Genetic reduction of tyramine ß hydroxylase suppresses Tau toxicity in a Drosophila model of tauopathy.

Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, Tau. These diseases are associated with degeneration and dysfunction of the noradrenergic system, a critical regulator of memory, locomotion, and the fight or flight response. Though Tau pathology accumulates early in noradrenergic neurons, the relationship between noradrenaline signaling and tauopathy pathogenesis remains unclear. The fruit fly, Drosophila melanogaster, is a valuable model organism commonly used to investigate factors that promote Tau-mediated degeneration. Moreover, Drosophila contain the biogenic amine, octopamine, which is the functional homolog to noradrenaline. Using a Drosophila model of tauopathy, we conducted a candidate modifier screen targeting tyramine ß hydroxylase (tßh), the enzyme that controls the production of octopamine in the fly, to determine if levels of this enzyme modulate Tau-induced degeneration in the fly eye. We found that genetic reduction of tßh suppresses Tau toxicity, independent of Tau phosphorylation. These findings show that reduction of tßh, a critical enzyme in the octopaminergic pathway, suppresses Tau pathogenicity and establishes an interaction that can be further utilized to determine the relationship between noradrenergic-like signaling and Tau toxicity in Drosophila.

New Homozygous Missense MSMO1 Mutation in Two Siblings with SC4MOL Deficiency Presenting with Psoriasiform Dermatitis.

Sterol-C4-methyl oxidase (SC4MOL) deficiency was recently described as an autosomal recessive cholesterol biosynthesis disorder caused by mutations in the MSMO1 (sometimes also referred to as SC4MOL) gene. To date, 5 patients from 4 unrelated families with SC4MOL deficiency have been reported. Diagnosis can be challenging as the biochemical accumulation of methylsterols can affect global development and cause skin and ocular pathology. Herein, we describe 2 siblings from a consanguineous Turkish family with SC4MOL deficiency presenting with psoriasiform dermatitis, ocular abnormalities (nystagmus, optic hypoplasia, myopia, and strabismus), severe intellectual disability, and growth and motor delay. We undertook whole-exome sequencing and identified a new homozygous missense mutation c.81A>C; p.Asn27Thr in MSMO1. Segregation analysis in all available family members confirmed recessive inheritance of the mutation. The siblings were treated with a combination of oral and topical statin and cholesterol which resulted in clinical improvement. This study demonstrates how genomics-based diagnosis and therapy can be helpful in clinical practice.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm.

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

Variant analysis of HPD genes from two families showing elevated tyrosine upon newborn screening by tandem mass spectrometry (MS/MS).

Background Alterations in the structure and activity of 4-hydroxyphenylpyruvate dioxygenase (HPD) are causally related to two different metabolic disorders: recessively inherited tyrosinemia type III and dominantly inherited hawkinsinuria. The aim of this study was to provide a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria. Case presentation A full-term newborn baby born after a safe pregnancy and childbirth with a birth weight of 3200 g and another full-term baby born after a safe pregnancy and childbirth with a birth weight of 2800 g are reported and analysed. DNA extraction, next-generation sequencing, bioinformatics analysis, Sanger sequencing and biochemical analysis were performed. One patient with a heterozygous HPD gene (NM_002150.2) c.460G > A mutation and one patient with a heterozygous HPD gene (NM_002150.2) c.248delG mutation showing elevated tyrosine levels upon newborn screening by tandem mass spectrometry (MS/MS) are reported. Conclusions The HPD gene may not be a strictly autosomal recessive pathogenic gene, which provides a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria.

Hawkinsinuria clinical practice guidelines: a Mexican case report and literature review.

Hawkinsinuria is an autosomal dominant disorder of tyrosine metabolism. Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) result in an altered HPD enzyme, causing hawkinsin and tyrosine accumulation. Persistent metabolic acidosis and failure to thrive are common features in patients with hawkinsinuria. We present the first known Latin American patient diagnosed with hawkinsinuria, and the tenth reported patient in the literature. We aim to establish clinical practice guidelines for patients with hawkinsinuria. The patient's plasma tyrosine level was 21.5 mg/dL, which is several times higher than the reference value. Mutation analysis indicated heterozygosity for V212M and A33T variants in HPD. In the case of altered tyrosine levels found during newborn screening, we propose exclusive breastmilk feeding supplemented with ascorbic acid. Amino acid quantification is useful for monitoring treatment response. If tyrosinemia persists, protein intake must be decreased via a low-tyrosine diet. Molecular studies can be used to confirm a patient's disease etiology. Further reports are required to elucidate new pathogenic and phenotypic variations to enable the development of an appropriate therapeutic approach.

Human species-specific loss of CMP-N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms.

Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD-risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr-/- mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr-/- mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a "xeno-autoantigen" via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc "xeno-autoantibodies" potentiate chronic inflammation ("xenosialitis"). Cmah-/-Ldlr-/- mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah-/-Ldlr-/- mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

In-Silico analysis of missense SNPs in Human HPPD gene associated with Tyrosinemia type III and Hawkinsinuria.

HPPD gene codes a dioxygenase enzyme involved in catalysis of different molecules such as tyrosine and phenylalanine by oxidizing them to produce energy. A single change in protein can trigger serious genetic disorders like Tyrosinemia type III and Hawkinsinuria. This study aims to identify the functional missense SNPs of the HPPD gene by using multiple computational tools. All deleterious missense SNPs retrieved from Ensembl and OMIM database were evaluated through six different software. Ultimately, out of 148 missense SNPs, only 27 were confirmed as diseasecausing SNPs by developing a consensus approach. These damaging SNPs were further examined to evaluate their impact on protein stability and energy including their evolutionary conservation. Native and mutated proteins structures were also designed and superimposed by I-TASSER and PyMol respectively. This work results in narrowing down missense SNPs which are still not confirmed experimentally and demands the confirmation by GWAS data. Thus, these missense SNPs could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein.

Absence of Neu5Gc and Presence of Anti-Neu5Gc Antibodies in Humans-An Evolutionary Perspective.

The glycocalyx of human cells differs from that of many other mammals by the lack of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and increased abundance of its precursor N-acetylneuraminic acid (Neu5Ac). Most humans also have circulating antibodies specifically targeting the non-human sialic acid Neu5Gc. Recently, several additional mammalian species have been found to also lack Neu5Gc. In all cases, loss-of-function mutations in the gene encoding the sialic acid-modifying enzyme CMAH are responsible for the drastic change in these species. Unlike other glycan antigens, Neu5Gc apparently cannot be produced by microbes, raising the question about the origin of these antibodies in humans. Dietary exposure and presentation on bacteria coating themselves with Neu5Gc from the diet are distinct possibilities. However, the majority of the non-human species that lack Neu5Gc do not consume diets rich in Neu5Gc, making it unlikely that they will have been immunized against this sialic acid. A notable exception are mustelids (ferrets, martens and their relatives) known for preying on various small mammal species rich in Neu5Gc. No studies exist on levels of anti-Neu5Gc antibodies in non-human species. Evolutionary scenarios for the repeated, independent fixation of CMAH loss-of-function mutations at various time points in the past include strong selection by parasites, especially enveloped viruses, stochastic effects of genetic drift, and directional selection via female immunity to paternal Neu5Gc. Convergent evolution of losses of the vertebrate-specific self-glycan Neu5Gc are puzzling and may represent a prominent way in which glycans become agents of evolutionary change in their own right. Such change may include the reconfiguration of innate immune lectins that use self-sialic acids as recognition patterns.

Justification of specific genetic modifications in pigs for clinical organ xenotransplantation.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.

The desirable donor pig to eliminate all xenoreactive antigens.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

Human-like Cmah inactivation in mice increases running endurance and decreases muscle fatigability: implications for human evolution.

Compared to other primates, humans are exceptional long-distance runners, a feature that emerged in genus Homo approximately 2 Ma and is classically attributed to anatomical and physiological adaptations such as an enlarged gluteus maximus and improved heat dissipation. However, no underlying genetic changes have currently been defined. Two to three million years ago, an exon deletion in the CMP-Neu5Ac hydroxylase (CMAH) gene also became fixed in our ancestral lineage. Cmah loss in mice exacerbates disease severity in multiple mouse models for muscular dystrophy, a finding only partially attributed to differences in immune reactivity. We evaluated the exercise capacity of Cmah-/- mice and observed an increased performance during forced treadmill testing and after 15 days of voluntary wheel running. Cmah-/- hindlimb muscle exhibited more capillaries and a greater fatigue resistance in situ Maximal coupled respiration was also higher in Cmah null mice ex vivo and relevant differences in metabolic pathways were also noted. Taken together, these data suggest that CMAH loss contributes to an improved skeletal muscle capacity for oxygen use. If translatable to humans, CMAH loss could have provided a selective advantage for ancestral Homo during the transition from forest dwelling to increased resource exploration and hunter/gatherer behaviour in the open savannah.

Deletion of the fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish.

Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.

Immune Responses of HLA Highly Sensitized and Nonsensitized Patients to Genetically Engineered Pig Cells.

BACKGROUND: We investigated in vitro whether HLA highly sensitized patients with end-stage renal disease will be disadvantaged immunologically after a genetically engineered pig kidney transplant. METHODS: Blood was drawn from patients with a calculated panel-reactive antibody (cPRA) 99% to 100% (Gp1, n = 10) or cPRA 0% (Gp2, n = 12), and from healthy volunteers (Gp3, n = 10). Serum IgM and IgG binding was measured (i) to galactose-α1-3 galactose and N-glycolylneuraminic acid glycans by enzyme-linked immunosorbent assay, and (ii) to pig red blood cell, pig aortic endothelial cells, and pig peripheral blood mononuclear cell from α1,3-galactosyltransferase gene-knockout (GTKO)/CD46 and GTKO/CD46/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (CMAHKO) pigs by flow cytometry. (iii) T-cell and B-cell phenotypes were determined by flow cytometry, and (iv) proliferation of T-cell and B-cell carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. RESULTS: (i) By enzyme-linked immunosorbent assay, there was no difference in IgM or IgG binding to galactose-α1-3 galactose or N-glycolylneuraminic acid between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pig peripheral blood mononuclear cell. CONCLUSIONS: (i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

A rare case of sterol-C4-methyl oxidase deficiency in a young Italian male: Biochemical and molecular characterization.

Inborn defects of cholesterol biosynthesis are metabolic disorders presenting with multi-organ and tissue anomalies. An autosomal recessive defect involving the demethylating enzyme C4-methyl sterol (SC4MOL) has been reported in only 4 patients so far. In infancy, all patients were affected by microcephaly, bilateral congenital cataracts, growth delay, psoriasiform dermatitis, immune dysfunction, and intellectual disability. Herein, we describe a new case of SC4MOL deficiency in which a 19-year-old Italian male was affected by bilateral congenital cataracts, growth delay and learning disabilities, behavioral disorders and small stature, but not microcephaly. Our patient had abundant scalp dandruff, without other skin manifestations. Analysis of the blood sterol profile showed accumulation of C4-monomethyl and C4-dimethyl sterols suggesting a deficiency of the SC4MOL enzyme. Sequencing of the MSMO1 gene (also known as the "SC4MOL" gene) confirmed mutations in each allele (c.731A>G, p.Y244C, which is already known, and c.605G>A, p.G202E, which is a novel variant). His father carried c.731A>G mutation, whereas his mother carried c.605G>A. Thus, the combination of multiple skills and methodologies, in particular, blood sterol profiling and genetic analysis, led to the diagnosis of a new case of a very rare defect of cholesterol biosynthesis. Consequently, we suggest that these two analyses should be performed as soon as possible in all undiagnosed patients affected by bilateral cataracts and developmental delay.

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans.

Our group previously investigated the levels of anti-Gal and anti-nonGal IgM and IgG in a cohort of 75 healthy humans of various backgrounds, and found some significant differences related to factors such as age, gender, ABO blood group, diet, vaccination history, and geographic location during childhood. We have now expanded our cohort (n = 84) to investigate the levels of anti-Neu5Gc and anti-nonGal/nonNeu5Gc antibodies in healthy humans. Anti-nonGal and anti-nonGal/nonNeu5Gc human IgM and IgG binding to pRBCs and pAECs from GTKO/CD46 and GTKO/CD46/Neu5GcKO pigs were measured by flow cytometry. Anti-Gal and anti-Neu5Gc IgM and IgG levels were measured by ELISA. In summary, (i) the great majority (almost 100%) of humans had anti-Neu5Gc IgM and IgG antibodies that bound to pAECs and approximately 50% had anti-Neu5Gc antibodies that bound to pRBCs, (ii) there was significantly less human antibody binding to pig cells that did not express either Gal or Neu5Gc compared with those that did not express Gal alone, (iii) the levels of both IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs and pAECs were low, (iv) the level of anti-Neu5Gc IgG was higher in men than women, (v) the level did not change with age or diet, and there was some variability associated with (vi) previous vaccination history and (vii) the geographic region in which the individual spent his or her childhood. Our study confirms that human antibody binding to RBCs and AECs from GTKO/CD46/Neu5GcKO pigs is greatly reduced compared to binding to GTKO/CD46 cells. However, all humans appear to have a low level of antibody that binds to pAECs that is not directed to either Gal or Neu5Gc. Our findings require consideration in planning clinical trials of xenotransplantation.

Chitosan signaling in guard cells requires endogenous salicylic acid.

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.

Biochemical, Cellular, Physiological, and Pathological Consequences of Human Loss of N-Glycolylneuraminic Acid.

About 2-3 million years ago, Alu-mediated deletion of a critical exon in the CMAH gene became fixed in the hominin lineage ancestral to humans, possibly through a stepwise process of selection by pathogen targeting of the CMAH product (the sialic acid Neu5Gc), followed by reproductive isolation through female anti-Neu5Gc antibodies. Loss of CMAH has occurred independently in some other lineages, but is functionally intact in Old World primates, including our closest relatives, the chimpanzee. Although the biophysical and biochemical ramifications of losing tens of millions of Neu5Gc hydroxy groups at most cell surfaces remains poorly understood, we do know that there are multiscale effects functionally relevant to both sides of the host-pathogen interface. Hominin CMAH loss might also contribute to understanding human evolution, at the time when our ancestors were starting to use stone tools, increasing their consumption of meat, and possibly hunting. Comparisons with chimpanzees within ethical and practical limitations have revealed some consequences of human CMAH loss, but more has been learned by using a mouse model with a human-like Cmah inactivation. For example, such mice can develop antibodies against Neu5Gc that could affect inflammatory processes like cancer progression in the face of Neu5Gc metabolic incorporation from red meats, display a hyper-reactive immune system, a human-like tendency for delayed wound healing, late-onset hearing loss, insulin resistance, susceptibility to muscular dystrophy pathologies, and increased sensitivity to multiple human-adapted pathogens involving sialic acids. Further studies in such mice could provide a model for other human-specific processes and pathologies involving sialic acid biology that have yet to be explored.