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 β‐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 factor

Evidence that phosphorylation of the alpha-subunit of eIF2 does not essentially inhibit mRNA translation in wheat germ cell-free system.

A mechanism based on reversible phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) has been confirmed as an important regulatory pathway for inhibition of protein synthesis in mammalian and yeast cells, while plants constitute the significant exception. We studied the induction of TaeIF2α phosphorylation in germinated wheat (Triticum aestivum) embryos subjected to different adverse conditions. Data confirmed that formation of TaeIF2(αP) was not a general response, as no phosphorylation was observed under salt, oxidative or heat stress. Nevertheless, treatment by salicylic acid, UV-light, cold shock and histidinol did induce phosphorylation of TaeIF2α of wheat, as has been established previously for AteIF2α in Arabidopsis (Arabidopsis thaliana). Influence of TaeIF2α phosphorylation on translation of reporter mRNA with different 5′-untranslated regions (5′UTRs) was studied in wheat germ cell-free system (WG-CFS), in which TaeIF2α was first phosphorylated either by heterologous recombinant human protein kinase, HsPKR (activated by double-stranded (ds)RNA), or by endogenous protein kinase TaGCN2 (activated by histidinol). Pre-treatment of WG-CFS with HsPKR in the presence of dsRNA or with histidinol resulted in intense phosphorylation of TaeIF2α; however, the translation levels of all tested mRNAs decreased by only 10–15% and remained relatively high. In addition, factor OceIF2 from rabbit (Oryctolagus cuniculus) bound GDP much more strongly than the homologous factor TaeIF2 from wheat germ. Furthermore, factor OceIF2B was able to stimulate guanine nucleotides exchange (GDP→GTP) on OceIF2 but had no effect on the similar exchange on TaeIF2. These results suggest that the mechanism of stress response via eIF2α phosphorylation is not identical in all eukaryotes and further research is required to find and study in detail new plant-specific mechanisms that may inhibit overall protein synthesis in plants under stress

Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i>

Ribosomal protein S6 (RPS6) is the only phosphorylatable protein of the eukaryotic 40S ribosomal subunit. Ribosomes with phosphorylated RPS6 can selectively translate 5’TOP-(5’-terminal oligopyrimidine)-containing mRNAs that encode most proteins of the translation apparatus. The study of translational control of 5’TOP-mRNAs, which are preferentially translated when RPS6 is phosphorylated and cease to be translated when RPS6 is de-phosphorylated, is particularly important. In Arabidopsis thaliana, AtRPS6 is phosphorylated by kinase AtRPS6K2, which should in turn be phosphorylated by upper level kinases (AtPDK1 – at serine (S) 296, AtTOR – at threonine (T) 455 and S437) for full activation. We have cloned AtRPS6K2 cDNA gene and carried out in vitro mutagenesis replacing codons encoding S296, S437 and T455 by triplets of phosphomimetic glutamic acid (E). After the expression of both natural and mutated cDNAs in Escherichia coli cells, two recombinant proteins were isolated: native AtRPS6K2 and presumably constitutively active AtRPS6K2(S296E, S437E, T455E). The activity of these variants was tested in vitro. Both kinases could phosphorylate wheat (Triticum aestivum L.) TaRPS6 as part of 40S ribosomal subunits isolated from wheat embryos, though the non-mutated variant had less activity than phosphomimetic one. The ability of recombinant non-mutated kinase to phosphorylate TaRPS6 can be explained by its phosphorylation by bacterial kinases during the expression and isolation steps. The phosphomimetically mutated AtRPS6K2(S296E, S437E, T455E) can serve as a tool to investigate preferential translation of 5’TOP-mRNAs in wheat germ cell-free system, in which most of 40S ribosomal subunits have phosphorylated TaRPS6. Besides, such an approach has a biotechnological application in producing genetically modified plants with increased biomass and productivity through stimulation of cell growth and division

Real-Time PCR Detection of <i>Bacillus anthracis</i> by Lambda_Ba03 Prophage Genes

The aim of the study was to develop a set of primers and fluorescent probes for the detection of two chromosomal targets of Bacillus anthracis using real-time PCR based on the lambda_Ba03 prophage genes.Materials and methods. BLAST analysis of B. anthracis chromosomal DNA identified two target genes in the region of lambdaBa03 prophage, BA_5358 (AE016879.1: 4852332..4853642) and BA_5361 (AE016879.1: 4855298..4856278). The designed primers and fluorescent hydrolysable TaqMan probes for simultaneous detection of B. anthracis chromosomal DNA by two stated genes were tested in qPCR for sensitivity and specificity.Results and discussion. Studies performed on chromosomal DNA samples of closely related bacteria (B. cereus, B. thuringiensis, B. subtilis, B. clausii) have shown 100 % specificity of the developed sets of primers/probes. The sensitivity of the devised multiplex kit, tested on DNA samples of the m55-VNIIVViM vaccine strain and archival DNA samples of B. anthracis, reached 100 fg of bacterial DNA, which sets the limit of sensitivity at 17 genomes per reaction. The developed multiplex kit can be used as a separate tool for research laboratories studying anthrax

Glyphosate treatment mediates the accumulation of small discrete 5^′ and 3^′-terminal fragments of 18S rRNA in plant cells

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stressinduced small RNAs, which are 5’and 3’-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5’-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 μM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5’and 3’-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops

Phosphorylation of the alpha-subunit of plant eukaryotic initiation factor 2 prevents its association with polysomes but does not considerably suppress protein synthesis

Phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis is a major survival response to different stresses in animal and yeast cells. However, the role of this regulatory mechanism in plants is not unambiguously established to date. Here we describe a slight reduction of polysome abundance in Nicotiana benthamiana after the transient expression of a cDNA, AteIF2α(S56D), encoding a phosphomimetic form of Arabidopsis thaliana eIF2α. In contrast, the expression of a cDNA, AteIF2α(S56A), encoding a non-phosphorylatable form of AteIF2α caused slightly elevated polysome formation compared to the control. Recombinant AteIF2α(S56A) was detected in association with 40S ribosomal subunit-containing complexes and also in the polysomal fraction, while recombinant AteIF2α(S56D) was detected mainly in complex with 40S subunits. Intentional phosphorylation of wheat (Triticum aestivum) TaeIF2α induced by L-histidinol in a wheat germ cell-free extract did not reduce the abundance of polysomes. Phosphorylated TaeIF2(αP) was not detected in the polysomal fraction, similar to AteIF2α(S56D) in the in vivo experiment. Using mRNAs containing a ‘Strepto-tag’ in the 3′ untranslated region, 48S pre-initiation complexes isolated from histidinol-treated wheat germ extracts were shown to contain phosphorylated TaeIF2(αP). Thus, the phosphorylation of plant eIF2 does not greatly affect its ability to participate in the initiation of mRNA translation, in contrast to the situation in animals and yeast, in which eIF2α phosphorylation results in profound suppression of protein synthesis

Lethal pulmonary embolism in a pregnant woman with severe acute respiratory syndrome coronavirus-2 receiving prophylactic anticoagulation: a case report

Abstract Background A limited number of studies have described thrombotic complications in pregnant women with COVID-19. Here we report on fatal pulmonary embolism in a pregnant woman with laboratory confirmed SARS-CoV-2 infection. Case presentation A 28-year-old Kazakh woman was hospitalized with muscle pain, dry cough and a temperature of 37.5 °C at the 29th week of gestation. Upon admission, a blood test demonstrated elevated neutrophil-to-lymphocyte ratio, decreased levels of erythrocytes and hemoglobin, as well as prolonged prothrombin and activated partial thromboplastin time. Within 14 days of admission, she experienced respiratory distress and underwent transfer to the intensive care unit, intubation and a cesarean section. The patient received intravenous antibiotics, antiviral medications, systemic corticosteroids and dual anticoagulation with aspirin and enoxaparin. Death outcome was reported on day 18 of illness despite aggressive supportive care. Histological analysis demonstrated that obstruction of the main pulmonary arthery and disseminated intravascular coagulation were the causes of death. Conclusions This case demonstrates that in the management of pregnancy and childbirth in patients with suspected or confirmed COVID-19 infection, special attention should be paid to coagulation system parameters and timely appropriate prophylaxis of thromboembolic complications, which has yet to be determined

Two case reports of neuroinvasive West Nile virus infection in the Almaty region, Kazakhstan

Background: West Nile virus (WNV) is a member of the genus Flavivirus, which transmitted to humans mainly by mosquitoes. Recent pilot serosurveillance data from the Almaty region, Kazakhstan, suggest widespread WNV circulation in this area. This report includes two cases of neuroinvasive WNV infection in the same family living in a rural area near Tekeli city, Eskeldinsky district, Almaty region, Kazakhstan. Occurring concurrently and manifesting as WNV infection with febrile illness and symptoms of meningoencephalitis. Methods: The study performed retrospective analysis of clinical histories and achieved serum samples obtained from patients with febrile and meningoencephalitic syndromes of unknown origin in the Almaty region spanning from April 1 to October 31, 2019. All sera samples obtained from patients with clinically suspected cases of acute WNV infection were retrospectively tested for WNV and tick-borne encephalitis virus by commercial immunoassays. Two cases were selected. Cases presentation: We report two cases that occurred in August 2019 in a rural area near Tekeli city. Previously healthy 28- and 19-year-old husband and wife with febrile illness and neurological manifestations were hospitalized with the diagnosis of meningoencephalitis of unknown etiology and treated empirically. Retrospective serological analysis showed the presence of high titers of IgG against WNV on day 9 after onset of symptoms in cases. Conclusions: This is the first report of aseptic meningitis with WNV infection in the background in Kazakhstan. The obtained data suggest circulation of WNV in the Almaty region and emphasize importance of laboratory testing for WNV in suspicious cases occurring in the region

Molecular and seroepidemiological investigation of Сoxiella burnetii and spotted fever group rickettsiae in the southern region of Kazakhstan

Ticks are involved in the circulation of a number of human pathogens, including spotted fever group (SFG) Rickettsia spp. and Coxiella burnetii. Little is known about the occurrence of these microorganisms in the southern region of Kazakhstan. In 2018–2022, a total of 726 ticks were collected from bitten humans, livestock, and vegetation in four oblasts of the southern region of Kazakhstan and subjected to DNA extraction. The overall infection rate of Coxiella spp. and Rickettsia spp. in the ticks was 3.3% (24/726) and 69.9% (300/429), respectively. Phylogenetic analysis of ompA and gltA genes revealed the presence of three pathogenic SFG rickettsiae: Candidatus R. tarasevichiae, R. aeschlimannii and R. raoultii in ticks collected from bitten humans. In addition, Candidatus R. barbariae was detected in six Rhipicephalus turanicus ticks for the first time in Kazakhstan. To determine the seroprevalence of C. burnetii infection, we performed a serological analysis of samples collected from 656 domestic ruminants (cattle, sheep, and goats) in the region. Overall, 23.5% (154/656) of the animals tested were positive for IgG against C. burnetii. Seroprevalence at the herd level was 54% (28/52). Goats (43%; 12/28; odds ratio (OD) = 28.9, p < 0.05) and sheep (31.9%; 137/430; OD = 18.1, p < 0.05) had higher seroprevalence than cattle (2.5%; 5/198). Among the risk factors considered in this study, age (p = 0.003) and the oblast in which the animals were sampled (p = 0.049) were statistically associated with seropostivity for Q fever in sheep, according to the results of multivariate logistic regression analysis. Seroprevalence ranged from 0% to 55.5% in animals in different districts of the southern region of Kazakhstan. Active C. burnetii bacteremia was detected in four of 154 (2.6%) seropositive animals. The data obtained provide strong evidence of the presence of pathogenic rickettsiae and C. burnetii in the southern region of Kazakhstan and emphasize the need to improve epidemiological surveillance in the region

Stages of functional treatment fractures of the distal humerus

Проведений аналіз функціонального лікування 54 хворих з переломами дистального відділу плечової кістки. У всіх хворих застосовано екстрамедулярний металоостеосинтез. Виділені окремі етапи медичної реабілітації, визначені певні завдання та особливості лікувальних заходів на кожному етапі. Визначені причини основних ускладнень, запропоновані шляхи їх профілактики; Проведенный анализ функционального лечения 54 больных с переломами дистального отдела плечевой кости. У всех больных применен экстрамедуллярный металлоостеосинтез. Выделены отдельные этапы медицинской реабилитации, определенны определённые задачи и особенности лечебных мероприятий на каждом этапе. Определены причины основных осложнений, предложены пути их профилактики; The analysis of functional treatment of 54 patients with fractures of the distal humerus was performed. In all patients, extramedullary metalloesteosynthesis was used. Separate stages of medical rehabilitation are singled out, certain tasks and features of medical measures at each stage are certain. The causes of the main complications are determined, the ways of their prevention are suggested