TNFAIP3) mutations.Inflammation and inflammatory cytokines have been shown to exert both positive and negative effects on hematopoietic stem cells (HSCs) and hematopoiesis. While the significance of inflammation driven hematopoiesis has begun to unfold, molecular players that regulate this phenomenon remain largely unknown. In the present study, we identified A20 as a critical regulator of inflammation controlled hematopoietic cell fate decisions of HSCs. A20 deficiency in HSCs leads to increased differentiation of myeloid cells and myeloproliferation. Analysis of erythroid lineage cells of A20 deficient mice indicated a striking reduction of erythrocytes in the bone marrow (BM), but elevated numbers in the spleen. Loss of A20 in HSCs causes a severe blockade of B cell differentiation in the BM and absence of peripheral B cells in the spleen, liver and blood. T cell differentiation studies revealed a reduction of both T cell progenitors and differentiated T cells in the thymus and altered T cell numbers in the spleens of A20 mutant mice. Analysis of lineage committed progenitors of the myeloid, erythroid and lymphoid lineages specified an altered composition in the A20 deficient BM. Genetic studies identified that specific loss of A20 in the myeloid lineage cells results in myeloproliferation. Bone marrow transplantation studies and mixed bone marrow chimera studies suggested an involvement of inflammatory cytokines, particularly interferon (IFN)- γ, in the onset of myeloproliferation and lymphopenia of A20 deficient mice. Finally, ablation of IFNγ signals in A20 deficient mice rescued the hematopoietic defects. In essence, these studies highlight a previously unknown role for A20 in the restriction of inflammation driven pathologic hematopoiesis. We believe that our studies based on A20 mutant mice will be helpful in understanding the pathophysiology and in the treatment of patients with A20 ( During myelopoiesis, HSCs differentiate into Multipotent Progenitors (MPPs), which further differentiate into common myeloid progenitors (CMPs). These CMPs give rise to either granulocyte monocyte progenitors (GMPs), that differentiate into granulocytes, monocyte/macrophages and dendritic cells, or Megakaryocyte erythrocyte progenitors (MEPs) that differentiate into either erythrocytes or megakaryocytes3. Differentiation of majority of these myeloid lineage cells occurs in the BM. On the other hand, during lymphopoiesis, MPPs differentiate into common lymphoid progenitors (CLPs), which either differentiate into B cell and natural killer (NK) cell progenitors in the BM or migrate to the thymus to generate early thymic progenitors (ETPs), which give rise to both cytotoxic and helper T cells, and NKT cells through a series of differentiation steps4.Hematopoiesis is a process through which blood cells are constantly generated and replenished in the body. A tight regulation on proliferation and differentiation of HSCs into lineage committed progenitors is vital for maintaining a balance between myeloid and lymphoid lineage cells in the blood. Indeed defective regulation of HSC proliferation and/or differentiation can lead to detrimental consequences, including myelodysplasia, lymphopenia, immunodeficiencies, anemia, myeloproliferation, leukemia and lymphoma6. We and others have shown the significance of post-translational modifications of proteins, especially ubiquitylation, in hematopoiesis. Loss functions mediated by the E3 ubiquitin ligase c-Cbl results in compromised HSC functions7, age related myeloproliferation and lymphopenia8, and the onset of acute myeloid leukemia9. Similarly, deficiency of another HECT type E3 ubiquitin ligase-Itch causes abnormal hematopoiesis10. More recently, we have shown that a deficiency of the ubiquitin editing enzyme-A20 causes increased NF-κB activation that results in premature death, due to compromised HSC pool and functions11.A constellation of cell intrinsic and extrinsic factors regulate these specific stages of differentiation from HSCs. To date various cell intrinsic factors, including transcription factors, cell cycle regulators, microRNAs and signal transducers, and extrinsic factors, such as cytokines, chemokines, and signals from the BM “niche”, have been shown to control hematopoiesis13. A20 is induced by NF-κB signals and is regulated at both transcriptional and post-transcriptional levels14. It plays a critical role in determining the duration and intensity of signaling by many components of the NF-κB pathway. A20 has been shown to interact with a variety of signaling molecules including TRAF1, TRAF2, TRAF6 and NEMO, therefore believed to regulate many other inflammatory pathways15. Mice deficient for A20 exhibit hypersensitivity to TNF and premature death due to severe inflammation and cachexia16. Deletion of A20 in specific cells of the immune system, including B cells17, T cells18, dendritic cells19, and myeloid cells20 resulted in a variety of multi-organ inflammation and immune pathologies14. Consistently, Mx1-Cre or ERT2-Cre mediated ablation of A20 in mice resulted in increased myeloid differentiation and rapid B cell apoptosis21. While all these studies have highlighted the importance of A20 in the functions of specific immune cell types, it remained unclear if A20 has any roles on hematopoietic differentiation, especially at the earlier stages of myeloid, erythroid and lymphoid lineages. Even though we have recently shown that deficiency of A20 leads to loss of quiescence and maintenance of HSCs due to exaggerated IFNγ signals11, it was unknown if exaggerated IFNγ mediated signals are responsible for the onset of myeloproliferation and lymphopenia caused by A20 deficiency. In the present study we provide evidence that A20 deficiency in myeloid lineage cells is sufficient to cause myeloproliferation and reduction of peripheral B cells. In addition, our data demonstrate that ablating IFNγ signals is sufficient to rescue the pathologic hematopoiesis caused by A20 deficiency.A20 (Tnfaip3) is a broadly expressed cytoplasmic protein that was originally identified as an inhibitor of TNF-induced NF-κB activityHemKO (Tnfaip3flox/flox mice crossed with Vav-iCre mice) mice were previously described11. VAVCre/+ mice, CD19cre/+ mice, LysMcre/+ mice and IFN-γ−/− mice were purchased from the Jackson laboratory. CD45.1 congenic animals were purchased from the National Cancer Institute. Mice were maintained under specific pathogen–free conditions and used according to the protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Columbia University Medical Center and University of Maryland School of Medicine.A20Hem-KO mice were analyzed at 14 days after birth (P14), unless otherwise specified. Bone marrow cells were isolated from the tibias and femurs by inserting a 23-gauge needle/1 mL syringe to the bone cavities and flushed with PBS 2%FCS until the bones become pale. Single cell suspensions were made through rigorous pipetting. Red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 nM nylon mesh. Bone marrow cells were then counted with a hemacytometer and trypan blue (Amresco) negative cells were counted as live cells. Peripheral blood cells were lysed with ice cold H20 and followed by ice cold 2.7% Nacl. Cells were filtered using a 70 nM nylon mesh.A20Cells were analyzed by flow cytometry with FACSForettsa or LSR II (BD) and FACSDiva software (BD Biosciences) or FlowJo software (Tree Star). The following monoclonal antibodies were used: anti-B220 (RA3–6B2), anti- CD19 (1D3), anti- CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-CD34 (RAM34), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD48 (HM48-1), anti-CD117 (2B8), anti-Flt3 (A2F10.1), anti-Gr-1 (RB6-8C5), anti-Sca-1 (D7), anti-CD71 (C2), anti-CD44 (IM7), anti-CD62L (MEL-14) and anti-Ter119 (TER119); from BD Biosciences; anti-CD25 (PC61) and anti-CD150 (TC15- 12F12.2) from Biolegend; anti-CD16/32 (93), anti-Ly6D (49-H4), anti-CD11C (N418) anti-NK1.1 (PK136)anti-CD41 (MWReg30), anti-CD105 (MJ7/18) and anti-CD127 (A7R34) from eBioscience. Cells incubated with biotinylated monoclonal antibodies were incubated with fluorochrome-conjugated streptavidin–Peridinin Chlorophyllprotein–Cyanine 5.5  or Streptavidin-Allophycocyanin-Cy7 . In all the FACS plots, indicated are the relative frequencies (%) of the gated fraction.Hem-KO mice were fixed and permeabilized using Fixation/Permeabilization solution kit (BD biosciences). Cells were treated with IFNγ-PE (XMG1.2) antibodies to detect intracellular IFNγ levels in various immune subsets.Surface stained splenocytes from control and A20Apoptotic cells were detected with the Annexin V PE Apoptosis Detection kit or Annexin V FITC Apoptosis Detection kit (BD) according to the manufacturer’s instructions.6 of bone marrow cells were injected into lethally irradiated (10 Gy) congenic (CD45.1+) recipient mice. For competitive-repopulation experiments, 1 × 106 of bone marrow cells from either A20 control or A20Hem-KO mice were mixed with 2 × 105 of wild type  bone marrow cells  and were injected into lethally irradiated congenic WT (CD45.1+) recipient mice.1 × 104 cells/1 mL in 3 cm dish) or purified hematopoietic progenitors (100 cells/1 ml in 3 cm dish) were mixed with methylcellulose medium with recombinant cytokines for mouse cells . Cells were plated in 3 independent dishes, cultured and myeloid colonies were scored after 10 days as described in the manual (Stemcell Technologies).Either Total BM , then cDNA was synthesized with oligo(dT) primer and Maxima reverse transcriptase (Thermo Scientific). Real-time PCR was performed using gene specific primers 22. Our analysis of A20Hem-KO mice revealed that A20 deficiency leads to perinatal lethality and loss of HSPC pool and functions.In an attempt to identify the functions of the ubiquitin editing enzyme A20 in HSCs, we previously generated mice that specifically lack A20 in HSCs , by crossing A20 11 specified the significance of A20 in HSC quiescence and functions, importance of A20 in hematopoietic differentiation remains to be explored. To this end, we performed immunophenotyping studies to study myeloid, erythroid and lymphoid differentiation in the absence of A20. Our analysis of peripheral blood indicated ~48% of CD11b+ cells in control animals, whereas ~99% of CD11b+ cells were noticed in A20Hem-KO mice  of Ly6G−CD11b+ subset and a decrease (~70% in control and ~50% in A20Hem-KO) of Ly6G+CD11b+ granulocytes in the BM of A20Hem-KO mice  of CD11b+ cells in the spleen of A20Hem-KO mice , both in relative and absolute numbers in the livers of A20Hem-KO mice  cells were reduced in the BM of A20Hem-KO mice , EryB (Ter119highCD71highFSClow) and EryC (Ter119highCD71lowFSClow)23, based on expression levels of Ter119 and CD71, and in combination with forward scatter (FSc) parameter23. Accordingly, EryA corresponds to baso-EBs; EryB corresponds to late baso-EBs and polychromatic-EBs; EryC are orth-EBs and reticulocytes. Our analysis of BM from A20Hem-KO mice indicated while EryA and EryB subsets were reduced  of CD19+ B cells in the peripheral blood of A20Hem-KO mice , in both relative and absolute numbers, of CD19+ cells in the BM of A20Hem-KO mice ; Fraction B ; Fraction C ; Fraction D ; Fraction E ; and Fraction F . Our analysis of A20Hem-KO mice revealed normal frequencies of Fraction A, Fraction C and Fraction E, modestly reduced frequencies of Fraction B and increased frequencies of Fraction D, and a strikingly reduced frequencies of Fraction F , however, the frequencies of apoptotic cells were remarkably increased at latter stages of differentiation (Fractions D-F) in the BM of A20Hem-KO mice  and Pro B (AA4.1+IL-7Ra+B220intc-Kit−) cells of the BM. Following this scheme of characterization, we identified that the relative frequencies of AA4.1+IL-7Ra+ cells were increased and the absolute numbers were decreased, and the relative frequencies and absolute numbers of AA4.1+IL-7Ra+B220int were reduced in the BM of A20Hem-KO mice , and a decrease in both relative and absolute numbers of ProB cells in the BM of A20Hem-KO mice  in the livers of A20Hem-KO mice , CD4+CD8+ , CD4+ CD8−  and CD4− CD8+  subsets, even though a modest increase of SP8 subset was noticed in A20Hem-KO mice , CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4), of which T cell differentiation proceeds from DN1 to DN2 to DN3 and to DN429. Analysis of relative numbers of DN subsets indicated normal frequencies of DN1, and augmented frequencies of DN2, DN3 and DN4 subsets in the thymus of A20Hem-KO mice  cells were reduced, the absolute numbers of CD3ε+ were normal in the spleens of A20Hem-KO mice , effector (CD44+CD62L+) and memory (CD44+CD62L−) revealed normal frequencies of naïve, decreased frequencies of effector and increased frequencies of memory T cells subsets in both CD4+ and CD8+ T cell fractions , Granulocyte-Monocyte Progenitors  and Megakaryocyte Erythrocyte Progenitors  indicated a decrease in the proportion of CMPs and MEPs, and an increased proportion of GMPs in A20Hem-KO mice  & pre-colony forming unit (CFU)-E, increased frequencies of GMP and CFU-E/pro-Ery, and reduced frequencies of megakaryocyte progenitors (MkPs) . Analysis of LMPPs revealed a reduction in the frequencies of cells that expressed IL7Rα in A20Hem-KO mice , a bona-fide precursor of the lymphoid lineage, indicated an increase in relative numbers, but a decrease in absolute numbers in the BM of A20Hem-KO mice  and B Lymphoid Progenitors . Interestingly, these data indicated normal relative and absolute numbers of ALPs and BLPs in the BM of A20Hem-KO mice  Fig.  in A20 dice Fig. . Consistice Fig. . Furtherice Fig. . Finallyice Fig. . These dHem-KO mice, we crossed A20floxed mice with either CD19cre or LysMcre transgenic mice, to specifically ablate A20 in B cells and myeloid lineage cells, respectively. Surprisingly, our analysis indicated normal frequencies of CD19+B220+ B cells , in the BM and spleen of A20F/FCD19cre/+ mice  experiments, as we reported earlier11. Even though recipients that received BM of A20Hem-KO mice showed poor engraftment in most of the experiments, due to HSC defects11, there were a few A20Hem-KO recipients in which the engraftment levels in the BM were ~90% and spleen were ~68%, at 3 weeks after transplantation  cells and B (CD19+) cells (that include both donor (CD45.2+) and recipient (CD45.1+) derived) in these mice indicated a complete loss of B cell differentiation and increased myeloid differentiation in the BM  WT cells, which were capable of differentiating into B lineage cells.To identify if the hematopoietic defects of A20ion Fig. . Analysi BM Fig.  and a se BM Fig.  of A20HeHem-KO derived cells secrete some soluble factors (such as inflammatory cytokines) that are responsible for altered hematopoietic differentiation. To test this hypothesis, we analyzed hematopoietic differentiation in recipients that had very low levels of A20Hem-KO derived hematopoiesis in the BM (~5%) and spleen (~20%)  and spleen (67% in control recipients and 11% in A20Hem-KO recipients)  cells in the BM. To further strengthen these findings, we generated recipients with mixed BM chimera containing Control:WT and A20Hem-KO:WT cells. Analysis of these mice revealed increased frequencies of myeloid cells in the BM, Spleen and Peripheral Blood  Fig. . Surprists) Fig. . Furtherts) Fig. . These dood Fig. , and sevood Fig.  of recipHem-KO donor cells in the BM). Real-Time PCR studies on the expression levels of inflammatory cytokines indicated elevated expression levels of IL1β, IL6, TNFα and IFNγ, and reduced levels of IFNα in the BM of WT recipients that received A20Hem-KO BM   that is responsible for the increased IFNγ levels in A20Hem-KO mice, we performed flow cytometry based intracytoplasmic staining analyses. As expected, both the relative frequencies and the absolute numbers of IFNγ+ cells were augmented in the spleen of A20Hem-KO mice , dendritic (CD11b+CD11c+) cells, B (CD19+) cells and NK (CD19+) cells were reduced, myeloid/monocytes (CD11b+) and CD4+ T cells were normal and CD8+ T cells were remarkably increased in A20Hem-KO mice  Fig. . InteresBM) Fig. , whereasice Fig. . Furtherice Fig. . These fHem-KO mice, by crossing A20Hem-KO mice with IFNγ−/− mice to generate A20Hem-KOIFNγ−/− double KO (DKO) mice, as described earlier11. Interestingly, analysis of DKO mice indicated a rescue of splenomegaly, thymic atrophy and hepatomegaly phenotype  assays using purified LMPPs, GMPs and MEPs from the BM of A20Hem-KO mice. These studies documented that A20 deficient lineage restricted progenitors lack intrinsic capacities to form increased numbers of myeloid colonies in response to hematopoietic cytokines  and these defects are largely reversible in the absence of IFNγ. Based on these findings we hypothesize that in our model, IFNγ does not suppress lymphoid differentiation capacities of HSCs, instead it suppresses B cell differentiation by directly acting on B cell progenitors, especially at late stages of differentiation. Of note, a similar phenomenon was observed earlier in a murine model of IFNγ mediated suppression of B cells34. Even though the molecular mechanism responsible for this suppression has not been elucidated in our studies, based on the previous studies40, we speculate that IL7 signaling may be compromised due to increased IFNγ in A20 deficient mice. Indeed, transgenic expression of IFNγ results in reduced IL7 response in lymphocytes32. Recent studies demonstrated that IFNγ signaling results in elevated expression of SOCS1 during B cell development and it thereby inhibits IL7 responses40. Similarly, the striking reduction in overall cellularity of the thymus from A20 deficient mice11 can be explained by defective IL7 signaling caused by IFNγ. While mechanistic insights on the link between elevated IFNγ and perturbed thymopoiesis are missing, it is tempting to speculate that defective IL7 signaling caused by IFNγ (similar to the model established in B cells40) might be responsible for the severe thymic defects of A20 deficient mice.In the present study, we demonstrate that lack of A20 during hematopoiesis causes aberrant differentiation of lymphoid cells and increased differentiation of myeloid lineage cells. Interestingly, based on mixed BM chimera and depletion of A20 in lineage specific cells, our studies demonstrated that the differentiation defects are mainly caused by cell extrinsic mechanisms, especially due to exaggerated IFNγ signals. In keeping with previous findings40. Mechanistic studies indicated that IFNγ induces expression of SOCS3, which in turn inhibit G-CSF induced activation STAT3, in GMPs that results in suppression of emergency granulopoiesis39 and activates expression of PU.1 and IRF-8 in GMPs to promote monocyte differentiation41. In addition to its direct role, IFNγ has also been shown to indirectly promote monocyte differentiation by inducing mesenchymal stromal cells to produce IL639. IFNγ has also shown to inhibit erythrocyte differentiation by activating expression of PU.1, which physically interacts with Gata1 to suppress its functions in erythroid progenitors39. While the alteration of myeloid and erythroid differentiation in A20 deficient mice can be explained by elevated IFNγ signals, there are some findings that we believe are unique to A20 deficiency and are independent of IFNγ. These include; 1. A20 deficient mice show myeloproliferation, with increased numbers of both myelo/monocyte and granulocyte lineage cells; 2. Splenic erythropoiesis is augmented in A20 deficient mice; 3. Myeloid specific deletion of A20 results in myeloproliferation; and 4. Loss of both IFNγ and A20 shows a modest increase of myeloid cells in both bone marrow and spleen, although the myeloproliferation phenotype is rescued.During myeloid differentiation, IFNγ plays a critical role and affects developmental checkpoints between neutrophils and monocytesIn essence, our study demonstrates that A20 plays an indispensable role in maintaining the myeloid vs. lymphoid differentiation potential of HSPCs, mainly by restricting decontrolled expression of pro-inflammatory cytokines. Our data also identified IFNγ as the key driver of A20 deficient myeloproliferation, anemia and lymphopenia. Even though we provide evidence that deregulated IFNγ signals are responsible for defective hematopoiesis, we believe that there are other distinct mechanisms, such as other direct targets of NF-κB, through which A20 may influence hematopoiesis. Future studies will be conducted to elucidate the molecular consequences that arise due to A20 deficiency during the development of immune system.Supplementary information


Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy with a relatively high frequency in malaria-endemic regions. In Eritrea, there is scanty knowledge of G6PD deficiency. The aim of the study was to characterize and determine the prevalence of four common G6PD allelic variants.G6PD A (A376G), G6PD A-(G202A), and G6PD Mediterranean (C563T) variants. To confirm the RFLP results, samples positive for A376G but negative for G202A variants were subjected to Sanger sequencing and a subset of PCR products (exon 5) directly sequenced to identify A376G and other mutations.Three hundred and fourteen dried blood spot samples from unrelated microscopically diagnosed malaria patient Eritrean ethnic groups living in five zobas (regions) of Eritrea were analysed by PCR-RFLP method to identify the G6PD B, G6PD B was detected in 87.5% and A376G detected in 12.5% of malaria patients, whereas G202A and C563T were absent. Bivariate Statistical analysis showed a statistically significant association between G6PD genotypes and zoba (P < 0.004 < 0.05). Sequencing revealed the expected A376G variant. In exon 5, four common (A376G) mutations, three uncommon mutations rs782669677 (535G→A) and one potentially new mutation (451G→C), relative to the reference, mRNA NM_001042351 were detected. Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function.For G6PD genotyping, G6PD B and G6PD A genotypes are prevalent in Eritrea. Similar findings were reported in neighboring countries. Further studies including phenotype analysis are needed to corroborate the observed results.This is the first report indicating that  Plasmodium falciparum is responsible for 67% of malaria cases and Plasmodium vivax for 30%; there is a small percentage of cases due to mixed infections [Anopheles arabiensis primary vector, which is difficult to control using conventional interventions method due to its facultative indoor and/or outdoor feeding and resting behavior [G6PD gene encoding the G6PD enzyme is located on X chromosome and is highly polymorphic with more than 400 variants, described based on biochemical diagnosis [Malaria remains to be a public health concern with 429,000 reported associated deaths in 2015 in spite of the effort to control and eventually eliminate it has recorded huge success in the last decades worldwide . Eritreabehavior  and funciagnosis . G6PD deiagnosis  present iagnosis  with an iagnosis . As X-liiagnosis .Plasmodium falciparum malaria [Plasmodium falciparum and Plasmodium vivax infections in Eritrea. This study aimed at molecular characterization and determination of the prevalence of common G6PD variants, namely, B, A, A- and Mediterranean, in microscopically diagnosed malaria patient Eritrean ethnic groups.Although most individuals with G6PD deficiency are clinically asymptomatic, this defect causes neonatal jaundice, mild hemolytic anemia and chronic non-spherocytic hemolytic anemia triggered by infections, specific foods (fava beans), or drugs . On the  malaria . Recent  malaria . To date malaria , 20,21.  malaria . G6PD*A- malaria . G6PD Me malaria . In EritStudy sites and study population: this cross-sectional study was undertaken at 10 study sites located in five zobas of Eritrea  were made from a drop of finger-prick blood. After the slides were air-dried, the thin smear fixed in methanol and both smears were stained with 10% Giemsa for 15 minutes. The stained slides were subsequently air-dried and viewed under 100X oil immersion microscope . An experienced microscopist read the slides. Species identification was done by visualizing thin blood smear. Parasitaemia was determined as the percent of malaria parasites observed per 200 white blood cells [od cells .Dried blood spot sample collection for genetic analysis: three hundred and fourteen finger-prick dried blood spot (DBS) samples were collected on Whatman 903TM filter paper cards from microscopically diagnosed malaria patients  Eritrean ethnic groups. None of the samples contain any personnel identifiers. Therefore, DBS samples were collected from Tigrigna (n=145), from Tigre (n=67), from Nara (n=35), from Saho (n=23), from Hidareb (n=15), from Kunama (n=14), from Bilen (n=14), from Rashaida (n=1) ethnic groups. Notably Tigrigna is the largest group followed by Tigre, while Rashaida is the smallest ethnic group in Eritrea. After properly air-dried at room temperature and individually packaged in a small zip-lock plastic bag with two desiccant bags, DBS samples were stored at 4°C during the fieldwork, and then transported to the National Health Laboratory (NHL), in Asmara, Eritrea for storage at -20°C. Subsequently all DBS samples were transported to the Laboratory of Molecular Anthropology in the Department of Biological, Environmental and Geological Sciences (BiGeA), University of Bologna, Italy for genetic analysis.Ethical consideration: ethical approval for this study was obtained from the Health Research Proposal Review and Ethical Clearance Committee of Eritrea and the Ministry of Local Government of all six zobas of Eritrea. Informed consent was obtained from all participants or from parents for children aged less than 15 years. All molecular laboratory activities were performed with permission from the Department of BiGeA, University of Bologna, Italy.DNA extraction and genotyping: three punched out circles were placed in a 1.5 ml microcentrifuge tube and DNA was extracted using QIAamp DNA blood Mini Kit  according to the manufacturer's recommendation for dried blood spots. DNA was eluted in 100 μL of elution buffer and used as templates to search for the more common G6PD genetic variants whose mutations are located within the exons, 3 + 4, 5 and 6 of the G6PD gene. A polymerase chain reaction (PCR) using three primer pairs as described by Tishkoff et al. [® Flexi Buffer, 10 mM of each dNTP, 10 μM of each primer, 25 mm of MgCI2, and 1.25 units of GoTag DNA polymerase using the Promega GoTaq® Flexi DNA Polymerase Kit . Genomic DNA was first amplified exon 5 for A376G: G6PD5F (5'-CAAAGAGAGGGGCTGACATC-3'and G6PD5R (5'-CTCATAGAGTGGTGG GAGC-3'). PCR thermo-cycling condition was performed as follows: initial denaturation at 94°C for 2 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 30 sec and a final extension step at 72 °C for 5 min. PCR products were analyzed by 1% agarose gel electrophoresis and midori green dye staining, and cleaved to perform the restriction fragment length polymorphism (RFLP) analysis with 10 units of FokI, NlaIII, and MboII endonucleases under conditions recommended by the manufacturer . Digestions were performed in 96 well microwell plates in a final volume of 35.5 µl. PCR products were incubated at 37°C for 1 hour with FokI to identify the G6PD A (A376G) variant. All samples showing positive for A376G were further amplified using primers for G202A: G6PD3/4F (5'-AACCACACACCTGTTCCCTC-3') and G6PD3/4R (5'-GCTGGTAGAGAGG GCAGAAC. Amplicons were incubated at 37°C for 50 min with NlaIII to identify the G6PD A- (G202A) variant. DNA was amplified using primers, G6PD6F (5'-TGCAGCTGTGATCCTCACTC-3' and G6PD6R (5'-AGGTGGAGGAACTGACCTTG-3'). PCR products were then incubated at 37°C for 40 minutes with MboII to identify the G6PD Mediterranean (C563T) variant. All RFLP products were separated by 3% nusieve-agarose gels stained with midori green dye at occurrence of 100 bp DNA size marker. Gels were visualized using UV light and sex was determined by the number of bands .f et al.  to ampliDNA sequencing: to confirm RFLP analysis results, samples showing positive for A375G, but negative for G202A variants were further analyzed by DNA sequencing in one direction [et al. [irection . A subse [et al. . PCR thePhylogenetic analysis: phylogenetic analysis was performed using the nucleotide sequences obtained from G6PD exon 5 PCR products. The nucleotide sequences were first manually edited by removing ambiguous sequences at the ends and generating reverse complements, which were then aligned using a Geneious version 11.1 [G6PD exon 5 sequence was extracted from NM_001042351.2 and GRC38/hg38 reference sequences in the NCBI nucleotide and human genome databases were used as wildtype allele B controls. Sample sequences with more than two mismatches to wildtype were assumed to contain sequence errors whereas sample sequences with only one or two mismatches to wildtype were assumed possibly to represent genuine variants. Multiple sequence alignments by Clustal Omega version 1.2.4 were generated using a previously identified uncommon Ethiopian G6PD variant (rs782669677) from West Ethiopia [ion 11.1 . Human GEthiopia  to highlData analysis: data was entered in Microsoft Excel sheet and transferred to SPSS version 23.0  for statistical analysis. Descriptive statistics such as proportions was used to summarize categorical v ariables. Bivariate logistic regression analysis was used to measure the strength of association between G6PD genotypes and demographic data. A P value <0.05 was considered statistically significant.P. falciparum infections, 28 P. vivax, and two P. falciparum and P. vivax mixed infection  samples were obtained from 314 microscopically diagnosed malaria patients  residing in five zobas (regions) of Eritrea. The microscopic examination revealed 284 nfection . The medMolecular findings and prevalence of G6PD variants: the PCR-RFLP analysis showed that 272 (87.5%) out of 311 clinically determined malaria patients carried the normal G6PD allele and 39 (12.5%) the G6PD A(A376G) variant. No G6PD A- and G6PD Mediterranean variants were found. African G6PD variants were confirmed by DNA sequencing. Notably, those individuals who lacked both FokI and NIaIII restriction sites were classified as B [G6PD B genotype, 88.1% (119/135) were females and 86.9% (153/176) were males, and no significant association between G6PD B and sex was observed . Overall prevalence of G6PD deficiency was 12.5% (39/311) in malaria patients. Of the 39 genotyped malaria patients with G6PD A variant, 15 (4.8%) were A heterozygous females (type AB), 1 (0.3%) was an A homozygous female (type AA), and 23(7.4%) were A hemizygous males (type A). The highest proportion of G6PD A variant was observed in males (13.1%) than in females (11.9%). Bivariate logistic regression analysis revealed no statistically significant difference between G6PD A variant and sex (P > 0.05). The prevalence of the G6PD A variant frequency with respect to ethnic groups was recorded: 14.3% in Bilen, 0% in Hidareb, 21.4% in Kunama, 2.9% in Nara, 8.7% in Saho, 7.4% in Rashaida/Tigre and 18.3% in Tigrigna. Highest prevalence was showed in malaria patients of Nara ethnic group with a frequency of 21.4%. In contrast, no A376G variant was observed in malaria patients in Hidareb ethnic group. For the validity analysis, the study has also grouped the ethnic groups based on their ethno-linguistic origins and analyzed. It was observed that the prevalence of A376G variant was 7.7% in Cushitic, 8.2% in Nilotic and 14.8% in Semitic; No statistically significant differences were assessed between G6PD genotypes and ethnic groups and ethno-linguistic origins (P > 0.05). With regards to zobas, the prevalence G6PD A (A376G) variant frequency was 8.3% in Maekel, 10.0% in Anseba, 12.4% in Gash Barka, 28.3% in Debub, and 3.6% in Semienawi Keyih Bahri. The highest frequency of A376G variant was observed in individuals residing in zoba Debub (28.3%), and a statistically significant difference of A376G variant and zoba was observed (P < 0.004 < 0.05). The distribution of G6PD B and G6PD A (A376G) with respect to demographic data in malaria patient Eritrean ethnic groups is summarized in G6PD A- (G202A) and Mediterranean (C563T) variants were also successfully genotyped in the analyzed population. Of the 311 genotyped malaria patients, 0.00% (0/311) carried the G202A and C563T variants.ied as B . Of the DNA sequencing results: thirty one out of 39 sequenced products of exons, 3+4 had the expected A376G mutation (T183C with respect to the PCR fragment) and eight samples were unreadable. Of the 68 out of 70 sequenced products of exon 5, 3/68 (4.41%) of individuals carried common A376G mutations  and 3/68 (4.41%) of individuals had uncommon mutations . The mutation 535G→A results in an Alanine→Threonine amino acid change at position 149 in protein isoform B (Accession No NP_001035810.1) from mRNA v. 2 (Accession No NM_001042351.2). And 1/68 (1.47%) of individual had a novel mutation .G6PD B and G6PD A (A376G) variants are quite prevalent in individuals with predominantly P. falciparum and P. vivax infections. The work enabled us to understand the genetic basis of G6PD deficiency in Eritrean populations. Eritrea is a multilingual and multiethnic nation. Its various ethnic groups are unequally distributed throughout the country. The prevalence of the normal G6PD B allele was 87.5% (272/311) among the malaria patients, which is in line with the literature that the wild type G6PD B allele is the most common variant in Africa and worldwide [G6PD B genotype in Burkina Faso (74.5%) [G6PD A variant was 12.5% observed in the analyzed microscopically diagnosed malaria patients. Of the 39 (12.5%) genotyped individuals with A376G variant, 4.8% were heterozygous females , 0.32% was a homozygous female, and 7.4% were homozygous males for A376G variant, which is in agreement with previously reported results from Ethiopia, of which 23.26% (20/86) genotyped malaria patients with A376G variant, 19 were heterozygous females and one hemizygous male carried the A376G mutation [G6PD A variant by sex was 11.9% in females and 13.1% in males in this study, but these difference are not statistically significant (P > 0.05). The higher prevalence of G6PD deficiency in male is consistent with other studies conducted in Afghanistan [G6PD A (A376G) variant prevalence was found to be associated with individuals residing in zoba Debub (28.3%) followed by zoba Gash Barka (12.4%) and these differences were statistically significant (P < 0.004 < 0.05) which is in agreement with previous findings in Ethiopia and Nepal [G6PD did differ by zoba (region) due to differing ethnic make-up of each zoba. As conclusion, the frequency of G6PD deficiency differed across the world depending on the ethnic groups and regions [This is the first study performed to genotype and determine the prevalence of four common G6PD variants by PCR-RFLP in microscopically confirmed malaria patients living in five zobas of Eritrea. It was found that the orldwide . Similar.5% 272/3 among th (74.5%) . The ovemutation . The prehanistan  and Solohanistan . The mal7.5% 272/1 among tnd Nepal , 34. Not regions , 35.G6PD A- (202GA) frequency estimates considerably lower than those generally found in sub-Saharan Africa [G6PD A- deficient variant was higher in Uganda (20.41%) and in Mali (7.9%) respectively [G6PD A- mutation in this study could be due to geographical location and the molecular heterogeneity of G6PD deficiency among different populations [G6PD Mediterranean (C563T) mutation in terms of frequency was zero (0.00%) in this study, which is in accordance with studies done in Angola [G6PD gene. In contrast, among 86 sequenced malarial samples from Ethiopia there were 20/86 (23%) common A376G mutations, and 2/86 (2.3%) uncommon mutations  mutations is exclusively the cause of G6PD deficiency. Few studies reported n Africa  and confn Africa . The pren Africa , 36 and 6G mutatiectively , 39. Theulations . G6PD Meganda 20.% and in ulations . The pren Angola  and in En Angola . In contn Angola . This mun Angola . The absfunction .G6PD B and G6PD A (A376G) variants while the G202A and C563T variants were absent in this study. The discovery of uncommon mutations one potentially novel mutation gives the impetus for further investigation into G6PD variation in Eritrea. Our findings can contribute in filling the knowledge gap to the overall molecular characterization and prevalence of G6PD genotypes in Eritrea and in world populations. The presented data suggest the use of primaquine treatment without considerable safety concerns in Eritrea. Further studies including phenotypic approaches are needed to confirm the observed results. Some limitations exist in the study. The sample size of certain ethnic groups was very small in this study, thus more data is required from each ethnic groups to accurately estimate the prevalence of G6PD genotypes in Eritrean population. There was lack of positive internal quality control material for G6PD deficient C563T variant and was not confirmed by sequencing due to fund constraint.This study is the first report on molecular characterization and the prevalence of G6PD allelic variants in microscopically confirmed malaria patients in Eritrea. PCR-RFLP results and DNA sequencing showed the prevalence of non-deficient Glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked hereditary defect affects over 400 million people particularly males;G6PD has over 186 mutations responsible for G6PD deficiency;G6PD deficiency is the most common in malaria endemic areas and has a protective power against malaria.This study is the first of its kind carried out to characterize and determine prevalence of common G6PD variants in Eritrea;Assessed the distribution of G6PD variants among the ethnic groups;The outcome of this study shed light on appropriateness of current existing malaria treatment in Eritrea.The authors declare no competing interest.


A USP15–mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1′ binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail–binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1′ region likely to confer specificity, whereas the S1 ubiquitin–binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone  Ubiquitin-specific proteases (USPs)12–15–A). These ancillary domains affect the catalytic function in different ways, whereby they regulate the catalytic activity of USP4 , with the insert harboring a predicted UBL domain replaced by a short linker . This showed that USP15-D1D2 fully retained its activity and suggests that the ancillary domains do not play a significant regulatory role in catalysis. USP15-D1D2 was then subjected to sparse matrix crystallization screening, and crystals were obtained in conditions using 0.1 m Tris-Cl, pH 8.5, 20% PEG 2000 . After optimization, crystals of space group P21 diffracted to 2 Å resolution and contained one molecule in the asymmetric unit. The structure was solved by molecular replacement using coordinates of paralog USP4 . A C-terminal tail structure unique to USP15 is rich in aromatic residues and folds back onto the catalytic core in the structure and is located on the opposite face to the distal ubiquitin S1 binding pocket. The active-site loop regions between helices α5 and α6, also referred to as the switching loop , and residues between β12 and β13, referred to as blocking loop 1  that engage in USP distal ubiquitin binding are partially flexible . As a consequence, the free enzyme structure of USP15 has a largely open catalytic channel conformation for accommodating the distal ubiquitin tail that harbors the two C-terminal glycines. Modeling of ubiquitin into the central binding cavity by superposition of the USP2–ubiquitin complex structure . Mapping B-factors indicative of mobile regions onto the structure reveals that residues likely to undergo conformational changes upon substrate binding also display higher B-factors .To determine the structure of the USP15 catalytic domain, we designed a construct spanning the catalytic core region . Enzymetry 2Y6E ) as a setry 2Y6E . The USPtry 2HD5 ) reveals269) in the catalytic cleft loop between β1 and α1  located ∼10 Å away from the catalytic histidine (His862) (C). The CC loop also displays higher B-factors compared with surrounding residues, indicating that this region can readily adopt different conformations. Equally, residues in the blocking loop 2 between β15 and β16  are partially flexible and associated with higher B-factors, suggestive of higher mobility. In contrast to Cys269, the side chains of catalytic triad residues His862  and Asp879 (end of β17) are “pre-aligned” for catalysis and within hydrogen-bonding distance (C).Interestingly, the USP15 structure shows the catalytic triad in an inactive conformation with the catalytic cysteine (Cys(His862) C. The CCdistance C.2Y6E (A and B). These differences in the catalytic competency states of the catalytic triad coincide with different conformations of USP15 Phe270 compared with USP4 Phe312, the neighboring residue to the catalytic cysteine in the CC loop (B and C). In our USP15 structure, the switching loop is partially flexible (C), whereas in the USP4 structure it is ordered, possibly aided by interactions with neighboring molecules in the crystal lattice . In USP15, Gln349 in the SL is flexible, but in USP4, the equivalent Gln391 reaches across the channel possibly aided by interactions with the BME molecule involving main chain NH and carbonyl groups from Gln391 and Gln392 and Val879 in the BL2. As a result, the ubiquitin C-terminal GG tail–binding channel is mostly open in USP15, whereas it is closed in the USP4 structure (C and D).USP15 shares 56.9% sequence identity with paralog USP4 over the entire length of its sequence, 57.8% sequence identity in the catalytic domain, and 77.4% in the catalytic core region D1D2. We therefore compared our structure with the available structure of the USP4 catalytic core  by supe2Y6E , A and B CC loop , B and Cflexible C, wherea lattice . In addih Ser394 B. In USPtructure , C and D340 (USP15) adopts a conformation (“in”), which is different from Phe382 in USP4 (“out”), whereas USP15 Pro342 and Gln343 adopt conformations similar to those of their USP4 counterparts. In turn, USP15 Met305, which packs against the side chain of Phe340, also adopts different conformations compared with USP4 (Met347 and Phe382) (C). These differences also coincide with different side chain conformations of USP15 Phe358 (out) and USP4 Phe400 (in) in helix α6, respectively (C). The preceding α5 helix is located further “in” in USP15, whereas in USP4, the ordered SL contains a helical turn (USP4 residues 384–387 PQFS), whereby the USP4 Phe386 side chain packs against residues Leu397, Phe312, and Phe400 in the core. The corresponding residues of the same amino acid sequence in the USP15 structure (Pro342–Ser345) are partly defined in the electron density due to flexibility. A USP15–USP4 amino acid sequence alignment in the SL region reveals the only difference to be a cysteine residue (USP15 Cys352), which replaces a serine (Ser394) in USP4 . USP15 SL residue Cys352 is conserved across an alignment of USP15 amino acid sequences, but in the crystal structure, it is not well-defined and therefore was not modeled and assumed to be flexible.At the N-terminal end of the SL, the side chain of Phe Phe382) C. These ectively C. The prD). Greater variability in amino acids is observed in the S1′ binding region, which engages a proximal ubiquitin moiety in a polyubiquitin substrate or a different target substrate conjugated to the C-terminal tail of ubiquitin (D). Of note is BL2, which in USP15 (GGMGG) is highly mobile due to the presence of four glycines, whereas BL2 in USP4 contains fewer glycine residues (GAMGV) and adopts a different conformation , although both display high B-factors, highlighting their mobility. Substitution of glycine for Val879 in USP4 may contribute to different BL2 conformations, as Val879 is positioned at the rim of the ubiquitin GG tail–binding channel . Other differences in the S1′ area cluster in the loop region between α4 and α5 (USP15 324–329 (KFSYVT) versus USP4 366–371 (RDAHVA)), which is close to the linker region that connects the catalytic core to the N-terminal UBL domain. There, USP15 Phe325, Ser326, and Tyr327 are replaced by USP4 Asp367, Ala368, and His369, respectively. Other changes in this area include USP15 Ser263 (USP4 Gly305), USP15 Ser882 (USP4 Asn901) and USP15 Thr885 (USP4 Leu904) . In addition to the S1′ region, other significant structural differences occur on the surface of helix α7 at the opposite site of the distal ubiquitin-binding pocket with a charge reversal (USP15 Glu391versus USP4 Lys433) and differences in the location of hydrophobic and hydrophilic residues (USP15 Leu398-Lys399versus USP4 Arg440-Leu441).We then mapped all residues that differ between USP15 and USP4 across the catalytic core onto the USP15 surface and vice versa, which revealed that residues in the distal ubiquitin-binding pocket are highly conserved between USP15 and USP4 D. Greatebiquitin D. Of notH and ΔS for the interaction of monoubiquitin with the USP15-D1D2 G860V and USP15-D1D2 bl2usp4 (G857A/G860V) mutants gradually changed with the stepwise substitution of the glycines in the BL2 approaching those obtained for USP4-D1D2 .To evaluate the substrate- and product-binding behavior of the USP15 and USP4 catalytic cores, we measured dissociation constants of inactive mutants USP15-D1D2 C269S and USP4-D1D2 C311S with monoubiquitin and linear diubiquitin . Remarkably, the results showed that monoubiquitin binds significantly tighter to USP4, whereas for linear diubiquitin, the dissociation constant for the interaction with USP15 was of the same order of magnitude compared with USP4 . InteresSP4-D1D2 . The dif1NB8, 2F1Z, 4M5X , US5X (2AYN ), CYLD (YN (2VHF ), USP8 (HF (2GFO ), USP18 FO (5CHT ), and USand 5K16 ). Among A and B). In our USP15 structure, the distance between the catalytic cysteine thiol group (Cys269) and the catalytic histidine imidazole (His862) is about 10 Å, which is even slightly farther away than the catalytic cysteine in the USP7 free enzyme structures (C) and is part of an extended CC loop. In USP7, the conformational changes between free and ubiquitin-bound forms involve different conformations of the aromatic residue C-terminal to the catalytic cysteine in the CC loop (Phe270 in USP15 and Phe312 in USP4)  and is ordered in the free as well as ubiquitin suicide inhibitor–bound forms possibly aided by crystal contacts, whereas in the USP15 structure, the SL is flexible and largely not visible in the electron density. In a CYLD structure (PDB code 2VHF), the SL is also observed as flexible in the crystal structure, but otherwise, the SL is ordered in other USP structures. The only highly conserved residue in the SL region among these USPs is the glutamine residue  , 42. USP QQD box ) that cl QQD box , USP7 . Co-crystallization trials in the presence of excess mitoxantrone yielded crystals of intense blue color that diffracted to 2.1 Å resolution . The catalytic triad in this different crystal form is also misaligned in both USP15 copies present in the asymmetric unit in a similar configuration to the USP15-free enzyme structure . This USP15–mitoxantrone complex structure revealed predominantly hydrophobic interactions between mitoxantrone and USP15 residues Tyr855, Gly856, Gly860, His862, which are located in the BL2 region . In addition, one of mitoxantrone's side arms contacts the CC loop Asn264, although the density is weaker compared with the mitoxantrone anthracenedione core . There are no interactions of mitoxantrone with other neighboring USP15 molecules in the crystal lattice. This binding site coincides with the S1′ region of USP15, suggesting a rationale for the inhibition observed . In this position, mitoxantrone is expected to compete with substrate interactions, as indicated by modeling a diubiquitin molecule into the binding site based on a superposition with a USP30 C77A Lys6-linked diubiquitin structure . Superposition of the USP15-free and mitoxantrone-bound structures revealed that the BL2 becomes ordered, but no other major conformational changes are observed upon mitoxantrone binding.We subsequently tested whether USP15 is inhibited by mitoxantrone using a diubiquitin gel shift cleavage assay, as this agent has previously been shown to inhibit the homolog USP11 . We detetructure , the clo269 and His862 separated by a large distance and active-site loop conformations not previously observed for any other USP. Our USP15 structure compared with USP4 , and crystal packing interactions may have promoted order in the USP4 switching loop. Substrate binding is likely to result in active-site rearrangement and alignment of the catalytic residues, which at least in part may coincide with conformational changes observed between the two structures.The USP15 structure reported here displays a catalytically incompetent open conformation of the ubiquitin C-terminal tail–binding channel with active-site loops not engaged in crystal contacts, whereas a “closed” form of USP4 was previously captured with a pre-aligned catalytic triad . The str354 repositioning to form a salt bridge with ubiquitin Arg72. The flexible USP15 Cys352 would be expected to adopt an α-helical conformation extending α6, as in USP4 the equivalent residue Ser394 is located on an additional α6 helical turn and faces inward, forming a hydrogen bond with Ser871 (USP15 Ser852). In this conformation, USP15 Cys352 would displace the Phe270 side chain in the CC loop to adopt a similar conformation as USP4 Phe312. This in turn would require displacement of the catalytic Cys269 to avoid steric clashes and would result in the alignment of the catalytic triad. As is typical for USP structures, the USP15 SL Gln349 is expected to reposition to close over the ubiquitin GG tail–binding channel. (iii) At the N-terminal end of the SL, a flexible-to-order transition of USP15 Phe344 (USP4 Phe386) could result in repacking of other hydrophobic residues, predominantly USP15 Phe358, Phe340, and Met305, and pushing helix α5 outward. The hydrophobic residues involved are highly conserved in USP15 and USP4 across species, and a USP4 SL F386G mutant was shown to increase the catalytic activity of the USP4 catalytic domain (USP4-CD) activity by affecting ubiquitin on and off rates  flexible-to-order transition of BL1 and (ii) flexible-to-order transition in parts of the C-terminal end of the switching loop, including Gluff rates .versus palm regions will need to occur upon substrate binding to avoid steric clashes. Further investigations, such as the determination of a USP15 substrate- or product-bound structure and molecular dynamics simulations will be required to confirm this mechanism.A hypothetical model of this proposed activation mechanism upon ubiquitin binding for USP15 is shown in Interestingly, differences between free and ubiquitin-bound forms of USP7 also rely on conformational changes in the switching and CC loops involving aromatic side chains, but the involved residues and adopted conformations of the loop regions, especially the switching loop, are different compared with the proposed changes between USP15-free and substrate-bound forms . ExampleOn the other hand, we noted clear differences between USP15 and USP4 that could contribute to different modes of regulation and substrate specificities in line with previous reports . The pro879 packs against Glu391 and is close to the expected position of the ubiquitin tail. When we substituted the corresponding Gly860 in USP15 with a valine, the USP15-D1D2 G860V mutant ubiquitin-binding parameters were altered, confirming that this residue is involved in ubiquitin binding. A USP15-D1D2 G857A/G860V mutant (USP15-D1D2 bl2usp4) displayed a further change in the ΔH and ΔS parameters associated with ubiquitin binding toward those obtained for USP4 with a more favorable enthalpic contribution, but less favorable entropic contribution, which may reflect additional order in the BL2 through interactions with the ubiquitin tail (352 to USP4 Ser394) had little impact on the ubiquitin-binding properties . It is evident that additional residues at the interface, and/or noninteracting regions through long-range effects, contribute to USP4 and USP15's ubiquitin-binding behavior.However, we identified that the BL2 influences the binding parameters. The USP15 BL2 is more flexible due to the presence of an additional two glycine residues compared with USP4. Within the USP4 BL2, Valtin tail . The BL2tin tail . The difIn USP4, the switching loop is ordered, possibly aided by crystal contacts, and interacts with the N-terminal DUSP domain , whereasThe only other USP shown to have a misaligned catalytic triad in a structure to date that requires significant conformational rearrangements for activation is the catalytic domain of USP7 (USP7-CD) , 38. How46–F). Mitoxantrone is a promiscuous binder with several targets described to date, including DNA-bound topoisomerase II β  and typ41 (4G0V ), mitoxaTogether, our data shed new light onto the conformational diversity of ubiquitin-free USP structures, differences between close paralogs, regulation through incompetent conformational states, and modes of small-molecule interactions, which enhances our molecular understanding of their modes of action and will aid structure-based molecular probe and drug design efforts.857–Gly860; GMGG) for the corresponding USP4 sequence AMGV. The USP4-D1D2 catalytic core (residues 294–963 Δ484–775) with the insertion removed and replaced with the USP8 ASTSK sequence analogously to USP15 was cloned into the pProEx-HTb expression vector using BamHI and HindIII restriction sites. The USP4 C311S mutant was created using primers GGAAACCTGGGAAACACCAGCTTCATGAACTCCGCT/AGCGGAGTTCATGAAGCTGGTGTTTCCCAGGTTTCC.Based on bioinformatical analysis, a human USP15 catalytic core domain construct, USP15-D1D2 (residues 255–919 Δ440-756) was designed and cloned into pET21d via NcoI and NotI restriction sites. A small linker (ASTSK) corresponding to the USP8 sequence was used to link the two catalytic subdomain halves, D1 and D2, and replace the insertion of ∼315 residues that is predicted to contain a high percentage of disorder. FL-USP15 (UniProtKB identifier Q9Y4E8-2) was cloned via SacI and HindIII restriction sites into the pCold-I expression vector. USP15 mutants C269S, C352S, and G860V were created using primers GTAACTTGGGAAATACGTCTTTCATGAACTCAGCTATTCAG/CTGAATAGCTGAGTTCATGAAAGACGTATTTCCCAAGTTAC, AAGCTAACAGTTCTTGACTGTCTTGCTGCTGATATCC/GGATATCAGCAGCAAGACAGTCAAGAACTGTTAGCTT, and CTATGGAGGGATGGGAGTAGGACACTATACTGCTTTTG/CAAAAGCAGTATAGTGTCCTACTCCCATCCCTCCATAG, respectively, following the QuikChange mutagenesis protocol. The USP15-D1D2 bl2usp4 loop swap mutant (G857A/G860V) was created by swapping USP15 or 48 h at 10 °C (FL-USP15) after induction. Cells were harvested, lysed by sonication, and clarified by centrifugation. USP proteins were lysed into 50 mm Tris-Cl, pH 7.5, 300 mm NaCl, 5 mm imidazole, and 1% glycerol (10% for FL-USP15). Proteins were then loaded onto a HiTrap chelating column precharged with nickel ions and eluted by an imidazole gradient. This was followed by size-exclusion chromatography using a buffer of 20 mm Tris-Cl, pH 7.5, 150 mm NaCl, and 1% glycerol on a Superdex200 16/60 column . Fractions were analyzed by SDS-PAGE, and relevant fractions were combined and concentrated for further use.All USP constructs were expressed in 2YT broth medium using the m ubiquitin-AMC as the fluorogenic substrate and 38 nm FL-USP15 or USP15-D1D2 in 150 mm NaCl, 50 mm Tris-Cl, pH 7.5, 1 mm DTT. Measurements were taken in 30-μl final volumes in triplicate in 384-well black plates (Nunc) read with an EnVision 2104 multilabel plate reader at 25 °C using an excitation wavelength of 355 nm and an emission wavelength of 426/428 nm. Measurements were taken every minute for the first 20 min and then every 2 min for the next 30 min and subsequently at increasing intervals of 5, 10, and 20 min. Curves measured in triplicate were fitted using nonlinear regression analysis in GraphPad prism software ) to establish Khalf (referred to as mK throughout for consistency with the literature) and kcat values.Kinetic parameters for FL-USP15 and USP15-D1D2 were derived from deubiquitinating assays with concentrations in the range of 0.1–1.5 μm Tris-Cl, pH 7.5, 300 mm NaCl, 1% glycerol, and 1 mm DTT. Mitoxantrone was solubilized in the assay buffer, and its concentration was determined by measuring the absorbance at 682 nm using an extinction coefficient of 8360 m−1 cm−1. Reactions were initiated by the addition of linear diubiquitin to FL-USP15, resulting in final concentrations of 5 μm linear diubiquitin and 400 nm USP15, respectively. The reaction was stopped by the addition of SDS-PAGE loading buffer. Linearity tests were completed to establish the linear range of the reaction. For the IC50 curve, mitoxantrone was added to FL-USP15 at specified concentrations in the range of 0.5–800 μm and pre-incubated for 30 min before the addition of linear diubiquitin. Reactions were stopped after 30 min and analyzed on 18% SDS-polyacrylamide gels. After staining, gels were scanned and then analyzed with the ImageJ software where relative amounts of diubiquitin and monoubiquitin for each point were determined. The mean monoubiquitin percentage was calculated and plotted using GraphPad Prism software using nonlinear regression analysis.Gel shift linear diubiquitin cleavage assays were carried out in triplicate in two independent experiments at 25 °C in 50 mm) were titrated into the sample cell containing 20–60 μm USP15-D1D2, USP15-D1D2 C269S, USP15-D1D2 C352S, USP15-D1D2 G860V, USP15-D1D2 bl2usp4, USP4-D1D2, or USP4-D1D2 C311S samples in 150 mm NaCl, 50 mm Tris-Cl, pH 7.5, and 1% glycerol at a temperature of 25 or 37 °C. The spacing was typically 180 s, and a stirring speed of 750 rpm was used. Data were analyzed using the PEAK ITC analysis software  fitting to a one-site binding model.ITC data were measured on a PEAQ ITC instrument . Monoubiquitin or linear diubiquitin samples ethylamino]anthracene-9,10-dione) in gel filtration buffer at a ∼1:10 molar ratio and cryoprotected by soaking in a mother liquor supplemented with 10% glycerol, 5% ethylene glycol, 1.75% dioxane, and 1.5 mm pan-USP inhibitor PR-619. Crystals belonged to monoclinic space group P21 with unit cell parameters a = 48.51 Å, b = 62.62 Å, c = 62.04 Å, and β = 104.97° and contained one molecule in the asymmetric unit. Data of these crystals were collected at the ESRF beamline ID30A-1 at a wavelength of 0.966 Å and 100 K. Data were processed using XDS  as a seDS  was collected at beamline I04 at Diamond Light Source at a wavelength of 0.97951 Å and a temperature of 100 K. Data collection statistics for both data sets are summarized in USP15 crystals were also obtained by incubating the protein with a ∼1:15 molar excess of mitoxantrone before crystallization in the condition 0.1 B). The final USP15–mitoxantrone complex structure consists of 331 and 309 residues in the two copies, respectively, with residues in the BL1 and SL regions and the C terminus not modeled due to flexibility. Further electron density indicated stacking interactions of additional mitoxantrone molecules against the USP15-interacting mitoxantrone molecule, but these were not modeled, as their orientation was unclear. In the final models, there are no Ramachandran outliers with 98% of residues (USP15-free) and 96% of residues (USP15–mitoxantrone) located in favored regions, respectively. Refinement statistics are summarized in Model building and adjustments were conducted using COOT . StructuFig. 6S. J. W., H. E. G., J. E., and I. D. formal analysis; S. J. W., S. K. M., and S. G. C. validation; S. J. W., H. E. G., P. I., C. M., R. M., and S. K. M. investigation; S. J. W. methodology; S. J. W., J. E., and I. D. writing-original draft; I. D. conceptualization; I. D. data curation; I. D. supervision; I. D. funding acquisition; I. D. project administration.


Discovery of physiological roles of H2S as a neurotransmitter by Kimura and colleagues triggered an intensive research in the biological roles of H2S in the past decades. Manipulation of H2S levels by inhibiting H2S synthesis or administration of H2S-releasing molecules revealed beneficial as well as harmful effects of H2S. As a result, it is now established that H2S levels are tightly controlled and too much or too little H2S levels cause harm. Nonetheless, translation of sulfide-based therapy to clinical practice has been stymied due to the very low therapeutic index of sulfide and the incomplete understanding of endogenous sulfide metabolism. One potential strategy to circumvent this problem is to use a safe and stable sulfide metabolite that may mediate effects of H2S. Alternatively, endogenous sulfide levels may be controlled using specific sulfide scavengers. In this review article, the role of endogenous H2S production and catabolism will be briefly reviewed followed by an introduction of thiosulfate and H2S scavengers as novel pharmacological tools to control H2S-dependent signaling.Hydrogen sulfide (H Manipulation of H2S levels by inhibiting H2S synthesis or administration of H2S-releasing molecules revealed beneficial as well as harmful effects of H2S [2S levels appear to cause harm. For example, deficiency in CBS or another H2S-synthesis enzyme cystathionine γ-lyase (CSE or CTH) causes hypertension in mice [2S), a H2S-donating compound, exaggerates ischemic brain injury, low-dose Na2S or inhibitors of CSE or CBS decreases ischemic stroke size [2S catabolizing enzyme, is a cause of ethylmalonic encephalopathy, which is characterized by abnormally high H2S levels in tissues and blood [2S metabolism may be pathogenic. However, controlling sulfide levels has proven to be very difficult using chemical H2S donors or inhibitors of H2S-producing enzymes. In recent studies, we revealed thiosulfate, an oxidative metabolite of H2S, may hold promise as a low toxicity sulfide donor. We also developed specific H2S scavenger to control local concentration of H2S. We will review the role of endogenous H2S production and catabolism followed by a focused discussion of thiosulfate and H2S scavengers as emerging pharmacological strategies to control H2S-dependent signaling.Hydrogen sulfide (Hollutant . Recentlide (CO) . In 1996g enzyme . Intensiotection . These ss of H2S . Too muc in mice , 16. Whioke size . Deficieoke size , whereas2S-producing pathways  and the remaining four fifth consist mostly of H2S with little amount of S2− in physiological fluids  according to the Henderson-Hasselbalch equation [Studies have revealed enzymatic and non-enzymatic Hays Fig. . In enzydirectly –23. Apprequation .Fig. 1P2S. CBS and CSE produce H2S from cysteine and homocysteine requiring a cofactor pyridoxal 5′-phosphate (PLP) via the transsulfuration pathway. CBS and CSE are mainly localized in cytosol while some reports suggest that CBS or CSE could translocate into mitochondria under hypoxic stress or conditions that increased intracellular free calcium, respectively [2S oxidation is significant electron source for electron transport chain (ETC) as described below in this review [2S produced by CBS and CSE in maintenance of ATP production in hypoxia. Deficiency in CBS or CSE causes marked hyperhomocysteinemia and hypertension in mice [2S [The transsulfuration pathway is a metabolic pathway where transfer of sulfur from homocysteine to cysteine occurs . Productectively , 27. Dris review –30. Tran in mice , 16. Dismice [2S . Deficiemice [2S . CSE defmice [2S .2S. 3-Mercaptopyruvate, a substrate of 3-MST, is produced by cysteine aminotransferase (CAT). 3-MST produces sulfane sulfur transferring sulfur in sulfane group of 3-mercaptopyruvate to other sulfur acceptor using zinc as a cofactor. H2S is subsequently released from sulfane sulfur [3-MST is a sulfurtransferase that produces sulfane sulfur, a sulfur atom with six valence electrons, rather than He sulfur . 3-MST le sulfur . Deficiee sulfur .2S, CARS increases H2S levels indirectly. Homozygous disruption of CARS-2 is embryonic lethal [+/− mouse remain to be elucidated because CARS knockout mice have been generated very recently [CARSs have been found initially as an enzyme that mediates translation of proteins in prokaryotes and eukaryotes including mammals . CARS-1 c lethal . Heterozrecently .2S synthesizing enzymes have suggested the biological role of endogenous H2S production. However, most of the currently available inhibitors are far from ideal [dl-propargyl glycine (PAG or PGG) or aminooxyacetic acid (AOAA) is the most frequently used compound as a CSE or CBS inhibitor, respectively. Despite only l-isomer of PAG, but not d-isomer, inhibits CSE and d-isomer may be a nephrotoxin, many studies have used the mixture of both isomers [50 (40 μM) possibly because of the limited cell permeability [50 of AOAA for CSE and CBS are in the similar range , AOAA is not a specific CBS inhibitor [2S synthesizing enzymes.Studies using inhibitors of Hom ideal . For exa isomers –41. PAG mers [50  μM possieability , 43. Becnhibitor . More sp2S productions reported. Iron (Fe3+) generates H2S from cysteine in a vitamin B6- and pyridoxal (or PLP)-dependent manner [2S production via this pathway may contribute to pathophysiology of conditions with iron dysregulation such as hemolysis, iron overload, and hemorrhagic disorders. H2S can also be generated from thiosulfate, one of H2S oxidation products, in the presence of an endogenous reductant  without enzymes [2S production via thiosulfate is augmented in hypoxic conditions [There have been a number of non-enzymatic Ht manner . Regulat enzymes . Interesnditions .2S is catabolized via both enzymatic and non-enzymatic pathways , a cluster of mitochondrial enzymes. SOU consists of sulfide: quinone oxidoreductase (SQR or SQOR), ETHE1 or sulfide dioxygenase (SDO), TST, and sulfite oxidase (SO) [2S is oxidized by SQR to generate sulfane sulfur (S0) which has six valence electrons and no charge forming persulfide (SQRS-S). SQR utilizes the oxidized form of coenzyme Q (CoQ) as an electron acceptor through the H2S oxidation to produce the reduced form of CoQ which is consumed to drive ETC. As the next step, SQR transfers sulfane sulfur to sulfur acceptors such as glutathione (GSH), sulfite (SO32−), cysteine, and homocysteine while SQR utilizes GSH as a dominant sulfur acceptor in the physiological conditions to produce glutathione persulfide (GSSH) which is the main product of H2S oxidation mediated by SQR. ETHE1 converts GSSH into sulfite and GSH consuming oxygen. Sulfite is further catabolized by TST or SO to produce thiosulfate (S2O32−) or sulfate (SO42−), respectively. Thiosulfate can be converted back to H2S via catabolism by 3-MST and TST [Hase (SO) , 46. H2S and TST .Fig. 2P2S catabolic pathways play some roles. H2S undergoes auto-oxidation both in aerobic and anaerobic conditions. High or low sulfide/oxygen ratio results in polysulfide or thiosulfate production in the buffer at physiological pH, respectively [2S also binds to heme iron of methemoglobin (MetHb) to be converted into thiosulfate and polysulfide [2S level (~ 0.2 μM) [2S at physiologically low levels. Myoglobin can also exert similar capacity of H2S oxidation as MetHb to generate thiosulfate and polysulfide [2S can bind to other globin species , which indicates the possible H2S oxidation by these globin species [Non-enzymatic Hectively . H2S alsysulfide . MetHb i 0.2 μM) , 49. Thiysulfide . H2S can species –53.Impairment of sulfide catabolism could be pathogenic. For example, mutation in ETHE1 is responsible for ethylmalonic encephalopathy , 54–56. Deficiency in TST markedly exacerbates, whereas TST activation by thiosulfate administration ameliorates, diabetes in mice . TST exp2S were initially examined using simple sulfide salts . For example, intra-left ventricular administration of Na2S at 50 μg/kg attenuated myocardial ischemic injury by preserving mitochondrial function in mice [2S at 7 μmol/kg IV improves survival rate and attenuates brain injury after cardiac arrest and cardiopulmonary resuscitation in mice via nitric oxide synthase 3-dependent manner [2S concentration instantly while H2S levels return to the baseline quickly due to the short half-life of H2S  [2S after administration were developed [2S-releasing compound, exerts beneficial effects of H2S even with wide therapeutic window (0.1–5 mM) and has been used frequently in both in vitro and in vivo experiments [2S-induced neurotoxicity may be mediated via enhancement of N-methyl-d-aspartate receptor (NMDAR) activation [2S-releasing molecule, S-memantine, which is a combination drug of slowly H2S-releasing molecule chemically conjugated with memantine which is a moderate NMDAR antagonist and approved for the treatment of Alzheimer’s disease patients. S-memantine exerts lower toxicity and greater therapeutic effects against cerebral ischemic injury in vitro and in vivo than do H2S-releasing molecule alone or sulfide salt [2S-releasing compounds have been tested in clinical trials [2S-releasing moiety, ATB-346, inhibits COX-2 as well as naproxen with less gastrointestinal damage than naproxen  [2S levels and to reduce markers of oxidative stress in heart failure patients in the phase II clinical trial (NCT01989208).The effects of administration of exogenous H in mice . Systemit manner . Systemie blood) , 60, 61.eveloped , 38, 62.eriments . Becausetivation –66, we dl trials –69. Wall3291418) . Sodium 2S oxidation. While sodium thiosulfate (STS) has been used as an antidote for cyanide poisoning, our discovery may expand its indication for other critical conditions.In a series of experimental studies, we unexpectedly uncovered therapeutic effects of thiosulfate in models of critical illnesses. Thiosulfate has traditionally been considered as an inert end product of H2S in a murine model of endotoxin-induced systemic inflammation and shock. Before our study, some studies showed pro-inflammatory effects of H2S whereas other studies reported anti-inflammatory effects of H2S. Our study revealed that endotoxin challenge decreased plasma sulfide concentration in mice. On the other hand, breathing H2S after endotoxin challenge restored sulfide levels and increased thiosulfate concentrations in plasma /lipopolysaccharide (LPS)-induced ALF in CSE-deficient mice on the C57BL6 background. A combination of GalN/LPS has been widely used to induce ALF in animal models. GalN sensitizes the liver toward other stimuli in part reflecting the role of uridine-containing compounds in hepatic biotransformation. Coadministration of LPS and GalN potentiates hepatic damage, leading to hepatocyte apoptosis. Given the protective effects of physiological levels of H2S against systemic inflammation, we hypothesized that CSE deficiency aggravates GalN/LPS-induced liver injury in mice. Unexpectedly, we observed that CSE deficiency attenuates liver injury and mortality in mice subjected to GalN/LPS-challenge, and prevents cell death in primary hepatocytes incubated with GalN/tumor necrosis factor (TNF)-α. Beneficial effects of CSE deficiency were associated with markedly elevated homocysteine and thiosulfate levels, upregulation of NF-E2 p45-related factor 2 (Nrf2) and antioxidant proteins, and markedly increased 3-MST and SQR expression in the liver. Upregulation of 3-MST seemed to compensate the decrease in sulfide production by CSE deficiency. Because upregulated 3-MST and SQR in CSE-deficient mice may accelerate H2S oxidation to thiosulfate, we again examined effects of STS in GalN/LPS-induced acute liver injury. We confirmed the robust cytoprotective effects of STS against acute liver failure  injury in a variety of organs including the brain, whether it is endogenously produced or exogenously administered as H2S gas or donor compounds  [2S were incompletely defined. In particular, since H2S has very short half-life in biological fluids including cell culture medium and blood, how H2S reaches its presumed targets in the cells, and in the target tissues in the body when given in vivo, has been poorly understood. In this study, we showed that H2S is mostly and quickly converted to thiosulfate in vitro and in vivo. While removal of thiosulfate from cell culture medium abolished the cytoprotective effects of Na2S against oxygen glucose deprivation, replacement of thiosulfate restored the protection. These results suggest that thiosulfate is not only required but sufficient for the cytoprotective effects of H2S. We observed that thiosulfate inhibits the mitochondrial apoptosis cascade and caspase-3 activity. The cytoprotective effects of thiosulfate were associated with increased persulfidation of cleaved caspase-3 at Cys163. The protective effect of Na2S or STS was facilitated by sodium sulfate cotransporter 2 -mediated transportation of thiosulfate across the cell membrane. Systemic administration of STS improved survival and neurological function of mice subjected to global cerebral I/R injury. Beneficial effects of STS, as well as Na2S, were associated with marked increase of thiosulfate, but not H2S, in plasma and brain tissues. These results suggest that thiosulfate is a circulating “carrier” molecule of cytoprotective effects of H2S.Another evidence that supports beneficial effects of thiosulfate came from our recent studies examining the mechanism of neuroprotective effects exerted by H2S donors. A number of studies suggest that Hor NaHS) , 71–73. 2S- or reactive sulfur species-related compounds. STS has also been used to treat calciphylaxis, a potentially lethal complication of hemodialysis [− and persulfide/polysulfide directly or indirectly [Since STS is an inexpensive compound with low toxicity and proven safety track record of clinical use as an antidote for cyanide intoxication, STS is one of the most clinically relevant Hdialysis . Effectsdirectly –77. It i2S) is a highly toxic chemical hazard. Workers in industries including agriculture, petroleum, and sewage processing have been exposed to high concentration of H2S accidentally [2S can be easily and inexpensively made at home from materials found in local stores, it has been increasingly used for suicide [2S is the second most common cause of death after CO [2S poisoning varies depending on the gas concentration breathed. When H2S gas at 1000 ppm or higher concentration was inhaled, victims become unconscious and their respiratory center paralyzed instantly with only one or two breath. This is so-called knockdown and caused by instant paralysis of the central nervous system (CNS). One-time exposure to H2S can lead to long-term neurological deficits [2S a high priority chemical threat, both industrially and as a potential weapon of mass destruction by terrorists. Mechanism of H2S poisoning is incompletely understood, and there is no antidote for H2S intoxication. Sodium nitrite, hydroxocobalamin, thiosulfate, hyperbaric oxygen, and hypothermia have been used after acute H2S poisoning with limited efficacy [Hydrogen sulfide  fail to achieve this goal. These observations prompted us to explore the role of sulfide catabolism in cellular respiration and survival.Toxic effects of HS levels . H2S accxidation , 83, 84. in mice . They deerfusion , 85, 86.g brains , 85, 87.g brains , 59, 85.g brains . Translag brains , 60, 89 2S poisoning, we recently launched a project to develop novel H2S-specific scavengers in collaboration with Xian laboratory [2S scavengers to control endogenous H2S levels have not been explored or reported. It should be noted that H2S scavengers are well-known in industrial settings as the removal of H2S or related sulfur-containing compounds in industrial processes has been extensively studied [2S scavengers. Unfortunately, these industrial sulfide scavengers cannot be applied into biological systems because of toxicity. Several compounds are known and used clinically as antidotes for H2S poisoning, but their specificity for H2S and applications for H2S-related pathologies have not been studied. For example, hydroxocobalamin (HC) has been investigated as an antidote for H2S poisoning, but it also scavenges cyanide, NO, CO, and ROS [2S scavengers by exploiting the library of existing specific chemical H2S sensors and conducting extensive in vitro and in vivo screening. Sulfonyl azide compounds exhibit fast reaction time with H2S, high specificity against sulfide, low cellular toxicity, and capability to remove H2S in cellular systems. Systemic administration of SS20, one of these sulfonyl azide-based H2S scavengers, prevented death in mice subjected to acute H2S poisoning  are important research tools as well as potential therapeutic agents. At the same time, it has become evident that currently available pharmacological tools are not sufficiently specific or versatile to elucidate the precise role of H2S in biology. Our discovery that thiosulfate may be an important carrier molecule of the biological effects of H2S may aid future research on the systemic effects of H2S. Recent development of specific H2S scavengers will enable more mechanistic studies by removing H2S from cellular milieu as well as propose a novel countermeasures against H2S poisoning. Because therapeutic window of H2S is narrow, to clarify how changes in balance of H2S production and catabolism play in illness must lead to further strategy of H2S-based therapies. This balance alteration seems to depend on the type of tissues and illness due to the diversity of expression levels related to H2S metabolism in tissues. For example, CNS is very sensitive to H2S poisoning due to the minimal level of H2S catabolizing capacity. Therefore, CNS seems to readily be affected by H2S toxicity in illness that increases H2S production [2S-production/catabolism balance in illness as well as development of novel pharmacological tools will undoubtedly advance our understanding of this fascinating gaseous molecule.Intensive research in the last decade established that Hdiseases –98. Consoduction , 99. Fur


In ferret immunization, two doses of inactivated whole virus antigens (3 μg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development. Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012  and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 10 Avian influenza (AI) viruses may not readily infect humans and cause diseases; however, mutations or reassortment give AI viruses a chance to escape the host barrier. AI H5, H6, H7, H9, and H10 subtypes are considered to have pandemic potential because of the occurrence of human infections and low level of population immunity . VaccinaIn late 2003, low-pathogenic avian influenza (LPAI) H5N2 viruses emerged in poultry farms in Taiwan. The H5N2 viruses carry a unique gene constellation containing HA and NA genes from American-lineage H5N2 viruses and six internal genes from local H6N1 viruses. The H5N2 viruses hit poultry farms again in 2008 and evolved into highly pathogenic AI (HPAI) strains in 2010 . A serolHA and NA genes of the novel H5N2 reassortant viruses belong to the American lineage which is antigenically different from the current H5N1 vaccines of the Eurasian lineage  6]. The. The6]. The embryonated egg-based vaccine production platform is a mature technology and has been widely used since 1945. However, the supply of the eggs may be influenced by the HPAI outbreaks. Moreover, an egg-induced antigenic drift during vaccine production may lead to poor vaccine effectiveness . TherefoThe current master donor virus used in the WHO reference labs, A/Puerto Rico/8/34 (PR8), was developed for the egg-based platform but not for Vero cell culture system. Therefore, a new master donor virus was required for the Vero cell-based platform. In 2010, a Vero cell-adapted high-growth H5N1 vaccine virus, Vero-15, was generated in our laboratory . Th. Th16]. 6 Vero cells were suspended in 300 μL of OPTI-MEM  and then mixed with eight plasmids (3 μg of each plasmid). The cell mixture was transferred into a 4-mm electroporation cuvette and then electroporated by two 220V square waves (20 ms duration time of each pulse and 0.1 s span between pulses). After electroporation, the cell mixture was transferred into a T25 flask which contained 5 mL pre-warmed M199 + 10% FBS and then incubated at 37 °C, 5% CO2 for overnight. The next day, the medium was replaced by M199 with 2 μg/mL TPCK-trypsin . Transfected cells were incubated at 37 °C, 5% CO2, and 1μg/mL TPCK-trypsin was added to the medium every day. Generated viruses were harvested when the cells showed 90% CPE or on day 7 after the transfection. The viruses may need further passages or plaque purification in Vero cells. Virus titre was determined by haemagglutination assay (HA assay) and 50% Tissue Culture Infective Dose (TCID50) following the WHO standard procedures [Eight plasmids which contained NA and modified HA genes from an H5N2 virus and six internal genes from the Vero-15 virus were transfected into Vero cells to generate H5N2 vaccine viruses by electroporation . A quantity of 5 × 10Viral RNA was extracted by MagNA Pure Compact Nucleic Acid Isolation Kit , and then the extracted viral RNAs were amplified by the one-step RT-PCR . Primers for sequencing are listed in Antigenicity was analysed by using hemagglutinin inhibition assay (HI assay). Tested antigens were diluted to 4 HAU and incubated with ferret antisera, anti-R3 and anti-A/CK/CH/1209/2003 (anti-CH/2003). These two antisera were generated in the previous study to evaluate the antigenicity of H5N2 viruses isolated from 2003 to 2012 . Turkey 6 Vero cells/well was cultured in 6-well plates at 37 °C, 5% CO2 overnight. Before virus infection, the cells were washed with 1X PBS. Tested viruses were diluted with M199 or M199 containing 2 μg/mL TPCK-trypsin and then added to wells with a cell layer. The cells and viruses were incubated at 37 °C, 5% CO2 for an hour, and then washed with 1X PBS to remove unattached viruses. In a trypsin-contained group, the cells were covered with M199 containing 0.3% agarose and 2 μg/mL TPCK-trypsin, and in counterpart group, the cells were covered with M199 containing 0.3% agarose  without TPCK-trypsin. The cells were incubated at 37 °C, 5% CO2 for three days. The plaques were fixed in 3.7% formaldehyde and stained with 0.5% crystal violet [A concentration of 1 × 10l violet .Six-week-old SPF chickens were provided by AHRI. Before injection, viruses were tested for sterilization and 10-fold diluted. Ten chickens were injected intravenously with 100 μL of diluted viruses. The chickens were examined daily for 10 days and scored .Four- to six-month-old ferrets were obtained through a laboratory-breeding program at Institute of Preventive Medicine, National Defence Medical Centre. Prior to the animal study, microchips capable of measuring temperature were implanted beneath the skin between the shoulder blades. Ferrets were numbered and housed separately in animal biosafety level 3 (ABSL3) laboratories with individual temperature control at 22–24 °C and 55–60% relative humidity throughout the experiment. All study procedures and animal care were conducted in accordance with the guidelines and under the supervision of Institutional Committee on Animal Care and Use, Institute of Preventive Medicine, National Defence Medical Centre.For the pathogenicity test, each ferret was intranasally infected with 0.5 mL of viruses, and each group had four ferrets . After the infection, the weight and temperature of the ferrets were monitored every day. Two ferrets  were sacrificed at DPI (day post-infection) 3, and the nasal turbinate, upper respiratory tract, lower respiratory tract, lung, and Hilar lymph node were collected for virus titration. Based on a pilot test, the H5N2 viruses were not pathogenic at DPI 7. Therefore, the other two ferrets were sacrificed at DPI 14 using the same procedure. 3 . Each dose contained 3 μg of HA protein and 300 μg of Al(OH)3. The ferrets were boosted 14 days after the first immunization. The sera were collected 23 days after the first immunization. For immunization study, four ferrets  were intramuscularly injected with two doses of inactivated whole virus vaccine adjuvanted with Al(OH)® BLU Single-Use Bioreactor, International Labmate Ltd, St Albans, Hertfordshire, UK) and infected with viruses (0.0001 MOI). The supernatant of virus culture was harvested on day 4. The harvest was inactivated with 0.01% formaldehyde and purified with flow-through chromatography [For standard antigen preparation, Vero cells were grown in VP-SFM with 4 g/L microcarrier using the 14-L bioreactor  and purified by using sucrose gradient . A goat t −20 °C .® ME Agarose, Lonza, Basel, Switzerland) and 1× PBS with 0.05% NaN3. The gel was mixed with the purified goat anti-E7HA serum. The ratio of serum to agarose was 1:75. The gel was placed in a circular mould and wells were generated by gel puncher. The purified viruses were treated with 1/10 V of 10% Zwittergent® 3-14  solution for 30 min and then diluted with 1× PBS with 0.05% NaN3. The diluted antigens were added to the wells and left overnight. After that, the gel was washed and then dried at 37 °C. Finally, the gel was stained with Coomassie blue to show the precipitin rings [Agarose gel (1%) was prepared with agarose powder  based on the Institutional Animal Care Committee Guidebook published by the US Office of Laboratory Animal Welfare under the supervision of IACUC, Animal Health Research Institutes , and IACUC, Institute of Preventive Medicine, National Defence Medical Centre .7 TCID50/mL). Growth efficiency of the vaccine virus was evaluated in Vero cells, which peaked to 5.62 × 107 TCID/mL and 512 HAU at DPI 2 . In trypsin dependency test, E7-V15 C11 virus formed plaques when trypsin was supplied in the culture medium, but it lost the ability without trypsin . Nine chickens were intravenously injected with the vaccine candidate virus, E7-V15 C11, and monitored for ten days. The chickens did not show any syndromes and were alive (IVPI = 0). The sera were collected for the HI test on the 10th day after infection. The HI titres of eight chicken sera were 16 or higher . The ser7 TCID50/mL, 0.5 mL/ferret). The physical condition of the ferrets was monitored for 14 days. The body temperature of all infected ferrets was lower than 39.5 °C. The body weight change was lower than 5%  were injected intramuscularly with two doses of the H5N2 E7 Ag (3 μg of each dose) adjuvanted with alum hydroxide. Interestingly, the male ferrets had a much lower serum HI titre against two wild-type H5N2 viruses compared with female ones . Since tAlthough avian influenza viruses are considered as a risk of a future pandemic, local circulating viruses sometimes are ignored, such as the novel American-lineage H5N2 reassortant viruses. However, considering the uncertainty of disease outbreak, the potential of influenza viruses causing human infection should receive more attention. In previous studies, the H5N2 viruses were found to cause asymptomatic infection in poultry workers . TherefoIn this study, we focused on an establishment of Vero cell-based reverse genetics and production platform. Because small countries usually do not have enough space and financial support to build many factories for each kind of vaccines, a multi-functional platform is reasonable . Vero ceThe first step of influenza vaccine production is to generate a high-growth vaccine virus. In this study, we generated two reassortant viruses, but only one (E7-V15) had detectable HA titres. Based on the receptor preference assay with turkey and horse RBCs, the E7 virus can bind 2,3- and 2,6-sialic acid receptor, but the R3 virus could only bind 2,3-sialic acid receptor (data not shown). Therefore, the low growth efficiency of the other reassortant virus (R3-V15) may be due to the receptor preference. Overall, it is desirable to generate multiple reassortant viruses bearing different receptor binding preferences.6 TCID50/mL, respectively), we adapted E7-V15 in Vero cells to generate high-growth vaccine viruses. After 16 passages and three times of plaque purification in Vero cells, the HA and live virus titre of E7-V15 C11 increased to 128 HAU and 3.16 × 107 TCID50/mL, respectively, and adapted mutations appeared on HA, NA and PA proteins. There were four adapted mutations on HA protein, N155D, T231K, G323R and I381V, and a mutation on NA protein, NA-N4K. The HA-N155D is located near a reported glycosylation site HA-154N [NVYR and NDVYR, respectively, so there was no glycosylation at HA-154 and HA-155 before and after adaptation. The NA mutation, NA-N4K, was located in the cytoplasmic tail of NA protein (M NPNQK). The cytoplasmic tail of NA protein is important for lipid raft targeting, virus assembly and morphology [Because the HA and live virus titres of E7-V15 were too low for commercial production  helps the decision of vaccine development ,43. For


Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During  Proper genome duplication is essential for normal development and tissue homeostasis. In diploid cells, genome duplication and cell proliferation occur via canonical G1→S→G2→M cell cycles in which origins of replication are specified during G1 phase, DNA replication occurs during S phase and chromosome segregation and cell division occur during M phase ,4. AlthoDrosophila larval salivary gland have long served as a model experimental tissue for understanding endoreplication . I. IH3K9R  control C. This dome arms . In agre control D,E. H3K9R mutants, but not H4K16R mutants, are defective in under-replication. To assess this possibility more directly, we next identified under-replicated domains in H3K9R, H4K16R and HWT using CNVnator, a highly sensitive method for copy number variation detection based on a statistical analysis of read depth of short reads [Drosophila [HWT control salivary glands, 86 and 98 of which overlap with domains identified in H3K9R and H4K16R mutants, respectively. The size distribution of under-replicated domains in each of the three genotypes were similar, with sizes ranging from 11 kb to over a megabase and medians ranging from 127 kb to 138 kb  , which as noted above most likely results from the way the sequencing data was analyzed rather than a biological phenomenon  B. In addenomenon B. In con mutants B. These HWT into those located on chromosome arms and those located within pericentric heterochromatin. For this purpose, pericentric heterochromatin was defined by high levels of H3K9me2 enrichment as described previously [H3K9R mutants (p < 2.2 × 10−16) relative to HWT control  decrease in copy number in H3K9R mutants relative to HWT control  a decrease in the S phase index, (ii) a biphasic shift in replication timing toward both earlier and the very latest  patterns and (iii) a reduction in the level of under-replication at pericentric heterochromatin but not along chromosome arms. Here, we utilized a genetic platform for histone gene replacement to interrogate the function of replication-dependent H3K9 and H4K16 in H3K9R mutant salivary glands reach the same size as control, consistent with completion of the endoreplication program. In addition, the slower development of H3K9R mutant animals may result in longer G periods between endo S phases, thus further reducing the S phase index. Another possibility is that the endoreplication timing program is generally condensed in H3K9R mutants such that S phase occurs more quickly, accounting for the change in distribution of RT categories and contributing to the decrease in S phase index. These changes unlikely indirectly result from changes in transcription, as we did not detect significant changes in the expression of protein coding genes, including those encoding replication factors, in the replication-dependent H3K9R mutant [A decrease in the S phase index could result from a reduction in the number or duration of endo-S phases, an increase in the duration of G phase, or both. The R mutant ,48. H3K9R mutant salivary gland nuclei with a very late replication pattern, represented by PCNA staining of the chromocenter, results from more extensive replication of pericentric heterochromatin each endocycle. Consistent with such a failure of normal under-replication, in H3K9R mutants we detected an increase in DNA copy number at pericentric heterochromatin by whole genome sequencing, as well as altered chromocenter cytology [H3K9R mutation disrupts HP1a recruitment, nucleosome occupancy and transposon repression at pericentric heterochromatin in diploid wing imaginal discs, without appreciably affecting the function of euchromatin [H4K16R analyses cannot rule out a small contribution from the replication-independent His4r gene, which resides outside the replication-dependent histone gene cluster and encodes a protein identical to replication-dependent H4 [The latest replicating sequences of polyploid salivary gland cells are not fully replicated each endocycle, yielding regions of the genome with decreased copy number, particularly in pericentric heterochromatin but also at specific loci along chromosome arms . We suggcytology . Intereshromatin ,48,49. Andent H4 ,57.H3K9R diploid wing discs revealed only a small number of advanced RT changes within pericentric heterochromatin [H3K9R mutant polyploid salivary glands. These data suggest that replication-dependent H3K9 plays a more significant role in regulating under-replication during salivary gland endoreplication than in regulating late replication during the canonical diploid cell cycle. Alternatively, these processes might be controlled by distinct mechanisms. The biological function of under-replication is not known and thus the consequence of losing pericentric under-replication is uncertain. Interestingly, Rif1 mutants, which lack under-replication altogether [Our previous analysis of hromatin . In conttogether , are viatogether ,58.Drosophila salivary glands [H3K9R mutants, under-replication in SuUR mutants is reduced both along chromosome arms and at pericentric heterochromatin [H3K9R mutants is the loss of pericentric HP1a [What might be the mechanism by which H3K9 promotes under-replication of pericentric heterochromatin? Previous studies established SuUR as a key regulator of under-replication in polyploid genomes of y glands ,33,34,35hromatin . SuUR fohromatin . In addihromatin ,36. Thusric HP1a . In the Drosophila polyploid cells. In conclusion, our data indicate that under-replication at salivary gland pericentric heterochromatin occurs through an H3K9-dependent mechanism and therefore suggest that the hallmarks of constitutive heterochromatin, H3K9me and HP1a, are critical regulators of under-replication in