MA-MIL: Sampling point-level abnormal ECG location method via weakly supervised learning.

BACKGROUND AND OBJECTIVE: Current automatic electrocardiogram (ECG) diagnostic systems could provide classification outcomes but often lack explanations for these results. This limitation hampers their application in clinical diagnoses. Previous supervised learning could not highlight abnormal segmentation output accurately enough for clinical application without manual labeling of large ECG datasets. METHOD: In this study, we present a multi-instance learning framework called MA-MIL, which has designed a multi-layer and multi-instance structure that is aggregated step by step at different scales. We evaluated our method using the public MIT-BIH dataset and our private dataset. RESULTS: The results show that our model performed well in both ECG classification output and heartbeat level, sub-heartbeat level abnormal segment detection, with accuracy and F1 scores of 0.987 and 0.986 for ECG classification and 0.968 and 0.949 for heartbeat level abnormal detection, respectively. Compared to visualization methods, the IoU values of MA-MIL improved by at least 17 % and at most 31 % across all categories. CONCLUSIONS: MA-MIL could accurately locate the abnormal ECG segment, offering more trustworthy results for clinical application.

Collectin-K1 Plays a Role in the Clearance of Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus).

Collectin-K1 (CL-K1) is a multifunctional C-type lectin that has been identified as playing a crucial role in innate immunity. It can bind to carbohydrates on pathogens, leading to direct neutralization, agglutination, and/or opsonization, thereby inhibiting pathogenic infection. In this study, we investigated a homolog of CL-K1 (OnCL-K1) in Nile tilapia (Oreochromis niloticus) and its role in promoting the clearance of the pathogen Streptococcus agalactiae (S. agalactiae) and enhancing the antibacterial ability of the fish. Our analysis of bacterial load displayed that OnCL-K1 substantially reduced the amount of S. agalactiae in tissues of the liver, spleen, anterior kidney, and brain in Nile tilapia. Furthermore, examination of tissue sections revealed that OnCL-K1 effectively alleviated tissue damage and inflammatory response in the liver, anterior kidney, spleen, and brain tissue of tilapia following S. agalactiae infection. Additionally, OnCL-K1 was found to decrease the expression of the pro-inflammatory factor IL-6 and migration inhibitor MIF, while increasing the expression of anti-inflammatory factor IL-10 and chemokine IL-8 in the spleen, anterior kidney, and brain tissues of tilapia. Moreover, statistical analysis of survival rates demonstrated that OnCL-K1 significantly improved the survival rate of tilapia after infection, with a survival rate of 90%. Collectively, our findings suggest that OnCL-K1 plays a vital role in the innate immune defense of resisting bacterial infection in Nile tilapia. It promotes the removal of bacterial pathogens from the host, inhibits pathogen proliferation in vivo, reduces damage to host tissues caused by pathogens, and improves the survival rate of the host.

Structurally Diverse Bisbenzylisoquinoline Alkaloids with Antiadipogenic Activity through PPARγ Downregulation from the Embryo of Nelumbo nucifera Seeds.

Six undescribed and six known bisbenzylisoquinoline alkaloids were isolated from the embryo of Nelumbo nucifera seeds. Their structures were fully characterized by a combination of 1H, 13C NMR, 2D NMR, and HRESIMS analyses, as well as ECD computational calculations. The antiadipogenic activity of 11 alkaloids was observed in a dose-responsive manner, leading to the suppression of lipid accumulation in 3T3-L1 cells. Luciferase assay and Western blot analysis showed that the active alkaloids downregulated peroxisome proliferator-activated receptor gamma (PPARγ, a key antiadipogenic receptor) expression in 3T3-L1 cells. Analysis of the structure-activity relationship unveiled that a 1R,1'S configuration in bisbenzylisoquinoline alkaloids led to a notable enhancement in antiadipogenic activity. The resistance level against lipid accumulation highlighted a consistent pattern with the suppressive effect on the PPARγ expression. These activity results indicate that alkaloids from the embryo of N. nucifera seeds have a potential of antiobesity effects through PPARγ downregulation.

Adoptive cell therapy for solid tumors beyond CAR-T: Current challenges and emerging therapeutic advances.

Adoptive cellular immunotherapy using immune cells expressing chimeric antigen receptors (CARs) is a highly specific anti-tumor immunotherapy that has shown promise in the treatment of hematological malignancies. However, there has been a slow progress toward the treatment of solid tumors owing to the complex tumor microenvironment that affects the localization and killing ability of the CAR cells. Solid tumors with a strong immunosuppressive microenvironment and complex vascular system are unaffected by CAR cell infiltration and attack. To improve their efficacy toward solid tumors, CAR cells have been modified and upgraded by "decorating" and "pruning". This review focuses on the structure and function of CARs, the immune cells that can be engineered by CARs and the transformation strategies to overcome solid tumors, with a view to broadening ideas for the better application of CAR cell therapy for the treatment of solid tumors.

Hollow Copper Sulfide Photothermal Nanodelivery Platform Boosts Angiogenesis of Diabetic Wound by Scavenging Reactive Oxygen Species.

Sharply rising oxidative stress and ineffectual angiogenesis have imposed restrictions on diabetic wound healing. Here, a photothermal-responsive nanodelivery platform (HHC) was prepared by peroxidase (CAT)-loaded hollow copper sulfide dispersed in photocurable methacrylamide hyaluronan. The HHC could scavenge reactive oxygen species (ROS) and promote angiogenesis by photothermally driven CAT and Cu2+ release. Under near-infrared light irradiation, the HHC presented safe photothermal performance (<43 °C), efficient bacteriostatic ability against E. coli and S. aureus. It could rapidly release CAT into the external environment for decomposing H2O2 and oxygen generation to alleviate oxidative stress while promoting fibroblast migration and VEGF protein expression of endothelial cells by reducing intracellular ROS levels. The nanodelivery platform presented satisfactory therapeutic effects on murine diabetic wound healing by modulating tissue inflammation, promoting collagen deposition and increasing vascularization in the neodermis. This HHC provided a viable strategy for diabetic wound dressing design.

An integrated wearable sticker based on extended-gate AlGaN/GaN high electron mobility transistors for real-time cortisol detection in human sweat.

Cortisol hormone imbalances can be detected through non-invasive sweat monitoring using field-effect transistor (FET) biosensors, which provide rapid and sensitive detection. However, challenges like skin compatibility and integration with sweat collection have hindered FET biosensors as wearable sensing platforms. In this study, we present an integrated wearable sticker for real-time cortisol detection based on an extended-gate AlGaN/GaN high electron mobility transistor (HEMT) combined with a soft bottom substrate and flexible channel for sweat collection. The developed devices exhibit excellent linearity (R2 = 0.990) and a high sensitivity of 1.245 µA dec-1 for cortisol sensing from 1 nM to 100 µM in high-ionic-strength solution, with successful cortisol detection demonstrated using authentic human sweat samples. Additionally, the chip's microminiature design effectively reduces bending impact during the wearable process of traditional soft binding sweat sensors. The extendedgate structure design of the HEMT chip enhances both width-to-length ratio and active sensing area, resulting in an exceptionally low detection limit of 100 fM. Futhermore, due to GaN material's inherent stability, this device exhibits long-term stability with sustained performance within a certain attenuation range even after 60 days. These stickers possess small, lightweight, and portable features that enable real-time cortisol detection within 5 minutes through direct sweat collection. The application of this technology holds great potential in the field of personal health management, facilitating users to conveniently monitor their mental and physical conditions.

CL-K1 Promotes Complement Activation and Regulates Opsonophagocytosis of Macrophages with CD93 Interaction in a Primitive Vertebrate.

Collectin is a crucial component of the innate immune system and plays a vital role in the initial line of defense against pathogen infection. In mammals, collectin kidney 1 (CL-K1) is a soluble collectin that has recently been identified to have significant functions in host defense. However, the evolutionary origins of immune defense of CL-K1 and its mechanism in clearance of pathogenic microorganisms remain unclear, especially in early vertebrates. In this study, the Oreochromis niloticus CL-K1 (OnCL-K1) protein was purified and identified, which was capable of binding to two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila. Interestingly, OnCL-K1 exhibited direct bactericidal activity by binding to lipoteichoic acid or LPS on cell walls, disrupting the permeability and integrity of the bacterial membrane in vitro. Upon bacterial challenge, OnCL-K1 significantly inhibited the proliferation of pathogenic bacteria, reduced the inflammatory response, and improved the survival of tilapia. Further research revealed that OnCL-K1 could associate with OnMASPs to initiate and regulate the lectin complement pathway. Additionally, OnCD93 reduced the complement-mediated hemolysis by competing with OnMASPs for binding to OnCL-K1. More importantly, OnCL-K1 could facilitate phagocytosis by collaborating with cell surface CD93 in a lectin pathway-independent manner. Moreover, OnCL-K1 also promoted the formation of phagolysosomes, which degraded and killed ingested bacteria. Therefore, this study reveals the antibacterial response mechanism of CL-K1 in primitive vertebrates, including promoting complement activation, enhancing opsonophagocytosis, and killing of macrophages, as well as its internal links, all of which provide (to our knowledge) new insights into the understanding of the evolutionary origins and regulatory roles of the collectins in innate immunity.

Intraoperative Protection of Pharyngeal Autonomic Nerves: Preventing Dysphagia After Anterior Cervical Decompression and Fusion Surgery.

OBJECTIVE: The study aims to investigate whether intraoperative protection of the pharyngeal autonomic nerve can effectively reduce the incidence of postoperative dysphagia following anterior cervical decompression and fusion surgery (ACDF). METHODS: A retrospective analysis was conducted on 130 cases that underwent ACDF from January 2018 to June 2022 at our hospital. Divided into nonautonomic neuroprotection (NANP) group and autonomic neuroprotection group based on whether receive protective measures for the pharyngeal autonomic nerve during surgery. General data were recorded and compared between the 2 groups. Postoperative outcomes were evaluated using Neck Disability Index, Japanese Orthopaedics Association (JOA) score, and JOA improvement rate. The incidence and severity of postoperative dysphagia were assessed using Bazaz dysphagia assessment criteria and swallowing-quality of life questionnaire. RESULTS: There were no significant differences in general data (P > 0.05). The average operation time and intraoperative blood loss also showed no significant differences (P > 0.05). Both groups showed significant improvements in Neck Disability Index and JOA scores at all follow-up time points compared to preoperative scores (P < 0.01). The incidence of postoperative dysphagia in the autonomic neuroprotection group was significantly lower than that in the NANP group at all follow-up time points (P < 0.05). Both group showed a significant reduction in scores 3 days postoperatively compared to preoperative scores (P < 0.01), and the NANP group also showed significant reductions in scores at 3 month and 1 year postoperative follow-up time points compared to preoperative scores (P < 0.01). CONCLUSIONS: The adoption of pharyngeal autonomic nerve protective measures during ACDF can effectively lower the probability of postoperative dysphagia.

Molecular Evolution of Antiparathion Nanobody with Enhanced Sensitivity and Specificity Based on Structural Analysis.

Nanobody (Nb) has gained significant attention in immunoassays owing to its numerous advantages, particularly its ease of molecular evolution. However, the limited understanding of how high sensitivity and specificity attained for antihapten Nbs hamper the development of high-performance Nbs. Herein, the antiparathion Nb (Nb9) we prepared previously was chosen as the model, and an approach based on X-ray crystallography, molecular docking, and rational site-directed saturation mutation for constructing a rapid and effective platform for nanobody evolution was described. Based on the structural analysis, two mutants, namely Nb-D5 (IC50 = 2.4 ± 0.2 ng/mL) and Nb-D12 (IC50 = 2.7 ± 0.1 ng/mL), were selected out from a six-sites directed saturation mutation library, 3.5-fold and 3.1-fold sensitivity enhancement over Nb9 to parathion, respectively. Besides, Nb-D12 exhibited improved sensitivity for quinalphos, triazophos, and coumaphos (5.4-35.4 ng/mL), indicating its broader detection potential. Overall, our study advances an effective strategy for the future rational evolution of Nbs with desirable performance.

ß-Glucans in particulate and solubilized forms elicit varied immunomodulatory and apoptosis effects in teleost macrophages in a dosedependent manner.

ß-Glucans are a group of heterogeneous glucose polymers that possess immunomodulatory activities. The complex nature of their structures, uncertainty regarding the doses, and variable immune effects pose a challenge to comprehensive understanding. In this study, we investigated the immune responses and apoptosis effects in Nile tilapia (Oreochromis niloticus) head kidney macrophages (MФ) upon exposure to two ß-Glucans (Paramylon and Laminarin) at low and high doses. Our results demonstrate that Paramylon elicits more robust immune responses than Laminarin, albeit with a dose-limiting effect. We also observed that the high-dose Paramylon induces apoptosis, whereas no such effect was detected in Laminarin treatment. Mechanistically, high-dose Paramylon activates the intrinsic apoptosis pathway, with significantly up-regulation of intrinsic apoptosis-related genes and impaired mitochondrial function. On the other hand, Laminarin triggers metabolic reprogramming in MФ, resulting in the enrichment of the metabolite α-Ketoglutarate, which protects the MФ from apoptosis. Overall, our findings highlight the importance of identifying the optimal dose range for ß-Glucans, based on sources or structures, to achieve maximal immunomodulatory effects. These results have important implications for the design and optimization of ß-Glucans-based drugs or adjuvants in immunotherapies.

Long-chain pentraxin 3 possesses agglutination activity and plays a role in host defense against bacterial infection in Oreochromis niloticus.

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule in the innate immune system that has multiple functions. It is involved in resisting pathogen infection. However, the functions of PTX3 in teleost fish are not well understood. In this study, we identified and characterized PTX3 in Nile tilapia (Oreochromis niloticus) (OnPTX3). The open reading frame of OnPTX3 was found to be 1305 bp, encoding 434 aa. We conducted spatial mRNA expression analysis and found that the expression of OnPTX3 was significantly increased after infection with Streptococcus agalactiae and Aeromonas hydrophila, both in vivo and in vitro. We also observed that recombinant OnPTX3 protein could bind and agglutinate bacterial pathogen. Furthermore, OnPTX3 enhanced the phagocytosis of bacteria (S. agalactiae and A. hydrophila) by head kidney macrophages. Additionally, OnPTX3 was found to influence the expression of inflammatory cytokines, suggesting its involvement in the regulation of the inflammatory response. Moreover, OnPTX3 was shown to promote complement-mediated hemolysis and possess antibacterial activity. In conclusion, our research demonstrates that OnPTX3 has bacterial binding and agglutination activities, enhances phagocytosis, and regulates inflammation. It plays a crucial role in the defense of Nile tilapia against pathogenic bacteria, providing valuable insights for the prevention and control of aquatic diseases in the future.

Real-time free spectral range measurement based on a correlated resonance-tracking technology.

In this paper, we present a real-time measurement technology for the free spectral range (FSR) of an ultrahigh-aspect-ratio silicon nitride (Si3N4) waveguide ring resonator (WRR). Two different correlated resonant modes were tracked by two optical single-sideband frequency-shifted lights to eliminate interference noise in the Pound-Drever-Hall error signals. A relative precision of 0.1474 ppm was achieved for a 35 mm WRR with FSR = 1,844,944.5 kHz and finesse (F) = 13.2. Furthermore, a cross-correlation of 0.913 between FSR-calculated and thermistor-measured temperatures indicated a high correlation between the real-time FSR and room temperature. We believe this technology is currently the best way to realize low-finesse (F < 50) real-time FSR measurements in the GHz range.

Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.

Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.

Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

Mannose-binding lectin suppresses macrophage proliferation through TGF-ß1 signaling pathway in Nile tilapia.

Mannose-binding lectin (MBL) is a multifunctional pattern recognition molecule, which not only mediates the recognition of pathogenic microorganisms and their products, playing an important role in innate immune defense, but also participates in adaptive immune responses of mammalian. However, it's related immune mechanism remains limited, especially the regulation of cell proliferation in early vertebrates. In this study, OnMBL was found to bind to kidney macrophages (MФ) from Nile tilapia (Oreochromis niloticus). Interestingly, OnMBL was able to reduce the proliferation of activated-MФ by regulating the cell cycle, arresting a large number of cells in the G0/G1 phase, and increasing the probability of apoptosis. More importantly, we found that the inhibition of cell proliferation by OnMBL was closely related to the evolutionarily conserved canonical transforming growth factor-beta 1 (TGF-ß1) signaling pathway. Mechanistically, OnMBL could significantly increase the expression of TGF-ß1, activate and regulate the downstream Smad-dependent pathway to reduce the MФ proliferation, thereby maintaining cellular homeostasis in the body's internal environment. This study represents the first description regarding the regulatory mechanisms of the MBL on cell proliferation in teleost fish, which provides a novel perspective on the understanding of the multiple function and evolutionary origins of C-type lectins in the immune system.

Endothelial Stat3 activation promotes osteoarthritis development.

The mechanism of the balance between subchondral angiogenesis and articular damage within osteoarthritis (OA) progression remains a mystery. However, the lack of specific drugs leads to limited clinical treatment options for OA, frequently failing to prevent eventual joint destruction in patients. Increasing evidence suggests that subchondral bone angiogenesis precedes cartilage injury, while proliferating endothelial cells (ECs) induce abnormal bone formation. Signal transducer and activator of transcription 3 (Stat3) is triggered by multiple cytokines in the OA microenvironment. Here, we observed elevated Stat3 activation in subchondral bone H-type vessels. Endothelial Stat3 activation will lead to stronger cell proliferation, migration and angiogenesis by simulating ECs in OA. In contrast, either Stat3 activation inhibition or knockdown of Stat3 expression could relieve such alterations. More interestingly, blocking Stat3 in ECs alleviated angiogenesis-mediated osteogenic differentiation and chondrocyte lesions. Stat3 inhibitor reversed surgically induced subchondral bone H-type vessel hyperplasia in vivo, significantly downregulating vessel volume and vessel number. Due to the reduced angiogenesis, subchondral bone deterioration and cartilage loss were alleviated. Overall, our data suggest that endothelial Stat3 activation is an essential trigger for OA development. Therefore, targeted Stat3 blockade is a novel promising therapeutic regimen for OA.

Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

Discovery of anti-allergic components in Guomingkang Formula using sensitive HEMT biochips coupled with in vitro and in vivo validation.

BACKGROUND: Allergic rhinitis (AR) is a prevalent allergic disease, which seriously affects the sufferers' life quality and increases the socioeconomic burden. Guominkang (GMK), a well-known prescription for AR treatment, showed satisfactory effects; while its anti-allergic components remain to be disclosed. AlGaN/GaN HEMT biochip is more sensitive and cost-effective than other binding equipments, indicating its great potential for screening of active ingredients from herbal medicines. METHODS: AR mouse models were first established to test the anti-allergic effect of GMK and discover the ingredients absorbed into blood by ultra-high performance liquid chromatography-mass spectra (UHPLC-MS). Then, novel Syk/Lyn/Fyn-functionalized high electron mobility transistor (HEMT) biochips with high sensitivity and specificity were constructed and applied to screen the active components. Finally, the results from HEMT biochips screening were validated via in silico (molecular docking and molecular dynamics simulation), in vitro (RBL-2H3 cells), and in vivo (PCA mice model) assays. RESULTS: GMK showed a potent therapeutic effect on AR mice, and fifteen components were identified from the medicated plasma. Furthermore, hamaudol was firstly found to selectively inhibit the Syk and Lyn, and emodin was to selectively inhibit Lyn, which were further confirmed by isothermal titration calorimetry, molecular docking, and molecular dynamics simulation analyses. Suppression of the activation of FcεRI-MAPK signals might be the possible mechanism of the anti-allergic effect of hamaudol. CONCLUSIONS: The targets of emodin and hamaudol were discovered by HEMT biochips for the first time. This study provided a novel and effective strategy to discover active components in a complex herbal formula by using AlGaN/GaN HEMT biochips.

A phase I trial of riluzole and sorafenib in patients with advanced solid tumors: CTEP #8850.

BACKGROUND: Overexpression of metabotropic glutamate receptor 1 (GRM1) has been implicated in the pathogenesis of multiple cancers. Riluzole, an inhibitor of glutamate release, showed synergistic antitumor activity in combination with the multi-kinase inhibitor sorafenib in preclinical models. This phase I trial identified the toxicity profile, dose-limiting toxicities, maximum tolerated dose (MTD), and pharmacokinetic and pharmacodynamic properties of riluzole combined with sorafenib in patients with advanced cancers. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled utilizing a 3+3 dose-escalation design. Riluzole was given at 100 mg PO BID in combination with sorafenib, beginning at 200 mg PO daily and escalating in 200 mg increments per level in 28-day cycles. Restaging evaluations were performed every 2 cycles. RESULTS: 35 patients were enrolled over 4 dose levels. The MTD was declared at dose level 3 (riluzole: 100 mg PO BID; sorafenib: 400 mg AM/200 mg PM). Pharmacokinetic analyses did not reveal definitive evidence of drug-drug interactions. Consistent decreases in phospho-forms of ERK and AKT in tumor tissue analyses with accompanying decrease in GRM1 expression and increase in pro-apoptotic BIM suggest target engagement by the combination. Best responses included a partial response in 1 (2.9%) patient with pancreatic acinar cell carcinoma with a KANK4-RAF1 fusion, and stable disease in 11 (36%) patients. CONCLUSION: Combination therapy with riluzole and sorafenib was safe and tolerable in patients with advanced solid tumors. The partial response in a patient with a RAF1 fusion suggests that further exploration in a genomically selected cohort may be warranted.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.