Molecular Probe Method Research Articles

Ex vivo susceptibilities to ganaplacide and diversity in potential resistance mediators in Ugandan Plasmodium falciparum isolates.

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P. falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC50s (31/750 with IC50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.

(Invited) Chemical Kinetic Method for Active-Site Quantification in Fe-N-C Catalysts and Correlation with Molecular Probe and Spectroscopic Site-Counting Methods

Heterogeneous catalysts consisting of iron cations incorporated into nitrogen-doped carbon (“Fe-N-C”) have received extensive attention as leading alternatives to Pt for the electrochemical oxygen reduction reaction (ORR). Fe-N-C catalysts host mononuclear nitrogen-ligated active centers, FeNx, that frequently coexist with agglomerated Fe species. Although FeNx are the dominant active centers for ORR and other electrocatalytic reactions, the relative accuracies of methods to quantify them are still debated. Here, we develop a kinetic probe-reaction approach to quantify FeNx centers and compare it with spectroscopic and molecular probe methods.Model Fe-N-C catalysts were synthesized to contain mononuclear FeNx species at low loadings (0.1–0.4 wt% bulk Fe) on solvent-accessible surfaces using a postsynthetic metalation approach and their Fe speciation was confirmed by low-temperature 57Fe Mössbauer spectroscopy. The initial rate of oxidation of a water-soluble hydroquinone molecule (per gcatalyst) catalyzed by these model materials correlates linearly with their density of FeNx centers, reflecting their intrinsic turnover frequency (TOF) for this reaction. This TOF, in turn, enables the estimation of the active-site density on any other Fe-N-C catalyst through a simple rate measurement. Kinetically determined FeNx site densities are measured on a suite of fourteen Fe-N-C catalysts with diverse synthetic origins and Fe speciation (0.3–8.4 wt% bulk Fe) and are compared with those estimated by low-temperature 57Fe Mössbauer spectroscopy, CO pulse chemisorption, and electrochemical stripping of NO derived from NO2 −. Kinetic quantifications of FeNx centers correlate well with those obtained from the CO pulse chemisorption method and Mössbauer spectroscopy. The broad survey of Fe-N-C materials also reveals the presence of outliers and challenges associated with each site quantification method. The kinetic method developed here does not require pretreatments that may alter active-site distributions nor specialized equipment beyond reaction vessels and standard analytical instrumentation, offering an attractive complementary approach. Figure 1

Revisiting the application of molecular probe diagnostics on quantifying aqueous OH radicals in plasma–liquid systems

AbstractWe revisit one of the most used techniques for quantifying the aqueous OH radicals (OHaq) in plasma–liquid systems, the molecular probe method which obtains the [OHaq] by measuring a stable material formed through a rapid reaction between the molecular probe and the OHaq. In this study, we used disodium terephthalate (NaTA) as the molecular probe; the experimental results with a theoretical analysis suggest that to obtain the correct OHaq concentration, the concentration of the molecular probe should be greater than a certain value, which depends on the types of the plasma–liquid systems. However, this is not the case in most of the existing reports in which the NaTA is often much less than the requisite value.

Chemical Kinetic Method for Active-Site Quantification in Fe-N-C Catalysts and Correlation with Molecular Probe and Spectroscopic Site-Counting Methods.

Mononuclear Fe ions ligated by nitrogen (FeNx) dispersed on nitrogen-doped carbon (Fe-N-C) serve as active centers for electrocatalytic O2 reduction and thermocatalytic aerobic oxidations. Despite their promise as replacements for precious metals in a variety of practical applications, such as fuel cells, the discovery of new Fe-N-C catalysts has relied primarily on empirical approaches. In this context, the development of quantitative structure-reactivity relationships and benchmarking of catalysts prepared by different synthetic routes and by different laboratories would be facilitated by the broader adoption of methods to quantify atomically dispersed FeNx active centers. In this study, we develop a kinetic probe reaction method that uses the aerobic oxidation of a model hydroquinone substrate to quantify the density of FeNx centers in Fe-N-C catalysts. The kinetic method is compared with low-temperature Mössbauer spectroscopy, CO pulse chemisorption, and electrochemical reductive stripping of NO derived from NO2- on a suite of Fe-N-C catalysts prepared by diverse routes and featuring either the exclusive presence of Fe as FeNx sites or the coexistence of aggregated Fe species in addition to FeNx. The FeNx site densities derived from the kinetic method correlate well with those obtained from CO pulse chemisorption and Mössbauer spectroscopy. The broad survey of Fe-N-C materials also reveals the presence of outliers and challenges associated with each site quantification approach. The kinetic method developed here does not require pretreatments that may alter active-site distributions or specialized equipment beyond reaction vessels and standard analytical instrumentation.

Identification of the active triple-phase boundary of a non-Pt catalyst layer in fuel cells.

The rational design of non-Pt oxygen reduction reaction (ORR) catalysts and catalyst layers in fuel cells is largely impeded by insufficient knowledge of triple-phase boundaries (TPBs) in the micropore and mesopore ranges. Here, we developed a size-sensitive molecular probe method to resolve the TPB of Fe/N/C catalyst layers in these size ranges. More than 70% of the ORR activity was found to be contributed by the 0.8- to 2.0-nanometer micropores of Fe/N/C catalysts, even at a low micropore area fraction of 29%. Acid-alkaline interactions at the catalyst-polyelectrolyte interface deactivate the active sites in mesopores and macropores, resulting in inactive TPBs, leaving micropores without the interaction as the active TPBs. The concept of active and inactive TPBs provides a previously unidentified design principle for non-Pt catalyst and catalyst layers in fuel cells.

Emerging Electrochemical Techniques for Probing Site Behavior in Single-Atom Electrocatalysts.

Single-atom catalysts (SACs) have aroused tremendous interest over the past decade, particularly in the community of energy and environment-related electrocatalysis. A rapidly growing number of recent publications have recognized it as a promising candidate with maximum atomic utilization, distinct activity, and selectivity in comparison to bulk catalysts and nanocatalysts. However, the complexity of localized coordination environments and the dispersion of isolated sites lead to significant difficulties when it comes to gaining insight into the intrinsic behavior of electrocatalytic reactions. Furthermore, the low metal loadings of most SACs make conventional ensemble measurements less likely to be accurate on the subnanoscale. Thus, it remains challenging to probe the activity and properties of individual atomic sites by available commercial instruments and analytical methods. In spite of this, continuing efforts have lately focused on the development of advanced measurement methodologies, which are very useful to the fundamental understanding of SACs. There have recently been a number of in situ/operando techniques applied to SACs, such as electron microscopy, spectroscopy, and other analysis methods, which support relevant functions to identify the active sites and reaction intermediates and to investigate the dynamic behavior of localized structures of the catalytic sites.This Account aims to present recent electrochemical probing techniques which can be used to identify single-atomic catalytic sites within solid supports. First, we describe the basic principles of molecular probe methods for the study and analysis of electrocatalytic site behavior. In particular, the in situ probing technique enabled by surface interrogation scanning electrochemical microscopy (SI-SECM) can measure the active site density and kinetic rate with high resolution. An alternative electrochemical probing technique is further demonstrated on the basis of single-entity electrochemistry, which allows the unique electrochemical imaging of the size and catalytic rate of single atoms, molecules, and clusters. The merits and limitations of different electrochemical techniques are then discussed, along with perspectives for future prospects. Apart from this, we further showcase the powerful capability of emerging electrochemical probing techniques for determining significant effects and properties of SACs for various electrocatalytic reactions, including oxygen reduction and evolution, hydrogen evolution, and nitrate reduction. Overall, electrochemical techniques with atomic resolution have greatly increased opportunities for observing, measuring, and understanding the surface and interface chemistry during energy conversion. In the future, it is anticipated that the development of electrochemical probing techniques will be advanced with innovative perspectives on the behavior and features of SACs. We hope that this Account can contribute in several ways to promoting the fundamental knowledge and technical progress of emerging electrochemical measurements for studying SACs.

Temperature and interlayer coupling induced thermal transport across graphene/2D-SiC van der Waals heterostructure

Graphene based two-dimensional (2D) van der Waals (vdW) materials have attracted enormous attention because of their extraordinary physical properties. In this study, we explore the temperature and interlayer coupling induced thermal transport across the graphene/2D-SiC vdW interface using non-equilibrium molecular dynamics and transient pump probe methods. We find that the in-plane thermal conductivity κ deviates slightly from the 1/T law at high temperatures. A tunable κ is found with the variation of the interlayer coupling strength χ. The interlayer thermal resistance R across graphene/2D-SiC interface reaches 2.71 times 10–7{text{Km}}^{2} /{text{W}} at room temperature and χ = 1, and it reduces steadily with the elevation of system temperature and χ, demonstrating around 41% and 56% reduction with increasing temperature to 700 K and a χ of 25, respectively. We also elucidate the heat transport mechanism by estimating the in-plane and out-of-plane phonon modes. Higher phonon propagation possibility and Umklapp scattering across the interface at high temperatures and increased χ lead to the significant reduction of R. This work unveils the mechanism of heat transfer and interface thermal conductance engineering across the graphene/2D-SiC vdW heterostructure.

Elucidation of Detailed Pore Structure of (NH4)4SiW12O40 Sponge Crystal

Abstract Pore structure of (NH4)4SiW12O40 sponge crystal was successfully elucidated by means of Ar adsorption isotherm analysis combined with molecular probe method and molecular modeling. The pores consist of spherical voids or “cages” with diameter of ca. 12 Å and these are connected by smaller “windows”. These “cages” and “windows” are clearly resolved in the pore size distribution analysis for the first time. Molecular probe method elucidated the diameter of the “windows” is larger than 8.5 Å and smaller than 10.2 Å, being consistent with molecular modeling.

Active facet determination of layered double hydroxide for oxygen evolution reaction

Correlation of edge area ratio and OER activity, cyanate inhibiting-probe and DFT calculation demonstrate that edge facet of LDHs is the real active facet in comparison with plane facet. Oxygen evolution reaction (OER) plays an indispensable role in developing renewable clean energy resources. One of the critical bottlenecks for the reaction is the development of highly efficient electrocatalyst to decrease the high overpotentials of four-electron transfer process of OER. Recently, layered double hydroxides (LDHs) have been widely investigated among the most promising electrocatalysts for OER due to their high intrinsic activity, excellent stability as well as low-cost. However, it remains unclear how the exposed facet of the LDHs affects their electrocatalytic activity. Here we elucidate the active edge facet of LDHs towards OER by combining the finely control of edge facet ratio coupled with molecular probe method and computational calculation. The LDHs with higher edge facet area ratio show superior activity with low onset potential as well as decreased Tafel slope. The active edge site is further proved by blocking the unsaturated edge sites with cyanate probe anion, of which the adsorption largely inhibits OER activity. Furthermore, based on density functional theory (DFT) calculation, two-dimensional map of theoretical overpotentials as a function of Gibbs free energy reveals that the edge (100) facet exhibits a much higher OER activity than basal plane (001) facet.

Macrostructure of anisotropic shape memory polymer films studied by the molecular probe method

AbstractAn anisotropic copolymer film of vinyl chloride and vinyl acetate, which has shape memory properties, was investigated by the molecular probe method, thermomechanical analysis, and scanning calorimetry. It was found that the amorphous‐crystalline structure and the stress–strain state of the surface layer of the film differ from the structure and state of the inner layers. The molecular probe (limited diffusion of a solvent into the surface layer of the film) revealed the localization of the elastic deformation energy of a film, which was rapidly cooled in a stressed state during production. It is shown that destroying/crushing the stressed macrostructure of the film surface via plasticization and dissolution of the copolymer, eliminates the effects of the elastic energy release during thermally stimulated shrinkage.

Application of Magnetic Resonance Imaging Molecular Probe in the Study of Pluripotent Stem Cell–Derived Neural Stem Cells for the Treatment of Posttraumatic Paralysis of Cerebral Infarction

The feasibility and efficacy of magnetic resonance imaging molecular probe application and pluripotent stem cell-derived neural stem cell (NSC) transplantation for the treatment of hind limb paralysis in mice with cerebral infarction were studied. A model of middle cerebral artery infarction using adult mice was established to stimulate hind limb reactions. After the model was successfully established, the mice were first divided into an experimental group and a control group, with 25 mice in each group. Cultured neural cells were obtained from the cerebral cortex and hippocampus of a mouse 15 days pregnant to prepare pluripotent stem cells. Pluripotent stem cell-derived NSCs were identified by positive expression of Nestin. The experimental group was injected with 1 μL of NSC suspension through the tail vein, and the control group was injected with 1 μL of saline through the tail vein. The neurologic function of mice in each group was scored 1 day, 3 days, 7 days, 14 days, and 28 days after transplantation according to the Garcia 18 subscale. Finally, the differentiation, migration, and integration of pluripotent stem cell-derived NSCs after transplantation were observed using a magnetic resonance imaging molecular probe method. The results showed that the neurologic function scores of the ischemic transplantation group were significantly higher than those of the control group, and the results were significantly different (P < 0.05). Through research, it was found that after transplantation of pluripotent stem cell-derived NSCs, the transplanted cells migrated and differentiated around the body at 28 days and participated in angiogenesis, and the blood vessels in the infarcted area were obviously proliferated. The NSCs cultured invitro were transplanted to the small infarction after cerebral infarction. In rats, it plays a positive role in the repair of nerve function in mice with cerebral infarction. NSCs cultured invitro can survive, migrate, and differentiate in the brain tissue of mouse ischemic models and play a positive role in the repair of neurologic function in mice with cerebral infarction. Magnetic resonance imaging molecular probes have a good adjuvant effect on the use of pluripotent stem cell-derived NSCs to treat hind limb paralysis in mice with cerebral infarction.

The role of sensitivity to estrogen change in the development of perimenopausal depressive symptoms

The feasibility and efficacy of magnetic resonance imaging molecular probe application and pluripotent stem cell–derived neural stem cell (NSC) transplantation for the treatment of hind limb paralysis in mice with cerebral infarction were studied. A model of middle cerebral artery infarction using adult mice was established to stimulate hind limb reactions. After the model was successfully established, the mice were first divided into an experimental group and a control group, with 25 mice in each group. Cultured neural cells were obtained from the cerebral cortex and hippocampus of a mouse 15 days pregnant to prepare pluripotent stem cells. Pluripotent stem cell–derived NSCs were identified by positive expression of Nestin. The experimental group was injected with 1 μL of NSC suspension through the tail vein, and the control group was injected with 1 μL of saline through the tail vein. The neurologic function of mice in each group was scored 1 day, 3 days, 7 days, 14 days, and 28 days after transplantation according to the Garcia 18 subscale. Finally, the differentiation, migration, and integration of pluripotent stem cell–derived NSCs after transplantation were observed using a magnetic resonance imaging molecular probe method. The results showed that the neurologic function scores of the ischemic transplantation group were significantly higher than those of the control group, and the results were significantly different (P < 0.05). Through research, it was found that after transplantation of pluripotent stem cell–derived NSCs, the transplanted cells migrated and differentiated around the body at 28 days and participated in angiogenesis, and the blood vessels in the infarcted area were obviously proliferated. The NSCs cultured in vitro were transplanted to the small infarction after cerebral infarction. In rats, it plays a positive role in the repair of nerve function in mice with cerebral infarction. NSCs cultured in vitro can survive, migrate, and differentiate in the brain tissue of mouse ischemic models and play a positive role in the repair of neurologic function in mice with cerebral infarction. Magnetic resonance imaging molecular probes have a good adjuvant effect on the use of pluripotent stem cell–derived NSCs to treat hind limb paralysis in mice with cerebral infarction.

Structural and Thermodynamic Investigation into the Protein-Binding Properties of a Natural Product Crytotanshinone

Crytotanshinone (CTSO) is a Chinese herbal medicine active ingredient isolated from Salvia miltiorrhiza. In this work, the interaction of CTSO and human serum albumin (HSA) was studied by fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra, molecular probe and molecular modeling methods. The results showed that the endogenous fluorescence of HSA was quenched by CTSO through a static mechanism. The number of binding sites, equilibrium constants, and thermodynamic parameters of the reaction were calculated at three different temperatures. The positive enthalpy change (ΔHθ) and entropy change (ΔSθ) revealed that the interaction was an endothermic as well as an entropy-driven process, where hydrophobic power played the major role in stabilizing the structure of the new complex. Site-selective binding experiments were carried out using warfarin and ibuprofen as probes, which proved that CTSO binds to Sudlow’s site II in subdomain IIIA of the HSA molecule. Circular dichroism (CD) spectra was employed to detect the α-helix and β-strand contents in HSA before and after the binding of CTSO. Based on the experimental results, the structure of the CTSO–HSA complex was calculated by docking CTSO to the proven site using molecular modeling. The study obtained comprehensive information on structure and thermodynamics, which is essential for understanding the bioaffinity, delivery process and pharmacological mechanism.

On the quantification of the dissolved hydroxyl radicals in the plasma-liquid system using the molecular probe method

Hydroxyl (OH) radical is one of the most important reactive species produced by plasma-liquid interactions, and the OH in liquid phase (dissolved OH radical, OHdis) takes effect in many plasma-based applications due to its high reactivity. Therefore, the quantification of the OHdis in a plasma-liquid system is of great importance, and a molecular probe method usually used for the OHdis detection might be applied. Herein, we investigate the validity of using the molecular probe method to estimate the [OHdis] in the plasma-liquid system. Dimethyl sulfoxide is used as the molecular probe to estimate the [OHdis] in an air plasma-liquid system, and usually the estimation of [OHdis] is deduced by quantifying the OHdis-induced derivative, the formaldehyde (HCHO). The analysis indicates that the true concentration of the OHdis should be estimated from the sum of three terms: the formed HCHO, the existing OH scavengers, and the H2O2 formed from the OHdis. The results show that the measured [HCHO] needs to be corrected since the HCHO consumption is not negligible in the plasma-liquid system. We conclude from the results and the analysis that the molecular probe method generally underestimates the [OHdis] in the plasma-liquid system. If one wants to obtain the true concentration of the OHdis in the plasma-liquid system, one needs to know the consumption behavior of the OHdis-induced derivatives, the information of the OH scavengers (such as hydrated electron, atomic hydrogen besides the molecular probe), and also the knowledge of the H2O2 formed from the OHdis.

Investigation on the protein-binding properties of icotinib by spectroscopic and molecular modeling method

Icotinib is a highly-selective epidermal growth factor receptor tyrosine kinase inhibitor with preclinical and clinical activity in non-small cell lung cancer, which has been developed as a new targeted anti-tumor drug in China. In this work, the interaction of icotinib and human serum albumin (HSA) were studied by three-dimensional fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra, molecular probe and molecular modeling methods. The results showed that icotinib binds to Sudlow's site I in subdomain IIA of HSA molecule, resulting in icotinib-HSA complexes formed at ground state. The number of binding sites, equilibrium constants, and thermodynamic parameters of the reaction were calculated at different temperatures. The negative enthalpy change (ΔHθ) and entropy change (ΔSθ) indicated that the structure of new complexes was stabilized by hydrogen bonds and van der Waals power. The distance between donor and acceptor was calculated according to Förster's non-radiation resonance energy transfer theory. The structural changes of HSA caused by icotinib binding were detected by synchronous spectra and circular dichroism (CD) spectra. Molecular modeling method was employed to unfold full details of the interaction at molecular level, most of which could be supported by experimental results. The study analyzed the probability that serum albumins act as carriers for this new anticarcinogen and provided fundamental information on the process of delivering icotinib to its target tissues, which might be helpful in understanding the mechanism of icotinib in cancer therapy.

Molecular inversion probe-based single base mutations of hepatitis B virus resistance gene analysis

Objective To establish a molecular inversion probe（MIP）method for detection of single base drug-resistance mutation in Hepatitis B virus（HBV）gene. Methods The HBV wild type and YVDD mutant strain were isolated by Daping Hospital of the Third Military Medical University.The MIP was designed and applied to detect the HBV drug-resistance YVDD mutation in one case of wild type and one case of YVDD mutant HBV strain isolated previously.The results of MIP method were compared with that of sequencing to evaluate the detection accuracy. Results Thermal cycling single-base extension and connection reaction performed by Taq DNA Ligase and Ampligase DNA Ligase could ensure the specificity of the detection.The optimum probe concentration of MIP was 1 nmol/L.Through detection of the target gene with different DNA concentrations, the detection sensitivity of MIP was determined as 1 nmol/L. The results of MIP were consistent with that of sequencing method in detection of the clinical samples. Conclusion MIP is successfully used to detect single-base drug-resistance mutation in HBV gene.（Chin J Lab Med，2014,37：337-341） Key words: Hepatitis B virus; Drug resistance, viral; Molecular probe techniques; Genotype; Polymorphism, single nucleotide

Characterization of microporous activated carbons using molecular probe method

Accurate knowledge of an adsorbent’s porosity is fundamental for scientific and industrial applications of adsorption technology. Over the last decades many approaches have been established to assess porosity of adsorbent materials by analyzing their nitrogen uptake at 77K with volumetric measurement devices. Despite using highly sophisticated physical models, all approaches make assumptions on pore shape as well as on the interactions between adsorbent and adsorptive molecules. Subsequently, significant differences in pore size distributions are observed depending on which modeling parameters were used. The molecular probe method presented in this paper therefore restrains to a minimum of approximations by measuring isotherms of chemically similar substances of increasing molecular size. Differences in pore volume can be reduced to sterical limitations in micropores below the size of adsorptive, leading to a high-resolution pore size distribution below 0.7nm where only few comparable methods exist. The analytical procedure was customized to take account of the amorphous and heterogeneous pore structure of activated carbon. By measuring adsorption isotherms of N2, n-hexane, iso-octane and cyclohexane on various activated carbons, it is shown that differences in pore accessibility of tested adsorptives are specific for each adsorbent. Using molecular probe molecules hence appears to be a promising method for a complementary porosity analysis of activated carbons.

Characterization of Microporous Activated Carbons Using Molecular Probe Method

AbstractDie genaue Kenntnis der Porensysteme von Adsorbentien ist für wissenschaftliche und industrielle Anwendungen der Adsorption von großer Bedeutung. Trotz ihrer teils erheblichen Komplexität vereinfachen die aktuellen physikalischen Modelle zur Interpretation der N2‐Isothermen den tatsächlichen Adsorptionsprozess wodurch völlig unterschiedliche Porengrößenverteilungen berechnet werden. Die vorgestellte Probemolekülmethode ermöglicht durch die Vermessung von Reinstoffisothermen chemisch ähnlicher Probemoleküle steigender Größe unter Einführung weniger Approximationen die direkte Bestimmung eines Ausschnitts der Porengrößenverteilung im Bereich &lt; 0,7 nm.

Multiplex Targeted Sequencing Identifies Recurrently Mutated Genes in Autism Spectrum Disorders

Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes-CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1-may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a β-catenin-chromatin-remodeling network to ASD etiology.

Hyperhomocysteinemia decreases intestinal motility leading to constipation

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine β-synthase (CBS(+/-)), MMP-9(-/-), and MMP-9(-/-) + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS(+/-) mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS(+/-) mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS(+/-) mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS(+/-) mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS(+/-) mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.