Microbial lectome versus host glycolipidome: How pathogens exploit glycosphingolipids to invade, dupe or kill.

Glycosphingolipids (GSLs) are ubiquitous components of the cell membranes, found across several kingdoms of life, from bacteria to mammals, including humans. GSLs are a subclass of major glycolipids occurring in animal lipid membranes in clusters named "lipid rafts." The most crucial functions of GSLs include signal transduction and regulation as well as participation in cell proliferation. Despite the mainstream view that pathogens rely on protein-protein interactions to survive and thrive in their hosts, many also target the host lipids. In particular, multiple pathogens produce adhesion molecules or toxins that bind GSLs. Attachment of pathogens to cell surface receptors is the initial step in infections. Many mammalian pathogens have evolved to recognize GSL-derived receptors. Animal glycosphingolipidomes consist of multiple types of GSLs differing in terminal glycan and ceramide structures in a cell or tissue-specific manner. Interspecies differences in GSLs dictate host specificity as well as cell and tissue tropisms. Evolutionary pressure exerted by pathogens on their hosts drives changes in cell surface glycoconjugates, including GSLs, and has produced a vast number of molecules and interaction mechanisms. Despite that abundance, the role of GSLs as pathogen receptors has been largely overlooked or only cursorily discussed. In this review, we take a closer look at GSLs and their role in the recognition, cellular entry, and toxicity of multiple bacterial, viral and fungal pathogens.

One of the two N-glycans on the human Gb3/CD77 synthase is essential for its activity and allosterically regulates its function.

N-glycosylation is a posttranslational modification that influences many protein properties, such as bioactivity, folding or solubility. The same principles apply to key enzymes in glycosylation pathways, including glycosyltransferases, that also undergoing N-glycosylation, changes in which may affect their activity. Human Gb3/CD77 synthase (encoded by A4GALT) is a Golgi-resident glycosyltransferase, which catalyzes the synthesis of Galα1→4Gal disaccharide on glycosphingolipid- and glycoprotein-derived acceptors, creating Gb3 or P1 antigens and P1 glycotopes (Galα1→4Galß1→4GlcNAc-R), respectively. The molecules that contain Galα1→4Gal serve as receptors for pathogens and Shiga toxins, which are the major virulence factors of Shiga toxin-producing Escherichia coli (STEC). Human Gb3/CD77 synthase contains two N-glycosylation sites at positions N121 and N203. Using the recombinant soluble glycovariants of human Gb3/CD77 synthase with mutated N-glycosylation sequons expressed in HEK293E cells, we show that the glycovariants devoid of N-glycan at position N203 or simultaneously at N121 and N203 sites reveal no enzymatic activity. In contrast, the N-glycan at position N121 plays a negligible role, whereas the presence of both N-glycans is required for efficient secretion of the enzyme. Moreover, utilizing specific glycosidases, we have found that the fully N-glycosylated enzyme contains one complex and one hybrid/oligomannose N-glycan, while single mutants contain only the complex type. Finally, in silico analysis using the AlphaFold enzyme model showed that N-glycan attached to N203 sequon is located in a protein motif near the active site and may allosterically influence the activity. All these findings highlight the prerequisite role of N-glycosylation in human Gb3/CD77 synthase activity (N203 sequon) and solubility (both N121 and N203), with a particularly prominent role of N-glycan at position N203 in the regulation of enzyme activity.

Ludwik Hirszfeld: A pioneer of transfusion and immunology during the world wars and beyond.

Ludwik Hirszfeld (1884-1954) was a Polish physician, immunologist and microbiologist. Together with Emil von Dungern, he showed that blood groups are heritable traits and established the terminology of the ABO blood group system. He discovered A1 and A2 blood groups, and showed for the first time, in a large-scale population study, that blood group frequency differs between populations. During World War I, he volunteered as an army physician. In the interwar period, he helped to create the National Institute of Hygiene in Warsaw and was instrumental in developing transfusion centres in Poland. During World War II, which he barely survived, he co-organized secret medical courses in the Warsaw Ghetto and played a major role in containing the typhus epidemic that ran rampant there since 1941. After the war, he was the first in Poland to put the theory of serological conflict between mother and foetus into clinical practice, saving the lives of almost 200 children by introducing exchange transfusions.

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates.

Most glycosyltransferases show remarkable gross and fine substrate specificity, which is reflected in the old one enzyme-one linkage paradigm. While human Gb3/CD77 synthase is a glycosyltransferase that synthesizes the Galα1→4Gal moiety mainly on glycosphingolipids, its pigeon homolog prefers glycoproteins as acceptors. In this study, we characterized two Gb3/CD77 synthase paralogs found in pigeons (Columba livia). We evaluated their specificities in transfected human teratocarcinoma 2102Ep cells by flow cytofluorometry, Western blotting, high-performance thin-layer chromatography, mass spectrometry and metabolic labelling with 14C-galactose. We found that the previously described pigeon Gb3/CD77 synthase (called P) can use predominately glycoproteins as acceptors, while its paralog (called M), which we serendipitously discovered while conducting this study, efficiently synthesizes Galα1→4Gal caps on both glycoproteins and glycosphingolipids. These two paralogs may underlie the difference in expression profiles of Galα1→4Gal-terminated glycoconjugates between neoavians and mammals.

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable.

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants resulted from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

How glycosylation affects glycosylation: the role of N-glycans in glycosyltransferase activity.

N-glycosylation is one of the most important posttranslational modifications of proteins. It plays important roles in the biogenesis and functions of proteins by influencing their folding, intracellular localization, stability and solubility. N-glycans are synthesized by glycosyltransferases, a complex group of ubiquitous enzymes that occur in most kingdoms of life. A growing body of evidence shows that N-glycans may influence processing and functions of glycosyltransferases, including their secretion, stability and substrate/acceptor affinity. Changes in these properties may have a profound impact on glycosyltransferase activity. Indeed, some glycosyltransferases have to be glycosylated themselves for full activity. N-glycans and glycosyltransferases play roles in the pathogenesis of many diseases (including cancers), so studies on glycosyltransferases may contribute to the development of new therapy methods and novel glycoengineered enzymes with improved properties. In this review, we focus on the role of N-glycosylation in the activity of glycosyltransferases and attempt to summarize all available data about this phenomenon.

Duffy blood group system - the frequency of Duffy antigen polymorphisms and novel mutations in the Polish population.

Duffy blood group genes are highly polymorphic with the distribution of alleles varying between different populations and ethnic groups. The aim of this study was to genotype Duffy blood group antigens and to establish FY alleles frequency in the Polish population and screen for novel FY gene mutations. Duffy phenotype and genotype frequencies analysis was based on studies of 596 persons. All these subjects were genotyped by high-resolution melting (HRM) method. It was shown that phenotype Fy(a+b+), defined by genotypes FY*A/FY*B (33%), FY*A/FY*B298A (13%), and FY*A/FY*02W.01 (2.8%) was the most common in Polish population (˜49%), followed by Fy(a-b+), ˜29%, determined by genotypes arising from FY*B allele and all its variants. Fy(a+b-) phenotype occurred with a frequency of 21.3% and was defined by the following genotypes: FY*A/A (21%), and FY*A/02N.01 (0.3%). Among the Polish population the frequencies of FY*A, FY*B, and FY*B298A alleles were 45.7%, 36% and 15.5%, respectively. The alleles FY*B298A and FY*B combined together, represented higher frequency (51%) than FY*A. Alleles FY*02W.01 and FY*02N.01 had frequencies 2.51% and 0.25%, respectively. The distribution of Duffy genotypes in the Polish population was in accordance with Hardy-Weinberg equilibrium (p = 0.9682). Alleles in the genotypes are independent from each other (r = 0.0278, R2 = 0.00077). New mutations identified in the promoter region (c.-79T > C) and the coding region of the FY gene (c.147C > A and c.175 G > A) did not affect the Duffy antigen expression on erythrocyte. Although FY alleles frequency is known in different populations, no data for Polish population is available.

The role of MMP-12 gene polymorphism - 82 A-to-G (rs2276109) in immunopathology of COPD in polish patients: a case control study.

BACKGROUND: Major symptoms of chronic obstructive pulmonary disease (COPD) are chronic bronchitis and emphysema leading from lung tissue destruction, that is an effect of an imbalance between metalloproteinases (MMPs) and their tissue inhibitors activity. As potential factor involved in this COPD pathogenesis, MMP-12 is considered. We investigated the role of genetic polymorphism and protein level of MMP-12 in the COPD development among Poles. METHODS: We analyzed - 82 A > G SNP in the promoter region of MMP-12 gene (rs2276109) among 335 smoked COPD patients and 309 healthy individuals, including 110 smokers. Additionally, 60 COPD patients and 61 controls (23 smokers) were tested for serum levels of MMP-12 using ELISA. All subjects were analyzed for lung function using spirometry (FEV1% and FEV1/FVC parameters). RESULTS: We observed that -82G allele and -82GG homozygous genotype frequencies of the SNP rs2276109 were significantly lower in COPD patients than in controls (12.5% vs 16.9%, respectively; χ2 = 4.742, p = 0.02 for allele and 0.5% vs 3.9%, respectively; χ2 = 9.0331, p = 0.01 for genotype). Moreover, -82G allele was more frequent in controls smokers than in non-smokers (22.3% vs 14.1%, χ2 = 6.7588, p = 0.01). Serum level of MMP-12 was significantly higher in COPD patients than in controls groups (6.8 ng/ml vs 3.3 ng/ml, respectively; F = 7.433, p < 0.0001), although independently of analyzed gene polymorphisms. Additionally, no correlation between parameters of lung function (FEV1% and FEV1/FVC) and protein level was found. CONCLUSIONS: We found that -82G allele of SNP rs2276109 was associated with reduced risk of COPD, and COPD patients released more MMP-12 than healthy individuals, but independently on this SNP.

Pediatric Dog Bite Injuries in Central Texas.

PURPOSE: Identify children at greatest risk for dog bite injuries and to provide injury prevention recommendations. METHODS: A retrospective review of patients aged ≤18 years treated for dog bite injuries from October 2011 to October 2016 was performed. Data collected included patient demographics, parental presence, time of injury, dog breed and ownership status, injury location and characteristics, need for operative intervention, and hospitalization. RESULTS: One-hundred and two patients met the inclusion criteria. The mean age was 5.84 years, and 43.1% were preschool-aged (2-5 years). Parental presence was reported in 43.6% of cases, and most attacks occurred in the evening (46.8%). Injuries often involved the head-neck region (92.1%), and 72.5% were of major severity. Pet dogs were responsible for 42% of injuries, and pit bull was the most-identified breed (36.2%). Most injuries occurred while the child was at home (57.8%) and was petting or playing with the dog (28.4%). Intervention in the operating room was required in 34.3% of patients. Major injury was more likely to require operative intervention (p = 0.015) but was not associated with patient age, sex, pet status, or the need for hospitalization. CONCLUSIONS: Preschool-aged children are more likely to be injured by dog bites, and dog bites can result in major injury to the head and neck region. Prevention efforts should focus on dog training, public education (children and adults), vigilant adult supervision, and a zero-tolerance policy. LEVEL OF CLINICAL EVIDENCE: Level IV-case series with no comparison group. TYPE OF STUDY: Prognostic.

RT-qPCR analysis of human melanoma progression-related genes - A novel workflow for selection and validation of candidate reference genes.

The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes. NormFinder software was used to identify the best reference gene or pair of reference genes from five candidate housekeeping genes, on the basis of expression stability in a given experimental model. The suitability of references was validated by normalizing the transcriptional activities of E-cadherin (CDH1), N-cadherin (CDH2) and endoplasmic reticulum aminopeptidase 1 (ERAP1) target genes. It has been shown that the relative expression of CDH2 and ERAP1 target genes in a given cell line may vary between experimental models, leading to biological misinterpretation. In view of this, we devised a strategy for improved selection of the best stable reference and for obtaining biologically consistent results. This strategy avoided experimental model- and normalizer-dependent conclusions concerning the relative expression of target gene, in the examined cell lines.

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system.

Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.

The Gerbich blood group system: old knowledge, new importance.

Antigens of the Gerbich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), minor sialoglycoproteins of human erythrocytes. GPC and GPD help maintain erythrocyte shape of and contributes to the stability of its membrane. There are six high-prevalence Gerbich antigens: Ge2, Ge3, Ge4, GEPL (GE10), GEAT (GE11), GETI (GE12) and five low-prevalence Gerbich antigens: Wb (GE5), Lsa (GE6), Ana (GE7), Dha (GE8), GEIS (GE9). Some Gerbich antigens (Ge4, Wb, Dha, GEAT) are expressed only on GPC, two (Ge2, Ana) are expressed only on GPD, while others (Ge3, Lsa, GEIS, GEPL, GETI) are expressed on both GPC and GPD. Antibodies recognizing GPC/GPD may arise naturally (so-called "naturally-occurring RBC antibodies") or as the result of alloimmunization, and some of them may be clinically relevant. Gerbich antibodies usually do not cause serious hemolytic transfusion reactions (HTR); autoantibodies of anti-Ge2- or anti-Ge3 specificity can cause autoimmune hemolytic anemia (AIHA).

Impact of Matrix Metalloproteinase 9 on COPD Development in Polish Patients: Genetic Polymorphism, Protein Level, and Their Relationship with Lung Function.

Chronic obstructive pulmonary disease (COPD) is characterized by a decline of lung function and symptoms such as chronic bronchitis and emphysema leading from lung tissue destruction. Increased activity of matrix metalloproteinases (MMPs) and an imbalance between MMPs and their tissue inhibitors (TIMPs) are considered as factors influencing the pathogenesis of COPD. We investigated the role of genetic polymorphism and expression level of MMP-9 and concentration of its complexes with TIMPs in the development of COPD among Polish patients. We analyzed SNP in the promoter region of MMP-9 gene (rs3918242) using PCR-RFLP method among 335 COPD patients and 309 healthy individuals. Additionally, 60 COPD patients and 61 controls were tested for copy number variants (CNV) of MMP-9 (by quantitative real-time PCR) and serum levels of MMP-9 and its complexes with TIMP1 and TIMP2 (using ELISA). All subjects were analyzed for lung function using spirometry (FEV1% and FEV1/FVC parameters). We observed that allele and genotype frequencies of the SNP rs3918242, as well as the number of gene copies, were similar in COPD patient and controls groups. Serum levels of MMP-9 and MMP-9/TIMP1 complex were significantly higher in COPD patients in comparison to controls groups, although independently of analyzed gene polymorphisms. Additionally, the significant inverse relationships between parameters of lung function (FEV1% and FEV1/FVC) and proteins level were found in ridge regression models, especially we found that FEV1% decreased when MMP-9 level increased in controls and patients with COPD group. In conclusion, we found that COPD patients were predisposed to produce more MMP-9 and MMP-9/TIMP1 complex than healthy individuals. This phenomenon is probably associated with the disease-related lung environment but not with genetic features of the MMP-9.

Plasmodium reichenowi EBA-140 merozoite ligand binds to glycophorin D on chimpanzee red blood cells, shedding new light on origins of Plasmodium falciparum.

BACKGROUND: All symptoms of malaria are caused by the intraerythrocytic proliferation of Plasmodium merozoites. Merozoites invade erythrocytes using multiple binding ligands that recognise specific surface receptors. It has been suggested that adaptation of Plasmodium parasites to infect specific hosts is driven by changes in genes encoding Plasmodium erythrocyte-binding ligands (EBL) and reticulocyte-binding ligands (RBL). Homologs of both EBL and RBL, including the EBA-140 merozoite ligand, have been identified in P. falciparum and P. reichenowi, which infect humans and chimpanzees, respectively. The P. falciparum EBA-140 was shown to bind human glycophorin C, a minor erythrocyte sialoglycoprotein. Until now, the erythrocyte receptor for the P. reichenowi EBA-140 remained unknown. METHODS: The baculovirus expression vector system was used to obtain the recombinant EBA-140 Region II, and flow cytometry and immunoblotting methods were applied to characterise its specificity. RESULTS: We showed that the chimpanzee glycophorin D is the receptor for the P. reichenowi EBA-140 ligand on chimpanzee red blood cells. CONCLUSIONS: We propose that the development of glycophorin C specificity is spurred by the P. falciparum lineage. We speculate that the P. falciparum EBA-140 evolved to hijack GPC on human erythrocytes during divergence from its ape ancestor.

Normocephalic Pancraniosynostosis: A Report of a Surgical Technique.

Normocephalic pancraniosynostosis is defined as the premature fusion of 3 or, more major sutures in the absence of another primary etiology, including primary, microcephaly, ventriculoperitoneal shunting, hypothyroidism, rickets, mucopolysaccharidoses, or other lysosomal storage diseases. It is very rare, thus far only 6 patients have been reported in the literature. Patients tend to present much later than those with single sutural, synostoses, and up to half have evidence of elevated intracranial pressure. The authors wish to present another patient, with emphasis on a unique treatment approach.

CD1: A Singed Cat of the Three Antigen Presentation Systems.

Contrary to general view that the MHC Class I and II are the kapellmeisters of recognition and response to antigens, there is another big player in that part of immunity, represented by CD1 glycoproteins. In contrast to MHC Class I or II, which present peptides, CD1 molecules present lipids. Humans express five CD1 proteins (CD1a-e), four of which (CD1a-d) are trafficked to the cell surface, where they may display lipid antigens to T-cell receptors. This interaction may lead to both non-cognate and cognate T cell help to B cells, the latter eliciting anti-lipid antibody response. All CD1 proteins can bind a broad range of structurally different exogenous and endogenous lipids, but each shows a preference to one or more lipid classes. This unorthodox binding behavior is the result of elaborate architectures of CD1 binding clefts and distinct intracellular trafficking routes. Together, these features make CD1 system a versatile player in immune response, sitting at the crossroads of innate and adaptive immunity. While CD1 system may be involved in numerous infectious, inflammatory, and autoimmune diseases, its involvement may lead to opposite outcomes depending on different pathologies. Despite these ambiguities and complexity, CD1 system draws growing attention and continues to show glimmers of therapeutic potential. In this review, we summarize the current knowledge about CD1 proteins, their structures, lipid-binding profiles, and roles in immunity, and evaluate the role of CD1 proteins in eliciting humoral immune response.

The Incidence of Ocular Injuries in Isolated Orbital Fractures.

BACKGROUND: Prompt identification of significant ocular injuries in patients who sustain an orbital fracture is important to prevent any potential long-term visual sequelae. The true incidence of these injuries has not been determined, however. As a consequence, most surgeons choose to have all patients evaluated by an ophthalmologist. The objective of this study was to conclusively identify the incidence of significant ocular injuries in patients with isolated orbital fractures and to determine their predictors to guide more efficient patient care. METHODS: A prospective cohort study powered to detect a 15% incidence of ocular injuries was designed. All patients presenting to our center with computed tomography findings of an isolated orbital fracture were included and evaluated by plastic surgery and ophthalmology services. Patients were followed up for a minimum of 1 week to identify any delayed injuries. RESULTS: Eighty patients were enrolled from 2012 to 2014. There were 46 men and 34 women with a mean age of 42.8 years. Assault was the most common mechanism of injury. There were 8 ocular injuries (10%): ruptured globe (1), uveal prolapse (1), retrobulbar hemorrhage (2), hyphema (2), hemorrhagic glaucoma with hyphema (1), and scleral tear (1). Predictors for significant ocular injuries were grossly abnormal visual acuity and abnormal pupillary reactivity of the affected eye. CONCLUSIONS: The incidence of significant ocular injuries in isolated orbital fractures is lower than previously reported. Patients presenting with grossly abnormal visual acuity or abnormal pupillary reactivity are at high risk and should receive prompt ophthalmology service evaluation.

Can mutations in the gene encoding transcription factor EKLF (Erythroid Krüppel-Like Factor) protect us against infectious and parasitic diseases?

Transcription factor EKLF (Erythroid Krüppel-Like Factor) belongs to the group of Krüppellike factors, which regulate proliferation, differentiation, development and apoptosis of mammalian cells. EKLF factor is present in erythroid cells, where it participates in regulation of hematopoiesis, expression of genes encoding transmembrane proteins (including blood group antigens), and heme biosynthesis enzymes. It is also a key factor in downregulation of γ-globins and activation of ß-globin gene expression. The EKLF factor consists of two domains: proline-rich transactivation domain and DNA-binding domain containing three zinc finger motifs, which recognize DNA. EKLF can act as a transcription activator (for example in the case of ß-globin gene) or repressor, which depends on the type of posttranslational modification (phosphorylation, SUMOylation, ubiquitination and acetylation). Mutations in the gene encoding EKLF may cause hemoglobinopathies, such as hereditary persistence of fetal hemoglobin and ß-thalassemia intermedia, and congenital dyserythropoietic anemia type IV, which is a hematopoietic disorder. These changes may impede invasion of red blood cells by malaria merozoites and cause faster removal of invaded erythrocytes. In addition, mutations in KLF1 may decrease the number of erythrocyte surface antigens that belong to blood group systems such as MN, P1PK, Lutheran, Duffy, Diego and OK. Such antigens can be receptors for protozoans (such as Plasmodium falciparum or Plasmodium vivax), bacteria (like uropathogenic strains of Escherichia coli, Neisseria meningitidis), and toxins (Shiga toxins), which may cause several dangerous diseases including malaria, pyelonephritis, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and meningitis. Here, we propose a hypothesis on possible liaisons between mutations in the gene encoding EKLF and resistance to pathogens.

Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.