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Abstract Magnaporthe oryzae is a devastating fungal pathogen that causes the rice blast disease worldwide. The post-translational modification of ADP-ribosylation holds significant importance in various fundamental biological processes. However, the specific function of this modification in M. oryzae remains unknown. This study revealed that Poly(ADP-ribosyl)ation (PARylation) executes a critical function in M. oryzae. M. oryzae Poly(ADP-ribose) polymerase 1 (PARP1) exhibits robust PARylation activity. Disruption of PARylation by PARP1 knock-out or chemical inhibition reveals its involvement in M. oryzae virulence, particularly in appressorium formation. Furthermore, we identified two M. oryzae 14-3-3 proteins, GRF1 and GRF2, as substrates of PARP1. Deletion of GRF1 or GRF2 results in delayed and dysfunctional appressorium, diminished plant penetration, and reduced virulence of the fungus. Biochemical and genetic evidence suggest that PARylation of 14-3-3s is essential for its function in M. oryzae virulence. Moreover, PARylation regulates 14-3-3 dimerization and is required for the activation of the mitogen-activated protein kinases (MAPKs), Pmk1 and Mps1. GRF1 interacts with both Mst7 and Pmk1, and bridges their interaction in a PARylation-dependent manner. This study unveils a distinctive mechanism that PARylation of 14-3-3 proteins controls appressorium formation through MAPK activation, and could facilitate the development of new strategies of rice blast disease control.

Diabetes causes a loss of sensation in the skin, so diabetics are prone to burns when using heating devices. Diabetic scalded skin is often difficult to heal due to the microenvironment of high glucose, high oxidation, and low blood perfusion. The treatment of diabetic scald mainly focuses on three aspects: 1) promote the formation of the epithelium; 2) promote angiogenesis; and 3) maintain intracellular homeostasis. In response to these three major repair factors, we developed a cadherin-responsive hydrogel combined with FGF21 and dental pulp stem cells (DPSCs) to accelerate epithelial formation by recruiting cadherin to the epidermis and promoting the transformation of N cadherin to E cadherin; promoting angiogenesis to increase wound blood perfusion; regulating the stability of lysosomal and activating autophagy to maintain intracellular homeostasis in order to comprehensively advance the recovery of diabetic scald.

The poor performance of recycled concrete aggregate (RCA) leads to greater creep in recycled aggregate concrete (RAC) compared to natural aggregate concrete (NAC). To enhance the quality of RCA, this paper utilizes a 2% concentration of a nano-SiO2 (NS) solution for pre-soaking RCA. This study aims to replace natural aggregate (NA) with NS-modified recycled aggregate (SRCA) and investigate the creep and shrinkage properties of NS-modified recycled aggregate concrete (SRAC) at various SRCA replacement rates. Subsequently, the creep and shrinkage strains of NAC, SRAC, and RAC are simulated using the finite element method. Finally, a comparative analysis is conducted with the predicted creep and shrinkage strains from CEB-FIP, ACI, B3, and GL2000 models. The experimental results indicate that the creep and shrinkage deformation of SRAC increases with the SRCA replacement rate. Compared to NAC, the creep and shrinkage deformation of SRAC at replacement rates of 30%, 50%, 70%, and 100% increased by 2%, 7%, 13%, and 30%, respectively. However, when 100% of the natural aggregate is replaced with SRCA, the creep and shrinkage deformation decreases by 7% compared to RAC. Moreover, the CEB-FIP and ACI models can predict the creep and shrinkage deformation of concrete reasonably well.

Recycled aggregate concrete (RAC) possesses different mechanical properties than ordinary concrete because of inherent faults in recycled aggregates (RAs), such as the old interfacial transition zone (ITZ). However, the application of nano-SiO2 presents an effective methodology to enhance the quality of RA. In this study, nano-SiO2-modified recycled aggregate (SRA) was used to replace natural aggregate (NA), and the stress–strain relationships and cyclic behavior of nano-SiO2-modified recycled aggregate concrete (SRAC) with different SRA replacement rates were investigated. After evaluating the skeleton curve of SRAC specimens, the existing constitutive models were compared. Additionally, the study also proposed a stress–strain model designed to predict the mechanical behavior of concrete in relation to the SRA replacement rate. The results show that compared with RAC, the axial compressive strength of SRAC specimens showed increases of 40.27%, 29.21%, 26.55%, 16.37%, and 8.41% at specific SRA replacement rates of 0%, 30%, 50%, 70%, and 100%, respectively. Moreover, the study found that the Guo model’s calculated results can accurately predict the skeleton curves of SRAC specimens.

Summary: Poration of the outer mitochondrial membrane by the effector BCL-2 proteins BAK and BAX initiates apoptosis. BH3-only initiators BID and BIM trigger conformational changes in BAK and BAX transforming them from globular dormant proteins to oligomers of the apoptotic pores. Small molecules that can directly activate effectors are being sought for applications in cancer treatment. Here, we describe the small molecule SJ572946, discovered in a fragment-based screen that binds to the activation groove of BAK and selectively triggers BAK activation over that of BAX in liposome and mitochondrial permeabilization assays. SJ572946 independently kills BAK-expressing BCL2allKO HCT116 cells revealing on target cellular activity. In combination with apoptotic inducers and BH3 mimetics, SJ572946 kills experimental cancer cell lines. SJ572946 also cooperates with the endogenous BAK activator BID in activating a misfolded BAK mutant substantially impaired in activation. SJ572946 is a proof-of-concept tool for probing BAK-mediated apoptosis in preclinical cancer research.

Abstract Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose‐limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR‐induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human‐induced pluripotent stem cell‐derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR’s antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Patient-derived xenografts provide a resource for basic and translational cancer research. Here, the authors generate multiple pediatric high-grade glioma xenografts, use omics technologies to show that they are representative of primary tumours and use them to assess therapeutic response.

Aiming at solving the issue of blurred images and difficult recognition of digital meters encountered by inspection robots in the inspection process, this paper proposes a deep-learning-based method for blurred image restoration and LED digital identification. Firstly, fast Fourier transform (FFT) is used to perform blur detection on the acquired images. Then, the blurred images are recovered using spatial-attention-improved adversarial neural networks. Finally, the digital meter region is extracted using the polygon-YOLOv5 model and corrected via perspective transformation. The digits in the image are extracted using the YOLOv5s model, and then recognized by the CRNN for digit recognition. It is experimentally verified that the improved adversarial neural network in this paper achieves 26.562 in the PSNR metric and 0.861 in the SSIM metric. The missing rate of the digital meter reading method proposed in the paper is only 1% and the accuracy rate is 98%. The method proposed in this paper effectively overcomes the image blurring problem caused by the detection robot during the detection process. This method solves the problems of inaccurate positioning and low digital recognition accuracy of LED digital meters in complex and changeable environments, and provides a new method for reading digital meters.

Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer’s disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 TauRD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.

Testis-restricted melanoma antigen (MAGE) proteins function as substrate adapters for E3 ubiquitin ligases. Biochemical and structural analyses of MAGE-A11 provide insight into the substrate binding mode of MAGE proteins and enable discovery of potent, cytotoxic inhibitors of MAGE-A11:substrate interaction.

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases.

The significance of protein nanoclustering in cell polarization is unclear. Here Pan et al. show that auxin-induced TMK1/sterol nanoclustering as well as microtubule-based positive feedback regulation of the TMK1/sterol nanoclusters is critical for cell polarity formation in Arabidopsis.

Abstract Background Current tools for diagnosing latent TB infection (LTBI) detect immunological memory of past exposure but are unable to determine whether exposure is recent. We sought to identify a whole-blood transcriptome signature of recent TB exposure. Methods We studied household contacts of TB patients; healthy volunteers without recent history of TB exposure; and patients with active TB. We performed whole-blood RNA sequencing (in all), an interferon gamma release assay (IGRA; in contacts and healthy controls) and PET/MRI lung scans (in contacts only). We evaluated differentially-expressed genes in household contacts (log2 fold change ≥1 versus healthy controls; false-discovery rate 0.19). Conclusions Transcriptomics can detect TB exposure and, with further development, may be an approach of value for epidemiological research and targeting public health interventions.

Abstract Background There is a need for better tools to evaluate new or repurposed TB drugs. The whole blood bactericidal activity (WBA) assay has been advocated for this purpose. We investigated whether transcriptional responses in the WBA assay resemble TB responses in vivo, and whether the approach might additionally reveal mechanisms of action. Results 1422 of 1798 (79%) of differentially expressed genes in WBA incubated with the standard combination of rifampicin, isoniazid, pyrazinamide and ethambutol were also expressed in sputum (P

Random flaps are widely used in tissue reconstruction, attributed to the lack of vascular axial limitation. Nevertheless, the distal end of the flap is prone to necrosis due to the lack of blood supply. Notoginseng triterpenes (NTs) are the active components extracted from Panax notoginseng, reducing oxygen consumption and improving the body’s tolerance to hypoxia. However, their role in random flap survival has not been elucidated. In this study, we used a mouse random skin flap model to verify that NT can promote cell proliferation and migration and that increasing blood perfusion can effectively improve the survival area of a skin flap. Our study also showed that the autophagy of random flaps after NT treatment was activated through the Beclin-1/VPS34/LC3 signaling pathway, and the therapeutic effect of NT significantly decreased after VPS34 IN inhibited autophagy. In conclusion, we have demonstrated that NT can significantly improve the survival rate of random flaps through the Beclin-1/VPS34/LC3 signaling pathway, suggesting that it might be a promising clinical treatment option.

Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer’s disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tauRD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer’s disease/tauopathies.

Summary: Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs.For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

Metal component surfaces are random textured and non-smooth. There are many stains on the surface of metal component that are similar to the gray scale of the scratches. The scratches have non-uniform gray distribution, various shapes, and low contrast in their background, posing challenges in accurate scratch detection. This paper presents a method for detecting weak scratches on metal component surfaces based on deep convolutional neural networks (DCNNs). First, a DCNN is trained using labeled scratch images. Then, the scratches and some faults are detected by the trained DCNN, and most of the faults can be removed through properly thresholding based on the size of connected regions. Finally, the scratch length united in the number of pixels is obtained by the skeleton extraction. The experimental results show that the proposed method can effectively deal with background noise, thereby achieving accurate scratch detection.

To solve the problem of inflexibility of offline hand–eye calibration in “eye-in-hand” modes, an online hand–eye calibration method based on the ChArUco board is proposed in this paper. Firstly, a hand–eye calibration model based on the ChArUco board is established, by analyzing the mathematical model of hand–eye calibration, and the image features of the ChArUco board. According to the advantages of the ChArUco board, with both the checkerboard and the ArUco marker, an online hand–eye calibration algorithm based on the ChArUco board is designed. Then, the online hand–eye calibration algorithm, based on the ChArUco board, is used to realize the dynamic adjustment of the hand–eye position relationship. Finally, the hand–eye calibration experiment is carried out to verify the accuracy of the hand–eye calibration based on the ChArUco board. The robustness and accuracy of the proposed method are verified by online hand–eye calibration experiments. The experimental results show that the accuracy of the online hand–eye calibration method proposed in this paper is between 0.4 mm and 0.6 mm, which is almost the same as the offline hand–eye calibration accuracy. The method in this paper utilizes the advantages of the ChArUco board to realize online hand–eye calibration, which improves the flexibility and robustness of hand–eye calibration.

For the task of obstacle detection in a complex traffic environment, this paper proposes a road-free space extraction and obstacle detection method based on stereo vision. The proposed method combines the advantages of the V-disparity image and the Stixel method. Firstly, the depth information and the V-disparity image are calculated according to the disparity image. Then, the free space on the road surface is calculated through the RANSAC algorithm and dynamic programming (DP) algorithm. Furthermore, a new V-disparity image and a new U-disparity image are calculated by the disparity image after removing the road surface information. Finally, the height and width of the obstacles on the road are extracted from the new V-disparity image and U-disparity image, respectively. The detection of obstacles is realized by the height and width information of obstacles. In order to verify the method, we adopted the object detection benchmarks and road detection benchmarks of the KITTI dataset for verification. In terms of the accuracy performance indicators quality, detection rate, detection accuracy, and effectiveness, the method in this paper reaches 0.820, 0.863, 0.941, and 0.900, respectively, and the time consumption is only 5.145 milliseconds. Compared with other obstacle detection methods, the detection accuracy and real-time performance in this paper are better. The experimental results show that the method has good robustness and real-time performance for obstacle detection in a complex traffic environment.

In the paper based on surface plasmon resonance (SPR) in a tilted fiber Bragg grating (TFBG), a novel algorithm is proposed, which facilitates demodulation of surrounding refractive index (SRI) via cladding mode interrogation and accelerates calibration and measurement of SRI. Refractive indices with a tiny index step of 2.2 × 10−5 are prepared by the dilution of glucose aqueous solution for the test and the calibration of this fiber sensor probe. To accelerate the calibration process, automatic selection of the most sensitive cladding mode is demonstrated. First, peaks of transmitted spectrum are identified and numbered. Then, sensitivities of several potentially sensitive cladding modes in amplitude adjacent to the left of the SPR area are calculated and compared. After that, we focus on the amplitudes of the cladding modes as a function of a SRI, and the highest sensitivity of −6887 dB/RIU (refractive index unit) is obtained with a scanning time of 15.77 s in the range from 1520 nm to 1620 nm. To accelerate the scanning speed of the optical spectrum analyzer (OSA), the wavelength resolution is reduced from 0.028 nm to 0.07 nm, 0.14 nm, and 0.28 nm, and consequently the scanning time is shortened to 6.31 s, 3.15 s, and 1.58 s, respectively. However, compared to 0.028 nm, the SRI sensitivity for 0.07 nm, 0.14 nm, and 0.28 nm is reduced to −5685 dB/RIU (17.5% less), −5415 dB/RIU (21.4% less), and −4359 dB/RIU (36.7% less), respectively. Thanks to the calculation of parabolic equation and weighted Gauss fitting based on the original data, the sensitivity is improved to −6332 dB/RIU and −6721 dB/RIU, respectively, for 0.07 nm, and the sensitivity is increased to −5850 dB/RIU and −6228 dB/RIU, respectively, for 0.14 nm.

Summary: Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.

Abstract COX-2 inhibition may be of benefit in the treatment of tuberculosis (TB) through a number of pathways including efflux pump inhibition (increasing intracellular TB drug levels) and diverse effects on inflammation and the immune response. We investigated celecoxib (a COX-2 inhibitor) alone and with standard anti-tuberculosis drugs in the whole-blood bactericidal activity (WBA) model. Healthy volunteers took a single dose of celecoxib (400 mg), followed (after 1 week) by a single dose of either rifampicin (10 mg/kg) or pyrazinamide (25 mg/kg), followed (after 2 or 7 days respectively) by the same anti-tuberculosis drug with celecoxib. WBA was measured at intervals until 8 hours post-dose (by inoculating blood samples with Mycobacterium tuberculosis and estimating the change in bacterial colony forming units after 72 hours incubation). Celecoxib had no activity alone in the WBA assay (cumulative WBA over 8 hours post-dose: 0.03 ± 0.01ΔlogCFU, p = 1.00 versus zero). Celecoxib did not increase cumulative WBA of standard TB drugs (mean cumulative WBA −0.10 ± 0.13ΔlogCFU versus −0.10 ± 0.12ΔlogCFU for TB drugs alone versus TB drugs and celecoxib; mean difference −0.01, 95% CI −0.02 to 0.00; p = 0.16). The lack of benefit of celecoxib suggests that efflux pump inhibition or eicosanoid pathway-related responses are of limited importance in mycobacterial killing in the WBA assay.

Alzheimer’s disease (AD) is a common neurodegenerative disease presenting with progressive memory and cognitive impairments. One of the pathogenic mechanisms of AD is attributed to the aggregation of misfolded amyloid β (Aβ), which induces neurotoxicity by reducing the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TRKB) and increasing oxidative stress, caspase-1, and acetylcholinesterase (AChE) activities. Here, we have found the potential of two novel synthetic coumarin derivatives, ZN014 and ZN015, for the inhibition of Aβ and neuroprotection in SH-SY5Y neuroblastoma cell models for AD. In SH-SY5Y cells expressing the GFP-tagged Aβ-folding reporter, both ZN compounds reduced Aβ aggregation, oxidative stress, activities of caspase-1 and AChE, as well as increased neurite outgrowth. By activating TRKB-mediated extracellular signal-regulated kinase (ERK) and AKT serine/threonine kinase 1 (AKT) signaling, these two ZN compounds also upregulated the cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of ZN014 and ZN015. A parallel artificial membrane permeability assay showed that ZN014 and ZN015 could be characterized as blood–brain barrier permeable. Our results suggest ZN014 and ZN015 as novel therapeutic candidates for AD and demonstrate that ZN014 and ZN015 reduce Aβ neurotoxicity via pleiotropic mechanisms.

ABCC4 is a chemotherapeutic drug exporter highly expressed in acute myeloid leukemia. Here, the authors demonstrate that MPP1 anchors ABCC4 to the outer cell membrane mediating drug resistance in leukemic cells and identify antimycin A as a chemical probe that disrupts such interaction and restores sensitivity.

A highly diastereoselective method for the synthesis of novel spiro-tetrahydroquinoline derivatives is reported here, using a one-pot reaction method. All compounds were characterized by 1H-nuclear magnetic resonance (NMR) and mass spectroscopy, and their stereo configurations were confirmed by X-ray analysis. These activities of these derivatives were then tested in human keratocyte cells. The responses of cells to treatment with selected compounds were studied using scratch analysis, and the compounds were tested in a mouse excision wound model. Three of the derivatives demonstrated significant wound-healing activities.

Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.

Abstract: New therapies for Philadelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL) patients are urgently needed. The genetic landscape of Ph-like ALL is characterized by a diverse array of kinase-activating alterations (including rearrangements, sequence mutations, and copy number alterations), suggesting that patients with Ph-like ALL are candidates for targeted therapy, similar to BCR-ABL1 ALL. We sought to investigate the functional role and targetability of the spectrum of kinase-activating alterations identified in Ph-like ALL. We demonstrate cytokine-independent growth and activation of JAK-STAT signaling pathways in Ba/F3 cells by all alterations tested. The development of murine Arf−/− pre-B ALL expressing RCSD1-ABL2 or SSBP2-CSF1R was accelerated with the presence of IK6, a dominant negative isoform of Ikaros common in Ph-like ALL, providing evidence that these fusions are leukemogenic. In vitro screening using a panel of tyrosine kinase inhibitors against 14 different kinase alterations identified the ABL1-inhibitor, dasatinib, as a potent inhibitor of ABL-class fusions (ABL1, ABL2, CSF1R, PDGFRB), whereas the JAK1/JAK2 inhibitor ruxolitinib, was most effective against JAK-STAT–activating alterations (JAK1, JAK2, JAK3, IL7R, IL2RB), but not TYK2. Evaluation of dasatinib or ruxolitinib against patient-derived xenograft models demonstrated superior antileukemic efficacy when combined with dexamethasone compared with either agent alone. These data provide the foundation for rationally designed clinical trials that assess the efficacy of targeted therapy in patients with Ph-like ALL.

The xenobiotic-activated human pregnane X receptor (hPXR) regulates drug metabolism. Here the authors develop hPXR modulators, which are of potential therapeutic interest and functionally and structurally characterize the antagonist SPA70 and the structurally related agonist SJB7.

Mycobacteria can adapt to the stress of human infection by entering a dormant state. Here the authors show that hypoxia-induced dormancy in M. bovisBCG involves the reprogramming of tRNA wobble modifications and copy numbers, coupled with biased use of synonymous codons in survival genes.

Organic cation transporters are important drug transporters that influence therapeutic outcomes. Here, the authors find that these transporters are regulated by tyrosine phosphorylation and propose that tyrosine kinase inhibitors can influence drug transporter function through post-translational mechanisms.

Ethionamide (ETH) is part of the drug arsenal available to treat multi-drug resistant tuberculosis. The current paradigm of this pro-drug activation involves the mycobacterial enzyme EthA and the transcriptional repressor, EthR. However, several lines of evidence suggest the involvement of additional players. The ethA/R locus was deleted in Mycobacterium bovis BCG and three Mycobacterium tuberculosis (MTB) strains. While complete resistance to ETH was observed with BCG ethA/R KO, drug susceptibility and dose-dependent killing were retained in the ethA/R KO MTB mutants, suggesting the existence of an alternative pathway of ETH bio-activation in MTB. We further demonstrated that this alternative pathway is EthR-independent, whereby re-introduction of ethR in ethA/R KO MTB did not lead to increased resistance to ETH. Consistently, ethA KO MTB (with intact ethR expression) displayed similar ETH susceptibility profile as their ethA/R KO counterparts. To identify the alternative ETH bio-activator, spontaneous ETH-resistant mutants were obtained from ethA/R KO MTB and whole genome sequencing identified single nucleotide polymorphisms in mshA, involved in mycothiol biosynthesis and previously linked to ETH resistance. Deletion of mshA in ethA/R KO MTB led to complete ETH resistance, supporting that the role of MshA in ETH killing is EthA/R-independent. Furthermore mshA single KO MTB displayed levels of ETH resistance similar or greater than those obtained with ethA/R KO strains, supporting that mshA is as critical as ethA/R for ETH killing efficacy.

A new highly efficient three-component reaction of alkyl acrylate, aldehyde and dialkyl malonate using ethyl diphenylphosphine as organocatalyst has been described. Various highly functional compounds bearing hydroxyl groups and the ester functions can be easily prepared in moderate to good yields according to our one-step procedure. The reactions are believed to proceed via Morita-Baylis-Hillman reactions of alkyl acrylate and aldehydes, followed by the Michael addition reactions of dialkyl malonates. Our reactions indicated that the intermediate species formed in the phosphine-catalyzed MBH reaction are an effective organic base to catalyze the Michael addition reactions of dialkyl malonates to the preformed MBH adducts.

p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca²⁺-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.

Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7-10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.