Kaposi's sarcoma-associated herpesvirus viral protein kinase augments cell survival.

Oncogenic viruses have developed various strategies to antagonize cell death and maintain lifelong persistence in their host, a relationship that may contribute to cancer development. Understanding how viruses inhibit cell death is essential for understanding viral oncogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different cancers in the human population, including Kaposi's sarcoma (KS), the most common cancer in HIV patients. Previous studies have indicated that the KSHV-encoded viral protein kinase (vPK) impacts many processes dysregulated in tumorigenesis. Here, we report that vPK protects cells from apoptosis mediated by Caspase-3. Human umbilical vein endothelial cells (HUVECs) expressing vPK (HUVEC-vPK) have a survival advantage over control HUVEC under conditions of extrinsic- and intrinsic-mediated apoptosis. Abolishing the catalytic activity of vPK attenuated this survival advantage. We found that KSHV vPK-expressing HUVECs exhibited increased activation of cellular AKT kinase, a cell survival kinase, compared to control cells without vPK. In addition, we report that vPK directly binds the pleckstrin homology (PH) domain of AKT1 but not AKT2 or AKT3. Treatment of HUVEC-vPK cells with a pan-AKT inhibitor Miransertib (ARQ 092) reduced the overall phosphorylation of AKT, resulting in the cleavage of Caspase-3 and the induction of apoptosis. Furthermore, vPK expression activated VEGF/VEGFR2 in HUVECs and promoted angiogenesis through the AKT pathway. vPK expression also inhibited the cytotoxicity of cisplatin in vitro and in vivo. Collectively, our findings demonstrate that vPK's ability to augment cell survival and promote angiogenesis is critically dependent on AKT signaling, which is relevant for future therapies for treating KSHV-associated cancers.

A nearby long gamma-ray burst from a merger of compact objects.

Gamma-ray bursts (GRBs) are flashes of high-energy radiation arising from energetic cosmic explosions. Bursts of long (greater than two seconds) duration are produced by the core-collapse of massive stars1, and those of short (less than two seconds) duration by the merger of compact objects, such as two neutron stars2. A third class of events with hybrid high-energy properties was identified3, but never conclusively linked to a stellar progenitor. The lack of bright supernovae rules out typical core-collapse explosions4-6, but their distance scales prevent sensitive searches for direct signatures of a progenitor system. Only tentative evidence for a kilonova has been presented7,8. Here we report observations of the exceptionally bright GRB 211211A, which classify it as a hybrid event and constrain its distance scale to only 346 megaparsecs. Our measurements indicate that its lower-energy (from ultraviolet to near-infrared) counterpart is powered by a luminous (approximately 1042 erg per second) kilonova possibly formed in the ejecta of a compact object merger.

Survived COVID-19 patient presented with death on arrival: A case report.

INTRODUCTION AND IMPORTANCE: Although Corona virus primarily infects respiratory system, several complications have been reported. The aim of this paper is to report a strange case of corona virus disease presented with death on arrival and survived after rigorous cardiopulmonary resuscitation. CASE PRESENTATION: A 35-year-old male present as a sudden loss of consciousness 10 min before admission. The patient was unconscious, pulseless, there was no sign of breathing, the pupils fixed, did not react to light. Blood sugar was 112 mg/dl. Cardiopulmonary resuscitation (CPR) commenced with insertion of two wide bore cannulas. The patient developed ventricular fibrillation. Later, he reverted to pulseless ventricular tachycardia. After several minutes of CPR, the patient returned back to sinus rhythm. He underwent percutaneous coronary intervention and became healthy one month after the intervention. CLINICAL DISCUSSION: Cardiac involvement in case of COVID-19 might be explained by the presence of the angiotensin-converting enzyme 2 (ACE2) receptor which is a transmembrane soluble protein regulating the local actions of the renin-angiotensin apparatus in cardio-vascular system. CONCLUSION: death on arrival could be the first presentation of COVID-19. Aggressive CPR is necessary to revive the victim.

Long non-coding RNA ANRIL polymorphisms in papillary thyroid cancer and its severity.

Background: Long non-coding RNAs are likely to have a role in the pathogenesis of many diseases, including cancer. We hypothesised an effect of certain ANRIL single nucleotide polymorphisms (SNPs) in papillary thyroid cancer. Methods: Genomic ANRIL SNPs in rs11333048, rs4977574, rs1333040 and rs10757274 were determined in 134 papillary thyroid cancer patients and 155 age- and sex-matched controls. Results: None of the ANRIL SNPs were individually linked to papillary thyroid cancer. However, the AAAC haplotype (A from rs11333048, A from rs4977574, A from rs1333040 and C from rs10757274, respectively) showed a protective effect from papillary thyroid cancer whilst the CAAC and CAGT haplotypes were associated with cancer. The rs1333048 CC variant was more frequent in patients with larger tumour size (≥1 cm) in a recessive model (OR 3.4 [95%CI, 1.1-11], P = 0.035). The rs4977574 AC variant was associated with smaller tumour size in an over-dominant model (OR 0.4 [95%CI, 0.2-1.0], P = 0.041). SNPs in rs10757274 (AA: p = 0.045) and rs1333040 (CC: p = 0.019) are linked to a lower likelihood of III-IV cancer stages in dominant or codominant models. Conclusions: Certain haplotypes of ANRIL SNPs are associated with papillary thyroid cancer. ANRIL rs1333048 and rs4977574 variants were associated with larger and smaller tumour sizes, respectively. rs10757274 and rs1333040 variants might lead to lower III-IV cancer stages. These SNPs may be important in the diagnosis of this form of thyroid cancer.

The effect of CASP3 rs4647610 and rs4647602 polymorphisms on tumour size and cancer stage in papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer whose incidence has increased in recent years. Dysregulated apoptosis is known in the pathogenesis of various cancers. Caspase-3 is an important apoptotic component and its abnormal function may play a key role in cancer pathogenesis. We tested the hypothesis of a link between CASP3 single nucleotide polymorphisms rs4647610 and rs4647602 on PTC and its clinical outcomes. MATERIAL AND METHODS: A total of 134 PTC patients and 151 healthy controls were genotyped for CASP3 rs4647610 and rs4647602 single nucleotide polymorphisms (SNPs) using PCR-RFLP method. RESULTS: Allele and genotype frequencies of both SNPs were not different between cases and controls. The combined genotypes and haplotypes were not linked to PTC. However, the frequencies of CASP3 rs4647610 GA and AA genotypes were higher in PTC patients with larger tumour size (≥1 cm), and the rs4647610 SNP was associated with increased tumour size in the dominant model (OR 3.4 [95% CI, 1.1-11], P = 0.04). The CASP3 rs4647602CA and AA genotypes were higher in PTC patients with lower TNM stage (I-II) compared to higher stages (III-IV). No association was observed between CASP3 polymorphisms and other PTC outcomes. CONCLUSION: Although CASP3 rs4647610 and rs4647602 SNPs are not associated with PTC, rs4647610 is linked to larger tumour size, and rs4647602 to lower stage of cancer.

An abundant 'Candidatus Phytoplasma solani' tuf b strain is associated with grapevine, stinging nettle and Hyalesthes obsoletus.

Bois noir (BN) associated with 'Candidatus Phytoplasma solani' (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of 'Ca. P. solani' in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated 'Ca. P. solani' strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. 'Candidatus Phytoplasma convolvuli' (bindweed yellows) was ascertained in nettle and bindweed samples.

Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti.

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue haemorrhagic fever and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an antipathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti heat shock protein 70 (hsp70) genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ~25-50-fold in whole adults by 4 h after heat-shock, with significant activity (~20-fold) remaining at 24 h. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ~2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of antipathogen molecules.

Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.).

A gene encoding a sulphur-rich, sunflower seed albumin (23% cysteine plus methionine) was modified to contain the promoter for the 35S RNA of cauliflower mosaic virus, in order to obtain leaf expression in transgenic plants. In addition, a sequence encoding an endoplasmic reticulum-retention signal was added to the 3' end of the coding region so as to stabilize the protein by diverting it away from the vacuole. The modified gene was introduced into subterranean clover (T. subterraneum L.) and its expression was detected by northern and western blots and by immunogold localization. The albumin was accumulated in the lumen of the endoplasmic reticulum, and, among six independent, transformed lines, it accumulated in the leaves of T0 transgenic plants at varying levels up to 0.3% of the total extractable protein. The level of accumulation of the sunflower albumin increased with increasing leaf age, and in the older leaves of the most highly expressing plants of the T1 generation it reached 1.3% of total extractable protein. Expression of the SSA gene was stable in the first and second generation progeny. These results indicate that there is potential for significantly improving the nutritional value of subterranean clover for ruminant animals such as sheep by expressing genes that code for sulphur-rich, rumen-stable proteins in leaves.

A biotechnological approach to improving the nutritive value of alfalfa.

The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep. We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds. Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT). Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves. The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit. The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein. This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage. Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily.

Structural and functional analysis of promoter from gliadin, an endosperm-specific storage protein gene of Triticum aestivum L.

To identify cis-regulatory elements of the gliadin gene, a study of the gliadin gene promoter was conducted by transient expression analysis of plasmid DNAs which were introduced into plant protoplasts by electroporation. The promoter region (-592 bp to +18 bp from the translational start) of this developmentally regulated gene, when fused upstream to the chloramphenicol acetyl transferase (CAT) reporter cassette was unable to direct significant CAT expression in wheat or tobacco suspension cells. Because this monocot gene promoter appeared to be under stringent tissue-specific control, a hybrid promoter approach using a nopaline synthase (nos) promoter was employed. A series of 3' deletions of the gliadin promoter were placed upstream of either a nonfunctional -101 nos or a nearly wild-type -155 nos promoter fused in turn to a CAT reporter gene cassette. Transient expression analysis of these plasmid DNAs in tobacco cells showed that the gliadin fragment could either restore the activity of the non-functional nos promoter (series I) or enhance the activity of the functional nos promoter (series II). The degree of restoration of the promoter function conferred by gliadin fragments of the first series was proportional to the enhancing effect of the same fragments in the second series of constructs. The transcriptional activity of the gliadin (-592 bp to -77 bp) -nos hybrid promoter was reduced by 26% upon 3' deletion of sequences in the region -141 bp to -77 bp, which contains both the TATA and CCAAT boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and expression of the rice glutelin multigene family.

A near full-length cDNA and three genomic clones for rice (Oryza sativa L.) glutelin were isolated and studied. Based on nucleic acid sequence and Southern blot analyses, the three isolated glutelin genomic clones were representative members of three gene subfamilies each containing five to eight copies. A comparison of DNA sequences displayed by relevant regions of these genomic clones showed that two subfamilies, represented by clones Gt1 and Gt2, were closely related and evolved by more recent gene duplication events. The 5'-flanking and coding sequences of Gt1 and Gt2 displayed at least 87% homology. In contrast, Gt3 showed little or no homology in the 5'-flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Conserved sequences in the 5'-flanking regions of these genes were identified and discussed in light of their potential regulatory role. The derived primary sequences of all three glutelin genomic clones showed significant homology to the legume 11 S storage proteins indicating a common gene origin. A comparison of the derived glutelin primary sequences showed that mutations were clustered in three peptide regions. One peptide region corresponded to the highly mutable hypervariable region of legume 11 S storage proteins, a potential target area for protein modification. Expression studies indicated that glutelin mRNA transcripts are differentially accumulated during endosperm development. Promoters of Gt2 and Gt3 were functional as they direct transient expression of chloramphenicol acetyltransferase in cultured plant cells.

Reversible Inactivation of Nitrate Reductase by NADH and the Occurrence of Partially Inactive Enzyme in the Wheat Leaf.

Nitrate reductase from wheat (Triticum aestivum L. cv Bindawarra) leaves is inactivated by pretreatment with NADH, in the absence of nitrate, a 50% loss of activity occurring in 30 minutes at 25 degrees C with 10 micromolar NADH. Nitrate (50 micromolar) prevented inactivation by 10 micromolar NADH while cyanide (1 micromolar) markedly enhanced the degree of inactivation.A rapid reactivation of NADH-inactivated nitrate reductase occurred after treatment with 0.3 millimolar ferricyanide or exposure to light (230 milliwatts per square centimeter) plus 20 micromolar flavin adenine dinucleotide. When excess NADH was removed, the enzyme was also reactivated by autoxidation. Nitrate did not influence the rate of reactivation.Leaf nitrate reductase, from plants grown for 12 days on 1 millimolar nitrate, isolated in the late photoperiod or dark period, was activated by ferricyanide or light treatment. This suggests that, at these times of the day, the nitrate reductase in the leaves of the low nitrate plants is in a partially inactive state (NADH-inactivated). The nitrate reductase from moisture-stressed plants showed a greater degree of activation after light treatment, and inactive enzyme in them was detected earlier in the photoperiod.