Improving the Duration and Rate of Home-Based Kangaroo Mother Care: A Before-and-After Intervention Study.

Introduction Kangaroo mother care (KMC) is an evidence-based, simple, time-tested, low-cost, and high-impact intervention for neonatal survival in hospitals and the community, particularly in resource-constrained areas. This has many beneficial effects on sick and stable low-birth-weight babies, lactating mothers, families, society, and the government. However, despite the World Health Organization (WHO) and United Nations International Children's Emergency Fund (UNICEF) recommendations for KMC, there is no satisfactory implementation of it in the community as well as in facilities. This study aimed to improve the duration of home-based kangaroo mother care (HBKMC). Material and methods  We conducted a before-and-after intervention hospital-based, single-center study in a level III neonatal intensive care unit (NICU) to improve the duration of HBKMC. The KMC duration was classified into four categories: short, extended, long, and continuous where KMC was provided for 4 hours/day, 5-8 hours/day, 9-12 hours/day, and more than 12 hours/day, respectively. All neonates with birth weight < 2.0 kg and their mothers/alternate KMC providers at a tertiary care hospital in India in the time period of five months from April 2021 to July 2021 were considered eligible for the study. We tested three sets of interventions by using the plan-do-study-act cycle (PDSA cycle). The first set of interventions was the sensitization of parents and healthcare workers regarding the benefits of KMC by comprehensive counseling to mothers and other family members using educational lectures, videos, charts, and posters. The second set of interventions was to reduce maternal anxiety/stress while maintaining maternal privacy by providing more female staff and teaching proper gown-wearing techniques. The third set of interventions was to solve lactation and environment temperature issues by providing antenatal and postnatal lactation counseling and warming of the nursery. The paired T-test and one-way analysis of variance (ANOVA) were used for statistical analysis, and p<0.05 was taken as significant.  Results  One hundred and eighty neonates were enrolled along with their mothers/alternate KMC providers in four phases, and three PDSA cycles were implemented. Out of 180 LBW infants, 21 (11.67%) infants received KMC < 4 hours/day. According to the KMC classification, 31% have continuous KMC in the institution, followed by 24% long KMC, 26% extended KMC, and 18% short KMC. After three PDSA cycles, HBKMC was 38.88% continuous KMC, followed by 24.22% long KMC, 20.55% extended KMC, and 16.11% short KMC. Continuous KMC was improved from 21% to 46% at the institute and 16% to 50% at home from phase 1 to phase 4 of the study after the implementation of three sets of interventions in three PDSA cycles. The phase-by-phase KMC rate and duration were improved after the application of the PDSA cycles, and this was maintained in HBKMC as well, but it was statistically not significant. Conclusion Sets of intervention packages based on needs analysis using the PDSA cycle were able to improve the rate and duration of KMC in the hospital and at home.

Thermodynamic reciprocity of the inhibitor binding to the active site and the interface binding region of IB phospholipase A2.

Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E(i)(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes (E(i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the E(i)I complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the E(i) complexes with PCU are qualitatively similar to those of E(i)(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E(2)(#) complex or, equivalently, the shift in the E(1)(#) to E(2)(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.

Allosteric effect of amphiphile binding to phospholipase A(2).

In the preceding paper, we showed that the formation of the second premicellar complex of pig pancreatic IB phospholipase A2 (PLA2) can be considered a proxy for interface-activated substrate binding. Here we show that this conclusion is supported by results from premicellar E(i)(#) (i = 1, 2, or 3) complexes with a wide range of mutants of PLA2. Results also show a structural basis for the correlated functional changes during the formation of E(2)(#), and this is interpreted as an allosteric T (inactive) to R (active) transition. For example, the dissociation constant K(2)(#) for decylsulfate bound to E(2)(#) is lower at lower pH, at higher calcium concentrations, or with an inhibitor bound to the active site. Also, the lower limits of the K(2)(#) values are comparable under these conditions. The pH-dependent increase in K(2)(#) with a pK(a) of 6.5 is attributed to E71 which participates in the binding of the second calcium which in turn influences the enzyme binding to phosphatidylcholine interface. Most mutants exhibited kinetic and spectroscopic behavior that is comparable to that of native PLA2 and DeltaPLA2 with a deleted 62-66 loop. However, the DeltaY52L substitution mutant cannot undergo the calcium-, pH-, or interface-dependent changes. We suggest that the Y52L substitution impairs the R to T transition and also hinders the approach of the Michaelis complex to the transition state. This allosteric change may be mediated by the structural motifs that connect the D48-D99 catalytic diad, the substrate-binding slot, and the residues of the i-face. Our interpretation is that the 57-72 loop and the H(48)DNCY(52) segment of PLA2 are involved in transmitting the effect of the cooperative amphiphile binding to the i-face as a structural change in the active site.

Effect of guggulsterone and cembranoids of Commiphora mukul on pancreatic phospholipase A(2): role in hypocholesterolemia.

Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown to be specific modulators of two independent sites that are also modulated by bile salts (1-6) to control cholesterol absorption and catabolism. Guggulsterone (7) antagonized the chenodeoxycholic acid (3)-activated nuclear farnesoid X receptor (FXR), which regulates cholesterol metabolism in the liver. The cembranoids did not show a noticeable effect on FXR, but lowered the cholate (1)-activated rate of human pancreatic IB phospholipase A2 (hPLA2), which controls gastrointestinal absorption of fat and cholesterol. Analysis of the data using a kinetic model has suggested an allosteric mechanism for the rate increase of hPLA2 by cholate and also for the rate-lowering effect by certain bile salts or cembranoids on the cholate-activated hPLA2 hydrolysis of phosphatidylcholine vesicles. The allosteric inhibition of PLA2 by certain bile salts and cembranoids showed some structural specificity. Biophysical studies also showed specific interaction of the bile salts with the interface-bound cholate-activated PLA2. Since cholesterol homeostasis in mammals is regulated by FXR in the liver for metabolism and by PLA2 in the intestine for absorption, modulation of PLA2 and FXR by bile acids and selected guggul components suggests novel possibilities for hypolipidemic and hypocholesterolemic therapies.

Contiguous binding of decylsulfate on the interface-binding surface of pancreatic phospholipase A2.

Pig pancreatic IB phospholipase A 2 (PLA2) forms three distinguishable premicellar E i (#) ( i = 1, 2, and 3) complexes at successively higher decylsulfate concentrations. The Hill coefficient for E 1 (#) is n 1 = 1.6, and n 2 and n 3 for E 2 (#) and E 3 (#) are about 8 each. Saturation-transfer difference nuclear magnetic resonance (NMR) and other complementary results with PLA2 show that decylsulfate molecules in E 2 (#) and E 3 (#) are contiguously and cooperatively clustered on the interface-binding surface or i-face that makes contact with the substrate interface. In these complexes, the saturation-transfer difference NMR signatures of (1)H in decylsulfate are different. The decylsulfate epitope for the successive E i (#) complexes increasingly resembles the micellar complex formed by the binding of PLA2 to preformed micelles. Contiguous cooperative amphiphile binding is predominantly driven by the hydrophobic effect with a modest electrostatic shielding of the sulfate head group in contact with PLA2. The formation of the complexes is also associated with structural change in the enzyme. Calcium affinity of E 2 (#) appears to be modestly lower than that of the free enzyme and E 1 (#). Binding of decylsulfate to the i-face does not require the catalytic calcium required for the substrate binding to the active site and for the chemical step. These results show that E i (#) complexes are useful to structurally characterize the cooperative sequential and contiguous binding of amphiphiles on the i-face. We suggest that the allosteric changes associated with the formation of discrete E i (#) complexes are surrogates for the catalytic and allosteric states of the interface activated PLA2.

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A(2): regulation of fat and cholesterol homeostasis.

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted DeltaPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k(cat)(*)-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E(*)B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.

Desolvation map of the i-face of phospholipase A2.

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.

Kinetic and structural properties of disulfide engineered phospholipase A2: insight into the role of disulfide bonding patterns.

The family of secreted 14 kDa phospholipase A(2) (PLA2) enzymes have a common motif for the catalytic site but differ in their disulfide architecture. The functional significance of such structural changes has been analyzed by comparing the kinetic and spectroscopic properties of a series of disulfide mutants engineered into the sequence of pig pancreatic IB PLA2 to resemble the mammalian paralogues of the PLA2 family [Janssen et al. (1999) Eur. J. Biochem. 261, 197-207, 1999]. We report a detailed comparison of the functional parameters of pig iso-PLA2, as well as several of the human homologues, with these disulfide engineered mutants of pig IB PLA2. The crystal structure of the ligand free and the active site inhibitor-MJ33 bound forms of PLA2 engineered to have the disulfide bonding pattern of group-X (eng-X) are also reported and compared with the structure of group-IB and human group-X PLA2. The engineered mutants show noticeable functional differences that are rationalized in terms of spectroscopic properties and the differences detected in the crystal structure of eng-X. A major difference between the eng-mutants is in the calcium binding to the enzyme in the aqueous phase, which also influences the binding of the active site directed ligands. We suggest that the disulfide architecture of the PLA2 paralogues has a marginal influence on interface binding. In this comparison, the modest differences observed in the interfacial kinetics are attributed to the changes in the side chain residues. This in turn influences the coupling of the catalytic cycle to the calcium binding and the interfacial binding event.

Cooperative binding of monodisperse anionic amphiphiles to the i-face: phospholipase A2-paradigm for interfacial binding.

Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A(2) (PLA2) to form the premicellar complexes (E(i)(#)) are characterized to discern the short-range specific interactions. Typically, E(i)(#) complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (E(i)(#), i = 1, 2, 3). In the presence of calcium, the dissociation constant K(1) for the E(1)(#) complex of PLA2 with decyl sulfate (CMC = 4500 microM) is 70 microM with a Hill coefficient n(1) = 2.1 +/- 0.2; K(2) for E(2)(#) is 750 microM with n(2) = 8 +/- 1, and K(3) for E(3)(#) is 4000 microM with an n(3) value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of E(i)(#); (c) K(i) and n(i) do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the E(i)(#) complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in E(i)(#). The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of E(i)(#). We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E and E(i)(#) aggregates is discussed.

Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex. The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase. The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex.

Interaction of monodisperse anionic amphiphiles with the i-face of secreted phospholipase A2.

Pancreatic IB phospholipase A(2) (PLA2) forms aggregates of defined size with monodisperse alkyl sulfates in the premicellar concentration range. As an extension of the interfacial kinetic paradigm, results are interpreted in terms of a model in which several amphiphile molecules bind along their polar headgroup to the interface binding region (i-face) of PLA2. The resulting complex, E(#), has a half-micellar structure, and it acts as an "amphiphile" in the aqueous phase. E(#) not only self-aggregates but also binds hydrophobic probes and interacts with hydrophobic surfaces. As expected, resonance energy transfer from the tryptophan donor in PLA2 to an acceptor probe partitioned in E(#) shows a biphasic dependence as the probe coexisting with PLA2 is diluted at higher alkyl sulfate concentrations. The gel-permeation behavior of PLA2 at premicellar alkyl sulfate concentrations is also biphasic. For example, above 1.2 mM decyl sulfate (CMC = 3.5 mM) PLA2 elutes as a single sharp peak, presumably the self-aggregate of E(#) with apparent molecular mass of 120-150 kDa. At 0.4-1 mM decyl sulfate the retention volume is even larger than that for the 14 kDa PLA2. This anomalous retention is attributed to the interaction of the hydrophobic region of E(#) with the hydrophobic patches on the gel-permeation matrix. Elution behavior of the self-aggregated E(#) form of site-directed mutants in dodecyl sulfate suggests that certain substitutions in the conserved hydrogen-bonding network have a significant effect on the aggregate size. These results suggest a role for the network in the amphiphile binding along the i-face of PLA2, presumably through a change in the anion coordination ligands.

Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive.

We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.

Crystal structure of human group X secreted phospholipase A2. Electrostatically neutral interfacial surface targets zwitterionic membranes.

The crystal structure of human group X (hGX) secreted phospholipase A2 (sPLA2) has been solved to a resolution of 1.97 A. As expected the protein fold is similar to previously reported sPLA2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca2+ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1-12)-N-terminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA2. Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA2; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and in k(cat*) allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.

Processive interfacial catalytic turnover by Bacillus cereus sphingomyelinase on sphingomyelin vesicles.

Sphingomyelinase (SMase), a water-soluble enzyme from Bacillus cereus, is shown to bind with high affinity to vesicles of sphingomyelin (SM) but not to vesicles of phosphatidylcholine (PC). The reaction progress by SMase bound to SM vesicles occurs in the scooting mode with virtually infinite processivity of the successive interfacial turnover cycles. Three conditions for the microscopic steady state during the reaction progress at the interface are satisfied: the bound SMase does not leave the interface even after all the SM in the outer layer is converted to ceramide; the SMase-treated vesicles remain intact; and the ceramide product does not exchange with SM present in excess vesicles or in the inner layer of the hydrolyzed vesicle. Within these constraints, on accessibility and replenishment of the substrate, the extent of hydrolysis in the scooting mode reaction progress is a measure of the number of vesicles containing enzyme. The slope of the Poisson distribution plot, for the enzyme per vesicle versus the logarithm of the fraction of the total accessible substrate remaining unhydrolyzed in excess vesicles, shows that a single 32 kDa subunit of SMase is fully catalytically active. The maximum initial rate of hydrolysis, at the limit of the maximum possible substrate mol fraction, X(S)*=1, is 400 s(-1) in H(2)O and 220 s(-1) in D(2)O, which is consistent with the rate-limiting chemical step. The integrated reaction progress suggests that the ceramide product does not codisperse ideally on the hydrolyzed vesicles. Furthermore, complex reaction progress seen with covesicles of SM+PC are attributed to slow secondary changes in the partially hydrolyzed SM vesicles.