New Constraint on the Local Relic Neutrino Background Overdensity with the First KATRIN Data Runs.

We report on the direct search for cosmic relic neutrinos using data acquired during the first two science campaigns of the KATRIN experiment in 2019. Beta-decay electrons from a high-purity molecular tritium gas source are analyzed by a high-resolution MAC-E filter around the end point at 18.57 keV. The analysis is sensitive to a local relic neutrino overdensity ratio of η<9.7×10^{10}/α (1.1×10^{11}/α) at a 90% (95%) confidence level with α=1 (0.5) for Majorana (Dirac) neutrinos. A fit of the integrated electron spectrum over a narrow interval around the end point accounting for relic neutrino captures in the tritium source reveals no significant overdensity. This work improves the results obtained by the previous neutrino mass experiments at Los Alamos and Troitsk. We furthermore update the projected final sensitivity of the KATRIN experiment to η<1×10^{10}/α at 90% confidence level, by relying on updated operational conditions.

Bound on 3+1 Active-Sterile Neutrino Mixing from the First Four-Week Science Run of KATRIN.

We report on the light sterile neutrino search from the first four-week science run of the KATRIN experiment in 2019. Beta-decay electrons from a high-purity gaseous molecular tritium source are analyzed by a high-resolution MAC-E filter down to 40 eV below the endpoint at 18.57 keV. We consider the framework with three active neutrinos and one sterile neutrino. The analysis is sensitive to the mass, m_{4}, of the fourth mass state for m_{4}^{2}≲1000 eV^{2} and to active-to-sterile neutrino mixing down to |U_{e4}|^{2}≳2×10^{-2}. No significant spectral distortion is observed and exclusion bounds on the sterile mass and mixing are reported. These new limits supersede the Mainz results for m_{4}^{2}≲1000 eV^{2} and improve the Troitsk bound for m_{4}^{2}<30 eV^{2}. The reactor and gallium anomalies are constrained for 100<Δm_{41}^{2}<1000 eV^{2}.

Continuous venovenous hemofiltration rapidly lowers toxic metabolites in a patient with MSUD and imminent cerebral herniation.

Maple syrup urine disease (MSUD) is an inborn error of amino acid metabolism. During catabolic stress encephalopathy and brain swelling that can culminate in brain herniation may occur. Beyond the neonatal period, these metabolic decompensations normally can be treated with a conservative dietary emergency regimen. Nevertheless in exceptionally severe cases also older patients may require extracorporeal interventions. We present the case of a 12-year-old patient with cerebral edema and imminent cerebral herniation. Continuous venovenous hemofiltration (CVVH) caused a prompt decrease of the toxic metabolite levels as well as an improvement of the patient's condition.

Intracranial hemorrhage in a female leading to the diagnosis of severe hemophilia A and Turner syndrome.

BACKGROUND: Severe hemophilia A (HA) in females is a very rare phenomenon. Ignoring HA as a possible diagnose can result in fatal complications. PATIENTS: We report a 3-month old girl suffering from severe hemophilia A, presenting with intracranial hemorrhage three weeks after drop down from an infant carrier. Recurrent bleeding after neurosurgery led to the diagnosis of a HA by findings of low levels of factor VIII coagulation activity (F8:C) below 1% and normal levels of factor von Willebrand activity. METHODS: Diagnosis of hemophilia A by one stage clotting test and proof by molecular studies via long - range - PCR. Chromosome analysis in metaphases from peripheral blood lymphocytes. RESULTS: Molecular analysis showed inversion of intron 22 as the result of a maternally inherited, distal, F8 gene inversion and chromosome analyses a 45,X karyotype indicative of Turner syndrome in our patient. Diagnosis was hampered by the female sex and the presence of neither a family history of bleeding disorders nor clinical signs of Turner syndrome. CONCLUSION: Our case shows that, although uncommon in female infants, x-linked genetic bleeding disorders like HA are a possible diagnosis by very different reasons. Rare bleeding disorders, although not expected, might be present and the combined clinical, laboratory and genetic analysis are needed to establish the final diagnosis. Repetitive prolonged aPTT and clinical bleeding signs should lead to further hemostasiological investigations. An algorithm for hemostasiological investigations in case of unexplained clinical bleeding is given.

Imaging stress and magnetism at high pressures using a nanoscale quantum sensor.

Pressure alters the physical, chemical, and electronic properties of matter. The diamond anvil cell enables tabletop experiments to investigate a diverse landscape of high-pressure phenomena. Here, we introduce and use a nanoscale sensing platform that integrates nitrogen-vacancy (NV) color centers directly into the culet of diamond anvils. We demonstrate the versatility of this platform by performing diffraction-limited imaging of both stress fields and magnetism as a function of pressure and temperature. We quantify all normal and shear stress components and demonstrate vector magnetic field imaging, enabling measurement of the pressure-driven [Formula: see text] phase transition in iron and the complex pressure-temperature phase diagram of gadolinium. A complementary NV-sensing modality using noise spectroscopy enables the characterization of phase transitions even in the absence of static magnetic signatures.

Transcriptional and translational control of gene expression in cauliflower mosaic virus.

Cauliflower mosaic virus sequences have developed as a powerful tool for the study of various aspects of gene expression in plants. Analysis of the promoter/enhancer region has led to the discovery of several transcription factors and factor-binding sites. Studies on RNA processing and polyadenylation reveal a viral strategy to obtain terminal redundancy of retrovirus pregenomic RNA. Striking differences between plant and vertebrate polyadenylation signals have been disclosed. The mechanisms for translation of the polycistronic 35S RNA are novel in the eukaryotic field and may give new insight to translational control in general.

Cauliflower Mosaic Virus Gene VI Controls Translation from Dicistronic Expression Units in Transgenic Arabidopsis Plants.

Transformed Arabidopsis plants were used to study the effect of the cauliflower mosaic virus (CaMV) inclusion body protein on translation of dicistronic RNA. Reporter plants contain a dicistronic transcription unit with CaMV open reading frame VII (ORF VII) as the first and the [beta]-glucuronidase (GUS) reporter ORF as the second cistron. "Transactivator plants" contain CaMV ORF VI under the control of the strong CaMV 35S promoter. The transactivator plants were difficult to regenerate and showed an abnormal phenotype. Expression of GUS activity in the reporter plants was very low, but high GUS activity could be induced by introduction of gene VI, either by crossing with plants containing gene VI as a transgene or by infection with CaMV. Histological GUS assays showed that transactivation occurred in all types of tissue and at all developmental stages. The practical implications of the induction of GUS expression from the dicistronic unit by virus infection are discussed.

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs.

The sequences of different plant viral leaders with known translation enhancer ability show partial complementarity to the central region of 18S rRNA. Such complementarity might serve as a means to attract 40S ribosomal subunits and explain in part the translation-enhancing property of these sequences. To verify this notion, we designed beta-glucuronidase (GUS) mRNAs differing only in the nature of 10 nt inserts in the center of their 41 base leaders. These were complementary to consecutive domains of plant 18S rRNA. Sucrose gradient analysis revealed that leaders with inserts complementary to regions 1105-1114 and 1115-1124 ('ARC-1') of plant 18S rRNA bound most efficiently to the 40S ribosomal subunit after dissociation from 80S ribosomes under conditions of high ionic strength, a treatment known to remove translation initiation factors. Using wheat germ cell-free extracts, we could demonstrate that mRNAs with these leaders were translated more than three times more efficiently than a control lacking such a complementarity. Three linked copies of the insert enhanced translation of reporter mRNA to levels comparable with those directed by the natural translation enhancing leaders of tobacco mosaic virus and potato virus Y RNAs. Moreover, inserting the same leaders as intercistronic sequences in dicistronic mRNAs substantially increased translation of the second cistron, thereby revealing internal ribosome entry site activity. Thus, for plant systems, the complementary interaction between mRNA leader and the central region of 18S rRNA allows cap-independent binding of mRNA to the 43S pre-initiation complex without assistance of translation initiation factors.

Pararetro- and retrovirus RNA: splicing and the control of nuclear export.

Splicing and nuclear export of RNA are obligatory steps in gene expression by eukaryotic cells. Not only have novel splicing events been identified during the replication cycle of retro- and pararetroviruses, but the resulting combination of spliced and unspliced products requires specialized mechanisms for nuclear export, which in turn is a key regulatory step for virus replication.

Alternative structures of the cauliflower mosaic virus 35 S RNA leader: implications for viral expression and replication.

The CaMV 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600 nts leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and probably also packaging. The structure of the leader has been characterized theoretically and experimentally. The predicted conformation, a low-energy elongated hairpin, base-pairing the two halves of the leader, with a cross-like structure at the top, is strongly supported by enzymatic probing, chemical modification, and phylogenetic comparison. The elongated hairpin is stabilized by strong base-pairing between the ends of the leader, regions which are important in allowing translation downstream of the leader via the ribosome shunt mechanism. At high ionic strength the 35 S RNA leader exhibits additional higher order structures of low electrophoretic mobility: (1) a long-range pseudoknot connecting central and terminal parts of the leader; (2) a dimer. Alternative structures of the CaMV 35 S RNA leader may co-exist and have specialized functions. Their potential impact on CaMV life cycle regulation is discussed.

Characterization of Novel Sesquiterpenoid Biosynthesis in Tobacco Expressing a Fungal Sesquiterpene Synthase.

The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabacum). Trichodiene was the sole sesquiterpene synthase product in enzyme reaction mixtures derived from unelicited transformant cell-suspension cultures, and both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor treatment. Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase only in transformant cell lines producing trichodiene. In vivo labeling with [3H]mevalonate revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell-suspension cultures. In a trichodiene-producing transformant, the level of 15-hydroxytrichodiene accumulation increased after elicitor treatment. In vivo labeling with [14C]acetate showed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment. Incorporation of radioactivity from [14C]acetate into capsidiol was reduced following elicitor treatment of a trichodiene-producing transformant as compared with wild type. These results demonstrate that sesquiterpenoid accumulation resulting from the constitutive expression of a foreign sesquiterpene synthase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene synthase to produce high levels of novel sesquiterpenoids.

Isolation and gene disruption of the Tox5 gene encoding trichodiene synthase in Gibberella pulicaris.

The trichodiene synthase gene (Tox5) was isolated from Gibberella pulicaris, and its nucleotide sequence was determined. Tox5 was disrupted through transformation with a plasmid carrying a doubly truncated copy of the coding region and a selectable marker for resistance to hygromycin B (Hygr). Analysis of 82 transformants for their ability to produce the trichothecene, 4,15-diacetoxyscirpenol (DAS), resulted in the identification of five DAS- strains. Southern hybridization analysis of DAS- Hygr transformants indicated that the plasmid integrated at the Tox5 locus. The disrupted Tox5 gene was shown to be mitotically stable. Analysis of nine tetrads revealed either the cosegregation of the disrupter plasmid and the DAS- phenotype or the loss of the disrupter plasmid. These results demonstrate the feasibility of using gene disruption in G. pulicaris and suggest a general method for obtaining Tox5- mutants in other trichothecene-producing fungi.

Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.

The production of trichothecene mycotoxins by some plant pathogenic species of Fusarium is thought to contribute to their virulence. Gibberella zeae (F. graminearum) is an important cereal pathogen that produces the trichothecene deoxynivalenol. To determine if trichothecene production contributes to the virulence of G. zeae, we generated trichothecene-deficient mutants of the fungus by gene disruption. The disrupted gene, Tri5, encodes the enzyme trichodiene synthase, which catalyzes the first step in trichothecene biosynthesis. To disrupt Tri5, G. zeae was transformed with a plasmid carrying a doubly truncated copy of the Tri5 coding region interrupted by a hygromycin B resistance gene. Tri5- transformants were selected by screening for the inability to produce trichothecenes and by Southern blot analysis. Tri5- strains exhibited reduced virulence on seedlings of Wheaton wheat and common winter rye, but wild-type virulence on seedlings of Golden Bantam maize. On Caldwell and Marshall wheat and Porter oat seedlings, Tri5- strains were inconsistent in causing less disease than their wild-type progenitor strain. Head blight developed more slowly on Wheaton when inoculated with Tri5- mutants than when inoculated with wild-type strains. These results suggest that trichothecene production contributes to the virulence of G. zeae on some hosts.

Nucleotide sequence of the dihydrofolate-reductase gene borne by the plasmid R67 and conferring methotrexate resistance.

The complete nucleotide sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R67 was determined. The gene is 234 bp long and codes for 78 amino acids. The polypeptide deduced from the DNA sequence is in perfect agreement with the previously published amino acid sequence. Comparison of the nucleotide sequence with the one determined for the R388-encoded DHFR indicates that 75% of the nucleotides are conserved in the two genes. The 3' end of the R67 gene can be modified without altering significantly the activity of the enzyme.

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence.

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.

Oligonucleotide directed mutagenesis of cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon.

Mutation of the initiation codon of the dispensible open reading frame, ORF VII, of cauliflower mosaic virus (CaMV) delayed the appearance of disease symptoms, but the mutants reverted with high frequency. This suggests a role of this start codon in viral expression. Oligonucleotide-directed mutagenesis, utilizing a novel, repair-resistant deoxyguanosine analogue, 2'-deoxy-7-deazainosine (dDI), highly improved the yield of mutants.

Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides.

The trichodiene synthase gene (Tox5) has been isolated from the fungus Fusarium sporotrichioides, and its nucleotide (nt) sequence determined. A lambda gt11 library of F. sporotrichioides DNA was screened with antiserum against trichodiene synthase (TS). DNA fragments were isolated which encode a portion of the Tox5 gene. In subsequent screening of the library we employed one of these DNAs as a probe and identified several recombinant phage containing the entire Tox5 gene. The gene consists of a 1182-nt open reading frame (ORF) which contains a 60-nt intron and specifies a Mr 43,999 protein. The deduced amino acid sequence of the ORF was identical to sequences determined for several CNBr peptides from purified TS. Southern and Northern analyses indicated that the Tox5 gene is present in a single copy and is transcribed into an mRNA of about 1450 nt. Upstream from the start codon, 'TATA'-like sequences and a short repeated sequence resembling the 'CCAAT' box were observed. The primary structure described for TS is the first such report for a member of the terpene cyclase group of enzymes.

Enhancement of a viral CaMV promoter by insertion of a BamHI linker.

The BamHI linker sequence, 5'-CCGGATCCGG, contains an element that acts as an enhancer for the cauliflower mosaic virus 19S promoter.

The instability of a recombinant plasmid, caused by a prokaryotic-like promoter within the eukaryotic insert, can be alleviated by expression of antisense RNA.

A region of the cauliflower mosaic virus genome was found to direct the expression of a nucleic-acid-binding protein in Escherichia coli. This protein is apparently toxic for the bacteria and leads to a destabilization of plasmids containing that region. Antisense RNA was used to diminish the unwanted expression and to stabilize the respective recombinant plasmids. The approach described may prove useful in other cases where problems with cloning of eukaryotic DNA arise.