Rare co-inherited alpha-thalassemia minor and beta-thalassemia minor with heterozygous H63D mutation mistaken as iron deficiency anemia: a case report.

The coexistence of alpha- and beta-thalassemia is not uncommon and neither is a single thalassemia subtype with a hemochromatosis H63D mutation, however the inheritance of all three diseases together has yet to be reported. We present this rare case of co-inherited alpha-thalassemia minor and beta-thalassemia minor initially misdiagnosed as iron deficiency anemia (IDA) in a reproductive aged female with a heterozygous H63D mutation. In our case report, a 27-year-old, Asian female presented with excessive lethargy and fatigue for the past 10 months. A year ago, she was prescribed Ferrous Sulfate 325 mg daily supplementation due to a suspicion of IDA secondary to a history of heavy menstruations. Although her reports displayed a low mean corpuscular volume (MCV) anemia, the patient declined therapy at that time due to abnormal labs, specifically regarding her urine and liver that subsequently lead to a hemochromatosis, heterozygous H63D diagnosis following genetic testing. Subsequently, the patient's anemia presenting in the setting of normal ferritin, high iron saturation, and elevated A2 fraction was most likely in accordance to carrying the alpha-thalassemia minor, beta-thalassemia minor, and heterozygous H63D gene mutations. Genetic testing further clarified two of the four alpha-globin genes were deleted, alpha3.7 and alpha4.2, consistent with alpha-thalassemia trait and a heterozygous, frameshift mutation of c.27dupG on the hemoglobin subunit beta (HBB) gene associated with beta-thalassemia minor. The initial diagnosis of IDA was inaccurate following the iron studies displaying normal ferritin levels. This is the first report of combined alpha- and beta-thalassemia with a hemochromatosis H63D mutation. Although the clinical presentation of our patient and laboratory values are stable, the course of inheriting all three diseases together is unknown and may inflate the risk of future complications beyond reported studies. Frequent monitorization of hemoglobin and iron studies will be conducted to follow this rare presentation and prevent life-threating iron overload.

Low-cost production and application of lipopeptide for bioremediation and plant growth by Bacillus subtilis SNW3.

At present time, every nation is absolutely concern about increasing agricultural production and bioremediation of petroleum-contaminated soil. Hence, with this intention in the current study potent natural surfactants characterized as lipopeptides were evaluated for low-cost production by Bacillus subtilis SNW3, previously isolated from the Fimkessar oil field, Chakwal Pakistan. The significant results were obtained by using substrates in combination (white beans powder (6% w/v) + waste frying oil (1.5% w/v) and (0.1% w/v) urea) with lipopeptides yield of about 1.17 g/L contributing 99% reduction in cost required for medium preparation. To the best of our knowledge, no single report is presently describing lipopeptide production by Bacillus subtilis using white beans powder as a culture medium. Additionally, produced lipopeptides display great physicochemical properties of surface tension reduction value (SFT = 28.8 mN/m), significant oil displacement activity (ODA = 4.9 cm), excessive emulsification ability (E24 = 69.8%), and attains critical micelle concentration (CMC) value at 0.58 mg/mL. Furthermore, biosurfactants produced exhibit excellent stability over an extensive range of pH (1-11), salinity (1-8%), temperature (20-121°C), and even after autoclaving. Subsequently, produced lipopeptides are proved suitable for bioremediation of crude oil (86%) and as potent plant growth-promoting agent that significantly (P < 0.05) increase seed germination and plant growth promotion of chili pepper, lettuce, tomato, and pea maximum at a concentration of (0.7 g/100 mL), showed as a potential agent for agriculture and bioremediation processes by lowering economic and environmental stress.

Granisetron Extended-Release Subcutaneous Injection versus Palonosetron Infusion for CINV Prevention: Cost Comparison of Unscheduled Hydration.

Background: Granisetron extended-release subcutaneous (SC) injection is a novel formulation of granisetron for the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Palonosetron is administered intravenously and is indicated for CINV prevention in acute and delayed phases after the use of moderately emetogenic chemotherapy (MEC) and in the acute phase after highly emetogenic chemotherapy (HEC). No data are available regarding the impact of SC granisetron on the cost of unscheduled hydration compared with other antiemetic drugs, specifically the older-generation palonosetron. Objective: To compare the costs of unscheduled hydration associated with breakthrough CINV after SC granisetron versus palonosetron administration in patients receiving MEC or HEC. Methods: This retrospective analysis was based on electronic medical records data from a single multicenter, community-based practice involving patients receiving MEC or HEC with a 3-drug antiemetic regimen, including a neurokinin-1 receptor antagonist, dexamethasone, and either SC granisetron or palonosetron. A cost-of-care analysis for SC granisetron and palonosetron was based on the maximum per-unit Medicare reimbursement amounts for the use of unscheduled hydration, administration of rescue antiemetic drugs, laboratory tests, and patient office evaluations. Results: A total of 182 patient records were evaluated, 91 for patients receiving SC granisetron and 91 receiving palonosetron. The mean per-patient cost of care related to unscheduled hydration in patients receiving HEC or MEC was significantly lower with SC granisetron ($296) than palonosetron ($837; P <.0001), including subset analysis of patients requiring additional care (SC granisetron [$691], N = 39; palonosetron [$1058], N = 72; P = .0260). The mean hydration costs per patient receiving HEC or MEC were lower with SC granisetron ($62) than with palonosetron ($253; P <.0001). The hydration costs per patient receiving only HEC were lower with SC granisetron ($66) than palonosetron ($280; P <.0001). The per-patient costs were lower when SC granisetron was administered than when palonosetron was administered as part of the antiemetic regimen, except for the cost of rescue antiemetic drug in patients receiving MEC. Fewer median unscheduled hydration therapies per patient were used with SC granisetron versus palonosetron (HEC, 3 vs 5; MEC, 2 vs 3). Conclusion: The use of SC granisetron reduced the total per-patient costs of care associated with unscheduled hydration compared with palonosetron in patients receiving HEC or MEC for breakthrough CINV events.

Granisetron Extended-Release Subcutaneous Injection versus Palonosetron Infusion for CINV Prevention: Cost Comparison of Unscheduled Hydration.

Background: Granisetron extended-release subcutaneous (SC) injection is a novel formulation of granisetron for the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Palonosetron is administered intravenously and is indicated for CINV prevention in acute and delayed phases after the use of moderately emetogenic chemotherapy (MEC) and in the acute phase after highly emetogenic chemotherapy (HEC). No data are available regarding the impact of SC granisetron on the cost of unscheduled hydration compared with other antiemetic drugs, specifically the older-generation palonosetron. Objective: To compare the costs of unscheduled hydration associated with breakthrough CINV after SC granisetron versus palonosetron administration in patients receiving MEC or HEC. Methods: This retrospective analysis was based on electronic medical records data from a single multicenter, community-based practice involving patients receiving MEC or HEC with a 3-drug antiemetic regimen, including a neurokinin-1 receptor antagonist, dexamethasone, and either SC granisetron or palonosetron. A cost-of-care analysis for SC granisetron and palonosetron was based on the maximum per-unit Medicare reimbursement amounts for the use of unscheduled hydration, administration of rescue antiemetic drugs, laboratory tests, and patient office evaluations. Results: A total of 182 patient records were evaluated, 91 for patients receiving SC granisetron and 91 receiving palonosetron. The mean per-patient cost of care related to unscheduled hydration in patients receiving HEC or MEC was significantly lower with SC granisetron ($296) than palonosetron ($837; P <.0001), including subset analysis of patients requiring additional care (SC granisetron [$691], N = 39; palonosetron [$1058], N = 72; P = .0260). The mean hydration costs per patient receiving HEC or MEC were lower with SC granisetron ($62) than with palonosetron ($253; P <.0001). The hydration costs per patient receiving only HEC were lower with SC granisetron ($66) than palonosetron ($280; P <.0001). The per-patient costs were lower when SC granisetron was administered than when palonosetron was administered as part of the antiemetic regimen, except for the cost of rescue antiemetic drug in patients receiving MEC. Fewer median unscheduled hydration therapies per patient were used with SC granisetron versus palonosetron (HEC, 3 vs 5; MEC, 2 vs 3). Conclusion: The use of SC granisetron reduced the total per-patient costs of care associated with unscheduled hydration compared with palonosetron in patients receiving HEC or MEC for breakthrough CINV events.

α-Actinin Anchors PSD-95 at Postsynaptic Sites.

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.

Cardiac Myocyte Exosome Isolation.

Exosomes are cell-derived small extracellular membrane vesicles (50-100 nm in diameter) actively secreted by a number of healthy and diseased cell types. Exosomes can mediate cellular, tissue, and organ level micro communication under normal and pathological conditions by shuttling proteins, mRNA, and microRNAs. Prior to vesicle molecular profiling, these exosomes can be isolated from conditioned cell media or bodily fluids such as urine and plasma in order to explore the contents and functional relevance. Exosome purification and analyses are a fast-growing research field. Regardless of several advances in exosome purification and analyses methods, research still faces several challenges. Despite tremendous interest in the role of extracellular vesicles, there is no general agreement on dependable isolation protocols. Therefore, there is an urgent need to establish reliable protocol of exosome purification and analysis. Here, we report a simple cost-effective isolation and analysis of cardiac myocyte exosomes from conditioned media.

Mission CaMKIIγ: shuttle calmodulin from membrane to nucleus.

Neuronal plasticity depends on plasma membrane Ca(2+) influx, resulting in activity-dependent gene transcription. Calmodulin (CaM) activated by Ca(2+) initiates the nuclear events, but how CaM makes its way to the nucleus has remained elusive. Ma et al. now show that CaMKIIγ transports CaM from cell surface Ca(2+) channels to the nucleus.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release.

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Heart failure and mitochondrial dysfunction: the role of mitochondrial fission/fusion abnormalities and new therapeutic strategies.

The treatment of heart failure (HF) has evolved during the past 30 years with the recognition of neurohormonal activation and the effectiveness of its inhibition in improving the quality of life and survival. Over the past 20 years, there has been a revolution in the investigation of the mitochondrion with the development of new techniques and the finding that mitochondria are connected in networks and undergo constant division (fission) and fusion, even in cardiac myocytes. This has led to new molecular and cellular discoveries in HF, which offer the potential for the development of new molecular-based therapies. Reactive oxygen species are an important cause of mitochondrial and cellular injury in HF, but there are other abnormalities, such as depressed mitochondrial fusion, that may eventually become the targets of at least episodic treatment. The overall need for mitochondrial fission/fusion balance may preclude sustained change in either fission or fusion. In this review, we will discuss the current HF therapy and its impact on the mitochondria. In addition, we will review some of the new drug targets under development. There is potential for effective, novel therapies for HF to arise from new molecular understanding.

Competition between α-actinin and Ca²âº-calmodulin controls surface retention of the L-type Ca²âº channel Ca(V)1.2.

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²âº channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²âº influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²âº-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²âº influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²âº-calmodulin triggers Ca²âº-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²âº influx.

SERINE proteinase like activity in apolipophorin III from the hemolymph of desert locust, Schistocerca gregaria.

Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology.

An apolipophorin III protein from the hemolymph of desert locust, Schistocerca gregaria.

Apolipophorin III (apoLp-III) from insects and apolipoprotein A-I from humans, are major component of the lipoprotein and share various properties. ApoLp-III is an abundant hemolymph protein. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. ApoLp-III was isolated and purified from the hemolymph of desert locust Schistocerca gregaria by ion-exchange and reversed-phase chromatography. The purity and the molecular weight of apoLp-III were determined at ∼19,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to similarities in the amino terminal sequence, molar mass and retention on reversed-phase analytical HPLC column, this protein is a Schistocerca gregaria homologue of Locusta migratoria apoLp-III.

Assembly of a beta2-adrenergic receptor--GluR1 signalling complex for localized cAMP signalling.

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding.

Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and alpha-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges alpha-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of alpha-actinin. Whereas alpha-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing alpha-actinin. Displacement of alpha-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.

Structure of human MIP-3alpha chemokine.

The structure of the human macrophage inflammatory protein-3alpha (MIP-3alpha) has been determined at 1.81 angstroms resolution by X-ray crystallography. The dimer crystallized in the tetragonal space group I4, with unit-cell parameters a = b = 83.99, c = 57.20 angstroms. The crystals exhibit two molecules in the asymmetric unit. The structure was solved by the molecular-replacement method and the model was refined to a conventional R value of 20.6% (R(free) = 25.7%). MIP-3alpha possesses the same monomeric structure as previously described for other chemokines. However, in addition to limited structural changes in the beta1-beta2 hairpin of monomer B, the electron density is fully defined for a few extra residues at the N- and C-termini of monomer A and the C-terminus of monomer B compared with MIP-3alpha in space group P6(1). As the N-terminal and loop regions have been shown to be critical for receptor binding and signaling, this additional structural information may help in determining the basis of the CCR6 selectivity of MIP-3alpha.

Structure and increased thermostability of Rhodococcus sp. naphthalene 1,2-dioxygenase.

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.

Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase.

One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB 12038.

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear non-heme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 A, alpha = beta = gamma = 90 degrees, and diffract to 2.3 A resolution.