Implication of chromosomal microarray analysis prior to in-utero repair of fetal open neural tube defect.

OBJECTIVE: In-utero repair of open neural tube defects (ONTD) is an accepted treatment option with demonstrated superior outcome for eligible patients. While current guidelines recommend genetic testing by chromosomal microarray analysis (CMA) when a major congenital anomaly is detected prenatally, the requirement for an in-utero repair, based on the Management of Myelomeningocele Study (MOMS) criteria, is a normal karyotype. In this study, we aimed to evaluate if CMA should be recommended as a prerequisite for in-utero ONTD repair. METHODS: This was a retrospective cohort study of pregnancies complicated by ONTD that underwent laparotomy-assisted fetoscopic repair or open-hysterotomy fetal surgery at a single tertiary center between September 2011 and July 2021. All patients met the MOMS eligibility criteria and had a normal karyotype. In a subset of the pregnancies (n = 77), CMA testing was also conducted. We reviewed the CMA results and divided the cohort into two groups according to whether clinically reportable copy-number variants (CNV) were detected (reportable-CNV group) or not (normal-CMA group). Surgical characteristics, complications, and maternal and early neonatal outcomes were compared between the two groups. The primary outcomes were fetal or neonatal death, hydrocephalus, motor function at 12 months of age and walking status at 30 months of age. Standard parametric and non-parametric statistical tests were employed as appropriate. RESULTS: During the study period, 146 fetuses with ONTD were eligible for and underwent in-utero repair. CMA results were available for 77 (52.7%) patients. Of those, 65 (84%) had a normal CMA and 12 (16%) had a reportable CNV, two of which were classified as pathogenic. The first case with a pathogenic CNV was diagnosed with a 749-kb central 22q11.21 deletion spanning low-copy-repeat regions B-D of chromosome 22; the second case was diagnosed with a 1.3-Mb interstitial deletion at 1q21.1q21.2. Maternal demographics, clinical characteristics, operative data and postoperative complications were similar between those with normal CMA results and those with reportable CNVs. There were no significant differences in gestational age at delivery or any obstetric and early neonatal outcome between the study groups. Motor function at birth and at 12 months of age, and walking status at 30 months of age, were similar between the two groups. CONCLUSIONS: Standard diagnostic testing with CMA should be offered when an ONTD is detected prenatally, as this approach has implications for counseling regarding prognosis and recurrence risk. Our results indicate that the presence of a clinically reportable CNV should not a priori affect eligibility for in-utero repair, as overall pregnancy outcome is similar in these cases to that of cases with normal CMA. Nevertheless, significant CMA results will require a case-by-case multidisciplinary discussion to evaluate eligibility. To generalize the conclusion of this single-center series, a larger, multicenter long-term study should be considered. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Structural Stability and Phase Transitions in Zeolite A: An In Situ High Pressure-High Temperature Investigation.

The high pressure-high temperature structural stability of Zeolite A (ZA) has been studied using the X-ray diffraction (XRD) method. Structural studies at high temperatures show a reduction in the oxygen occupancy, belonging to the water molecule, indicating thermal dehydration and subsequent expulsion of water molecules from the pores of the structure. ZA does not undergo structural phase transition with temperature. However, structural transitions are observed in in situ XRD studies at high pressure and high temperature. At 1.3 GPa and 300 °C, the cubic ZA concomitantly transformed to cubic sodalite (SOD) and tetragonal zeolite NaP (ZNP). This transition was completely forbidden at 2.7 GPa, where a temperature-induced amorphization was favored at 250 °C. The thermal studies at higher pressure reveal the marginal influence of pressure on the thermal expansion coefficients of hydrated ZA. Pressure evolution of the high pressure-high temperature phases indicates no further phase transitions up to 5.9 GPa. The equation of state fit to the pressure-volume data of these phases show that ZNP is less compressible, followed by SOD and ZA. In contrast to the behavior at 0.1 MPa, SOD shows a pressure-induced negative thermal expansion (NTE) at 5.9 GPa. On the other hand, the positive thermal expansion (PTE) observed along the direction of c axis is compensated by the NTE along the a axis leading to a negligible volume thermal expansion for the ZNP structure. The bulk moduli and thermal expansion coefficients of all of the observed phases are reported. The outcomes of this study have been consolidated as a pressure-temperature phase diagram, which provides an insight into the technological and industrial applications of ZA at extreme conditions.

Prevalence, molecular characterization, genetic heterogeneity and antimicrobial resistance of Listeria monocytogenes associated with fish and fishery environment in Kerala, India.

The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n = 97) shellfish (n = 19), ready-to-cook fish products (n = 47), ready-to-eat fish products (n = 10), dried fish (n = 11) and retail ice (n = 43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain.

High-Pressure Synthesis of Manganese Monocarbide: A Potential Superhard Material.

In this paper, we report for the first time formation of novel manganese monocarbide (MnC) using laser-heated diamond anvil cell (LHDAC). The synthesis was carried out at high pressure-high temperature (HPHT) and subsequently quenched to ambient condition. The formation and reproducibility have been confirmed in the pressure range of 4.7 to 9.2 GPa. Employing contribution of different probes viz.X-ray diffraction (XRD), selected area electron diffraction (SAED), and ab initio electronic structure calculation, the structure of MnC was found to be ZnS type i.e. a cubic lattice with a = 4.4294(2) Å. The bulk modulus has been determined to be 170(5) GPa from in situ high-pressure X-ray diffraction (HPXRD). Hardness of ZnS type MnC is estimated from an empirical relation to be about 40 GPa, making it a potential superhard material.

Synthesis of novel Ru2C under high pressure-high temperature conditions.

We report here, for the first time, synthesis of the Fe(2)N type hexagonal phase of ruthenium carbide by a high pressure-high temperature technique using a laser heated diamond anvil cell (LHDAC). The synthesis is carried out by laser heating a mixture of pure elements, Ru and C, at very low 'pressure' of 5 GPa and T ~ 2000 K. The structure of the temperature quenched high pressure phase is characterized by in situ high pressure x-ray diffraction (HPXRD) and is corroborated by ex situ TEM imaging and diffraction, carried out for the first time on the retrieved sample synthesized by LHDAC. The lattice parameters of Ru(2)C at ambient pressure are found to be a = 2.534 Å and c = 4.147 Å. In situ HPXRD studies up to 14.2 GPa yield a bulk modulus of 178(4) GPa. Electronic structure calculations reveal the system to be metallic in nature with a degree of covalence along the Ru-C bond. As ruthenium is isoelectronic to osmium, this result for Ru(2)C has significant implications in the synthesis and study of osmium carbides.

Cryogenic gas loading in a Mao-Bell-type diamond anvil cell for high pressure-high temperature investigations.

A simple system for loading argon fluid at cryogenic temperatures in a Mao-Bell-type diamond anvil cell (DAC) has been developed. It is done in a two step process in which the piston-cylinder assembly alone is submerged in the cryogenic chamber for trapping the liquefied inert gas. Liquid nitrogen is used for condensing the argon gas. This system is now being efficiently used for loading liquid argon in the DAC for high pressure-high temperature experiments. The success rate of trapping liquefied argon in the sample chamber is about 75%. The performance of the gas loading system is successfully tested by carrying out direct conversion of pyrolitic graphite to diamond under high pressure-high temperature using laser heated DAC facility.

Interposition arthroplasty for osteoarthritis of trapezio metacarpal joint: results of a modified incision and technique of interposing with early mobilisation.

Sixty flexor carpi radialis (FCR) tendon interposition arthroplasties were done using a modified incision from Froimson's approach for osteoarthritis (OA) of thumb carpo metacarpal joint (CMCJ) The tendon was made to resemble an anchovy fillet to preserve pillar length (average 7.5 mm). There was no incidence of injury to the superficial branch of the radial nerve. Graded mobilisation was commenced at two weeks. Our average follow-up for five and a half years shows good results, viz. pain relief (100%), power grip (21 kg), pinch grip (4.2 kg), tripod grip (5.5 kg), key grip (6.5 kg), ability to touch base and tip of little finger (91.6%) and (96.6%), respectively. Activities of daily living (ADL) without pain in turning a key (96.7%), opening jar top (100%), bottle top (93.4%), wringing cloth (86.7%), and using scissors (88.4%). None of them suffered reflex sympathetic dystrophy (RSD) and mobility was almost equal to the non-operated hand. Our experience with this modified incision and technique of interposing with early mobilisation has shown good functional outcome with no significant operative or postoperative complications.

Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.

We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity.

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.

"Dehydrogenase" and "oxidase" reactions of medium-chain fatty acyl-CoA dehydrogenase utilizing chromogenic substrates: role of the 3',5'-adenosine diphosphate moiety of the coenzyme A thioester in catalysis.

We undertook a comparative investigation of the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction utilizing indole-, furyl-, and 4-(dimethylamino)phenyl-substituted propionyl- and acryloyl-CoAs as potential substrate/product pairs. All these propionyl-CoA derivatives undergo MCAD-catalyzed conversion into their corresponding acryloyl-CoAs via both "dehydrogenase" (in the presence of "organic" electron acceptors) and "oxidase" (buffer-dissolved oxygen serving as the electron acceptor) pathways [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. The steady-state kinetic parameters for the enzyme utilizing these substrates reveal that the KmS (for the CoA substrates) and kcatS for the dehydrogenase reaction are at least an order of magnitude higher than those for the oxidase reaction. As with the CoA substrates, the enzyme catalyzes the conversion of indolepropionyl pantetheine phosphate (IPPP) into indoleacryloyl pantetheine phosphate (IAPP) via these two pathways. However, with IPPP as substrate, the Km (for IPPP) and kcat values of the dehydrogenase and oxidase reactions are the same. These, coupled with the spectral changes of the enzyme-product complexes as well as the binding affinities of the enzyme-substrate/product complexes, lead to the following conclusions: (1) The aromatic/heterocyclic group-containing substrates are converted into their corresponding products via both the dehydrogenase and the oxidase pathways. (2) The 3',5'-ADP moiety of the CoA thioester provides a significant fraction of the total binding energy in stabilizing the enzyme-substrate/product complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair: similarity in the microscopic pathways of octanoyl-CoA oxidation and octenoyl-CoA binding.

Of the different chain length fatty acyl-CoA substrates, octanoyl-CoA has been known as one of the most efficient (and physiological) substrates for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction of MCAD-FAD with octanoyl-CoA ([MCAD-FAD] << [octanoyl-CoA]), measured via the stopped-flow technique, at 5 degrees C was characterized by a biphasic decrease and increase in absorptions at 450 and 545 nm, respectively. The average values of the fast (1/tau 1) and slow (1/tau 2) relaxation rate constants, derived from the data at these wavelengths, were found to be 319.7 +/- 33.5 and 28.8 +/- 12.5 s-1, respectively, and both of these relaxation rate constants remained invariant between 8 and 200 microM concentrations of octanoyl-CoA. Under identical experimental conditions, we measured time courses for the interaction of MCAD-FAD with octenoyl-CoA ([MCAD-FAD] << [octenoyl-CoA]) by monitoring the absorption changes at 299, 394, and 440 nm. The binding profile was consistent with a biphasic decrease (at 440 nm) and increase (at 299 and 394 nm) in absorbance, with similar magnitudes of fast [1/tau 1 (average) = 382.3 +/- 39.8 s-1] and slow [1/tau 2 (average) = 14.3 +/- 7.4 s-1] relaxation rate constants. The observed relaxation rate constants were, once again, found to be invariant with changes in the octenoyl-CoA concentration from 40 to 150 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of the medium-chain fatty acyl-CoA dehydrogenase-catalyzed "oxidase" reaction: pH-dependent distribution of intermediary enzyme species during catalysis.

In a previous paper, we demonstrated that the medium-chain fatty acyl-CoA dehydrogenase-catalyzed (MCAD-catalyzed) reductive half-reaction of indolepropionyl-CoA proceeds via formation of a chromophoric intermediary species "X" (absorption maximum = 400 nm) and proposed that the decay of this species might limit the overall rate of the "oxidase" reaction [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013]. During this latter reaction, the buffer-dissolved O2 served as an electron acceptor [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. To ascertain whether the intrinsic stability of X influences the oxidase activity, we undertook a detailed kinetic investigation of this enzyme at different pH values. The time-resolved spectra for the reductive half-reaction (obtained via the rapid-scanning stopped-flow method) at different pH values reveal that the amplitude of the intermediary (X) spectral band is more pronounced at a lower pH (pH 6.4) than at a higher pH (pH 9.0). Single-wavelength transient kinetic data for the reductive half-reaction (in both the forward and the reverse direction) at all pH values are consistent with fast (1/tau 1) and slow (1/tau 2) relaxation rate constants. Of these, whereas the fast relaxation rate constant for the reaction in the forward direction (1/tau 1f) decreases with an increase in pH, the corresponding slow relaxation rate constant (1/tau 2f) increases with an increase in pH. The pH-dependent steady-state kinetic data reveal that, like 1/tau 2f, kcat for the MCAD-catalyzed oxidase reaction increases with an increase in the pH of the buffer media.(ABSTRACT TRUNCATED AT 250 WORDS)

Microscopic pathway for the medium-chain fatty acyl CoA dehydrogenase catalyzed oxidative half-reaction: changes in the electronic structures of flavin and CoA derivatives during catalysis.

In a previous communication, we demonstrated that the medium-chain fatty acyl CoA dehydrogenase (MCAD) catalyzed conversion of 3-indolepropionyl CoA (IPCoA) to trans-3-indoleacryloyl CoA (IACoA) proceeds via the formation of an intermediary species X that possesses the electronic properties of reduced flavin and highly conjugated CoA product. Since the steady-state turnover of the enzyme-catalyzed dehydrogenation reaction precisely matches with the rate of formation of X [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013], the latter species appeared to be the likely site for the transfer of electrons to external electron acceptors (e.g., ferricenium hexafluorophosphate, FcPF6). To probe the microscopic pathway for the oxidative half-reaction, we employed a sequential mixing stopped-flow technique utilizing IPCoA as the enzyme substrate and FcPF6 as the electron acceptor. The time-dependent changes in absorption at 450, 415, and 367 nm were measured upon mixing FcPF6 with previously mixed and aged solutions of MCAD-FAD+IPCoA in the stopped-flow syringes. The kinetic traces show an increase (1/tau 1) followed by a decrease (1/tau 2) in absorption at 450 and 415 nm, and a lag (corresponding to the time regime of 1 u 1) followed by an increase in absorption (1/tau 2) at 367 nm. The relaxation rate constants (1/tau's) thus measured remain unaffected, with variations in the aging time; however, the amplitudes of these phases increase up to the aging time of 5 s, after which the amplitudes attain maxima. For an aging time of 5 s, 1/tau 1 and 1/tau 2 show a linear and a hyperbolic dependence on the FcPF6 concentration, respectively. These, coupled with the complementary studies involving butyryl CoA as a nonchromophoric substrate for this enzyme, lead us to propose the following sequence of events during the MCAD-catalyzed oxidative half-reaction: (1) The enzyme-catalyzed oxidative half-reaction proceeds via the formation of a collision complex between X and FcPF6 during the fast (1/tau 1) relaxation phase. (2) The reduced flavin moiety of X is oxidized via (rapid) transfer of electrons to FcPF6 within the collision complex, without formation of a detectable (metastable) flavin semiquinone intermediate. (3) The transfer of electrons is accompanied by changes in the electronic structures of both the flavin and IACoA moieties within the enzyme-IACoA complex. The electronic structure of this newly formed complex is exactly the same as that formed upon isomerization of the MCAD-FAD-IACoA complex [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575].(ABSTRACT TRUNCATED AT 400 WORDS)

A novel spectroscopic titration method for determining the dissociation constant and stoichiometry of protein-ligand complex.

We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.

Conservative medical management of traumatic pharyngoesophageal perforations.

Traditionally, surgical treatment has been the acceptable management for perforation of the pharyngoesophageal tract secondary to blunt and penetrating trauma. From July 1983 to June 1990, we managed 10 patients with this type of lesion by a conservative medical management approach. Mirror or fiberoptic flexible laryngoscopy was performed in the majority of cases to ascertain the nature of the injury. An esophagogram is very helpful to locate and evaluate the extent of the injury. All patients were treated with broad-spectrum intravenous antibiotic therapy and no oral feeding. There were no complications or need for surgical treatment in any of the cases. The head and neck surgeon, in selected cases, should consider the possibility of using conservative management of pharyngoesophageal perforations. This approach has proven in our hands to be relatively safe and cost-effective, resulting in no disability or prolonged hospitalization of our patients. This study involves two institutions (two affiliated hospitals of Case Western Reserve University School of Medicine) with different surgeons selecting appropriate antibiotic therapy. It is a retrospective review. No controls were made by random selection of cases treated surgically. These cases, if not properly managed, may lead to fatal outcomes.