Glycosylphosphatidylinositol Research Articles

Investigation of Glycosylphosphatidylinositol (GPI)-Plasma Membrane Interaction in Live Cells and the Influence of GPI Glycan Structure on the Interaction.

Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI-cell membrane interaction. Eighty-eight unique membrane proteins, many of which are related to GPIs/GPI-anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI-related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.

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.

Candida krusei pneumonia in graft-versus-host disease after allogeneic hematopoietic stem cell transplant for paroxysmal nocturnal hemoglobinuria: a case report

Introduction and importance: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder caused by a somatic mutation of PIGA (phosphatidylinositol glycan anchor biosynthesis, class A) gene that leads to the destruction of blood cells. Allogeneic haematopoietic stem cell transplant (HSCT) is a treatment option for PHN, but it can cause graft-versus-host disease (GVHD). Long-term immunosuppression as a treatment of GVHD increases the risk for invasive fungal infections such as Candida krusei pneumonia. Case presentation: We present the case of a 22-year-old male with C. krusei pneumonia in a known case of chronic GVHD following HSCT for PNH undergoing long-term immunosuppressive therapy. The patient presented with progressive shortness of breath, productive cough, palpitations, and difficulty swallowing. On examination, he had skin rashes and oral lesions, along with signs of severe malnutrition. Diagnosis was made on the basis of radiological imaging and fungal culture. Discussion: The combination of PNH, GVHD, and HSCT created an immunocompromised state, making the patient susceptible to opportunistic infections, including fungal pneumonia. Early recognition of this condition is challenging due to its non-specific symptoms and potential overlap with other post-transplant complications. Timely diagnosis and appropriate treatment, including antifungal therapy and immunosuppression management, are crucial for optimising patient outcomes. Conclusion: This case highlights the importance of early recognition and timely treatment of fungal infections in patients with severe conditions such as GVHD following HSCT for PNH. Timely treatment with appropriate antifungals is necessary for optimal outcomes. Additionally, more research with long-term follow-up and monitoring is necessary to address the necessary knowledge gaps in this field.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

Unraveling the Clinical Spectrum: A Case Study of Classic and Aplasia-Related Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare nonmalignant clonal disorder of hematopoietic stem cells characterized by a phosphatidylinositol glycan protein A (PIGA) gene mutation. This mutation results in the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, on the cell surface, rendering red blood cells (RBC) susceptible to complement-mediated destruction. This leads to hemolytic anemia, thromboses, and bone marrow failure. Here we describe two cases of PNH presenting to a tertiary care center. Case 1 A 29-year-old male with a history of polysubstance use presented to the Emergency Department with a 2-day history of progressively worsening left lower quadrant abdominal pain. CT abdomen pelvis showed multiple renal infarcts and findings suggestive of pneumatosis intestinalis. A complete blood count (CBC) revealed pancytopenia with a leukocyte count (WBC) of 3 x 10 9/L, hemoglobin (Hb) of 94 g/L, and platelet count (Plt) of 48 x 10 9/L. Lactate dehydrogenase (LDH) was elevated (900 IU/L), haptoglobin level was low ( <8mg/dL), and direct antiglobulin test (DAT) was negative. Urine was positive for hemosiderin, and flow cytometry showed RBCs (6.7%), granulocytes (97.97%), and monocytes (89.02%) with PNH phenotype. Bone marrow biopsy (BMB) was negative for dysplasia and fibrosis with normal hematopoiesis but showed increased erythroid precursors. Enoxaparin was started for thromboses and levofloxacin for neutropenia. Treatment for PNH was initiated with a loading dose of ravulizumab and meningococcal vaccination. The patient was lost to follow-up after receiving the first maintenance dose. Case 2 An 81-year-old female with a medical history of hypertension and coronary artery disease was hospitalized for gradually worsening shortness of breath and fatigue ongoing for 2 months. She reported easy bruising but denied a history of obvious bleeding from any orifice. Physical exam was significant for petechial rash in her lower extremities. Labs revealed pancytopenia (WBC 1.44 x 10 9/L, Hb 68 g/L, Plt 8 x 10 9/L), low reticulocyte count, LDH of 276 IU/L, and negative DAT. BMB showed marked aplasia, and she became transfusion dependent. Subsequently, flow cytometry revealed PNH phenotype in 1.45% of RBCs, 54% granulocytes, and 54% monocytes. Eculizumab therapy was started for the diagnosis of PNH. However, her transfusion requirements did not decrease. She developed iron overload and showed positive conversion on DAT. Darbepoetin alfa and romiplostim were added. Her Hb stabilized at >70g/L in 3 years, following which she only required romiplostim to maintain Plt >20,000 ( Graph 1). After 5 years, she opted to discontinue all treatment. She is being followed up regularly and has since remained stable. Discussion The two cases above are reflective of how PNH can present in different ways, ranging from the classic triad of symptoms to those with subclinical findings associated with other underlying conditions, such as aplastic anemia or myelodysplastic syndrome. The introduction of complement inhibitors completely changed the natural history of PNH, as patients' median survival shifted from 15-20 years to align with age-matched controls. Indications for treatment include severe anemia, thrombosis, or PNH symptoms like dyspnea, fatigue, and painful paroxysms. Currently, eculizumab and ravulizumab are the only FDA-approved drugs for PNH treatment. Ravulizumab is non-inferior to eculizumab, with the added advantage of less frequent dosing (every 8 weeks versus 2 weeks). Regular monitoring for hemolysis is warranted with treatment. Many patients require long-term therapy, which continues to be a significant challenge, even with the spaced dosing of ravulizumab. Eculizumab protects from terminal complement-mediated damage preventing intravascular hemolysis. However, these cells are still deficient in CD55, making them vulnerable to opsonization by C3 and premature removal in the spleen. As a result, >50% of PNH patients receiving treatment show positive conversion on DAT, as seen in case 2. Heparin is preferred in acute thrombotic states. Most hematologists recommend anticoagulation till a few months after starting complement inhibition.

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.

Metagenomic Sequencing Analysis of the Effects of Acetylcysteine on the Pig Gut Microbiome

The gut microbiota is engaged in multiple interactions affecting host health, and gut dysbiosis can lead to many diseases. However, the effects of acetylcysteine (NAC) on the gut microbiome composition in pigs using metagenomic sequencing have not been reported. In this study, we used metagenome sequencing to study the effects of NAC on the pig gut microbiome. Sequencing results showed that microbial diversity was changed after NAC treatment. Antibiotic Resistance Genes Database (ARDB) analysis demonstrated that the main genes modified were macb, tsnr, norm, bl2be-per, vansb and pbp1b in the NAC group. Our data showed that NAC could affect microbial distribution at the phylum, gene and species levels. At the species level, NAC significantly increased the abundances of Megasphaera, Lactobacillus reuteri and Megasphaeraelsdenii and reduced the abundances of Phascolarctobacterium succinatutens, Prevotellacopri and Selenomonasbovis compared with the control group. In addition, Gene Ontology (GO) analysis revealed that in the NAC group, cellular process, metabolic process and single-organism process were the dominant terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that RNA transport, MAPK signaling pathway, cell cycle, glycosylphosphatidylinositol (GPI)-anchor biosynthesis and VEGF signaling pathway were the dominant signaling pathways in the NAC group. In conclusion, our results suggest that NAC may modify the piglet gut microbiome composition and these findings might provide a new strategy for maintaining animal and human health in the future.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Industrialization possibilities of purified pig sperm hyaluronidase.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

RALF22 promotes plant immunity and amplifies the Pep3 immune signal.

Rapid alkalinization factors (RALFs) in plantshave been reported to dampen pathogen-associated molecular pattern (PAMP)-triggered immunity via suppressing PAMP-induced complex formation between the pattern recognition receptor (PRR) and its co-receptor BAK1. However, the direct and positive role of RALFs in plant immunity remains largely unknown. Herein, we report the direct and positive roles of a typical RALF, RALF22, in plant immunity. RALF22 alone directly elicited a variety of typical immune responses and triggered resistance against the devastating necrotrophic fungal pathogen Sclerotinia sclerotiorum in a FERONIA (FER)-dependent manner. LORELEI (LRE)-like glycosylphosphatidylinositol (GPI)-anchored protein 1 (LLG1) and NADPH oxidase RBOHD were required for RALF22-elicited reactive oxygen species (ROS) generation. Themutation of cysteines conserved in the C terminus of RALFs abolished, while the constitutive formation of two disulfide bridges between these cysteines promoted the RALF22-elicited ROS production and resistance against S. sclerotiorum, demonstrating the requirement of these cysteines in the functions of RALF22 in plant immunity. Furthermore, RALF22 amplified the Pep3-induced immune signal by dramatically increasing the abundance of PROPEP3 transcript and protein. Supply with RALF22 induced resistance against S. sclerotiorum in Brassica crop plants. Collectively, our results reveal that RALF22 triggers immune responses and augments the Pep3-induced immune signal in a FER-dependent manner, and exhibits the potential to be exploited as an immune elicitor in crop protection.

The effect of lipid metabolism disorder on patients with hyperuricemia using Multi-Omics analysis

A multiomics study was conducted to investigate how lipid metabolism disorders affect the immune system in Xinjiang patients with hyperuricemia. The serum of 60 healthy individuals and 60 patients with hyperuricemia was collected. This study used LC–MS and HPLC to analyze differential lipid metabolites and enrichment pathways. It measured levels of immune factors tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), carnitine palmitoyltransferase-1 (CPT1), transforming growth factor-β1 (TGF-β1), glucose (Glu), lactic acid (LD), interleukin 10 (IL-10), and selenoprotein 1 (SEP1) using ELISA, as well as to confirm dysregulation of lipid metabolism in hyperuricemia. 33 differential lipid metabolites were significantly upregulated in patients with hyperuricemia. These lipid metabolites were involved in arachidonic acid metabolism, glycerophospholipid metabolism, linoleic acid metabolism, glycosylphosphatidylinositol (GPI)—anchor biosynthesis, and alpha-Linolenic acid metabolism pathways. Moreover, IL-10, CPT1, IL-6, SEP1, TGF-β1, Glu, TNF-α, and LD were associated with glycerophospholipid metabolism. In patients with hyperuricemia of Han and Uyghur nationalities, along with healthy individuals, significant differences in CPT1, TGF-β1, Glu, and LD were demonstrated by ELISA (P < 0.05). Furthermore, the levels of SEP1, IL-6, TGF-β1, Glu, and LD differed considerably between groups of the same ethnicity (P < 0.05). It was found that 33 kinds of lipid metabolites were significantly different in patients with hyperuricemia, which mainly involved 5 metabolic pathways. According to the results of further studies, it is speculated that CPT1, TGF-β1, SEP1, IL-6, Glu and LD may increase fatty acid oxidation and mitochondrial oxidative phosphorylation in patients through glycerophospholipid pathway, reduce the rate of glycolysis, and other pathways to change metabolic patterns, promote different cellular functions, and thus affect the disease progression in patients with hyperuricemia.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

The Multifaceted Functions of Prion Protein (PrPC) in Cancer.

The cellular prion protein (PrPC) is a glycoprotein anchored to the cell surface by glycosylphosphatidylinositol (GPI). PrPC is expressed both in the brain and in peripheral tissues. Investigations on PrPC's functions revealed its direct involvement in neurodegenerative and prion diseases, as well as in various physiological processes such as anti-oxidative functions, copper homeostasis, trans-membrane signaling, and cell adhesion. Recent findings have revealed the ectopic expression of PrPC in various cancers including gastric, melanoma, breast, colorectal, pancreatic, as well as rare cancers, where PrPC promotes cellular migration and invasion, tumor growth, and metastasis. Through its downstream signaling, PrPC has also been reported to be involved in resistance to chemotherapy and tumor cell apoptosis. This review summarizes the variance of expression of PrPC in different types of cancers and discusses its roles in their development and progression, as well as its use as a potential target to treat such cancers.

MHC class III lymphocyte antigens 6 as endogenous immunotoxins: Unlocking immunotherapy in proficient mismatch repair colorectal cancer.

A majority of cancers, including colorectal cancer (CRC) with intact DNA mismatch repair, exhibit a paralyzed antitumor immune response and resistance to immune checkpoint inhibitors. Members of MHC class III lymphocyte antigen 6G (LY6G) encode glycosylphosphatidylinositol (GPI) proteins anchored to the membrane. Snake venom neurotoxins and LY6G proteins share a three-finger (3F) folding domain. LY6 proteins such as LY6G6D are gaining a reputation as excellent tumor-associated antigens that can potently inhibit anti-tumor immunity in cancers with proficient mismatch repair. Thus, we called MHC class III LY6G endogenous immunotoxins. Since the discovery of LY6G6D as a tumor-associated antigen, T-cell engagers (TcEs) have been developed to simultaneously bind LY6G6D on cancer cells and CD3 on T cells, improving the treatment of metastatic solid tumors that are resistant to ICIs. We present a current understanding of how alterations in MHC class III genes inhibit antitumor immunity, and how these understandings can be turned into effective treatments for patients who are refractory to standard immunotherapy. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics Cancer > Molecular and Cellular Physiology.

The PIG-A gene mutation assay in human biomonitoring and disease.

The blood cell phosphatidylinositol glycan class A (PIG-A) gene mutation assay has been extensively researched in rodents for in vivo mutagenicity testing and is now being investigated in humans. The PIG-A gene is involved in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis. A single mutation in this X-linked gene can lead to loss of membrane-bound GPI anchors, which can be enumerated via corresponding GPI-anchored proteins (e.g., CD55) using flow cytometry. The studies published to date by different research groups demonstrate a remarkable consistency in PIG-A mutant frequencies. Moreover, with the low background level of mutant erythrocytes in healthy subjects (2.9-5.56 × 10-6 mutants), induction of mutation post genotoxic exposure can be detected. Cigarette smoking, radiotherapy, and occupational exposures, including lead, have been shown to increase mutant levels. Future applications of this test include identifying new harmful agents and establishing new exposure limits. This mutational monitoring approach may also identify individuals at higher risk of cancer development. In addition, identifying protective agents that could mitigate these effects may reduce baseline somatic mutation levels and such behaviors can be encouraged. Further technological progress is required including establishing underlying mechanisms of GPI anchor loss, protocol standardization, and the development of cryopreservation methods to improve GPI-anchor stability over time. If successful, this assay has the potential be widely employed, for example, in rural and low-income countries. Here, we review the current literature on PIG-A mutation in humans and discuss the potential role of this assay in human biomonitoring and disease detection.

A novel GPI-anchored dominant-negative TGF-β receptor II renders T cells unresponsive to TGF-β signaling

Transforming growth factor β (TGF-β) is a pleiotropic cytokine expressed by a wide range of cell types and is known for hampering the effectiveness of cancer immune cell therapeutic approaches. We have designed a novel construct containing the extracellular domain of the TGF-β receptor II linked to a glycosylphosphatidylinositol (GPI) anchor (GPI-ecto-TβRII) lacking the transmembrane and cytoplasmic signaling domain of TGF-β receptor II (TβRII). Tcells transduced with lentivirus expressing the GPI-ecto-TβRII construct show 5 to 15 times higher membrane expression compared with a previously established dominant-negative receptor carrying a truncated signaling domain. GPI-ecto-TβRII expression renders Tcells unresponsive to TGF-β-induced signaling seen by a lack of SMAD phosphorylation upon exogeneous TGF-β treatment. Transduced Tcells continue to express high levels of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), among other cytokines, in the presence of TGF-β while cytokine expression in untransduced Tcells is being markedly suppressed. Furthermore, Tcells expressing GPI-ecto-TβRII constructs have been shown to efficiently capture and inactivate TGF-β from their environment. These results indicate the potential benefits of GPI-ecto-TβRII expressing cytotoxic Tcells (CTLs) in future cell therapies.

Recent genome resequencing paraded COBRA-Like gene family roles in abiotic stress and wood formation in Poplar.

A cell wall determines the mechanical properties of a cell, serves as a barrier against plant stresses, and allows cell division and growth processes. The COBRA-Like (COBL) gene family encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that controls cellulose deposition and cell progression in plants by contributing to the microfibril orientation of a cell wall. Despite being studied in different plant species, there is a dearth of the comprehensive global analysis of COBL genes in poplar. Poplar is employed as a model woody plant to study abiotic stresses and biomass production in tree research. Improved genome resequencing has enabled the comprehensive exploration of the evolution and functional capacities of PtrCOBLs (Poplar COBRA-Like genes) in poplar. Phylogeny analysis has discerned and classified PtrCOBLs into two groups resembling the Arabidopsis COBL family, and group I genes possess longer proteins but have fewer exons than group II. Analysis of gene structure and motifs revealed PtrCOBLs maintained a rather stable motif and exon-intron pattern across members of the same group. Synteny and collinearity analyses exhibited that the evolution of the COBL gene family was heavily influenced by gene duplication events. PtrCOBL genes have undergone both segmental duplication and tandem duplication, followed by purifying selection. Promotor analysis flaunted various phytohormone-, growth- and stress-related cis-elements (e.g., MYB, ABA, MeJA, SA, AuxR, and ATBP1). Likewise, 29 Ptr-miRNAs of 20 families were found targeting 11 PtrCOBL genes. PtrCOBLs were found localized at the plasma membrane and extracellular matrix, while gene ontology analysis showed their involvement in plant development, plant growth, stress response, cellulose biosynthesis, and cell wall biogenesis. RNA-seq datasets depicted the bulk of PtrCOBL genes expression being found in plant stem tissues and leaves, rendering mechanical strength and rejoinders to environmental cues. PtrCOBL2, 3, 10, and 11 manifested the highest expression in vasculature and abiotic stress, and resemblant expression trends were upheld by qRT-PCR. Co-expression network analysis identified PtrCOBL2 and PtrCOBL3 as hub genes across all abiotic stresses and wood developing tissues. The current study reports regulating roles of PtrCOBLs in xylem differentiating tissues, tension wood formation, and abiotic stress latency that lay the groundwork for future functional studies of the PtrCOBL genes in poplar breeding.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.