Glycosylphosphatidylinositol Biosynthesis Research Articles

In-Depth Phenotyping of PIGW-Related Disease and Its Role in 17q12 Genomic Disorder

Glycosylphosphatidylinositol (GPI) biosynthesis defect 11 (GPIBD11), part of the heterogeneous group of congenital disorders of glycosylation, is caused by biallelic pathogenic variants in PIGW. This rare disorder has previously been described in only 12 patients. We report four novel patients: two sib fetuses with congenital anomalies affecting several organs, including the heart; a living girl with tetralogy of Fallot, global developmental delay, behavioral abnormalities, and atypic electroencephalography (EEG) without epilepsy; a girl with early-onset, treatment-resistant seizures, developmental regression, and recurrent infections, that ultimately passed away prematurely due to pneumonia. We also illustrate evolving facial appearance and biochemical abnormalities. We identify two novel genotypes and the first frameshift variant, supporting a loss-of-function pathogenic mechanism. By merging our cohort with patients documented in the literature, we deeply analyzed the clinical and genetic features of 16 patients with PIGW-related disorder, revealing a severe multisystemic condition deserving complex management and with uncertain long-term prognosis. We consider the role of PIGW within the critical 17q12 region, which is already associated with genomic disorders caused by deletion or duplication and characterized by variable expressivity. Finally, we discuss PIGW dosage effects and a second hit hypothesis in human development and disease.

Homozygous PGAP2 mutation cause hyperphosphatasia with mental retardation syndrome-3 (HPMRS3): Genetic and clinical evaluation of the ultra rare inherited glycosylphosphatidylinositol (GPI) biosynthesis defect

Introduction: Inherited glycosylphosphatidylinositol biosynthesis defect is considered a subset of the congenital glycosylation disorder that result from mutations in the genes encoding proteins participating in glycosylphosphatidylinositol biosynthesis and modification. Glycosylphosphatidylinositol anchor proteins play important roles in numerous cellular processes including neurogenesis, cell adhesion, immune response and signalling. Hyperphosphatasia with mental retardation syndrome-3 is one of the Glycosylphosphatidylinositol anchor defects, characterized by moderate to severe intellectual disability, dysmorphic features, hypotonia, seizures and persistent hyperphosphatasia. The aim of this study was to investigating the clinical implications of the PGAP2 gene and identifying the severe phenotype. Case Presentation: A male patient with dysmorphic features, neurodevelopmental delay, seizures, hearing loss, Hirschsprung disease, central fever and elevated alkaline phosphatase was included in the study. The magnetic resonance imaging showed cerebral atrophy, corpus callosum hypoplasia. The whole exome sequencing analysis of the individual, and Sanger sequencing were performed for segregation. Additionally, next generation sequencing, whole transcriptome sequencing and homology modelling and analysis were performed. Whole exome sequencing revealed a homozygous c.651C>G (p.His217Gln) in the PGAP2 gene. The Sanger sequencing confirmed the parents were heterozygous. There is no splicing variant was detected by whole transcriptome sequencing. The AlphaFold model was interpreted hypothetically. It was observed the substitution of histidine, with glutamine and may affect the stability of protein. Discussion: Homozygous PGAP2 mutations in the patient we reported in our study resulted in a severe clinic including, severe developmental delay and intellectual disability, severe epilepsia, dysmorphic features, central fever, biochemical, hormonal and immunological abnormalities. This patient would be the youngest case published in the literatur. We showed that, the instability of mutant PGAP2 protein that cause Hyperphosphatasia with mental retardation syndrome-3 lead to more severe phenotypes.

Innovative therapies for treatment of invasive fungal diseases

Invasive fungal diseases (IFD) are difficult to treat and pose a significant threat to immunocompromised individuals. Current antifungal agents face limitations, including antifungal resistance and adverse effects. This review aims to give a comprehensive overview of emerging treatment strategies.Novel drugs in development are Ibrexafungerp, an orally available triterpenoid inhibiting glucan synthesis, and Rezafungin representing the echinocandins with extended half-life and improved tissue penetration, both recently licensed for certain indications. Fosmanogepix targets glycosylphosphatidylinositol biosynthesis, while Olorofim, an orotomide, inhibits fungal nucleic acid synthesis, both currently assessed in advanced clinical trials.Immunotherapeutic approaches include immune checkpoint inhibitors to enhance immune response in immunosuppressed individuals and fungal-specific allogeneic CAR-T cell therapy. For prophylactic purpose in high-risk populations to develop IFD, monoclonal antibodies against different virulence factors of Candida spp. have been discovered but are not yet seen in clinical trials. Vaccines against distinct fungal antigens as well as pan fungal vaccines to prevent IFD are under development in preclinical stages, notably for Candida spp., Cryptococcus spp., and Aspergillus spp., however, their clinical value is still discussed.In summary, major advances to treat IFD have been observed, but challenges for their establishment in the clinical routine persist.

Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 2 Caused by a Novel PIGA Variant Not Associated with a Skewed X-Inactivation Pattern

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs.

The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

Innovative therapies for treatment of invasive fungal diseases

Invasive fungal diseases (IFD) are difficult to treat and pose a significant threat to immunocompromised individuals. Current antifungal agents face limitations, including antifungal resistance and adverse effects. This review aims to give a comprehensive overview of emerging treatment strategies.Novel drugs in development are Ibrexafungerp, an orally available triterpenoid inhibiting glucan synthesis, and Rezafungin representing the echinocandins with extended half-life and improved tissue penetration, both recently licensed for certain indications. Fosmanogepix targets glycosylphosphatidylinositol biosynthesis, while Olorofim, an orotomide, inhibits fungal nucleic acid synthesis, both currently assessed in advanced clinical trials.Immunotherapeutic approaches include immune checkpoint inhibitors to enhance immune response in immunosuppressed individuals and fungal-specific allogeneic CAR-T cell therapy. For prophylactic purpose in high-risk populations to develop IFD, monoclonal antibodies against different virulence factors of Candida spp. have been discovered but are not yet seen in clinical trials. Vaccines against distinct fungal antigens as well as pan fungal vaccines to prevent IFD are under development in preclinical stages, notably for Candida spp., Cryptococcus spp., and Aspergillus spp., however, their clinical value is still discussed.In summary, major advances to treat IFD have been observed, but challenges for their establishment in the clinical routine persist.

Role of PIGM and PIGX in glycosylphosphatidylinositol biosynthesis and human health (Review).

Glycosylphosphatidylinositol-glycan (GPI) is an anchor to specific cell surface proteins known as GPI-anchored proteins (APs) that are localized in lipid rafts and may act as cell co-receptors, enzymes and adhesion molecules. The present review investigated the significance of GPI biosynthesis class phosphatidylinositol-glycan (PIG)M and PIGX in GPI synthesis and their implications in human health conditions. PIGM encodes GPI-mannosyltransferase I (MT-I) enzyme that adds the first mannose to the GPI core structure. PIGX encodes the regulatory subunit of GPI-MT-I. The present review summarizes characteristics of the coding sequences of PIGM and PIGX, and their expression in humans, as well as the relevance of GPI-MT-I and the regulatory subunit in maintaining the presence of GPI-APs on the cell surface and their secretion. In addition, the association of PIGM mutations with paroxysmal nocturnal hemoglobinuria and certain types of GPI-deficiency disease and the altered expression of PIGM and PIGX in cancer were also reviewed. In addition, their interaction with other proteins was described, suggesting a complex role in cell biology. PIGM and PIGX are critical genes for GPI synthesis. Understanding gene and protein regulation may provide valuable insights into the role of GPI-APs in cellular processes.

AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models

Thirty genes are involved in the biosynthesis and modification of Glycosylphosphatidylinositol (GPI)-anchored proteins and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate central nervous system-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno associated virus (AAV). It expressed efficacy within 6 days and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for about a year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter.

No Benefits from Glucocorticoid Maintenance Therapy in Patients with PNH

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, complement-mediated hemolytic anemia resulting from a somatic phosphatidylinositol glycan class A ( PIGA) mutation which bugs the biosynthesis of glycosylphosphatidylinositol (GPI). The lack of GPI-anchored proteins, especially CD55 and CD59 molecules, prompts blood cells to be prone to complement attack and lysis. Targeted on the terminal complement inhibitor has been recommended as a first-line treatment, such as eculizumab (C5 inhibitors), which not only controls hemolytic anemia, reduces thrombosis and blood transfusion, but also improves the staging status of chronic kidney disease in PNH patients. Complement inhibitors are not yet widely administrated by Chinese PNH patients, so glucocorticoids are still mostly used for the first-line treatment. However, the efficacy of glucocorticoid maintenance therapy in PNH is uncertain. Methods We conducted a prospective self-controlled comparison of 40 PNH patients with high disease activity (HDA), collecting anemia, hemolysis, thrombosis and renal function indicators at baseline and 1st, 3rd and 6th month after treatment in the Chinese Eastern Collaboration Group of Anemia (CECGA). All enrolled patients were administrated with prednisone 1.0 mg/kg/d at first and then adjusted the dosage according to the situation. The clinical trial registration number is ChiCTR2100050945. Results A total of 40 patients with PNH were enrolled in this study, 21 (52.5%, 21/40) were male, 19 (47.5%, 19/40) were female and the median age was 33.5 years (range 18-69 years). 29 of 40 (72.5%) presented classical PNH and 11 patients (27.5%) were diagnosed as PNH associated with bone marrow disease (BMD)-10 were aplastic anemia and 1 was myelodysplastic syndrome. Four cases detected single-site thrombosis and one had multi-site thrombosis. The most common locations were portal vein thrombus (2/6, 33.33%) and lower extremity intermuscular thrombus (2/6, 33.33%), others included mesentery vein, hepatic vein, splenic vein and intracranial microthrombus. The laboratory indexes at baseline were presented in Table 1. Compared to the baseline (Table 2), at 1 st, 3 rd, and 6 th month after treatment, both hemoglobin and red blood cell (RBC) have improved, but hemoglobin and RBC didn't keep rising, and showed no difference when comparing 3 rd month with 1 st month and 6 th month with 3 rd month. 19 patients (19/40, 47.5%) required blood transfusion before treatment, and 11 patients (11/40, 27.5%) needed transfusion at 1 st month ( P=0.021). Then 13 patients (13/40, 32.5%) at 3 rd month, 15 patients (15/40, 37.5%) at 6 th month were transfusion dependent ( P=0.109; P=0.388). After 6-month treatment, 3 cases of severe anemia (Hb < 30 g/L) at baseline were still severe anemia (3/7, 42.86%), and 4 cases (4/7, 57.14%) were upgraded to moderate anemia (60 g/L ≤ Hb ≤ 90 g/L). Among the original moderate anemia, 20 (20/31, 64.52%) cases were still moderate anemia, 6 (6/31, 19.35%) cases decreased to severe anemia and 5 (5/31, 16.13%) cases were improved to mild anemia (Hb > 90 g/L). 1 (1/2, 50%) patient of mild anemia originally maintained mid anemia and one (1/2, 50%) patient decreased to moderate anemia. The levels of LDH, indirect bilirubin (IBIL) and reticulocyte (Ret) were not changed significantly at 1 st, 3 rd, 6 th month after treatment. At 1 st, 3 rd, 6 th month, only 1, 2 and 2 patients achieved hemolytic control (LDH < 1.5 ULN). The d-dimer and glomerular filtration rate (GFR) also were indicated no difference. At 1 st, 3 rd, 6 th month, 3 (3/11, 27.27%), 4 (4/11, 36.36%), 6 (6/11, 54.54%) patients were back to normal range, 1 (1/11, 9.09%), 2 (2/11, 18.18%), 4 (4/11, 36.36%) patient was newly elevated (D-dimer > 0.55 mg/L) separately. At baseline 2 patients' renal function was severely reduced (GFR＜60ml/min/1.73m 2), and at 1 st, 3 rd, 6 th month, 1, 1, 2 patients had declined respectively. Discussions and Conclusions The use of prednisone in PNH patients with HDA may initially improve anemia, but the improvement in anemia may also be due to self-remission after paroxysmal episodes of PNH itself, or transfusion. The maintenance therapy with prednisone cannot control hemolysis, improve thrombosis and renal function damage. Long-term glucocorticoid maintenance therapy for PNH is not recommended.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

Identification of the glycosylphosphatidylinositol-specific phospholipase A2 (GPI-PLA2) that mediates GPI fatty acid remodeling in Trypanosoma brucei

The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in the parasitic protozoan Trypanosoma brucei involves fatty acid remodeling of the GPI precursor molecules before they are transferred to protein in the endoplasmic reticulum. The genes encoding the requisite phospholipase A2 and A1 activities for this remodeling have thus far been elusive. Here, we identify a gene, Tb927.7.6110, that encodes a protein that is both necessary and sufficient for GPI-phospholipase A2 (GPI-PLA2) activity in the procyclic form of the parasite. The predicted protein product belongs to the alkaline ceramidase, PAQR receptor, Per1, SID-1, and TMEM8 (CREST) superfamily of transmembrane hydrolase proteins and shows sequence similarity to Post-GPI-Attachment to Protein 6 (PGAP6), a GPI-PLA2 that acts after transfer of GPI precursors to protein in mammalian cells. We show the trypanosome Tb927.7.6110 GPI-PLA2 gene resides in a locus with two closely related genes Tb927.7.6150 and Tb927.7.6170, one of which (Tb927.7.6150) most likely encodes a catalytically inactive protein. The absence of GPI-PLA2 in the null mutant procyclic cells not only affected fatty acid remodeling but also reduced GPI anchor sidechain size on mature GPI-anchored procyclin glycoproteins. This reduction in GPI anchor sidechain size was reversed upon the re-addition of Tb927.7.6110 and of Tb927.7.6170, despite the latter not encoding GPI precursor GPI-PLA2 activity. Taken together, we conclude that Tb927.7.6110 encodes the GPI-PLA2 of GPI precursor fatty acid remodeling and that more work is required to assess the roles and essentiality of Tb927.7.6170 and the presumably enzymatically inactive Tb927.7.6150.

What Do We Know about Surface Proteins of Chicken Parasites Eimeria?

Poultry is the first source of animal protein for human consumption. In a changing world, this sector is facing new challenges, such as a projected increase in demand, higher standards of food quality and safety, and reduction of environmental impact. Chicken coccidiosis is a highly widespread enteric disease caused by Eimeria spp. which causes significant economic losses to the poultry industry worldwide; however, the impact on family poultry holders or backyard production-which plays a key role in food security in small communities and involves mainly rural women-has been little explored. Coccidiosis disease is controlled by good husbandry measures, chemoprophylaxis, and/or live vaccination. The first live vaccines against chicken coccidiosis were developed in the 1950s; however, after more than seven decades, none has reached the market. Current limitations on their use have led to research in next-generation vaccines based on recombinant or live-vectored vaccines. Next-generation vaccines are required to control this complex parasitic disease, and for this purpose, protective antigens need to be identified. In this review, we have scrutinised surface proteins identified so far in Eimeria spp. affecting chickens. Most of these surface proteins are anchored to the parasite membrane by a glycosylphosphatidylinositol (GPI) molecule. The biosynthesis of GPIs, as well as the role of currently identified surface proteins and interest as vaccine candidates has been summarised. The potential role of surface proteins in drug resistance and immune escape and how these could limit the efficacy of control strategies was also discussed.

Discovery of a new chemical scaffold for the treatment of superbug Candida auris infections

ABSTRACT Candida auris has emerged as a serious threat of public health and caused global epidemic due to multi-drug resistance, remarkable transmissibility and high mortality. To tackle the challenging super fungus, novel benzoanilide antifungal agents were discovered by an integrated strategy of phenotypic screen, hit optimization, antifungal assays and mechanism exploration. The most promising compound A1 showed potent in vitro and in vivo efficacy against Candida auris infection. Mechanism investigation revealed that compound A1 blocked the biosynthesis of virulence factors and fungal cell walls through the inhibition of glycosylphosphatidylinositol (GPI) and GPI-anchored proteins. Thus, compound A1 represents a promising lead compound to combat drug-resistant candidiasis.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Overexpression of LAS21 in Cellulase-Displaying Saccharomyces cerevisiae for High-Yield Ethanol Production from Pretreated Sugarcane Bagasse

The valorization of lignocellulosic feedstocks into biofuels and biochemicals has received much attention due to its environmental friendliness and sustainability. However, engineering an ideal microorganism that can both produce sufficient cellulases and ferment ethanol is highly challenging. In this study, we have tested seven different genes that are involved in glycosylphosphatidylinositol (GPI) biosynthesis and remodeling for the improvement of cellulase activity tethered on the S. cerevisiae cell surface. It was found that the overexpression of LAS21 can improve β-glucosidase activity by 48.8% compared to the original strain. Then, the three cellulase genes (cellobiohydrolase, endoglucanase, and β-glucosidase) and the LAS21 gene were co-introduced into a diploid thermotolerant S. cerevisiae strain by a multiple-round transformation approach, resulting in the cellulolytic ECBLCCE5 strain. Further optimization of the bioprocess parameters was found to enhance the ethanol yield of the ECBLCCE5 strain. Scaling up the valorization of pretreated sugarcane bagasses in a 1 L bioreactor resulted in a maximum ethanol concentration of 28.0 g/L (86.5% of theoretical yield). Our study provides a promising way to improve the economic viability of second-generation ethanol production. Moreover, the engineering of genes involved in GPI biosynthesis and remodeling can be applied to other yeast cell surface display applications.

Prospective Self-Control Analysis of Glucocorticoid Maintenance Therapy for Paroxysmal Nocturnal Hemoglobinuria

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, complement-mediated hemolytic anemia caused by a somatic phosphatidylinositol glycan class A (PIGA) gene mutation on the X chromosome of hematopoietic stem cells. The mutation of PIGA gene destroys the biosynthesis of glycosylphosphatidylinositol (GPI), resulting in the lack of GPI anchor proteins, including CD55 and CD59 molecules, in hematopoietic stem cells and their progeny cells (erythrocytes, granulocytes, monocytes, lymphocytes). Erythrocyte membrane without CD55 and CD59 are prone to complement attack and lysis[1]. PNH presents as hemolytic anemia, bone marrow failure, and thrombosis, and so on. Targeting the terminal complement inhibitor, such as eculizumab (C5 inhibitors), could not only control hemolytic anemia, reduce thrombosis and transfusion, improve the staging status of chronic kidney disease, and prevents the deterioration of renal function in PNH patients. However, eculizumab has not yet been available in mainland of China, glucocorticoids is still used as the first-line therapy in China. Methods From September 2020 to April 2022, 26 PNH patients with high disease activity (HAD) were enrolled in the study. Oral prednisone was initiated at a dose of 1mg/kg/day and gradually tapered four weeks later. Clinical characteristics of patients was described in Table 1. Results The hemoglobin paired T-test results before treatment and at 1 month, 3 months, and 6 months after treatment were as follows: (69.12±14.83) vs (77.50±18.11), p＜0.05；(69.12±14.83) vs (74.15±18.23), p=0.15；(69.12±14.83) vs (75.54±19.91), p=0.09; paired chi-square test (McNemar Test) for anemia stratification (>90g/L, 60-90g/L, <60g/L) showed no marked changes: p=0.082, p=0.344, p=0.247; By Friedman Test, it showed reticulocyte percentage was also similar: (6.76±3.92) vs (6.23±4.08) vs (6.62±3.92) vs (7.11±4.73), p=0.457. Before treatment, 25 patients (25/26, 96.15%) were transfusion dependent. After treatment at 1 month, 3 months, 6 months, 19 patients (19/26, 73.08%), 22 patients (22/26, 84.62%), 21 patients (21/26, 80.77%) were transfusion dependent, respectively (p=0.031; p=0.375; p=0.219). Two patients progressed to transfusion dependent at 3 months, 6months, respectively. No patients achieved remission of hemolysis (LDH < 1.5 ⅹULN) at 1, 3, and 6 months; the level of lactate dehydrogenase and indirect bilirubin (IBIL) showed no changes by analysis with Friedman test (p=0.436, p=0.881). By Friedman test, the serum level of D-dimer also showed no changes at different times ( p=0.263). Level of D-dimer elevated (>0.55mg/L) in seven patients before treatment, and returned back within normal range in one patient at 1 month, two patients at 3 months, three patients at 6 months. But among the people with normal D level initially, one patient and three patients each had elevated D-dimer level at 3 months and 6 months, respectively. Two patients achieved renal function improvement at 3 months, 6months, respectively. But three patients had renal function deterioration after treatment. Glomerular filtration rate (GFR) was (114.15±42.98) ml/min/1.73m2 before treatment, and become (117.79±37.69) ml/min/1.73m2, (120.85±44.05) ml/min/1.73m2, (110.30±36.58) ml/min/1.73m2 at 1, 3, and 6 months (p=0.42, p=0.43, p=0.41). Discussion Previous studies have mainly compared the improvement of anemia after breakthrough of PNH by glucocorticoids therapy. The improvement of anemia could not be ruled out as a result of transfusion support or the reduction of temporary hemolysis after breakthrough of PNH. We found the level of LDH increased along with improvement of hemoglobin during prednisone treatment, and percentage of reticulocytes was not changed at same time. It suggested that the improvement of anemia in some patients may not be caused by controlling intravascular hemolysis or the compensatory hyperplasia of the bone marrow, but by transfusion. Conclusions It preliminarily showed prednisone maintenance therapy for PNH could not control hemolysis and thrombosis, and could not improve anemia and renal function. The efficacy of glucocorticoid maintenance therapy in PNH is uncertain. Reference: [1] Brodsky R A. How I Treat Paroxysmal Nocturnal Hemoglobinuria[J]. Blood, 2021, 137(10): 1304-1309. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rare multiple congenital anomalies-hypotonia-seizures syndrome type 1 (MCAHS1) – the clinical and molecular summary

Multiple congenital anomalies-hypotonia-seizures syndrome type 1 (MCAHS1) is a rare autosomal recessive genetic disease belonging to glycosylphosphatidylinositols biosynthesis defects (GPIBD), a group of recessive disorders characterized by intellectual disability, hypotonia, and seizures. Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor and remodel cell proteins. These processes are highly conserved and fundamental in the metabolism of all eukaryotes, including humans. Here, we have reported a male patient presenting with hypotonia, intellectual disability, and epilepsy, who underwent whole exome sequencing (WES). The analysis revealed the presence of two deleterious variants in PIGN that encodes GPI ethanolamine phosphate transferase-1 – one novel (c.1247_1251delAAGTG; p.Glu416Glyfs*22), and one that has been previously reported in the medical literature (c.1434+5G>A) resulting in MCAHS1. The detailed clinical assessment followed by the medical literature review also pointed out transient macrosomia and unreported in MCAHS1 advanced bone age and postnatal tall stature. These symptoms suggest that MCAHS1 shares a phenotypic overlap with disorders associated with overgrowth. To conclude, our case report and summary of the medical literature may be helpful for clinicians and geneticists who diagnose patients presenting with hypotonia accompanied by tall stature, advanced bone age, and transient macrosomia.

Vascular Diseases in PNH

PNH is a condition in which uncontrolled complement activity leads to systemic complications, principally through intravascular hemolysis and platelet activation. It arises through a somatic mutation of the phosphatidylinositol glycan A (PIG-A) gene in bone marrow stem cells, 1,2 resulting in disruption to glycosylphosphatidylinositol (GPI) biosynthesis, 3. Among the deficient proteins are the complement regulatory proteinsCD55 andCD59, resulting in increased complement sensitivity of PNH cells, intravascular hemolysis, promotion of inflammatory mediators, and systemic hemoglobin release4 . Patients with PNH can present with multisystemic clinical manifestations due to intravascular hemolysis, thrombosis and bone marrow failure5 . Symptoms are therefore often non-specific, ranging from loss of vision (due to retinal thrombosis), headache and nausea/vomiting (due to cerebral thrombosis), pulmonary hypertension (due to pulmonary embolism), anaemia, through to pain and swelling in the lower extremities (due to deep vein thrombosis), renal failure and other symptoms affecting different systems6 . Thromboembolism is the most common cause of mortality in patients with PNH and accounts for approximately 40% to 67% of deaths of which the cause is known. Further, 29% to 44% of patients with PNH have been reported to have at least 1 thromboembolic event during the course of their disease, although the reason(s) a thrombotic event may suddenly occur remains an enigma7,8,9. Platelet activation, complementmediated hemolysis, impaired nitric oxide (NO) bioavailability, impairment of the fibrinolytic system, and inflammatory mediators are all proposed mechanisms and thought to be responsible for the increased thrombotic risk in patients with PNH. Multiple factors are likely to contribute to any one thrombotic event in patients with PNH. 10 Therapeutic strategies include terminal complement blockade and bone marrow transplantation. Eculizumab, a monoclonal antibody complement inhibitor, is highly effective and the only licensed therapy for PNH.11 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.12,13 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment.14,15 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment.16,17

Application of plasma metabolome for monitoring the effect of rivaroxaban in patients with nonvalvular atrial fibrillation.

Rivaroxaban, an oral factor Xa inhibitor, has been used to treating a series of thromboembolic disorders in clinical practice. Measurement of the anticoagulant effect of rivaroxaban is important to avoid serious bleeding events, thus ensuring the safety and efficacy of drug administration. Metabolomics could help to predict differences in the responses among patients by profiling metabolites in biosamples. In this study, plasma metabolomes before and 3 hours after rivaroxaban intake in 150 nonvalvular atrial fibrillation (NVAF) patients and 100 age/gender-matched controls were analyzed by liquid chromatography coupled with mass spectrometry (LC–MS/MS). When compared with controls, a total of thirteen plasma metabolites were differentially expressed in the NVAF patients. Pathway analysis revealed that purine and lipid metabolism were dysregulated. A panel of three metabolites (17a-ethynylestradiol, tryptophyl-glutamate and adenosine) showed good predictive ability to distinguish nonvalvular atrial fibrillation with an area under the receiver operating characteristic curve (AUC) of 1 for the discovery phase and 1 for validation. Under rivaroxaban treatment, a total of seven metabolites changed, the lipid and glycosylphosphatidylinositol biosynthesis pathways were altered and the panel consisting of avocadene, prenyl glucoside and phosphatidylethanolamine showed predictive ability with an AUC of 0.86 for the discovery dataset and 0.82 for the validation. The study showed that plasma metabolomic analyses hold the potential to differentiate nonvalvular atrial fibrillation and can help to monitor the effect of rivaroxaban anticoagulation.

Investigating Celastrol's Anti-DCM Targets and Mechanisms via Network Pharmacology and Experimental Validation.

Methods Data from TCMSP and GEO databases were utilized to identify targets for Celastrol on DCM. The relationship between the major targets and conventional glycolipid metabolism was obtained with Spearman correlation analysis. Experiments on animals were conducted utilizing healthy control (HC), low-dose Celastrol interventions (CL), and no intervention groups (NC), all of which had 8 SD rats in each group. To study alterations in signaling molecules, RT-PCR was performed. Results There were 76 common targets and 5 major targets for Celastrol-DCM. Celastrol have been found to regulate AGE-RAGE, TNF, MAPK, TOLL-like receptors, insulin resistance, and other signaling pathways, and they are closely linked to adipocytokines, fatty acid metabolism, glycolipid biosynthesis, and glycosylphosphati-dylinositol biosynthesis on DCM. These five major targets have been found to regulate these pathways. Experiments on rats indicated that P38 MAPK was considerably elevated in the cardiac tissue from rats in the CL and NC groups compared to the HC group, and the difference was statistically significant (P < 0.01). Significant differences were seen between the CL and NC groups in P38 MAPK levels, with a statistical significance level of less than 0.05. Conclusion Celastrol may play a role in reversing energy remodeling, anti-inflammation, and oxidative stress via modulating p38 protein expression in the MAPK pathway, which have been shown in the treatment of DCM.