Top-Down, Knowledge-Based Genetic Reduction of Yeast Central Carbon Metabolism.

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterized by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of "minor" paralogs and complicates modular and combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 of the 111 genes yielded a "minimal CCM" strain which, despite the elimination of 32% of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes from the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodeling of yeast CCM. IMPORTANCE Fundamental questions regarding the minimal requirements for life have prompted scientists to embark on top-down efforts to reduce microbial genomes to the minimum set of genes and proteins necessary to sustain cell survival and division. While these efforts are generally focused on small, prokaryotic genomes, Saccharomyces cerevisiae, a popular industrial and model organism, has a typical eukaryotic genome characterized by a high genetic redundancy. The cellular function of redundant genes is generally poorly understood and is often investigated at the scale of a few genes. In this study, we explore genetic redundancy at large scale, encompassing the ~100 genes involved in central carbon metabolism, a part of metabolism essential for life and highly conserved among eukaryotes. This study reveals the remarkable resilience of this model eukaryote, as it was hardly affected, under a broad range of conditions, by a 32% reduction of its central carbon metabolism.

ß-Cyclodextrin complex improves the bioavailability and antitumor potential of cirsiliol, a flavone isolated from Leonotis nepetifolia (Lamiaceae).

Cirsiliol is a flavone found in many Lamiaceae species with high cytotoxic activity against tumor cell lines. Although cirsiliol is being used in cancer therapy, its pharmacological potential is limited by its low solubility and bioavailability. In this paper, a cirsiliol-ß-cyclodextrin inclusion complex was developed in order to increase its solubility and bioavailability. The formation of inclusion complex was proved by scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) and solubility increment was verified through the ultraviolet-visible (UV-Vis) method. The cytotoxic effect against tumor cells (PC3, HCT-116 and HL-60 human cell lines, and S-180 murine cell line) and the antitumor activity in mice bearing sarcoma S-180 were also investigated. The inclusion complex was obtained with 71.45% of total recovery and solubility 2.1 times higher compared to the compound in its free form. This increment in solubility was responsible by a tumor growth inhibition potentiation (1.5 times greater compared to compound in its free form). In addition, this study showed that cirsiliol and its inclusion complex in ß-cyclodextrin have strong antitumor potential at low doses without promoting side effects commonly observed for conventional drugs as doxorubicin.

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.

A fatal case of transplantation-mediated alloimmune thrombocytopenia following liver transplantation.

OBJECTIVE AND IMPORTANCE: Transplantation-mediated alloimmune thrombocytopenia (TMAT) occurs when leukocytes transferred in a donor organ from a patient with immune thrombocytopenia (ITP), mount a response against recipient platelets. We present the first fatal case of TMAT following liver transplantation and review its aetiology and treatment. CLINICAL PRESENTATION: The liver donor had ITP and died from an intracranial haemorrhage. The recipient platelet count fell to 2 × 109/l on post-operative day 2. Treatment refractory thrombocytopenia resulted in pulmonary haemorrhage and death. TMAT did not occur in a kidney recipient from the same ITP donor. INTERVENTION: Extramedullary haematopoiesis was identified in the donor liver biopsy. Antibodies against platelet GPIb/IX were demonstrated in both donor and recipient. The thrombocytopenia was refractory to platelet transfusions, intravenous immunoglobulin, methylprednisolone, rituximab, romiplostim, plasmapheresis, vincristine and splenic artery embolization. On review of the literature, severe thrombocytopenia (<10 × 109/l) has started within 3 days of transplantation in all reported TMAT cases. Serious non-fatal bleeding was observed in 3/5 previously reported cases. The optimal treatment is unclear. TMAT should resolve as donor lymphocytes are eliminated but re-transplantation may be required in severe refractory cases. TMAT has been reported in recipients of a liver but not kidney or heart transplant from ITP donors, probably because of the greater burden of co-transplanted lymphoid tissue. CONCLUSION: Before using the liver of an ITP donor, the recipient's fully informed consent is required. However, the risk of TMAT from an ITP donor is currently unknown and systematic review of donor registries is needed.

Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism.

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.

Immune neutropenia associated with anti-human neutrophil antigen-2a (NB1) antibodies following unrelated donor stem cell transplantation for chronic myeloid leukaemia: perpetuation by granulocyte colony-stimulating factor.

A case of immune neutropenia following unrelated stem cell transplantation for chronic myeloid leukaemia is described. The neutropenia developed following herpes zoster viral infection and was associated with antibodies to the human neutrophil antigen (HNA)-2a (formerly known as NB1). The neutropenia was prolonged, profound and unresponsive to granulocyte colony-stimulating factor (GCSF). The neutrophil count recovered after GCSF was discontinued. HNA-2a has been reported to be upregulated following GCSF administration. In the present case, it appears that the immune neutropenia may have been perpetuated by GCSF administration.

Case report: four donors with granulocyte-specific or HLA class I antibodies implicated in a case of transfusion-related acute lung injury (TRALI).

A 54-year-old female patient with a history of chronic liver disease and portal hypertension was admitted for an elective cholecystectomy. Preoperative evaluation revealed a prolonged prothrombin time of 17.4 seconds (control 12 to 15.5 seconds). Six units of fresh frozen plasma (FFP) were prescribed after failure of correction of the coagulopathy with intravenous vitamin K (10 mgs). During infusion of the fifth unit of FFP, the patient became acutely dyspneic. Arterial blood gas analysis revealed marked hypoxemia (PO(2) 6.58 kPa) and the chest X-ray showed new diffuse bilateral alveolar infiltrates. The patient remained hypoxemic with unstable oxygen saturations over the following 7 days, during which time she required 60 to 100 percent oxygen administered by face mask. Intravenous methylprednisolone (200 mgs) was given for 5 days. Mechanical ventilation was not required. The lung infiltrates gradually cleared over 3 to 4 days and the patient showed clinical improvement after 1 week. Four of the donors of the implicated units of plasma were female and all had a history of pregnancy. Two donors had HLA class I antibodies and two had granulocyte-specific antibodies detectable in their serum. In crossmatch studies, granulocyte-reactive antibodies from two donors bound to granulocytes from the patient, which suggested that these antibodies were clinically relevant. These clinical and serologic findings support a diagnosis of transfusion-related acute lung injury (TRALI).

Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus) for the detection of antibodies against human platelet antigens.

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.

Post-transfusion purpura (PTP) associated with anti-HPA-1a, anti-HPA-2b and anti-HPA-3a antibodies.

The serum from an 85-year-old man with a clinical diagnosis of post-transfusion purpura (PTP) was investigated for the presence of platelet-specific antibodies. Clinically, the case was typical of PTP but, unusually, the serum was found to contain multiple platelet-specific alloantibodies. Anti-HPA-1a, anti-HPA-2b and anti-HPA-3a antibodies were detected together with multispecific anti-HLA (class I) antibodies. Additional (but weaker) antibody reactivity was also observed with platelet glycoproteins (Gp) IIb/IIIa, GpIb and GpIa/IIa which lacked the antigens recognized by the alloantibodies, suggesting the presence of auto- or cross-reacting antibodies. The patient's genotype was HPA-1b/1b, HPA-2a/2a, HPA-3b/3b, HPA-5a/5b and was consistent with the platelet alloantibodies detected. The patient made a complete recovery following treatment with intravenous gamma-globulin.

Predicting the severity of haemolytic disease of the newborn: an assessment of the clinical usefulness of the chemiluminescence test.

The ability of the chemiluminescence test (CLT) to predict the severity of haemolytic disease of the newborn (HDN) was determined in 80 alloimmunized pregnant women who delivered antigen-positive babies. In 54 cases of alloimmunization to D, results from the CLT showed better correlation with fetal outcome than anti-D concentration measured by AutoAnalyzer (r = 0.70 and 0.36 respectively). Results from the CLT permitted a threshold level of antibody activity (30%) below which 15/20 babies were unaffected or had mild disease, and only one required transfusion therapy in utero. CLT results above 30% were associated with moderate or severe disease in all cases. Results from the AutoAnalyzer proved a less reliable predictor of disease severity; three women with anti-D levels > 20 iu/ml delivered unaffected babies, and two women with anti-D levels < 10 iu/ml delivered babies who had required transfusion in utero. The clinical usefulness of the CLT derives from the possibility of avoiding invasive monitoring procedures in women with high levels of anti-D which is relatively non-functional.

Prospective evaluation of the chemiluminescence test for the detection of granulocyte antibodies: comparison with the granulocyte immunofluorescence test.

A series of 213 neutropenic patients were tested for the presence of granulocyte antibodies using the granulocyte chemiluminescence test (GCLT) and the granulocyte immunofluorescence test (GIFT). Sera containing lymphocyte (HLA) antibodies were excluded from the study. A direct GIFT was performed on granulocytes from 56 patients. Samples were obtained from patients with a range of clinical conditions including primary adult autoimmune neutropenia, autoimmune neutropenia of infancy, autoimmune neutropenia secondary to Felty's syndrome, rheumatoid arthritis, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, proliferative disorders, bone marrow transplantation and patients with documented febrile or pulmonary transfusion reactions. Overall, granulocyte antibodies were detected in 52.1% of patient sera. Results for the GCLT and GIFT (IgG) were strongly correlated (p < 0.001) for both primary and secondary immune neutropenias. The results confirm the applicability of the GCLT in the granulocyte serology laboratory.

Predicting hemolytic disease of the newborn: a comparison of the monocyte monolayer assay and the chemiluminescence test.

The ability of the monocyte monolayer assay (MMA) and the chemiluminescence test (CLT) to predict the clinical significance of alloantibodies associated with hemolytic disease of the newborn (HDN) was assessed by the use of 22 well-characterized antisera--predominantly anti-D--from alloimmunized pregnant women. Seventeen sera were obtained before delivery from women whose infants were antigen positive for the antibody specificities identified in the maternal serum. With testing of these 17 sera by MMA, 10 results were in agreement with the presence or absence of HDN, but there were 5 false-positive and 2 false-negative results. With the CLT, 16 results were in agreement with the presence or absence of HDN, and there was 1 false-negative result. Five sera were obtained from women whose infants were antigen negative for the antibody specificities identified in the maternal serum. The CLT and the MMA were both subject to false-positive results with these sera. These results suggest that the CLT may be more valuable than the MMA as a noninvasive test for predicting the clinical significance of alloantibodies in HDN.

Autoimmune neutropenia of infancy.

AIM: Assessment of the clinical and haematological course of autoimmune neutropenia of infancy (ANI) in a defined childhood population in the south east of Scotland. METHODS: From January 1986 to February 1991 all children presenting with persistent neutropenia were examined serologically for evidence of antigranulocyte antibodies. The clinical course of those children found to have anti-granulocyte antibodies was then closely monitored. RESULTS: During the study period five children had serologically confirmed ANI, giving an annual incidence of approximately 1/100,000 in this population. All of these cases followed the classic benign course of the condition. The presenting illnesses were mild, often with superficial skin sepsis and the initial absolute neutrophil count (ANC) ranged from 0.00-0.87 x 10(9)/l. All have remained well with no serious infections. Two children attained a normal ANC after 14 and 24 months respectively, the others currently remain neutropenic. CONCLUSIONS: Autoimmune neutropenia of infancy is a condition which rests on a serological diagnosis. It follows a chronic benign course and all children eventually attain a normal ANC. The level of antigranulocyte antibody in the serum often begins to wane prior to improvement in the ANC and can give an indication of when recovery will begin to occur.

The use of interferon-gamma-treated U937 cells in chemiluminescence assays to detect red cell, platelet and granulocyte antibodies of potential clinical significance.

The chemiluminescent (CL) response of interferon-gamma-treated U937 (IFN-U937) cells to sensitized target cells has been used to detect red cell, platelet and granulocyte antibodies. A clone of U937 cells was selected which expressed Fc receptor I (Fc gamma RI) and which, after incubation with IFN-gamma for 72 h, was capable of generating high levels of lucigenin-enhanced CL. The CL responses of IFN-U937 cells and peripheral blood human monocytes to sensitized red cells, platelets or granulocytes were then compared. Assays using monocytes or IFN-U937 cells were of comparable sensitivity for detection of antibodies against all three types of target cell. In addition, the use of IFN-U937 cells reduced interassay variation and simplified assay performance. The potential clinical usefulness of these CL assays was suggested by the ability of both monocytes and IFN-U937 cells to respond to red cells, platelets or granulocytes sensitized with sera from pregnant women whose babies had either haemolytic disease of the newborn (HDN), alloimmune thrombocytopenia or alloimmune neutropenia respectively. In addition, monocytes and IFN-U937 cells both responded to red cells sensitized with antibodies against a variety of specificities of assumed (although not documented) clinical significance for blood transfusion recipients. In contrast, monocytes and IFN-U937 cells responded only weakly to red cells sensitized with either anti-D in sera from mothers of babies unaffected by HDN, or with antisera containing high titre antibodies with specificities not normally associated with significantly reduced red cell survival.