Complementation Tests Research Articles

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

Identification and validation of an ECERIFERUM2- LIKE gene controlling cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata L.).

A single nucleotide mutation of BoCER2 is the primary cause of the wax deficiency in cabbage. An effective allele-specific KASP marker was developed for marker-assisted selection of glossiness. TL28-1 is a novel spontaneous wax-deficient mutant with a glossy phenotype identified from cabbage. In this study, the genetic analysis suggested that the wax-deficient trait of TL28-1 was controlled by a single recessive gene. All wax monomers longer than 28 carbons were significantly decreased in TL28-1. Fine-mapping results showed that the wax-deficient locus wdtl28 was located at an 80-kb interval between BOL01-20 and BOL01-24 markers on chromosome 1. According to the genome annotation of B. oleracea, the ECERIFERUM2- LIKE (CER2-LIKE) gene, BoCER2, was identified as the candidate gene. Phylogenetic analysis showed that BoCER2 and other CER2-LIKEs from vascular plants formed a clade within the BAHD superfamily of acyltransferases. The BoCER2 transcript was detected in various tissues, including stem, leaf, flower, and silique, but not in the cabbage roots. Subcellular localization indicated that BoCER2 protein functions in the endoplasmic reticulum. Further sequence analysis showed that a single nucleotide mutation (G to A) is present in the BoCER2 coding sequence in TL28-1, leading to a stop codon (TGA), hence premature translation termination. Linkage analysis showed that the homozygotic mutational BoCER2 co-segregated with wax deficiency. Moreover, the complementation test suggested that BoCER2 from wild type can rescue the wax deficiency of TL28-1. These results indicate that BoCER2 mutation hinders the elongation of very-long-chain fatty acid precursors in TL28-1, leading to wax deficiency. The allele-specific KASP marker designed in this study could be effective for marker-assisted selection of glossiness.

A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiae's DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panel's 'Mosaic beer' clade and share an enrichment for C > A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C > A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C > A mutations if left unrepaired.

TRIM22 inhibits the proliferation of gastric cancer cells through the Smad2 protein

TRIM22 is involved in tumorigenesis and development, but its mechanism is not clear. In this study, we investigated the expression and biological role of TRIM22 in gastric cancer. We found that TRIM22 mRNA and protein expression was abnormally low in gastric cancer tissues and cells and correlated with tumor size and depth of invasion. Overexpression of TRIM22 significantly inhibited the proliferation, colony formation, and migration of gastric cancer cells and downregulated the expression of HSPA6. However, the HSPA6-siRNA complementation test showed that TRIM22 did not regulate cell proliferation through HSPA6. Furthermore, overexpression of TRIM22 downregulated the phosphorylation of Smad2 and Smad3. In addition, TRIM22 directly binds to Smad2, and overexpression of Smad2 can reverse the inhibition of cell proliferation and migration induced by TRIM22. In vivo, overexpression of TRIM22 significantly inhibited the growth of subcutaneous xenografts in nude mice. Our study indicates that TRIM22 has an important role in the development of gastric cancer and may inhibit the proliferation of gastric cancer cells through Smad2.

A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves

BackgroundPlant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined.ResultsHere an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14.ConclusionsThese data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

Gibberellins orchestrate panicle architecture mediated by DELLA–KNOX signalling in rice

SummaryPanicle architecture is a key determinant of grain yield in cereals, but the mechanisms governing panicle morphogenesis and organ development remain elusive. Here, we have identified a quantitative trait locus (qPA1) associated with panicle architecture using chromosome segment substitution lines from parents Nipponbare and 9311. The panicle length, branch number and grain number of Nipponbare were significantly higher than CSSL‐9. Through map‐based cloning and complementation tests, we confirmed that qPA1 was identical to SD1 (Semi Dwarf1), which encodes a gibberellin 20‐oxidase enzyme participating in gibberellic acid (GA) biosynthesis. Transcript analysis revealed that SD1 was widely expressed during early panicle development. Analysis of sd1/osga20ox2 and gnp1/ osga20ox1 single and double mutants revealed that the two paralogous enzymes have non‐redundant functions during panicle development, likely due to differences in spatiotemporal expression; GNP1 expression under control of the SD1 promoter could rescue the sd1 phenotype. The DELLA protein SLR1, a component of the GA signalling pathway, accumulated more highly in sd1 plants. We have demonstrated that SLR1 physically interacts with the meristem identity class I KNOTTED1‐LIKE HOMEOBOX (KNOX) protein OSH1 to repress OSH1‐mediated activation of downstream genes related to panicle development, providing a mechanistic link between gibberellin and panicle architecture morphogenesis.

Expanding Horizons in Complement Analysis and Quality Control.

Complement not only plays a key role in host microbial defense but also modulates the adaptive immune response through modification of T- and B-cell reactivity. Moreover, a normally functioning complement system participates in hematopoiesis, reproduction, lipid metabolism, and tissue regeneration. Because of its powerful inflammatory potential, multiple regulatory proteins are needed to prevent potential tissue damage. In clinical practice, dysregulation and overactivation of the complement system are major causes of a variety of inflammatory and autoimmune diseases ranging from nephropathies, age-related macular degeneration (AMD), and systemic lupus erythematosus (SLE) to graft rejection, sepsis, and multi-organ failure. The clinical importance is reflected by the recent development of multiple drugs targeting complement with a broad spectrum of indications. The recognition of the role of complement in diverse diseases and the advent of complement therapeutics has increased the number of laboratories and suppliers entering the field. This has highlighted the need for reliable complement testing. The relatively rapid expansion in complement testing has presented challenges for a previously niche field. This is exemplified by the issue of cross-reactivity of complement-directed antibodies and by the challenges of the poor stability of many of the complement analytes. The complex nature of complement testing and increasing clinical demand has been met in the last decade by efforts to improve the standardization among laboratories. Initiated by the IUIS/ICS Committee for the Standardization and Quality Assessment in Complement Measurements 14 rounds of external quality assessment since 2010 resulted in improvements in the consistency of testing across participating institutions, while extending the global reach of the efforts to more than 200 laboratories in 30 countries. Worldwide trends of assay availability, usage, and analytical performance are summarized based on the past years’ experiences. Progress in complement analysis has been facilitated by the quality assessment and standardization efforts that now allow complement testing to provide a comprehensive insight into deficiencies and the activation state of the system. This in turn enables clinicians to better define disease severity, evolution, and response to therapy.

Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress.

Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

Analysis of barley mutants ert-c.1 and ert-d.7 reveals two loci with additive effect on plant architecture

Main conclusionBoth mutant ert-c.1 and ert-d.7 carry T2-T3 translocations in the Ert-c gene. Principal coordinate analyses revealed the translocation types and translocation breakpoints. Mutant ert-d.7 is an Ert-cErt-d double mutant.Mutations in the Ert-c and Ert-d loci are among the most common barley mutations affecting plant architecture. The mutants have various degrees of erect and compact spikes, often accompanied with short and stiff culms. In the current study, complementation tests, linkage mapping, principal coordinate analyses and fine mapping were conducted. We conclude that the original ert-d.7 mutant does not only carry an ert-d mutation but also an ert-c mutation. Combined, mutations in Ert-c and Ert-d cause a pyramid-dense spike phenotype, whereas mutations in only Ert-c or Ert-d give a pyramid and dense phenotype, respectively. Associations between the Ert-c gene and T2-T3 translocations were detected in both mutant ert-c.1 and ert-d.7. Different genetic association patterns indicate different translocation breakpoints in these two mutants. Principal coordinate analysis based on genetic distance and screening of recombinants from all four ends of polymorphic regions was an efficient way to narrow down the region of interest in translocation-involved populations. The Ert-c gene was mapped to the marker interval of 2_0801to1_0224 on 3HL near the centromere. The results illuminate a complex connection between two single genes having additive effects on barley spike architecture and will facilitate the identification of the Ert-c and Ert-d genes.

Candidate Gene Test for Quantitative Trait Loci Conferring Hypercholesterolemia on Mouse Chromosome 1

ObjectivesThe TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a subcongenic mouse with TH donor segment, ∼25 Mb, on chromosome (Chr) 1 in a C57BL/6J (B6) background that harbors quantitative trait loci (QTL) conferring hypercholesterolemia, named Tchol1 (Tallyho Associated Cholesterol 1). The subcongenic mouse developed hypercholesterolemia compared to B6 mice demonstrating that distal segment of Chr 1 from TH genome is necessary to cause the hypercholesterolemia. In this study, we tested the candidacy of the apolipoprotein A2 (Apoa2) gene for Tachol1 by the quantitative complementation test. Apoa2, known regulator of cholesterol metabolism, maps to the Tchol1 locus. MethodsTo carry out the quantitative complementation test, both TH-homozygous Tachol1 subcongenic and B6-homozygous (B6) mice were mated to the Apoa2 knockout heterozygous [wild-type (wt)/null] mice to produce four types of animals; TH/wt, TH/null, B6/wt, and B6/null. Both male and female mice were weaned onto standard rodent chow and maintained. Blood was collected when animals were euthanized at 16 weeks of age. Total plasma cholesterol levels were determined using colorimetric assays. A two-way ANOVA was used to evaluate Apoa2 (null vs. wt) and Tachol1 (TH vs. B6) interaction effects for dependent variables, followed by the multiple comparison post test with Tukey correction using GraphPad Prism 8. ResultsTotal plasma cholesterol levels were: 137 ± 5 (TH/wt), 119 ± 8 (TH/null), 103 ± 8 (B6/wt), and 80 ± 4 (B6/null) for males, and 149 ± 8 (TH/wt), 130 ± 9 (TH/null), 98 ± 3 (B6/wt), and 103 ± 6 (B6/null) for females [mean ± s.e.m; mg/dl]. Two-way ANOVA revealed no significant interaction between Tchol1 and Apoa2 knockout alleles for total plasma cholesterol levels in both males and females. However, there were significant main effects of Tchol1 and Apoa2 knockout alleles on total plasma cholesterol levels in males, while significant main effects of Tchol1 on them in females. ConclusionsNo significant interaction effect between knockout and QTL alleles is interpreted as evidence that the knockout locus is not equal to the QTL. Our results suggest that the Apoa2 gene is not identical to the Tchol1 QTL. Funding SourcesAHA 18AIREA33960437, NIH 1 R15 DK113604-01A1, the WV-INBRE grant (P20GM103434), and the COBRE ACCORD grant (1P20GM121299).

Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice

BackgroundRibosomes responsible for transcription and translation of plastid-encoded proteins in chloroplasts are essential for chloroplast development and plant growth. Although most ribosomal proteins in plastids have been identified, the molecular mechanisms regulating chloroplast biogenesis remain to be investigated.ResultsHere, we identified albinic seedling mutant albino seedling lethality 4 (asl4) caused by disruption of 30S ribosomal protein S1 that is targeted to the chloroplast. The mutant was defective in early chloroplast development and chlorophyll (Chl) biosynthesis. A 2855-bp deletion in the ASL4 allele was verified as responsible for the mutant phenotype by complementation tests. Expression analysis revealed that the ASL4 allele was highly expressed in leaf 4 sections and newly expanded leaves during early leaf development. Expression levels were increased by exposure to light following darkness. Some genes involved in chloroplast biogenesis were up-regulated and others down-regulated in asl4 mutant tissues compared to wild type. Plastid-encoded plastid RNA polymerase (PEP)-dependent photosynthesis genes and nuclear-encoded phage-type RNA polymerase (NEP)-dependent housekeeping genes were separately down-regulated and up-regulated, suggesting that plastid transcription was impaired in the mutant. Transcriptome and western blot analyses showed that levels of most plastid-encoded genes and proteins were reduced in the mutant. The decreased contents of chloroplast rRNAs and ribosomal proteins indicated that chloroplast ribosome biogenesis was impaired in the asl4 mutant.ConclusionsRice ASL4 encodes 30S ribosomal protein S1, which is targeted to the chloroplast. ASL4 is essential for chloroplast ribosome biogenesis and early chloroplast development. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

LYSINE KETOGLUTARATE REDUCTASE TRANS-SPLICING RELATED 1 is involved in temperature-dependent root growth in rice.

Root length is an important root parameter directly related to the uptake of water and nutrients. However, the molecular mechanisms controlling root length are still not fully understood. Here, we isolated a short-root mutant of rice, dice2 (defective in cell elongation 2). The cell length and meristem size of the roots were decreased in dice2, but the root function in terms of mineral element uptake, root cell width, and root anatomy were hardly altered compared with wild-type (WT) rice. The root growth defect in dice2 could be partially rescued by high temperature. Map-based cloning combined with a complementation test revealed that the short-root phenotype was caused by a nonsense mutation in a gene which was annotated to encode Lysine Ketoglutarate Reductase Trans-Splicing related 1 (OsLKRT1). OsLKRT1, encoding a cytosol-localized protein, was expressed in all cells of the root tip and elongation region as well as the shoot. RNA-seq analysis showed that there was no difference between dice2 and the WT in the expression level of genes involved in root development identified so far. These results indicate that OsLKRT1 is involved in a novel pathway required for root cell elongation in rice, although its exact role remains to be further investigated.

Genetic basis and cross-resistance of Vip3Aa resistance in Spodoptera frugiperda (lepidoptera: Noctuidae) derived from Texas, USA

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an economically important crop pest and a major target of transgenic crops expressing Cry and/or Vip3Aa proteins. Widespread adoption of Bt crops has placed a strong selection pressure on the insect populations. Till now, seven lepidopteran and two coleopteran insect species have developed the field-evolved resistance to a Bt crop with practical consequences. Understanding the genetic basis of insect resistance to a Bt protein is essential for designing appropriate resistance management strategies. In this study, we firstly characterized the genetic basis of Vip3Aa resistance in S. frugiperda derived from Texas. Then, we compared the genetic basis of Vip3Aa resistance of this Texas population with a known Vip3Aa resistant population of S. frugiperda collected from Louisiana to determine whether the genetic basis of the resistance in these two resistant populations was the same. Finally, we evaluated the responses of the Vip3Aa-resistant population of S. frugiperda collected from Texas against Cry1F and Cry2Ab2 purified proteins. The results showed that the resistance to Vip3Aa protein in S. frugiperda collected from Texas was likely controlled by a single, functionally recessive and autosomal gene. In addition, the resistance to Vip3Aa in the Texas population of S. frugiperda was not cross-resistant to Cry1F and Cry2Ab2 proteins. The interstrain complementation tests between the Texas and Louisiana resistant populations indicated the genetic basis of the resistance to Vip3Aa in these two populations are different. Considering the rapid invasion of S. frugiperda and the adoption of Vip3Aa Bt crops in the United States and other countries, results generated from this study should provide important information for management of Vip3Aa resistance in S. frugiperda worldwide.

Background mutation in strain RB2126 affects the locomotion behavior of flp-1 mutants.

The FLP-1 neuropeptide is involved in locomotion, mechanosensation, and reproduction. Strain RB2126 is reported to contain the flp-1(ok2811) mutation, a prospective null allele. Here we report that strain RB2126, in addition to flp-1(ok2811), also contains two additional background mutations. One of the mutations gives the animals a constitutive dauer phenotype (Daf-c), and complementation testing confirms that it is in the Daf-c gene daf-11. The second mutation is isolated based on the fact that it partially suppresses the elevated body curvature of flp-1(ok2811) mutants, but it remains uncloned. The updated genotype of RB2126 strain is flp-1(ok2811); daf-11(yak155); yak156.

GRAS transcription factor LOSS OF AXILLARY MERISTEMS is essential for stamen and runner formation in wild strawberry.

Axillary bud development is a major factor that impacts plant architecture. A runner is an elongated shoot that develops from axillary bud and is frequently used for clonal propagation of strawberry. However, the genetic control underlying runner production is largely unknown. Here, we identified and characterized loss of axillary meristems (lam), an ethyl methanesulfonate-induced mutant of the diploid woodland strawberry (Fragaria vesca) that lacked stamens in flowers and had reduced numbers of branch crowns and runners. The reduced branch crown and runner phenotypes were caused by a failure of axillary meristem initiation. The causative mutation of lam was located in FvH4_3g41310, which encodes a GRAS transcription factor, and was validated by a complementation test. lamCR mutants generated by CRISPR/Cas9 produced flowers without stamens and had fewer runners than the wild-type. LAM was broadly expressed in meristematic tissues. Gibberellic acid (GA) application induced runner outgrowth from the remaining buds in lam, but failed to do so at the empty axils of lam. In contrast, treatment with the GA biosynthesis inhibitor paclobutrazol converted the runners into branch crowns. Moreover, genetic studies indicated that lam is epistatic to suppressor of runnerless (srl), a mutant of FveRGA1 in the GA pathway, during runner formation. Our results demonstrate that LAM is required for stamen and runner formation and acts sequentially with GA from bud initiation to runner outgrowth, providing insights into the molecular regulation of these economically important organs in strawberry.

Real-world data of thrombotic microangiopathy management: The key role of ADAMTS13 activity and complement testing

ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 motifs) activity is a key tool in differential diagnosis of thrombotic microangiopathies (TMAs). Due to the lack of availability of ADAMTS13 testing, PLASMIC/PLASIC scores have been suggested to predict ADAMTS13 deficiency. The importance of differentiating TTP from other complement-mediated TMAs is highlighted by the need to urgently start plasma exchange and utility of treatments such as caplacizumab or eculuzimab. Therefore, we aimed to evaluate ADAMTS13 activity, PLASMIC/PLASIC scores, and complement testing in guiding management of a real-world TMA cohort. We enrolled consecutive TMA patients with samples referred to our Center (01/2018–2020). If ADAMTS13 ​> ​10%, soluble C5b-9 was measured. Among 80 TMA patients, ADAMTS13 activity was ≤10% in 50 patients, while 28 had a relapsing disease. PLASMIC/PLASIC were excellent predictors of ADAMTS13 deficiency, especially in patients without secondary causes. Soluble C5b-9 levels were elevated (median 525 ​ng/ml, range 313–913 ​ng/ml) in 7 patients without secondary causes and ADAMTS13 ​> ​10% (hemolytic uremic syndrome/HUS). Two were shiga-toxin associated; while 5 atypical HUS. Only 1/5 patients received eculizumab and achieved TMA resolution implemented by guidance based on soluble C5b-9 levels. In transplant-associated TMA, 8/16 patients not responding to first-line treatment received eculizumab due to elevated C5b-9 levels (median 353 ​ng/ml, range 281–1252 ​ng/ml) and achieved TMA resolution. In conclusion, our real-world data confirm that ADAMTS13, complement testing, and PLASMIC/PLASIC are valuable tools in diagnosis and management of TMAs, but also highlight the unmet need of using available markers and treatments in clinical practice.

Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium.

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

Complementation Test of Draft Oxen in Dry Season with Harvest Residues in the Cotton Zone in Northwestern Benin

Complementation Test of Draft Oxen in Dry Season with Harvest Residues in the Cotton Zone in Northwestern Benin

Genome-wide analysis of cabbage cytochrome P450 genes and characterization of BoCYP704B1, a gene responsible for cabbage anther development

Plant cytochrome P450 monooxygenases (CYPs) make up a large superfamily of heme-thiolate enzymes involving in many primary and secondary metabolism reactions. In this study, a total of 279 Brassica oleracea CYP (BoCYP) genes were identified in the cabbage genome. These BoCYPs were classified into eight distinct clans with 44 families. Expression analyses of the BoCYP genes of different cabbage tissues suggested that some BoCYPs may play significant roles in reproduction. Recently, BoCYP704B1 has been identified as a candidate gene associated with male sterility in the cabbage line 83121A. Here, the functional mechanism of BoCYP704B1 was explored. Subcellular localization showed that BoCYP704B1 is located in the endoplasmic reticulum. RNA in situ hybridization indicated that BoCYP704B1 is predominantly expressed in the tapetum of cabbage anther, and the expression was maximal at the tetrad and early monocyte stage and weakened to the termination. Furthermore, functional complementation test results showed that BoCYP704B1 from wild type could rescue the male sterility of 83121A mutant. The results of this study increase our understanding of the functional mechanism of cabbage CYP genes and will facilitate the application of the BoCYP704B1 gene in cabbage molecular breeding.