Reoperation for Hirschsprung Disease: Two cases of Vanishing Ganglion Cells and Review of the Literature.

Hirschsprung disease (HD) is characterized by circumferential aganglionosis of the rectum with variable proximal bowel involvement. The underlying pathogenesis is due to failure of caudal migration of neural crest cells during pre-natal development, causing functional bowel obstruction. Definitive therapy is surgical resection; however, a subset of patients will require reoperation. An important cause of reoperation is the rare but distinct entity described as the ganglion cell "vanishing" phenomenon. In this phenomenon, affected patients have normal circumferential ganglion cells present at the proximal margin during primary resection. They undergo a variable asymptomatic period post-primary resection but ultimately develop recurrent symptoms. Upon reoperation, ganglion cells seemingly vanish and are no longer present in the previously functioning and ganglionated bowel proximal to the initial anastomotic site. To further characterize and investigate this poorly understood pathology, here we present 2 cases of HD patients who required reoperation. Our small series implicates that an immune component may contribute as patient 2 had a brisk neurotrophic eosinophilic infiltrate only present in the reoperation specimen. However, this was not observed in patient 1. Other possible etiologies include post-operative ischemia/hypoxia, visceral neuropathy, or signaling abnormalities within the residual ganglion cells themselves.

Structure, Function, and Thermodynamics of Lactate Dehydrogenases from Humans and the Malaria Parasite P. falciparum.

Temperature adaptation is ubiquitous among all living organisms, yet the molecular basis for this process remains poorly understood. It can be assumed that for parasite-host systems, the same enzymes found in both organisms respond to the same selection factor (human body temperature) with similar structural changes. Herein, we report the existence of a reversible temperature-dependent structural transition for the glycolytic enzyme lactate dehydrogenase (LDH) from the malaria parasite Plasmodium falciparum (pfLDH) and human heart (hhLDH) occurring in the temperature range of human fever. This transition is observed for LDHs from psychrophiles, mesophiles, and moderate thermophiles in their operating temperature range. Thermodynamic analysis reveals unique thermodynamic signatures of the LDH-substrate complexes defining a specific temperature range to which human LDH is adapted and parasite LDH is not, despite their common mesophilic nature. The results of spectroscopic analysis combined with the available crystallographic data reveal the existence of an active center within pfLDH that imparts psychrophilic structural properties to the enzyme. This center consists of two pockets, one formed by the five amino acids (5AA insert) within the substrate specificity loop and the other by the active site, that mutually regulate one another in response to temperature and induce structural and functional changes in the Michaelis complex. Our findings pave the way toward a new strategy for malaria treatments and drug design using therapeutic agents that inactivate malarial LDH selectively at a specific temperature range of the cyclic malaria paroxysm.

Insect Cell Glycosylation and Its Impact on the Functionality of a Recombinant Intracrystalline Nacre Protein, AP24.

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.

Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System.

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times, and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein-mineral polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process.

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase.

Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride-proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.

Pif97, a von Willebrand and Peritrophin Biomineralization Protein, Organizes Mineral Nanoparticles and Creates Intracrystalline Nanochambers.

The formation of the mollusk nacre layer involves the assembly and organization of mineral nanoparticles into fracture-toughened mesoscale-sized aragonite tablets that possess intracrystalline nanoporosities. At least one nacre protein family, known as the framework proteome, is strategically located as part of a macromolecular coating around each nacre tablet and is believed to participate in tablet formation. Here, we report new studies of a recombinant form (rPif97) of a unique Japanese pearl oyster (Pinctada fucata) nacre framework biomineralization protein, Pif97. This unique protein possesses both a von Willlebrand factor type A domain (vWA, F23-Y161) and a Peritrophin A chitin-binding domain (PAC, E234-D298). rPif97 self-associates or aggregates to form amorphous protein phases that organize both amorphous and single-crystal calcium carbonate nanoparticles in vitro. Further, in the presence of nucleating calcite crystals, rPif97 protein phases deposit onto these crystals and become occluded over time, forming nanochambers within the crystal interior. The formation of these mineral-modifying amorphous protein phases is linked to the presence of intrinsic disorder and amyloid-like cross-ß-strand aggregation-prone regions, and three-dimensional modeling indicates that both the vWA and PAC domains are accessible for intermolecular interactions. Thus, the vWA- and PAC-containing Pif97 protein exhibits key functionalities that would allow its participation in mollusk nacre layer tablet assembly and porosity formation.

An oligomeric C-RING nacre protein influences prenucleation events and organizes mineral nanoparticles.

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca2+. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31-N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1-T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization.

A nacre protein, n16.3, self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.

The mollusk shell is a complex biological material that integrates mineral phases with organic macromolecular components such as proteins. The role of proteins in the formation of the nacre layer (aragonite mineral phase) is poorly understood, particularly with regard to the organization of mineral deposits within the protein extracellular matrix and the identification of which proteins are responsible for this task. We report new experiments that provide insight into the role of the framework nacre protein, n16.3 (Pinctada fucata), as an organizer or assembler of calcium carbonate mineral clusters. Using a combination of biophysical techniques, we find that recombinant n16.3 (r-n16.3) oligomerizes to form amorphous protein films and particles that possess regions of disorder and mobility. These supramolecular assemblies possess an intrinsically disordered C-terminal region (T64-W98) and reorganize in the presence of Ca(2+) ions to form clustered protein oligomers. This Ca(2+)-induced reorganization leads to alterations in the molecular environments of Trp residues, the majority of which reside in putative aggregation-prone cross-ß strand regions. Potentiometric Ca(2+) titrations reveal that r-n16.3 does not significantly affect the formation of prenucleation clusters in solution, and this suggests a role for this protein in postnucleation mineralization events. This is verified in subsequent in vitro mineralization assays in which r-n16.3 demonstrates its ability to form gel-like protein phases that organize and cluster nanometer-sized single-crystal calcite relative to protein-deficient controls. We conclude that the n16 nacre framework proteome creates a protein gel matrix that organizes and dimensionally limits mineral deposits. This process is highly relevant to the formation of ordered, nanometer-sized nacre tablets in the mollusk shell.

The intrinsically disordered C-RING biomineralization protein, AP7, creates protein phases that introduce nanopatterning and nanoporosities into mineral crystals.

We report an interesting process whereby the formation of nanoparticle assemblies on and nanoporosities within calcite crystals is directed by an intrinsically disordered C-RING mollusk shell nacre protein, AP7. Under mineralization conditions, AP7 forms protein phases that direct the nucleation of ordered calcite nanoparticles via a repetitive protein phase deposition process onto calcite crystals. These organized nanoparticles are separated by gaps or spaces that become incorporated into the forming bulk crystal as nanoporosities. This is an unusual example of organized nanoparticle biosynthesis and mineral modification directed by a C-RING protein phase.

Malate dehydrogenase in parasitic protozoans: roles in metabolism and potential therapeutic applications.

The role of malate dehydrogenase (MDH) in the metabolism of various medically significant protozoan parasites is reviewed. MDH is an NADH-dependent oxidoreductase that catalyzes interconversion between oxaloacetate and malate, provides metabolic intermediates for both catabolic and anabolic pathways, and can contribute to NAD+/NADH balance in multiple cellular compartments. MDH is present in nearly all organisms; isoforms of MDH from apicomplexans (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium spp.), trypanosomatids (Trypanosoma brucei, T. cruzi) and anaerobic protozoans (Trichomonas vaginalis, Giardia duodenalis) are presented here. Many parasitic species have complex life cycles and depend on the environment of their hosts for carbon sources and other nutrients. Metabolic plasticity is crucial to parasite transition between host environments; thus, the regulation of metabolic processes is an important area to explore for therapeutic intervention. Common themes in protozoan parasite metabolism include emphasis on glycolytic catabolism, substrate-level phosphorylation, non-traditional uses of common pathways like tricarboxylic acid cycle and adapted or reduced mitochondria-like organelles. We describe the roles of MDH isoforms in these pathways, discuss unusual structural or functional features of these isoforms relevant to activity or drug targeting, and review current studies exploring the therapeutic potential of MDH and related genes. These studies show that MDH activity has important roles in many metabolic pathways, and thus in the metabolic transitions of protozoan parasites needed for success as pathogens.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and "branched-tail" oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors' IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure-activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 µM concentrations matching the effect of 30 µM roxadustat and 500 µM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.