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}.

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.

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.

Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin alpha.

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus--Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin alpha, suggesting that both CPs are imported into the nucleus via an importin alpha-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

Control of translation reinitiation on the cauliflower mosaic virus (CaMV) polycistronic RNA.

Translation of the polycistronic 35S RNA of CaMV (cauliflower mosaic virus) occurs via a reinitiation mechanism, which requires TAV (transactivator/viroplasmin). To allow translation reinitiation of the major open reading frames on the polycistronic RNA, TAV interacts with the host translational machinery via eIF3 (eukaryotic initiation factor 3) and the 60S ribosome. Accumulation of TAV and eIF3 in the polysomal fraction isolated from CaMV-infected cells suggested that TAV prevents loss of eIF3 from the translating ribosomes during the first initiation event. The TAV-eIF3-80S complex could be detected in vitro by sucrose-gradient-sedimentation analysis. The question is whether TAV interacts directly with the 48S preinitiation complex or enters polysomes after the first initiation event. eIF4B, a component of the 48S initiation complex, can preclude formation of the TAV-eIF3 complex via competition with TAV for eIF3 binding; the eIF4B- and TAV-binding sites on eIF3g overlap. eIF4B out-competes TAV for binding to eIF3 and to the eIF3-40S complex. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated transactivation of polycistronic translation. Our results thus indicate that eIF4B precludes TAV-eIF3-40S complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only the second and subsequent initiation events. We propose a model in which TAV enters the host translational machinery at the eIF4B-removal step to stabilize eIF3 within polysomes.

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.

Tissue-specific silencing of a transgene in rice.

In a transgenic rice line, a beta-glucuronidase reporter gene under the control of the rice tungro bacilliform virus promoter became gradually methylated, and gene activity was lost concomitantly. Methylation was observed only in the homozygous offspring and was initially restricted to the promoter region and accompanied by loss of expression in the vascular bundle tissue only. This expression pattern was similar to that of a promoter with a deletion of a vascular bundle expression element. The gene activity could be reestablished by treatment with 5-azacytidine. Methylation per se did not inhibit the binding to the promoter region of protein factors which also bound to the unmethylated sequence. Instead, promoter methylation enabled the alternative binding of a protein with specificity for sequence and methylation. In further generations of homozygous offspring the methylation spread into the transcribed region and gene activity was completely repressed also in nonvascular cells. The results indicate that different stages are involved in DNA methylation-correlated gene inactivation, and that at least one of them may involve the attraction of a sequence and methylation-specific DNA-binding protein.

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene.

Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

A novel regulatory gene, Tri10, controls trichothecene toxin production and gene expression.

We report here the characterization of Tri10, a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of Tri10 in Fusarium sporotrichioides abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (Tri4, Tri5, Tri6, and Tri101) and an apparent farnesyl pyrophosphate synthetase (Fpps) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and Fpps. Further analysis revealed that disruption of Tri10, and to a greater extent the disruption of Tri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved in Fusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10 acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10.

A plant viral "reinitiation" factor interacts with the host translational machinery.

The cauliflower mosaic virus transactivator, TAV, controls translation reinitiation of major open reading frames on polycistronic RNA. We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3 and the 60S ribosomal subunit. Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated reinitiation activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by reinitiation, overcoming the normal cell barriers to this process.

Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor.

Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.

Biochemical characterization of the helper component of Cauliflower mosaic virus.

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Tetramerization is a conserved feature of the virion-associated protein in plant pararetroviruses.

All plant pararetroviruses belong to the Caulimoviridae family. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus of Caulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.

Polyadenylation in rice tungro bacilliform virus: cis-acting signals and regulation.

The polyadenylation signal of rice tungro bacilliform virus (RTBV) was characterized by mutational and deletion analysis. The cis-acting signals required to direct polyadenylation conformed to what is known for plant poly(A) signals in general and were very similar to those of the related cauliflower mosaic virus. Processing was directed by a canonical AAUAAA poly(A) signal, an upstream UG-rich region considerably enhanced processing efficiency, and sequences downstream of the cleavage site were not required. When present at the end of a transcription unit, the cis-acting signals for 3'-end processing were highly efficient in both monocot (rice) and dicot (Nicotiana plumbaginifolia) protoplasts. In a promoter-proximal position, as in the viral genome, the signal was also efficiently processed in rice protoplasts, giving rise to an abundant "short-stop" (SS-) RNA. The proportion of SS-RNA was considerably lower in N. plumbaginifolia protoplasts. In infected plants, SS-RNA was hardly detectable, suggesting either that SS-RNA is unstable in infected plants or that read-through of the promoter-proximal poly(A) site is very efficient. SS-RNA is readily detectable in transgenic rice plants (A. Klöti, C. Henrich, S. Bieri, X. He, G. Chen, P. K. Burkhardt, J. Wünn, P. Lucca, T. Hohn, I. Potrylus, and J. Fütterer, 1999. Plant Mol. Biol. 40:249-266), thus the absence of SS-RNA in infected plants can be attributed to poly(A) site bypass in the viral context to ensure production of the full-length pregenomic viral RNA. RTBV poly(A) site suppression thus depends both on context and the expression system; our results suggest that the circular viral minichromosome directs assembly of a transcription-processing complex with specific properties to effect read-through of the promoter-proximal poly(A) signal.

The product of ORF III in cauliflower mosaic virus interacts with the viral coat protein through its C-terminal proline rich domain.

Using the yeast two-hybrid system, we show that the ORF III product of cauliflower mosaic virus (pIII) interacts through its C-terminus with the viral coat protein. The last five amino acids of pIII were essential for the interaction and virus infectivity. Deletion of the last three amino acids or the mutation F129A decreased the strength of the interaction by 90%. We further show that pIII is closely associated with virus particles found in the inclusion bodies of infected plants but not in viral particles released from the inclusion bodies by urea treatment.

Ribosome shunt is essential for infectivity of cauliflower mosaic virus.

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.