Full-length αIIbß3 cryo-EM structure reveals intact integrin initiate-activation intrinsic architecture.

Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryo-EM structures of the full-length αIIbß3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbß3 structure at 3 Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region. After the addition of a Mn2+ agonist, we resolve two coexisting structures representing two new states between inactive and active state. Our structures show conformational changes of the αIIbß3 activating trajectory and a unique twisting of the integrin legs, which is required for platelets accumulation. Our structure provides direct structural evidence for how the lower legs are involved in full-length integrin activation mechanisms and offers a new strategy to target the αIIbß3 lower leg.

Full-length αIIbß3 CryoEM structure reveals intact integrin initiate-activation intrinsic architecture.

Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation, thus pivotal for hemostasis, and arterial thrombosis as well as a proven drug-target for antithrombotic therapies. Here we resolve the cryoEM structures of the intact full-length αIIbß3, which covers three distinct states along the activation pathway. Here, we resolve intact αIIbß3 structure at 3Å resolution, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the head region ligand-binding domain tucked in a specific angle proximity to the TM region. In response to the addition of an Mn2+ agonist, we resolved two coexisting states, "intermediate" and "pre-active". Our structures show conformational changes of the intact αIIbß3 activating trajectory, as well as a unique twisting of the lower integrin legs representing intermediate state (TM region at a twisting conformation) integrin and a coexisting pre-active state (bent and opening in leg), which is required for inducing the transitioning platelets to accumulate. Our structure provides for the first time direct structural evidence for the lower legs' involvement in full-length integrin activation mechanisms. Additionally, our structure offers a new strategy to target the αIIbß3 lower leg allosterically instead of modulating the affinity of the αIIbß3 head region.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXß2.

Allosteric activation and silencing of leukocyte ß2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXß2 ligand-binding domain-the αX I-domain-corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH- and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXß2. This work highlights how intrinsically flexible pH- and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Hemophagocytic Lymphohistiocytosis in the Elderly.

Hemophagocytic lymphohistiocytosis (HLH) is a rare disease of massive, dysregulated cytokine release and secondary multiorgan failure and is associated with high mortality. Primary HLH occurs predominately in infants and young children with a genetic predisposition. Acquired HLH is less well characterized and usually occurs in younger adults in the setting of severe inflammation triggered by infection or malignancy. Little is known about the disease in elderly. We report 3 patients >50 years old who presented with multiorgan failure and shock without an identifiable source and were ultimately diagnosed with acquired HLH. We performed a literature review of HLH in adults >50 years of age and identified an additional 68 cases. Mean age was 62 years, with male predominance. Most cases were triggered by infection (49%) followed by malignancy (27%). Nineteen patients were treated with the HLH-94 protocol, 11 received corticosteroids and the remainder received non-HLH specific interventions. Overall mortality was 62%.

Infectious and Noninfectious Acute Pericarditis in Children: An 11-Year Experience.

OBJECTIVE: The study was undertaken to determine the etiology, review management, and outcome in children diagnosed with acute pericarditis during 11 years at tertiary pediatric institution. METHODS: Retrospective chart review of children diagnosed between 2004 and 2014. Patients with postsurgical pericardial effusions were excluded. RESULTS: Thirty-two children were identified (median age 10yr/11mo). Pericardiocentesis was performed in 24/32 (75%) patients. The most common cause of pericarditis was infection in 11/32 (34%), followed by inflammatory disorders in 9 (28%). Purulent pericarditis occurred in 5 children including 4 due to Staphylococcus aureus: 2 were methicillin resistant (MRSA). All patients with purulent pericarditis had concomitant infection including soft tissue, bone, or lung infection; all had pericardial drain placement and 2 required pericardiotomy and mediastinal exploration. Other infections were due to Histoplasma capsulatum (2), Mycoplasma pneumoniae (2), Influenza A (1), and Enterovirus (1). Pericarditis/pericardial effusion was the initial presentation in 4 children with systemic lupus erythematosus including one who presented with tamponade and in 2 children who were diagnosed with systemic onset juvenile inflammatory arthritis. Tumors were diagnosed in 2 patients. Five children had recurrent pericarditis. Systemic antibiotics were used in 21/32 (66%) and prednisone was used in 11/32 (34%) patients. CONCLUSION: Infections remain an important cause of pericarditis in children. Purulent pericarditis is most commonly caused by Staphylococcus aureus and is associated with significant morbidity, need of surgical intervention, and prolonged antibiotic therapy. Echocardiography-guided thoracocentesis remains the preferred diagnostic and therapeutic approach. However, pericardiotomy and drainage are needed when appropriate clinical response is not achieved with percutaneous drainage.

Fluorescence of tautomeric forms of curcumin in different pH and biosurfactant rhamnolipids systems: Application towards on-off ratiometric fluorescence temperature sensing.

Medicinal properties of curcumin are widely getting realized. For its applicability as a hydrophobic drug molecule and food spice interaction of curcumin with rhamnolipids, a biosurfactant, bears importance. Here we have explored interaction of curcumin with rhamnolipids biosurfactant and its aggregation behavior. The impact of pH on critical micelle concentration (cmc) of rhamnolipids has been studied using fluorescence of curcumin and found that cmc of rhamnolipids increases with increase in pH of the medium. In acidic, neutral and slightly alkaline medium (pH8), at λex=355nm (for ß-diketone form) curcumin undergoes excited state hydrogen transfer (ESHT) and emits solely from enol form both in the presence and absence of rhamnolipids, but first time we report that in extreme alkaline condition, at pH13, at λex=355nm curcumin emits from both ß-diketone as well as enolic ESHT forms in absence of rhamnolipids but in the presence of rhamnolipids ß-diketone is stabilized and the emission solely comes from ß-diketone by completely revoking ESHT process. Fluorescence quenching by hydrophobic cetylpyridinium bromide confirms curcumin penetrates deep inside the hydrophobic pocket of rhamnolipid aggregates/micelle by reducing the distance between N+-atom of pyridinium ion and curcumin. On the other hand hydrophobic molecule like pyrene stays near to the Stern layer of rhamnolipids facilitating electron transfer from pyrene to N+-atom of pyridinium ion. Even in neutral condition, in the presence of rhamnolipids the ß-diketone form, though in small proportions, can be stabilized in higher temperature in expense of enolic ESHT form, thus, offering an on off ratiometric fluorescence temperature sensing in solution, which bears significance as ratiometric probe molecules. Interaction of curcumin with rhamnolipids stabilizes curcumin in acidic, neutral and moderate alkaline condition but fails at extreme pH13.

Polyelectrolyte mediated nano hybrid particle as a nano-sensor with outstandingly amplified specificity and sensitivity for enzyme free estimation of cholesterol.

As a proof of concept, here it is established that curcumin integrated chitosan oligosaccharide lactate (COL) self-assembles on silica nanoparticle surface to form nano hybrid particles (NHPs). These NHPs have size in the ranges of 25-35nm with silica nanoparticle as its core and curcumin-COL as outer layer having thickness of 4-8nm. The fluorescence intensity of these NHPs are found to be quenched and emission maximum is ~50nm red shifted compared to free curcumin implying inner filter effect and/or homo-FRET between curcumin molecules present on the surface of individual nano hybrid particle. Although fluorescence of free curcumin is remarkably quenched by Hg2+/Cu2+ ions due to chelation through keto-enol form, the fluorescence of NHPs is unaffected by Hg2+/Cu2+ ion that boosts analytical selectivity. The fluorescence intensity is outstandingly enhanced in the presence of cholesterol but is not influenced by ascorbic acid, uric acid, glucose, albumin, lipid and other potential interfering substances that either obstruct during enzymatic reaction or affect fluorescence of free curcumin. Thus, NHPs outstandingly improve analytical specificity, selectivity and sensitivity during cholesterol estimation compared to free curcumin. The interaction between cholesterol and NHPs is found to be a combination of ground state electrostatic interaction through the free hydroxyl group of cholesterol along with hydrophobic interaction between NHPs and cholesterol and excited state interaction. The proposed cholesterol biosensor illustrates a wider linear dynamic range, 0.002-10mmolL-1, (upper limit is due to lack of solubility of cholesterol) needed for biomedical application and better than reported values during enzymatic reaction. In addition, the NHPs are found to be photo-stable potentially making it suitable for simple, quick and cost-effective cholesterol estimation and opening an alternative approach other than enzymatic reaction using nano hybrid structure to tune analytical specificity, selectivity and sensitivity of probe molecule.

Interaction of curcumin with 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine liposomes: Intercalation of rhamnolipids enhances membrane fluidity, permeability and stability of drug molecule.

Stability of curcumin in neutral and alkaline buffer conditions has been a serious concern for its medicinal applications. We demonstrate that the stability of curucmin can be improved in 1,2-Dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. Curcumin strongly partition into liquid crystalline phase compared to solid gel phase of DSPC liposomes. Variation of fluorescence intensity of curcumin associated with liposomes with temperature successfully determines phase transition temperature of DSPC liposomes. However, at higher molar ratio curcumin can influence phase transition temperature by intercalating into deep hydrophobic layer of liposomes and facilitating fusion of two membrane phases. Rhamnolipids (RLs) are recently being applied for various biomedical applications. Here, we have explored new insight on intercalation of rhamnolipids with DSPC liposomes. Intercalation of rhamnolipids exceptionally increases partition of curcumin into solid gel phase of DSPC liposomes, whereas this increase is moderate in liquid crystalline phase. Fluorescence quenching study establishes that permeability and fluidity of the DSPC liposomes are enhanced in the presence of RLs. Membrane permeability and fluidity can be improved further by increasing the percentage of RLs in DSPC liposomes. The phase transition temperature of DSPC liposomes decreases with increase in percentage of RLs in DSPC liposomes by encouraging fusion between solid gel and liquid crystalline phases. Intercalation of RLs is found to further boost stability of drug, curcumin, in DSPC liposomes. Thus, mixing RLs with DSPC liposomes could potentially serve as a good candidate for drug delivery application.

Encapsulation of curcumin in cyclodextrin-metal organic frameworks: Dissociation of loaded CD-MOFs enhances stability of curcumin.

Curcumin has been successfully encapsulated in cyclodextrin-metal organic frameworks (CD-MOFs) without altering their crystallinity. The interaction between curcumin and CD-MOFs is strong through hydrogen bond type interaction between the OH group of cyclodextrin of CD-MOFs and the phenolic hydroxyl group of the curcumin. Interestingly, dissolving the curcumin loaded CD-MOFs crystals in water results in formation of a unique complex between curcumin, γCD and potassium cations. In fact, the initial interaction between curcumin and CD-MOF is crucial for the formation of the latter. This new complex formed in alkaline media at pH 11.5 has maximum absorbance at 520nm and emittance at 600nm. Most importantly, the stability of curcumin in this complex was enhanced by at least 3 orders of magnitude compared to free curcumin and curcumin:γ-CD at pH 11.5. These results suggest a promising benign system of CD-MOFs, which can be used to store and stabilize curcumin for food applications.

Band, target, and onion patterns in Co(OH)2 Liesegang systems.

The study of morphology and shape development has gained considerable interest in certain sciences, notably biology and geology. Liesegang experiments producing Co(OH)2 stratification are performed here, in one, two, and three dimensions for comparison of the pattern morphologies. We obtain well-resolved bands in one dimension, target patterns (rings) in two dimensions, and onion patterns (spherical shells) in three dimensions. The morphological characteristics of the various patterns (spacing coefficients, rate of growth of ring spacing with distance) were measured. The spacing ratio of the strata in the different spatial dimensions was found to be anticorrelated with the surface-to-volume ratio of the gel domain. Some studies featuring the importance of morphology in Liesegang systems are briefly surveyed.