An unusual presentation of penile fracture with complete transection of urethra: a case report.

Penile fracture is one such urologic emergency that occurs when the penis is struck bluntly during sexual activity, and in less than 5-10% of cases, the concurrent urethral damage is evident, but complete transection is very rare. A 37-year-old male presented with a history of 'snap' sound and immediate detumescence of penis during intercourse, when he fell and hit the pubic bone of his partner. There was acute retention of urine, an attempt to pass a catheter failed and the patient underwent supra-pubic catheterization. On examination, there was classical 'eggplant deformity' of the penis with blood at the tip of the meatus. MRI showed a tunical tear on both sides at the penoscrotal junction with indistinct urethra and extensive hematoma in the proximal penile shaft. Surgical management was successfully done by anastomotic urethroplasty and cavernosal repair.

Magnesium Hydrogen Phosphate: An Efficient Catalyst for Acrylic Acid Production from Biorenewable Lactic Acid.

A series of Magnesium hydrogen phosphate (MgHP) catalysts with different magnesium to phosphorous (Mg/P) mole ratios at varying calcination temperatures has been synthesised, bearing in mind the effectiveness as well as the stability of MgHP to catalyse acrylic acid (AA) production from biorenewable lactic acid (LA), a synthetic process applicable to biomass conversion. The physicochemical properties of the MgHP catalysts have been thoroughly characterised and the formation of Mg(NH4)PO4, MgHPO4 and Mg2P2O7 with different structural and acidic properties have been reported. The high catalytic performance of MgHP catalysts with high AA yields (100% conversion and 85% selectivity) at high space velocities (WHSVLA = 3.13 h-1) have been achieved at 360 °C. NH3-Temperature programmed desorption (TPD) and pyridine FTIR have shown that the effectiveness of a catalyst is accounted for not primarily by the actual strength of acidic sites, but is due to the presence of Lewis acidic sites compared to Bronsted sites.

Efficient Vapor-Phase Selective Hydrogenolysis of Bio-Levulinic Acid to γ-Valerolactone Using Cu Supported on Hydrotalcite Catalysts.

In this work, Cu nanoparticles (Cu NPs, 2-20 nm) supported on Hydrotalcite catalysts exhibit enhanced selectivity for γ-valerolactone (GVL) during hydrogenolysis of levulinic acid (LA). At 260 °C, over 3 wt% Cu achieved 87.5% of LA conversion with a maximum GVL selectivity (95%). In contrast, LA hydrogenolysis over 3Cu/Hydrotalcite catalyst is highly active and stable toward the production of GVL due to balanced acido-basicity and higher Cu dispersion with ultrasmall particle sizes, which are investigated through the temperature programmed desorption (TPD) of ammonia, N2O titration, and transmission electron microscopy (TEM) analysis. Hydrotalcite in combination with inexpensive Cu catalyst is found to be an efficient and environmentally benign for LA hydrogenolysis.

Understanding regulation in substrate dependent modulation of growth and production of alcohols in Clostridium sporogenes NCIM 2918 through metabolic network reconstruction and flux balance analysis.

Flux Balance Analysis was performed for Clostridium sporogenes NCIM 2918 grown on sole glucose and glycerol or glucose-glycerol combinations at varied concentrations. During acidogenesis, glucose and glucose-glycerol combinations favored improved growth and butyric acid production. Glycerol fermentation was however marked by reduced growth and predominant ethanol synthesis. Further, with increase of glycerol fraction in glucose-glycerol blend, flux towards ethanol synthesis linearly increased with simultaneous decrease in butanol flux. Elevated ATP demand due to improved growth was satisfied by upregulated carbon flux towards butyric acid synthesis during both glucose and dual substrate fermentations. Possible repression of pyruvate carboxylase by glycerol resulting in downturn of carbon uptake flux towards TCA cycle through anaplerotic reaction may be responsible for reduced growth in glycerol fermentation. Ammonium acetate mediated induction of acetic acid utilization, during acidogenesis, led to excess acetyl-CoA generation and its subsequent metabolism to lesser reduced products, butyric acid or ethanol.

High dielectric permittivity and improved mechanical and thermal properties of poly(vinylidene fluoride) composites with low carbon nanotube content: effect of composite processing on phase behavior and dielectric properties.

The composite processing technique and nanofiller concentration and its functionalization significantly alter the properties of polymer nanocomposites. To realize this, multi-walled carbon nanotubes (CNT) were dispersed in a poly(vinylidene fluoride) (PVDF) matrix at carefully selected CNT concentrations by two illustrious methods, such as solution-cast and melt-mixing. Notwithstanding the processing method, CNTs induced predominantly the γ-phase in PVDF, instead of the commonly obtained ß-phase upon nanofiller incorporation, and imparted significant improvements in dielectric properties. Acid-treatment of CNT improved its dispersion and interfacial adhesion significantly with PVDF, and induced a higher γ-phase content and better dielectric properties in PVDF as compared to pristine CNT. Further, the γ-phase content was found to be higher in solution-cast composites than that in melt-mixed counterparts, most likely due to solvent-induced crystallization in a controlled environment and slow solvent evaporation in the former case. However, interestingly, the melt-mixed composites showed a significantly higher dielectric constant at the onset of the CNT networked-structure as compared to the solution-cast composites. This suggests the possible role of CNT breakage during melt-mixing, which might lead to higher space-charge polarization at the polymer-CNT interface, and in turn an increased number of pseudo-microcapacitors in these composites than the solution-cast counterparts. Notably, PVDF with 0.13 vol% (volume fraction, f c  = 0.0013) of acid-treated CNTs, prepared by melt-mixing, displayed the relative permittivity of ∼217 and capacitance of ∼5430 pF, loss tangent of ∼0.4 at 1 kHz and an unprecedented figure of merit of ∼10(5). We suggest a simple hypothesis for the γ-phase formation and evolution of the high dielectric constant in these composites. Further, the high-dielectric composite film showed marked improvements in mechanical and thermal properties over the neat PVDF film. These composites with exceptional dielectric properties and concomitant improvement in mechanical and thermal properties offer a great promise for use in flexible and mechanically robust charge storage devices.

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca(2+)-dependent and in a Ca(2+)-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca(2+) response and other basic biophysical properties, we have characterized the Ca(2+) filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca(2+) binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca(2+) binding process becomes more prominent in the presence of Mg(2+). Besides, the analysis of (15)N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca(2+) binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1.

Information on the low-energy excited states of a given protein is important as this controls the structural adaptability and various biological functions of proteins such as co-operativity, response towards various external perturbations. In this article, we characterized individual residues in both non-myristoylated (non-myr) and myristoylated (myr) neuronal calcium sensor-1 (NCS-1) that access alternate states by measuring nonlinear temperature dependence of the backbone amide-proton (¹H(N)) chemical shifts. We found that ~20% of the residues in the protein access alternative conformations in non-myr case, which increases to ~28% for myr NCS-1. These residues are spread over the entire polypeptide stretch and include the edges of α-helices and ß-strands, flexible loop regions, and the Ca²(+)-binding loops. Besides, residues responsible for the absence of Ca²(+)-myristoyl switch are also found accessing alternative states. The C-terminal domain is more populated with these residues compared to its N-terminal counterpart. Individual EF-hands in NCS-1 show significantly different number of alternate states. This observation prompts us to conclude that this may lead to differences in their individual conformational flexibility and has implications on the functionality. Theoretical simulations reveal that these low-energy excited states are within an energy band of 2-4 kcal/mol with respect to the native state.

Sequence specific 1H, 13C and 15N backbone resonance assignments of UVI31+ from Chlamydomonas reinhardtii.

The cDNA of UVI31+ was cloned from C. reinhardtii and expressed in E. coli from where the protein was purified to homogeneity. The purified protein exhibited beta-lactamase activity (Manuscript in preparation). However, UVI31+ has no homology with the known ß-lactamases. In order to understand the structural basis of the ability of UVI31+ to hydrolyze ß-lactam antibiotics, we in parallel, set out to structurally characterize it by NMR. Its ß-lactamase activity in relation to the solution structure by NMR is likely to provoke deeper understanding of its mechanism and facilitate the rationalization of other functions of the protein, if any. In this endeavor, we report almost complete sequence-specific backbone (1)H, (13)C and (15)N NMR assignments of UVI31+.

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea.

The sequence specific (1)H, (13)C and (15)N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for ß-rich conformation in the denatured state.

Resonance assignments of myristoylated and non-myristoylated neuronal calcium sensor-1(NCS-1) embedded in a membrane.

Neuronal Calcium Sensor-1 (NCS-1) is a member of calcium sensor family. It is originally identified as frequenin. NCS-1 has been found to interact with membrane and cytosolic proteins and its physiological role is governed by N-terminal myristoylation. In this paper, we report the NMR assignments of both myristoylated and non-myristoylated NCS-1 in the presence of a membrane.

Structural change in a B-DNA helix with hydrostatic pressure.

Study of the effects of pressure on macromolecular structure improves our understanding of the forces governing structure, provides details on the relevance of cavities and packing in structure, increases our understanding of hydration and provides a basis to understand the biology of high-pressure organisms. A study of DNA, in particular, helps us to understand how pressure can affect gene activity. Here we present the first high-resolution experimental study of B-DNA structure at high pressure, using NMR data acquired at pressures up to 200 MPa (2 kbar). The structure of DNA compresses very little, but is distorted so as to widen the minor groove, and to compress hydrogen bonds, with AT pairs compressing more than GC pairs. The minor groove changes are suggested to lead to a compression of the hydration water in the minor groove.

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions.

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.

Sequence specific 1H, 13C, and 15N resonance assignments of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily.

The sequence specific (1)H, (13)C, and (15)N resonance assignments of Hahellin, a putative member of betagamma-crystallin family, from Hahella Chejuensis, have been accomplished by NMR spectroscopy. The resonance assignments reveal that the protein adopts predominantly a beta-sheet conformation as in the case of betagamma-crystallin folds.

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double-stranded RNA binding domain of protein kinase R.

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common alpha-beta-beta-beta-alpha tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N-1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime.

Measurement of 1J(Ni,Calpha(i)), 1J(Ni,C'i-1), 2J(Ni,Calpha(i-1)), 2J(H(N)i,C'i-1) and 2J(H(N)i,Calpha(i)) values in 13C/15N-labeled proteins.

Use of partial or selective (13)C/(15)N labeling of specific amino acid residues in a given protein to measure the values of (1)J((15)N(i),(13)C(alpha) (i)), (2)J((1)H(N),(13)C(alpha) (i)), (2)J((15)N(i),(13)C(alpha) (i-1)), (1)J((15)N(i),(13)C'(i-1)) and (2)J((1)H(N),(13)C'(i-1)) is described. This was achieved by recording a sensitivity-enhanced 2D [(15)N-(1)H] HSQC experiment, without mixing the spin states of C(alpha) and C' during the course of entire experiment.

A novel approach for uniform (13)C and (15)N labeling of DNA for NMR studies.

A novel method is proposed for large-scale synthesis of (13)C- and (15)N-labeled DNA for NMR studies. In this methodology, endonuclease-sensitive repeat amplification (ESRA), a modified PCR strategy, has been used to amplify tandem repeats of the target DNA sequence. The design of the template is such that restriction enzyme (RE) sites separate repeats of the target sequence. The ESRA product is then cloned into a suitable vector. The Escherichia coli cells harboring the plasmid are grown in minimal medium containing [(13)C]glucose and (15)NH(4)Cl as the sole source of carbon and nitrogen, respectively. The target sequence is released by RE digestion of the plasmid, followed by purification using PAGE. Under optimized conditions, the yield ( approximately 5 mg/liter of culture) of (13)C/(15)N-labeled DNA prepared using this approach is found to be several times higher compared to other known enzymatic methods. Successful incorporation of the isotopes has been confirmed using 2D NMR techniques.

NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba Histolytica.

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger water exposed total hydrophobic surface area as compared to CaM and TnC. Further dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play crucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino acid composition is required for the function of the protein.

Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA.

Klenow-DNA complex is known to undergo a rate-limiting, protein conformational transition from an 'open' to 'closed' state, upon binding of the 'correct' dNTP at the active site. In the 'closed' state, Mg(2+) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3' hydroxyl of the primer terminus. The enzyme returns to the 'open' state upon the release of PPi and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the addition of the next dNTP, without the preceding chemical step, we studied the ternary complex (Klenow-DNA-dNTP) in the absence of Mg(2+). While the ternary complex is proficient in chemical addition of dNTPs in Mg(2+), as revealed by primer extensions, the same in Mg(2+)-deficient conditions lead to non-covalent (physical) sequestration of first two 'correct' dNTPs in the ternary complex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP-DNA complex is found to be stable even after the dissociation of KLENOW: This reveals the novel state of the dNTP-DNA complex where the complementary base is stacked in a DNA-helix non-covalently, without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg(2+)-deficient ternary complex exhibits a 'closed' conformation.

Selective 'unlabeling' of amino acids in fractionally 13C labeled proteins: an approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues.

A novel methodology for stereospecific NMR assignments of methyl (CH3) groups of Val and Leu residues in fractionally 13C-labeled proteins is presented. The approach is based on selective 'unlabeling' of specific amino acids in proteins while fractionally 13C-labeling the rest. A 2D [13C-1H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the 'unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH3 groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP).