensitive EGFR mutations, a head-to-head study was performed to compare chemotherapy and gefitinib-erlotinip, osimertinib treatment in combination or with either agent alone as first-line therapy, in terms of efficacy and safety.MethodsA total of 200 untreated patients with advanced lung adenocarcinoma who harbored sensitive EGFR mutations were randomly assigned to receive gefitinib-erlotinip combined with pemetrexed and carboplatin group, gefitinib-erlotinip osimertinib combined with pemetrexed and carboplatin group, pemetrexed plus carboplatin alone group, or gefitinib-erlotinip alone group, osimertinib alone group.ResultsThe progression-free survival (PFS) of patients in the gefitinib-erlotinip combination group Mean Survival Time PFS 22.00 month,95%CI[16.29,27.70] and osimertinib gefitinib-erlotinip combination group Mean Survival Time PFS 40.00 month,95%CI[28.12,51.87]was longer than that of patients in the chemotherapy alone group PFS10,81 months, 95% CI,[ 8.99–12.64],gefitinib-erlotinip alone group PFS14.00 month.95%CI[11.98-20.01], osimertinib alone group PFS 26.66 month 95%CI[24.77-29.22].The gefitinib-erlotinip osimertinib combinational resulted in longer overall survival (OS) than chemotherapy alone (HR = 0.46, p = 0.016) or gefitinib-erlotinip alone (HR = 0.36, p = 0.01). osimertinib alone (HR = 0.26, p = 0.01).ConclusionsOur finding suggested that treatment with pemetrexed plus carboplatin combined with gefitinib-erlotinip and pemetrexed plus carboplatin combined with gefitinib-erlotinip osimertinib group could provide better survival benefits for patients with lung adenocarcinoma harboring sensitive EGFR mutations.

In this perspective, we outline that a space borne gravitational wave detector network combining LISA and Taiji can be used to measure the Hubble constant with an uncertainty less than 0.5% in ten years, compared with the network of the ground based gravitational wave detectors which can measure the Hubble constant within a 2% uncertainty in the next five years by the standard siren method. Taiji is a Chinese space borne gravitational wave detection mission planned for launch in the early 2030 s. The pilot satellite mission Taiji-1 has been launched in August 2019 to verify the feasibility of Taiji. The results of a few technologies tested on Taiji-1 are presented in this paper. Gravitational wave astronomy has opened the door to test general relativity and the effect of gravity in the Universe. The authors present the capabilities of an overlap between space gravitational wave detectors LISA and Taiji to constrain the Hubble constant to 0.5%, in 10 years, and what can be learned from the satellite pilot Taiji-1 launched in 2019.

Ya-Hong Zheng,1,* Chong-Yang Ren,2,* Ying Shen,3 Jia-Bin Li,1,4– 6 Ming-Wei Chen7 1Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China; 2Department of Neurology (Sleep Disorder), The Affiliated Chaohu Hospital of Anhui Medical University, Chaohu 238000, People’s Republic of China; 3Department of Endocrinology, Nanjing Tongren Hospital, Nanjing 211102, People’s Republic of China; 4Department of Infectious Diseases, The Affiliated Chaohu Hospital of Anhui Medical University, Chaohu 238000, People’s Republic of China; 5Anhui Center for Surveillance of Bacterial Resistance, Hefei 230022, People’s Republic of China; 6Institute of Bacterial Resistance, Anhui Medical University, Hefei 230022, People’s Republic of China; 7Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ming-Wei ChenDepartment of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of ChinaEmail chmw1@163.comJia-Bin LiDepartment of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of ChinaEmail lijiabin@ahmu.edu.cnObjective: This study explored the changes in the levels of IL-6, IL-17, TNF-α, and TNF-β, whether such changes were associated with anxiety and depression in diabetic peripheral neuropathy (DPN), and what factors associated with the occurrence of DPN.Methods: Forty-four patients diagnosed with DPN comprised the DPN group, including DPN1 (mild diabetic peripheral neuropathy, 29 cases) and DPN2 groups (moderate-severe diabetic peripheral neuropathy, 15 cases). Thirty-seven individuals with type 2 diabetes mellitus constituted the diabetes mellitus with no neuropathy (NDPN) group. Electromyography was applied to confirm DPN, and the Toronto clinical scoring system (TCSS) score was used to assess the severity of DPN. All subjects’ emotions were evaluated using the self-rating anxiety scale (SAS) and self-rating depression scale (SDS). Triiodothyronine (T3), tetraiodothyronine (T4), and thyroid-stimulating hormone (TSH) levels were measured using chemiluminescent immunoassay. The relevant biochemical indicators were detected using an automatic biochemical analyzer. The plasma levels of cytokines were detected using quantitative sandwich enzyme-linked immunosorbent assay.Results: Patients with DPN had elevated levels of anxiety, IL-6, IL-17, and TNF-α. There were some positive associations between negative emotions and cytokines. The TCSS score positively correlated with IL-17, SAS score, and T3. DPN independently correlated with age, disease duration, fasting plasma glucose (FPG), and IL-17. The combination of IL-17 and TNF-α had higher diagnostic value for DPN than any single cytokine.Conclusion: Patients with DPN had elevated levels of inflammatory cytokines, which were associated with negative emotion, and IL-17 had independent correlation with DPN.Keywords: diabetic peripheral neuropathy, emotions, cytokines

The prevalence of Helicobacter pylori infection is high worldwide, while numerous research has focused on unraveling the relationship between H. pylori infection and extragastric diseases. Although H. pylori infection has been associated with thyroid diseases, including thyroid nodule (TN), the relationship has mainly focused on potential physiological mechanisms and has not been validated by large population epidemiological investigations. Therefore, we thus designed a case-control study comprising participants who received regular health examination between 2017 and 2019. The cases and controls were diagnosed via ultrasound, while TN types were classified according to the guidelines of the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS). Moreover, H. pylori infection was determined by C14 urea breath test, while its relationship with TN type risk and severity was analyzed using binary and ordinal logistic regression analyses. A total of 43,411 participants, including 13,036 TN patients and 30,375 controls, were finally recruited in the study. The crude odds ratio (OR) was 1.07 in Model 1 (95% CI = 1.03–1.14) without adjustment compared to the H. pylori non-infection group. However, it was negative in Model 2 (OR = 1.02, 95% CI = 0.97–1.06) after being adjusted for gender, age, body mass index (BMI), and blood pressure and in Model 3 (OR = 1.01, 95% CI = 0.97–1.06) after being adjusted for total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein on the basis of Model 2. Control variables, including gender, age, BMI, and diastolic pressure, were significantly correlated with the risk of TN types. Additionally, ordinal logistic regression results revealed that H. pylori infection was positively correlated with malignant differentiation of TN (Model 1: OR = 1.06, 95% CI = 1.02–1.11), while Model 2 and Model 3 showed negative results (Model 2: OR = 1.01, 95% CI = 0.96–1.06; Model 3: OR = 1.01, 95% CI = 0.96–1.05). In conclusion, H. pylori infection was not significantly associated with both TN type risk and severity of its malignant differentiation. These findings provide relevant insights for correcting possible misconceptions regarding TN type pathogenesis and will help guide optimization of therapeutic strategies for thyroid diseases.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor—RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens. In response to infections, the human body produces proteins called antibodies. Neutralizing antibodies (NAbs) are one type of such proteins that are capable of inactivating the target, such as the SARS-CoV-2 virus that causes COVID-19. Monitoring levels of NAb allows us to understand levels of protective immunity. However, current methods to measure NAb are laboratory-based and are not necessarily suitable for large scale NAb monitoring in a large population. We develop a rapid test to detect SARS-CoV-2 NAb in 10 min that can be operated outside a laboratory. Our test provides results that are comparable to lab-based tests, which require between 1 h and up to 2 days to get a result. Our test may be useful for large-scale monitoring of immunity, for example in populations that do not have routine access to a lab. Kongsuphol et al. develop a paper-based, vertical flow assay to detect SARS-CoV-2 neutralizing antibodies. The point-of-care assay has comparable performance to lab-based tests and provides results in 10 min.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population. Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This study is supported by National Health Innovation Singapore (NHIC) grant # NHIC-COVID19-2005004, National Research Foundation via CREATE Share grant #R571-002-021-592 and the Anti-microbial Resistance Interdisciplinary Research Group (AMR-IRG) of Singapore-MIT Alliance in Research and Technology (SMART). All samples acquired from National Centre for Infectious Diseases (NCID) were supported by Singapore Ministry of Health's National Medical Research Council COVID-19 Research Fund: COVID19RF-0008.

The rapid increase in camping activities and campsites has had negative environmental impacts in mountainous areas. Tourism policies may be an important factor in changing recreational behavior and increasing campsites. The purpose of this study was to explore the effects of tourism policies on campsite-related landscape changes in Taiwan. The study area consisted of 276 campsites in the Jianshi and Wufeng Townships in Hsinchu County. The tourism policy periods were divided into 2001-2007 (Taiwan's agri-tourism policy), 2008-2015 (China and Taiwan's travel permit policy), and 2016-2019 (China's travel restriction policy), based on a reference review and relative theories. The 2000, 2008, 2016, and 2019 campsite landscapes were classified into forestland and non-forestland through object-based classification. This study established a general linear model to analyze the effect of tourism policy period on campsite forestland and non-forestland landscape change, according to the 50, 100, 250, 500, and 1000 m radii of 276 campsites. The results showed that tourism policies had a significant effect on campsite forestland and non-forestland landscape changes. The effect sizes ranged from medium to large. The Chinese tourist travel permit policy was significantly associated with increased non-forestland in campsites from 2008 to 2016. This policy likely affected recreational behavior indirectly, promoting camping and increasing non-forestland through the crowding-out effects of the many Chinese tourists, which was not the original purpose of the policy. Tourism policy decision-makers should consider the potential negative landscape change effects of changes in recreational behavior, and provide supporting measures to maintain recreational quality and avoid crowding-out effects. Campsite development should also be regulated to prevent forestland changes and achieve sustainable management.

We investigate the gas-phase photochemistry of the enolone tautomer of acetylacetone (pentane-2,4-dione) following S2(ππ*) ← S0 excitation at λ = 266 and 248 nm, using three complementary time-resolved spectroscopic methods. Contrary to earlier reports, which claimed to study one-photon excitation of acetylacetone and found OH and CH3 as the only important gas-phase products, we detect 15 unique primary photoproducts and demonstrate that five of them, including OH and CH3, arise solely by multiphoton excitation. We assign the one-photon products to six photochemical channels and show that the most significant pathway is phototautomerization to the diketone form, which is likely an intermediate in several of the other product channels. Furthermore, we measure the equilibrium constant of the tautomerization of the enolone to diketone on S0 from 320 to 600 K and extract Δ H = 4.1 ± 0.3 kcal·mol-1 and Δ S = 6.8 ± 0.5 cal·mol-1·K-1 using a van't Hoff analysis. We correct the C-OH bond dissociation energy in acetylacetone, previously determined as 90 kcal·mol-1 by theory and experiment, to a new value of 121.7 kcal·mol-1. Our experiments and electronic structure calculations provide evidence that some of the product channels, including phototautomerization, occur on S0, while others likely occur on excited triplet surfaces. Although the large oscillator strength of the S2 ← S0 transition results from the (ππ*) excitation of the C═C-C═O backbone, similar to conjugated polyenes, the participation of triplets in the dissociation pathways of acetylacetone appears to have more in common with ketone photochemistry.

Three new monoterpenoid indole alkaloids, kopsiaofficines A–D (1–3), were isolated from the 95% EtOH extract of the aerial parts of Kopsia officinalis. Their structures were established on the basi...

BackgroundThe third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive lung adenocarcinoma patients and more recently for first-line treatment of EGFR-mutant T790M negative lung adenocarcinoma, Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance.In this study,we investigated a the combination of osimertinib/ gefitinib/ erlotinib with cytotoxic chemotherapy for EGFR-mutated positive lung adenocarcinoma patients in long-term survival outcomes. Method We enrolled Ⅲb-IV stage lung adenocarcinoma patients with an EGFR mutation，Patients receiving standard Osimertinib,Gefitinib,Erlotinip alone treatment and Osimertinib,Gefitinib and Erlotinip with cytotoxic chemotherapy treatment were retrospectively reviewed.The performance status were collected,The response rate, progression-free survival (PFS) and overall survival (OS) and toxicity profile were analyzed.ResultBetween January 2014 to Dec 2020,240 patients with Ⅲb-Ⅳstages lung adenocarcinoma were enrolled from a institution.All patients who received different standard treatment respectively,were divided into four groups,64 who received（gefitinib or Erlotinb)with cytotoxic chemotherapy, 60 who received single gefitinib or erlotinib.58 who received (Osimertinib) with cytotoxic chemotherapy,58 who received single (Osimertinib) were eligible for this study.First generation Chemical-TKItherapy group PFS vs First generation TKI therapy alone PFS.Pp p (HR = 0.56 [0.35–0.89], p ConclusionFirst-Third generation EGFR inhibitor combined with cytotoxic chemotherapy represents a suitable palliative treatment option in further therapy lines for elderly patients with advanced lung adenocarcinoma.The results obtained under real-life conditions add to our understanding of the benefits and risks of First-Third generation EGFR inhibitor combined with cytotoxic chemotherapy in routine clinical practice.

The Arg–Gly–Asp (RGD) peptide shows a high affinity for αvβ3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvβ3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45's cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition. Published version This work was supported by a grant awarded from the Singapore Ministry of Education to Ruowen Ge (MOE2014-T2-2-150). DCK was supported by the Singapore International Graduate Award from the Agency for Science, Technology & Research and the National University of Singapore.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome. Ministry of Education (MOE) Submitted/Accepted version This research was supported by the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2016T22097). Work in the A.M. laboratory was supported by the European Regional Development Fund through the Centre of Excellence in Molecular Cell Engineering, Estonia, 2014-2020.4.01.15-013, and by The Wellcome Trust (200171/Z/15/Z).

Selected vibronic bands of the B ← X laser-induced fluorescence (LIF) spectra of jet-cooled 2-pentoxy and 2-hexoxy, including the origin and CO-stretch bands, have been measured with rotational resolution and analyzed using (1) an effective Hamiltonian that comprises a rotational part and a spin-rotation (SR) part (the "isolated-states model") and (2) a recently developed Hamiltonian in which the nearly degenerate A and X states are treated together (the "coupled-states model") (see Liu, J., J. Chem. Phys. 2018, 148, 124112). The observed rotational and fine structures of the strongest vibronic bands have first been simulated using a genetic algorithm with the isolated-states model. The parameters for the simulation include rotational constants for both the X and B states, which can be calculated from the electronic structure theory, as well as the electronic SR constants of the X state and the transition dipole moments (TDMs), both of which are predicted based on their transferability in an "orbital-fixed coordinate system" using iso-propoxy as the reference molecule. Quantum chemistry calculations suggest that the lowest two electronic (X and A) states of secondary alkoxy radicals have small energy separations on the order of 100 cm-1 (see Part I of this series: J. Phys. Chem. A 2021, DOI: 10.1021/acs.jpca.0c10662). The electron configurations of these two nearly degenerate states have been determined by comparing the experimentally determined rotational constants and the TDMs to the ones predicted for the X and A states. The experimental LIF spectra were also simulated with the coupled-states model, in which the effective spin-orbit (SO) constants (aζed) and the SO-free separation between the A and the X states (ΔE0) have been determined. Molecular constants derived from fitting the rotational and fine structures of the experimental LIF spectra enabled unambiguous assignment of the observed vibronic bands to specific conformers of 2-pentoxy and 2-hexoxy as reported in Part I.

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG. Ministry of Education (MOE) We would like to thank the Singapore Ministry ofEducation Academic Research Fund Tier 1 (Rg137/15)for the funding to GG. BS thanks the Nanyang Tech-nological University, Singapore, for awarding her aPhD Scholarship during her studies.

Chemical proteomics methods have been used as effective tools to identify novel protein targets for small molecules. These methods have great potential to be applied as environmental toxicants to figure out their mode of action. However, these assays usually generate dozens of possible targets, making it challenging to validate the most important one. In this study, we have integrated the cellular thermal shift assay (CETSA), quantitative proteomics, metabolomics, computer-assisted docking, and target validation methods to uncover the protein targets of monoethylhexyl phthalate (MEHP). Using the mass spectrometry implementation of CETSA (MS-CETSA), we have identified 74 possible protein targets of MEHP. The Gene Ontology (GO) enrichment integration was further conducted for the target proteins, the cellular dysregulated proteins, and the metabolites, showing that cell cycle dysregulation could be one primary change due to the MEHP-induced toxicity. Flow cytometry analysis confirmed that hepatocytes were arrested at the G1 stage due to the treatment with MEHP. Subsequently, the potential protein targets were ranked by their binding energy calculated from the computer-assisted docking with MEHP. In summary, we have demonstrated the development of interactomics workflow to simplify the redundant information from multiomics data and identified novel cell cycle regulatory protein targets (CPEB4, ANAPC5, and SPOUT1) for MEHP. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Ministry of Health (MOH) Nanyang Technological University National Environmental Agency (NEA) National Research Foundation (NRF) This work is supported by the Singapore Ministry of Education Academic Research Fund Tier 1 (04MNP000567C120), the NTU-Harvard SusNano (M4082370.030), the National Environment Agency-Singapore (04SBS000714N025), and the Singapore Ministry of Health’s National Medical Research Council under its Clinician-Scientist Individual Research Grant (CS-IRG) (MOH-000141) and the Open Fund-Individual Research Grant (OFIRG/0076/2018) to M.F. and A-STAR core fundings and NRF-SiS grant by National Research Foundation to R.M.S.

[Image: see text] We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated K(V)1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to K(V)1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for K(V)1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with (18)F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10–100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) contains an N-terminal RNA helicase with both nucleotide triphosphatase and RNA triphosphatase activities, and a C-terminal cysteine protease that is responsible for polyprotein processing. Both N-terminal RNA helicase and C-terminal cysteine protease are connected through a flexible linker. Although the structure of the C-terminal cysteine protease has been solved, the structure and the conformational arrangement of full-length nsP2 remains elusive. Here, we determined the crystal structure of the helicase part of the CHIKV nsP2 (nsP2h) bound to the conserved 3′-end of the genomic RNA and the nucleotide analogue ADP-AlF4. The structure of this ternary complex revealed the molecular basis for viral RNA recognition and ATP hydrolysis by the nsP2h. Unique hydrophobic protein–RNA interactions play essential roles in viral RNA replication. We also determined the solution structure of full-length nsP2 using small-angle X-ray scattering (SAXS). The solution architecture of the nsP2 was modeled using the available high-resolution structures and program CORAL (complexes with random loops). The CORAL model revealed that nsP2 is partially unfolded and the N-terminal protease domain is arranged near the N-terminal domain of the helicase domain. These findings expand our knowledge of CHIKV and related alphaviruses and might also have broad implications for antiviral and vaccine developments against pathogenic alphaviruses.

Flavivirus is a genus of the Flaviviridae family which includes significant emerging and re-emerging human disease-causing arboviruses such as dengue and Zika viruses. Flaviviral non-structural protein 3 (NS3) protease-helicase plays essential roles in viral replication and is an attractive antiviral target. A construct which connects the cytoplasmic cofactor region of NS2B and NS3 protease with an artificial glycine-rich flexible linker has been widely used for structural, biochemical and drug-screening studies. The effect of this linker on the dynamics and enzymatic activity of the protease has been studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed and carried out a comparative study of constructs of NS2B cofactor joined to the full length DENV4 NS3 in three different ways, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich linker). We report the crystal structures of linked and unlinked NS2B47-NS3 constructs in their free state and in complex with bovine pancreatic trypsin inhibitor (BPTI). These structures demonstrate that the NS2B cofactor predominantly adopts a closed conformation in complex with full-length NS3. The glycine-rich linker between NS2B and NS3 may promote the open conformation which interferes with protease activity. This negative impact on the enzyme structure and function is restricted to the protease activity as the ATPase activity is not affected in vitro. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) This work was supported by (1) the start-up grant to DL lab from Lee Kong Chian School of Medicine, Nanyang Technological University, (2) Ministry of Education grant MOE2016-T2-2-097 to DL lab, (3) National Medical Research Council grant NMRC/CBRG/0103/2016 to SV lab, (4) National Research Foundation grant NRF2016NRFCRP001-063. Ms. Wint Wint Phoo was supported by Nanyang Research Scholarship, Nanyang Technological University.

To explore the effect of miR-100 on the migration of rat bone marrow mesenchymal stem cells(rBMMSC).The rBMMSC were isolated by density gradient centrifugation, cell surface epitopes of CD105, CD45, CD34, CD29 and CD44 were analyzed by flow cytometry. The rBMMSC were transfected with miR-100 mimic or inhibitor, then the expression of miR-100 in transfected cells was detected by real-time PCR. Migration test was used to observe the effect of miR-100 on cell migration ability. The secretion level of chemokine SDF-1 in culture supernatant of cells was quantitatively detected by ELISA.The isolated cells were identified as BMMSC. After rBMMSC were transfected with miR-100 mimic or inhibitor, as compared with control group,the expression of miR-100 in rBMMSC significantly increased or decreased respectively. In the migration experiment, the rBMMSC migration was significantly inhibited in the miR-100 mimic group (P0.01), while the rBMMSC migration was significantly enhanced in the miR-100 inhibitor group (P0.01). The concentration of SDF-1 in the supernatant of the miR-100 mimic group and the miR-100 inhibitor group did not change significantly compared with the control group (P0.05).miR-100 can significantly inhibit the migration of rBMMSC, but not significantly correlated with the SDF-1.miR-100对大鼠骨髓间充质干细胞迁移能力的影响.探讨MicroRNA-100（miR-100）对大鼠骨髓间充质干细胞（rBMMSC）迁移能力的影响.分离培养rBMMSC，采用流式细胞术检测rBMMSC表面标志物CD105、CD45、CD34、CD29和CD44的表达。用miR-100类似物或抑制剂转染rBMMSC，应用实时定量PCR检测转染后细胞miR-100表达量。迁移实验测定转染后细胞迁移能力，观察miR-100对细胞迁移能力的影响。ELISA法定量检测培养上清中趋化因子SDF-1的分泌水平.经鉴定所分离的细胞确定为rBMMSC。用miR-100类似物或抑制剂转染rBMMSC后，与对照组相比，转染miR-100类似物的细胞miR-100表达显著上调(P0.01)，转染miR-100抑制剂的细胞miR-100表达显著下调(P0.01)。在迁移实验中，转染miR-100类似物可显著抑制rBMMSC迁移(P0.01)，而转染miR-100抑制剂可显著增强rBMMSC迁移(P0.01)。转染miR-100类似物组、转染miR-100抑制剂组的培养上清中，SDF-1浓度与对照组相比未见明显变化（P＞0.05）.miR-100可显著抑制rBMMSC的迁移，但与趋化因子SDF-1无明显关系.

Background Lung adenocarcinoma (ADC) is a major form of lung cancer, which is a main cause of global cancer-related death in male and female patients. LncRNAs are implicated in tumor development. However, the functions and mechanisms of the LncRNA HOTAIRM1 in ADC are not known. Materials and Methods Here, the downregulated HOTAIRM1 in ADC was selected by TCGA analysis. Subsequently, qRT-PCR, CCK-8, EdU, cell apoptosis, cell cycle and cell invasion assays were utilized for evaluating the roles of HOTAIRM1 in ADC. Finally, we explored the mechanism of HOTAIRM1 in ADC. Results HOTAIRM1 expression was considerably decreased in ADC tissues. The knockdown of HOTAIRM1 promoted the cell cycle, growth, and invasion of ADC. Moreover, HOTAIRM1 competitively bound miR-498 to regulate the expression of WWOX. Conclusion HOTAIRM1 suppressed the proliferation and invasion of ADC cells via the modulation of miR-498/WWOX axis. This finding suggested that it might be clinically valuable as a biomarker for ADC. Furthermore, the findings suggest LncRNA HOTAIRM1 as a candidate therapeutic target in ADC.

A two dimensional silicon carbide fiber reinforced SiC matrix (2D SiC/SiC) composite fabricated by precursor infiltration pyrolysis (PIP) process used a liquid SiC ceramic precursor was obtained. Two key properties including matrix cracking stress and thermal residual stress were investigated for this PIP 2D SiC/SiC composites. Three methods were applied to determine the matrix cracking stress in order to obtained a trusted value, and the value of matrix cracking stress for SiC/SiC composite was 75±4 MPa. The thermal residual stress of the composites was calculated by linear regression line according to the loading-unloading-reloading stress-strain curve of the 2D SiC/SiC composite, and the result showed that the value of thermal residual stress of SiC matrix in composite was 20MPa, which means the PIP SiC matrix in the 2D SiC/SiC composite was under the compressive stress when the composite cooling down from the fabrication temperature to the room temperature.

A ZrB2-SiC multiphase ceramic was fabricated with a precursor pyrolysis method by using zirconium boride organic precursor and polycarbosilane. In order to study the pyrolysis process of complex phase ceramics, the pyrolysis process of the two precursors was studied respectively at the beginning. The precursor and the mixture with different weight ratios both pyrolyzed at 800°C. ~1500°C. under argon atmosphere. The specimens were characterized by thermo-gravimetric analysis, infra-red, X-ray diffraction. It was found that the pyrolysis products of PCS remained amorphous below 1000°C. and converted into crystallization with temperature increasing. ZrO2as the intermediate pyrolysis product of ZrB2precursor initiate convert to t-ZrO2from m-ZrO2at 1200°C.. Finally, all kinds of ZrB2-SiC multiphase ceramic are successful obtained via pyrolysis of precursor after 1500°C. And the 40wt%ZrB2precursor initial carbothermal reduction temperature below the ZrB2precursor reactive temperature and the crystal degree of SiC is higher than pyrolysis product of PCS.

In order to obtain the near net shaped SiC/SiC composite pins without the processing cost, four kinds of SiC fiber pin preforms including the knitting yarn and core yarn with two dimension braided structure were designed. The SiC/SiC composite pins were fabricated by precursor infiltration pyrolysis (PIP) process used a liquid SiC ceramic precursor. The results showed that, the near net shaped SiC/SiC composite pins could be obtained by using the designed SiC fiber preform and the graphite mould with curved groove. The macrostructure and microstructure of the SiC/SiC composite pins were observed by scanning electron microscope. Both the single shear stress-displacement curves and the double shear stress-displacement curves of the different kinds of SiC/SiC composite pins showed the saddle-shaped, which indicated the fracture model of the SiC/SiC composites pins was non-catastrophic, and the SiC/SiC composite pins in this paper possessed the secondary bearing capacity. This paper may provide some new ideas for fiber reinforced SiC ceramic matrix composites components designers.

Formation of the key general radical chain carriers, ˙OH and HO2˙, during pulsed-photolytic ˙Cl-initiated oxidation of tetrahydropyran and cyclohexane are measured with time-resolved infrared absorption in a temperature-controlled Herriott multipass cell in the temperature range of 500–750 K at 20 Torr. The experiments show two distinct timescales for HO2˙ and ˙OH formation in the oxidation of both fuels. Analysis of the timescales reveals striking differences in behavior between the two fuels. In both cyclohexane and tetrahydropyran oxidation, a faster timescale is strongly related to the “well-skipping” (˙R + O2 → alkene + HO2˙ or cyclic ether + ˙OH) mechanism and is expected to have, at most, a weak temperature dependence. Indeed, the fast HO2˙ formation timescale is nearly temperature independent both for cyclohexyl + O2 and for tetrahydropyranyl + O2 below 700 K. A slower HO2˙ formation timescale in cyclohexane oxidation is shown to be linked to the sequential ˙R + O2 → ROO˙ → alkene + HO2˙ pathway, and displays a strong temperature dependence mainly from the final step (with energy barrier ∼32.5 kcal mol−1). In contrast, the slower HO2˙ formation timescale in tetrahydropyran oxidation is surprisingly temperature insensitive across all measured temperatures. Although the ˙OH formation timescales in tetrahydropyran oxidation show a temperature dependence similar to the cyclohexane oxidation, the temperature dependence of ˙OH yield is opposite in both cases. This significant difference of HO2˙ formation kinetics and ˙OH formation yield for the tetrahydropyran oxidation can arise from contributions related to ring-opening pathways in the tetrahydropyranyl + O2 system that compete with the typical ˙R + O2 reaction scheme. This comparison of two similar fuels demonstrates the consequences of differing chemical mechanisms on ˙OH and HO2˙ formation and shows that they can be highlighted by analysis of the eigenvalues of a system of simplified kinetic equations for the alkylperoxy-centered ˙R + O2 reaction pathways. We suggest that such analysis can be more generally applied to complex or poorly known oxidation systems.