In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine–dithiazole (QCy–DT) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCy–DT exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCy–DT for DNA containing 5′-AATT-3′ sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5′-X(AATT)Y-3′ recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC₅₀ value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC₅₀ make QCy–DT a potential and commercially viable DNA probe.

Even though it is increasingly evident that post-transcriptional events like mRNA processing and splicing may regulate gene expression and proteome diversity of malaria parasite Plasmodium, molecular mechanisms that regulate events like mRNA splicing in malaria parasite are poorly understood. Protein kinases control a wide variety of cellular events in almost all eukaryotes, including modulation of mRNA splicing, transport, and stability. We have identified a novel splicing-related protein kinase from Plasmodium falciparum, PfSRPK1. PfSRPK1 when incubated with parasite nuclear extracts inhibited RNA splicing, suggesting that it may control mRNA splicing in the parasite. PfSR1, a putative splicing factor from P. falciparum, was identified as a substrate of PfSRPK1. PfSR1 interacts with RNA and PfSRPK1 modulates its RNA binding. Early in the parasite development, PfSRPK1 and PfSR1 are present in the nucleus. These studies provide useful insights into the function of two potentially key components of P. falciparum mRNA splicing machinery.

Radiation-induced lung injury is a major dose-limiting toxicity for thoracic radiotherapy patients. In experimental models, treatment with angiotensin converting enzyme (ACE) inhibitors mitigates radiation pneumonitis; however, the mechanism of action is not well understood. Here, we evaluate the direct role of ACE inhibition on lung immune cells. ACE expression and activity were determined in the lung immune cell compartment of irradiated adult rats following either high dose fractionated radiation therapy (RT) to the right lung (5 fractions × 9 Gy) or a single dose of 13.5 Gy partial body irradiation (PBI). Mitigation of radiation-induced pneumonitis with the ACE-inhibitor lisinopril was evaluated in the 13.5 Gy rat PBI model. During pneumonitis, we characterized inflammation and immune cell content in the lungs and bronchoalveolar lavage fluid (BALF). In vitro mechanistic studies were performed using primary human monocytes and the human monocytic THP-1 cell line. In both the PBI and fractionated RT models, radiation increased ACE activity in lung immune cells. Treatment with lisinopril improved survival during radiation pneumonitis (p=0.0004). Lisinopril abrogated radiation-induced increases in BALF MCP-1 (CCL2) and MIP-1α cytokine levels (p < 0.0001). Treatment with lisinopril reduced both ACE expression (p=0.006) and frequency of CD45⁺CD11b⁺ lung myeloid cells (p=0.004). In vitro, radiation injury acutely increased ACE activity (p=0.045) and reactive oxygen species (ROS) generation (p=0.004) in human monocytes, whereas treatment with lisinopril blocked radiation-induced increases in both ACE and ROS. Interestingly, radiation-induced ROS generation was blocked by pharmacological inhibition of either NADPH oxidase 2 (NOX2) (p=0.012) or the type 1 angiotensin receptor (AGTR1) (p=0.013). These data demonstrate radiation-induced ACE activation within the immune compartment promotes the pathogenesis of radiation pneumonitis, while ACE inhibition suppresses activation of pro-inflammatory immune cell subsets. Mechanistically, our in vitro data demonstrate radiation directly activates the ACE/AGTR1 pathway in immune cells and promotes generation of ROS via Nox2.

Calcium‐dependent protein kinases (CDPKs) are major effectors of calcium signaling in apicomplexan parasites like Toxoplasma and Plasmodium and control important processes of the parasite life cycle. Despite recently reported crystal structures of Toxoplasma gondii ( Tg )CDPKs, several important questions about their regulation remain unanswered. Plasmodium falciparum (Pf)CDPK1 has emerged as a key player in the life cycle of the malaria parasite, as it may be involved in the invasion of the host cells. Molecular modeling and site‐directed mutagenesis studies on Pf CDPK1 suggested that several residues in the regulatory domain play a dual role, as they seem to contribute to the stabilization of both the active and inactive kinase. Mass spectrometry revealed that Pf CDPK1 was autophosphorylated at several sites; some of these were placed at strategic locations and therefore were found to be critical for kinase activation. The N‐terminal extension of Pf CDPK1 was found to be important for Pf CDPK1 activation. Unexpectedly, an ATP binding site in the NTE of Pf CDPK1 was identified. Our studies highlight several novel features of Pf CDPK1 regulation, which may be shared by other members of the CDPK family. These findings may also aid design of inhibitors against these important targets, which are absent from the host.—Ahmed, A., Gaadhe, K., Sharma, G. P., Kumar, N., Neculai, M., Hui, R., Mohanty, D., Sharma, P. Novel insights into the regulation of malarial calcium‐dependent protein kinase 1. FASEB J. 26, 3212–3221 (2012). www.fasebj.org

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family which has been extensively studied for its roles in neural development, long-term memory, brain injury, and neurodegenerative diseases. BDNF signaling through tropomyosin receptor kinase B (TrkB) stimulates neuronal cell survival. For this reason, small molecule TrkB agonists are under pre-clinical develoment for the treatment of a range of neurodegenerative diseases and injuries. Our laboratory recently reported BDNF is secreted by pro-regenerative endothelial progenitor cells (EPCs) which support hematopoietic reconstitution following total body irradiation (TBI). Here we report BDNF-TrkB signaling plays a novel regenerative role in bone marrow and thymic regeneration following radiation injury. Exogenous administration of BDNF or TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following myelosuppressive radiation injury promoted faster recovery of mature blood cells and hematopoietic stem cells capable of multi-lineage reconstitution. BDNF promotes hematopoietic regeneration via activation of PDGFRα⁺bone marrow mesenchymal stem cells (MSCs) which increase secretion of hematopoietic cytokines interleukin 6 (IL-6) and leukemia inhibitory factor (LIF) in response to TrkB activation. These data suggest pharmacologic activation of the BDNF pathway with either BDNF or 7,8-DHF may be beneficial for treatment of radiation or chemotherapy induced myelosuppression.