Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor.

RyR1 is an intracellular Ca2+ channel important in excitable cells such as neurons and muscle fibers. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 Å resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.

Structural basis for DNA proofreading.

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

Thermal plasma processing of Moringa oleifera biochars: adsorbents for fluoride removal from water.

Anthropogenic activities accelerate fluoride contamination in groundwater, which largely affects public health. Though biochars have been explored for defluoridation, the plasma technology-based production of biochars has not received as considerable attention as other methods and it is also important that biochars be tested on groundwater samples. In the present study, for the first time, we report the preparation of biochars from different parts of Moringa oleifera using thermal plasma processing and demonstrate fluoride adsorption in both synthetic and contaminated groundwater. Water samples were collected from different locations in Nuapada district of Odisha such as Kotamal-Makardampada (20°24'46''N 82°37'19''E), Pandrapathar (20°34'41''N 82°39'25''E), Karlakot-Kadobhata (20°22'52''N 82°37'24''E), Kotamal-Jhakarpada (20°24'35''N 82°37'20''E), and Dohelpada (20°33'50''N 82°38'57''E). The Moringa leaf samples are processed at 1600 °C for 3 min in an inert atmosphere under a continuous flow of argon to get suitable biochars. The plasma-synthesized biochars contain larger exposed surfaces, which are efficient for the adsorption of fluoride. The prepared biochars were highly porous, amorphous, and contain > 72% carbon, which increases the efficiency of defluoridation due to the surface adsorbate site exposed. XRD of the samples showed the presence of calcium hydroxide, magnesium oxide, and calcium oxide, and large peaks of carbon. Raman data showed the double bond of carbon with oxygen in the form of carbonyl bonds, thioether, and sulfhydryl bonds, which contribute to the protonated site for the adsorption of fluoride, and assist in water penetration and swelling of biochars. The biochar of Moringa oleifera is very efficient for the adsorption of fluoride from standard samples as well as groundwater samples up to a concentration of 6 ppm. Conclusively, the present investigation shows that Moringa oleifera leaves are a good alternative adsorbent that could be used for the removal of fluoride from groundwater samples with > 85% removal in 18 h using 1 g biochar for 100 mL or 10 g biochar for 1 L water containing 4 ppm fluoride. To our knowledge, this is the first report on the thermal plasma-based production of Moringa biochars for the removal of fluoride from drinking water.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM.

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for a seamless transition from inactivated to closed conformations.

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells.

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.

miRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures.

Sulfatase enzymes catalyze sulfate ester hydrolysis, thus deficiencies of sulfatases lead to the accumulation of biomolecules resulting in several disorders. One of the important sulfatases is estrone sulfatase that converts inactive estrone sulfate to active estradiol. Posttranslational modification of highly conserved cysteine residue leads to unique formylglycine in the active site of sulfatases being critical for its catalytic activity. The essential factor responsible for this modification of sulfatase is Sulfatase-Modifying Factor 1 (SUMF1). The role of estrone sulfatase is well evident in breast cancer progression. However, the function and regulation of SUMF1 in cancer are not studied. In the present study, for the first time, we have assessed the expression of SUMF1 in breast cancer and report the oncogenic behavior upon overexpression of SUMF1. Although increased expression or activity of SUMF1 is anticipated based on its function, the expression of SUMF1 was found to be reduced in breast cancer cells at both mRNA and protein levels. An estrogen receptor (ER) dependent expression of SUMF1 was observed and higher SUMF1 expression is associated with improved breast cancer patient survival in ER-positive cases. However, high SUMF1 expression leads to reduced median survival in ER-negative breast cancer patients. Putative binding sites for miRNAs-106b-5p, 128-3p and 148b-3p were found at 3'-UTR of SUMF1. Since self-assembled branched DNA (bDNA) structures have emerged as a highly efficient strategy for targeting multiple miRNAs simultaneously, we studied the alteration in SUMF1 expression using bDNA nanostructures with a complementary sequence to miRNAs. The findings suggest the involvement of co-regulators and repressors in miRNA-mediated SUMF1 expression in breast cancer cells and reveal the therapeutic potential of SUMF1 in endocrine-related malignancies.

Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM.

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

Molecular and structural analysis of a mechanical transition of helices in the L. donovani coronin coiled-coil domain.

Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the 'ade' core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position 'a' in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.

Preparation of Stable Branched DNA Nanostructures: Process of Cooperative Self-Assembly.

The construction of functionalizable branched DNA (bDNA) relies on the designing of oligonucleotides and exploitation of their complementary chemistries. The stability of these structures largely depends on the hybridization specificity of the contributing oligonucleotides. However, most of the bDNA structures are not found suitable for in vivo application due to poor yield owing to uncharacterized hybridization efficiency and instability in biological fluids. In this report, our group has explored a mechanistic way for studying the hybridization pathway of genomic sequence derived oligonucleotides that are self-assembled to fabricate robust bDNA structures. The effect of change in nucleotide sequences on bDNA stability was studied by taking oligonucleotides derived from primers of different genes. Additionally, the stability of the bDNA in solutions with different pH, salts, and DNaseI which mimics physiological environment was reported. It was found that genomic sequence derived oligonucleotides self-assembled in a cooperative manner to yield the designed bDNAs, which are stable in physiological environment.

A cryo-EM-based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor.

Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain. Because the more flexible conformation may correspond to RyR2 with a phosphorylated P2 domain, we suggest that phosphorylation may set RyR2 in a conformation that needs less energy to transition to the open state. Comparison of RyR2 from cardiac muscle and RyR1 from skeletal muscle showed substantial structural differences between the two, especially in the helical domain 2 (HD2) structure forming the Clamp domain, which participates in quaternary interactions with the dihydropyridine receptor and neighboring RyRs in RyR1 but not in RyR2. Rigidity of the HD2 domain of RyR2 was enhanced by binding of FKBP12.6, a ligand that stabilizes RyR2 in the closed state. These results help to decipher the molecular basis of the different mechanisms of activation and oligomerization of the RyR isoforms and could be extended to RyR complexes in other tissues.

Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice.

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices.

Cerium chloride stimulated controlled conversion of B-to-Z DNA in self-assembled nanostructures.

DNA adopts different conformation not only because of novel base pairs but also while interacting with inorganic or organic compounds. Self-assembled branched DNA (bDNA) structures or DNA origami that change conformation in response to environmental cues hold great promises in sensing and actuation at the nanoscale. Recently, the B-Z transition in DNA is being explored to design various nanomechanical devices. In this communication we have demonstrated that Cerium chloride binds to the phosphate backbone of self-assembled bDNA structure and induce B-to-Z transition at physiological concentration. The mechanism of controlled conversion from right-handed to left-handed has been assayed by various dye binding studies using CD and fluorescence spectroscopy. Three different bDNA structures have been identified to display B-Z transition. This approach provides a rapid and reversible means to change bDNA conformation, which can be used for dynamic and progressive control at the nanoscale.

Lanthanum induced B-to-Z transition in self-assembled Y-shaped branched DNA structure.

Controlled conversion of right-handed B-DNA to left-handed Z-DNA is one of the greatest conformational transitions in biology. Recently, the B-Z transition has been explored from nanotechnological points of view and used as the driving machinery of many nanomechanical devices. Using a combination of CD spectroscopy, fluorescence spectroscopy, and PAGE, we demonstrate that low concentration of lanthanum chloride can mediate B-to-Z transition in self-assembled Y-shaped branched DNA (bDNA) structure. The transition is sensitive to the sequence and structure of the bDNA. Thermal melting and competitive dye binding experiments suggest that La(3+) ions are loaded to the major and minor grooves of DNA and stabilize the Z-conformation. Our studies also show that EDTA and EtBr play an active role in reversing the transition from Z-to-B DNA.

Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.

Congenital interparietal encephalocele: a case report.

Encephalocele is a mesodermal defect in the skull bones and duramater. Parietal encephalocele is a rare congenital anomaly of newborn with variable prognostic value. The authors report a case of a very large inter parietal encephalocele with no associated other system malformations. A midline inter parietal encephalocele is much rare, earlier reported cases were posterior parietal in location. Such cases can be successfully operated upon with a very good outcome. A unique case of a 18 day neonate, with swelling over scalp was evaluated by the neurosurgical team and the patient underwent neurosurgery. In planning the strategy for management of encephalocele, one needs to take into consideration the site, size, contents, patency of CSF pathway, neurological status and other associated anomalies. Inspite of such a big encephalocele in an atypical location, excision and repair gave excellent results.

Aneurysmal bone cyst of the pubis: a case report.

An aneurysmal bone cyst is considered as a locally aggressive benign tumour. Intra-lesional extended curettage and bone-grafting is the mainstay of the treatment for aneurysmal bone cysts. Grafting is used usually in cases where the lesion compromises the mechanical strength of the bone. However, the massive size of the highly vascular tumour and the relative inaccessibility of its deeper extensions into the femoral vessels and the intra-abdominal structures, especially the urinary bladder, make it a relatively challenging case to perform excision and curettage.Presenting a case of a 15 years old male patient with the complaint of a right inguinal swelling since the past eight months. The swelling had started growing since the past two months and it was associated with pain. X-ray showed a lytic blowout legion of the entire right pubic ramus. An intra-lesional curettage was done. Complete tumour excision which was done by intra-lesional curettage and biopsy yielded satisfactory results with low complications and low recurrence of aneurysmal bone cyst of the superior ramus of the pubis.

Giant cell tumor of lower end of tibia.

Introduction. Giant cell tumor of bones is an unusual neoplasm that accounts for 4% of all primary tumors of bone, and it represents about 10% of malignant primary bone tumors with its different grades from borderline to high grade malignancy. Case Report. A 35-year-old patient presented with complains of pain and swelling in left ankle since 1 year following a twisting injury to his left ankle. On examination, swelling was present over the distal and anterior part of leg and movements of ankle joint were normal. All routine blood investigations were normal. X-ray and CT ankle showed morphology of subarticular well-defined expansile lytic lesion in lower end of left tibia suggestive of giant cell tumor. Histopathology of the tissue shows multinucleated giant cells with uniform vesicular nucleus and mononuclear cells which are spindle shaped with uniform vesicular nucleus suggestive of GCT. The patient was treated by excision, curettage, and bone cement to fill the defect. Conclusion. The patient at 12-month followup is doing well and walking without any pain comfortably and with full range of motion at ankle joint with articular congruity maintained and no signs of recurrences.

Is closed manipulative reduction and percutaneous Kirschner wiring of supracondylar humeral fracture in children as day-care surgery a safe procedure ?

INTRODUCTION: Supracondylar fracture of the humerus is a common injury in children. It accounts for 60% of fractures around the elbow children. If the fracture is not treated properly it may give rise to many complications like malunion, Volkmann's ischemic contracture, nerve injury, arterial injury, skin slough, heterotopic bone formation , and stiffness of elbow. The management of displaced supracondylar fracture of the elbow is one of the most difficult of the many fractures seen in children. The purpose of the study was to evaluate the anatomical and functional results of treatment of supracondylar fractures of humerus with closed reduction and percutaneous 'K' wire fixation as a day care procedure and record associated complications, thus decreasing the cost of treating these fractures and hospitalization. METHODS: Fifty displaced closed extension type supracondylar fractures (Gartland's type III) of the humerus in children were treated by closed reduction and percutaneous fixation with Kirschner wires. All the patients selected for this study had been treated in a day care unit and were discharged in the same evening and followed up at 3 and 6 weeks and 3 months. Open fractures, fractures with neurovascular complications and children older than 15 yrs were excluded. The final results were evaluated by Flynn's criteria. RESULTS: The majority (72%), of the patients had fracture displaced posteomedially, Fourty one of the fifty patients had satisfactory results. The majority of the patients were male, and the average age was 8-9 years. CONCLUSION: Percutaneous fixation of supracondylar humerus done as a day care procedure is an acceptable modality of treatment and reduces the duration of hospital stay for the patient. KEY WORDS: Supracondylar humerus, K-wire fixation, day care procedure.

Remote sensing study on geomorphological degradation of Sarda Sagar reservoir.

An attempt was made to estimate the geomorphological degradation due to sedimentation of Sarda Sagar reservoir, located in Pilibhit and Udhamsingh Nagar, district of Uttar Pradash and Uttarakhand respectively. The study was conducted using multidated IRS LIISS III remote sensing data for the year 2006-2007. Using satellite images of different seasons during 2006-2007, a total of 45.23 million m3 volume of sedimentation was computed in-between the 183.704 m and 190.504 m elevation. The reservoir has lost 11.72 % of the total capacity of water storage and an average rate of sedimentation was calculated as 0.26 % per year. Due to this sedimentation the new feeder channel of Sarda Sagar is choked with silt and the water flow from this channel has almost stopped. The morphology of the reservoir has been changed due to sedimentation during the period 1962 to 2007. This has altered breeding ground of fishes since important indigenous fish species which need flowing water condition to perform the breeding. This study would be helpful for the planners to manage the reservoir and to assess the biological productivity.