Role of Heme Oxygenase in modulating expression of ROS-regulatory enzymes in Medicago truncatula

Heme Oxygenase (HO) is an enzyme universally found in animals, plants and microbes. In plants, the role of heme oxygenase in the synthesis of the phytochrome chromophore is well recognized and has been extensively studied; however its role in regulating reactive oxygen species (ROS) in plants is just beginning to be explored, particularly in legumes. Legumes interact with Rhizobium bacteria to form symbiotic nitrogen fixing nodules. ROS plays an important role in the development of roots as well as symbiotic nodules. In the model legume Medicago truncatula, ROS in the root is regulated in part by the LATD/NIP gene. The M. truncatula giraffe mutant has a deletion that removes the entire HO coding sequence. We have found that the M. truncatula GIRAFFE HO regulates expression of some of the LATD/NIP-regulated ROS genes such as RESPRATORY BURST OXIDASE HOMOLOG C (RBOHC) and a cell wall peroxidase (cwPRX2) in seedlings. This means that the wild-type function of GIRAFFE is to up-regulate expression of RBOHC and cwPRX2 in roots, in contrast to LATD/NIP, which down-regulates them. We also found that LATD/NIP and GIRAFFE do not regulate expression of each other in seedlings. Given that the highest expression of GIRAFFE HO is in a senescing nodule, we tested the expression of ROS-regulatory enzymes in senescing nodules. We found that GIRAFFE up-regulates expression of RBOHC during nitrate-induced nodule senescence. At present, with changing climatic conditions and exposure to various environmental stresses that can alter ROS homeostasis, characterizing the role of GIRAFFE in the antioxidant machinery of legumes can be useful in improving crop productivity and for enhancing soil fertility

SYNCHRONOUS SQUAMOUS CELL CARCINOMA OF EXTERNAL ACOUSTIC MEATUS FOLLOWING PIGMENTED BASAL CELL CARCINOMA OF CHEEK -A RARE OCCURRENCE

Introduction: A synchronous second primary malignancy as squamous cell carcinoma of external acoustic meatus following basal cell carcinoma of cheek is reported with their management and review of literature. Case Report: Pigmented basal cell carcinoma of cheek was managed successfully by wide local excision followed by flap reconstruction and subsequently diagnosed squamous cell carcinoma of external acoustic meatus by concurrent chemoradiation after initial surgical debridement. Conclusion: Second primary malignancy as squamous cell carcinoma of external ear canal is rare after basal cell carcinoma of cheek though there is anatomical vicinity. Surgery in case of basal cell carcinoma and concurrent chemoradiotherapy in case of external ear canal squamous cell carcinoma is the mainstay of treatment

Pooling of Nasopharyngeal (NP) Swab Samples to Overcome Global Shortage of rRT-PCR COVID-19 Test Kits

The global outbreak and rapid spread of SARS-CoV-2 has created an urgent need for large scale testing of populations. There is a demand for high throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled-PCR protocol for testing nasopharyngeal swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. Nasopharyngeal swabs were accurately identified as positive or negative for SARS-CoV-2 in pools of 5 (100% sensitivity; 100% specificity). Even though specificity remained unaffected (100%), the detection sensitivity was reduced (66.67%) when 10 samples were pooled together. Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results

Arabinosylated Lipoarabinomannan Skews Th2 Phenotype towards Th1 during Leishmania Infection by Chromatin Modification: Involvement of MAPK Signaling

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an antiinflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal–regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara- LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis

Graphene Nanocomposite Ink Coated Laser Transformed Flexible Electrodes for Selective Dopamine Detection and Immunosensing

Novel and flexible disposable laser-induced graphene (LIG) sensors modified with graphene conductive inks have been developed for dopamine and interleukin-6 (IL-6) detection. The LIG sensors exhibit high reproducibility (relative standard deviation, RSD = 0.76%, N = 5) and stability (RSD = 4.39%, N = 15) after multiple bendings, making the sensors ideal for wearable and stretchable bioelectronics applications. We have developed electrode coatings based on graphene conductive inks, poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (G-PEDOT:PSS) and polyaniline (G-PANI), for working electrode modification to improve the sensitivity and limit of detection (LOD). The selectivity of LIG sensors modified with the G-PANI ink is 41.47 times higher than that of the screen-printed electrode with the G-PANI ink modification. We have compared our fabricated bare laser-engraved Kapton sensor (LIG) with the LIG sensors modified with G-PEDOT (LIG/G-PEDOT) and G-PANI (LIG/G-PANI) conductive inks. We have further compared the performance of the fabricated electrodes with commercially available screen-printed electrodes (SPEs) and screen-printed electrodes modified with G-PEDOT:PSS (SPE/G-PEDOT:PSS) and G-PANI (SPE/G-PANI). SPE/G-PANI has a lower LOD of 0.632 μM compared to SPE/G-PEDOT:PSS (0.867 μM) and SPE/G-PANI (1.974 μM). The lowest LOD of the LIG/G-PANI sensor (0.4084 μM, S/N = 3) suggests that it can be a great alternative to measure dopamine levels in a physiological medium. Additionally, the LIG/G-PANI electrode has excellent LOD (2.6234 pg/mL) to detect IL-6. Also, the sensor is successfully able to detect ascorbic acid (AA), dopamine (DA), and uric acid (UA) in their ternary mixture. The differential pulse voltammetry (DPV) result shows peak potential separation of 229, 294, and 523 mV for AA–DA, DA–UA, and UA–AA, respectively

Ara-LAM induced MHC-II expression, antigen presentation ability of infected macrophages increased under SOCS3 silenced condition.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or SOCS3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 4 hr as mentioned above. After 24 hr of incubation treated macrophages were analyzed by flow cytometry for MHCII (FL2-H) expression as described in material method (A). Data are from 1 of 3 experiments conducted in the same way with similar results. In a separate set, normal and treated macrohages either uninfected or infected were pulsed with λR_12–26, and then were incubated with T-cell hybridoma 9H 3.5. The culture supernatants were analysed for the presence of IL-2 by ELISA as described in the methods (B). Incorporation of 3H-Thymidine in the IL-2 dependent cell line HT-2 was assessed in presence of the cultured supernatant. Results are expressed as mean</i> ± <i>SD of 5 replicate experiments (C). *P<.001, **P<.005 compared with infected macrophages.</i></p

SOCS3 silencing significantly enhances host protective proinflammatory response generation by Ara-LAM in infected macrophages.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or SOCS3-specific siRNA followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 h and assayed for the levels of TNF- α (A), IL-12 (B), and IL-10 (C) in the culture supernatant by ELISA, as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages. ***<.01 compared to Ara-LAM–pretreated infected macrophages. In a separate experiment macrophages were cultured and treated as described above and assayed for the levels of extracellular NO as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (D). *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages. In a separate set macrophages were transfected and treated with Ara-LAM as described above followed by Leishmania infection for 24 hr. Western blot analysis was performed to analyze the expression of inducible nitric oxide synthase. The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (E). Peritoneal macrophages were cultured and treated with Ara-LAM as described above followed by Leishmania infection for 3 hr. Changes in mRNA expression of IL-12p40, TNF- α, IL-10, NO were determined by real-time PCR analysis Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results; data are expressed as means</i> ± <i>standard deviations. (F). *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages.</i></p

STAT3 silencing potentiate Ara-LAM mediated abrogation of SOCS3 expression in <i>Leishmania-donovani</i> infected macrophages.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 hr. The cells were then lysed and subjected to Western blot with anti-SOCS3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (A). In a separate experiment macrophages were transfected with either control-siRNA or</i> STAT3 <i>specific siRNA, subsequently followed by Ara-LAM treatment for 3 hr and Leishmania infection for another 3 h. RNA was isolated and semi quantitative RT-PCR analyses for SOCS3 and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (B). Changes in expression of SOCS3 mRNA were also determined by quantitative real-time PCR. Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results (C). *P<.001 compared with infected macrophages. The comparison of SOCS3 expression between Ara-LAM treated infected macrophages and STAT3 siRNA group did not show any statistical significance.</i></p

Hstone H3 modifications at the IL-12 promoter in Ara-LAM treated infected macrophages.

<p> <i>Murine macrophages (1×10⁶cells/mL) were treated and infected as described in the </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#pone-0024141-g003" target="_blank"><i>Figure 3</i></a><i> legend. After 45 min of incubation, ChIP assays were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a>. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (A) or IP acetyl-H3 (B), and conventional RT-PCR or quantitative real-time PCR was performed using primers specific to the IL-12p40 promoter. *P<.001 compared with Ara-LAM–pretreated infected macrophages. In a separate experiment peritoneal macrophages were treated and infected by Leishmania for 15, 30 min as described in </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#pone-0024141-g003" target="_blank"><i>Figure 3</i></a><i> legend. Cytosolic and nuclear protein extracts were analyzed for the nuclear translocation of NF-κB (C). The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (i,ii) (inset). Peritoneal macrophages (1×10⁶ cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 45 min. Immunoprecipitations were conducted using NF-κB specific Abs. Conventional RT-PCR or quantitative real-time PCR was performed for amplifying the putative NF-κB binding sites of the IL-12p40 promoter. *P<.001 compared with Ara-LAM–pretreated infected macrophages.</i></p