Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.

The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence-associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild-type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

SummaryNudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.

Introduction: The topic of the article is "Analysis of cargo securing in road transport". It consists of an analytical part and a research part, the assumption of which is to conduct a survey among people who have passed a number of requirements allowing to obtain the title of a professional driver. The subject of the work is the analysis of the most important issues related to cargo securing and securing means used in road transport of cargo, which are supported by the relevant legal acts [1,2]. Aims: The aim of the research was to analyze the securing of loads in road transport and to check the knowledge of drivers of heavy goods vehicles professionally transporting loads by road on a given topic. The respondents are both drivers employed in nearby transport companies (Zakład Rolniczo-Przemysłowego "Farmutil HS", PHUS Eksport-Import Transpil Spedition Waldemar Bocheński, PUH "Gólcz i Sons" Mieczysław Gólcz, Martrans, AGA-Guderski Transport & Logistyka) and drivers, who are members of transport groups on Facebook The research lasted 3 months and covered the period from January 7, 2020 to April 7, 2020. Research Object: The subject of research is a number of issues related to loads on the means of transport, i.e. legal regulations, fastening accessories, methods of fastening. Drivers answering according to their knowledge reveal a practical perspective on securing and transporting loads. The three-month survey period resulted in 230 forms returned. The majority of respondents are men in the number of 214 people (93%). Only 16 women took part in the survey, which constitutes 7% of all respondents. The difference between the representatives of both sexes is great, which suggests that the profession of a driver is performed to a greater extent by men. Education can be a factor indicating the level of knowledge of the respondent. More than half of the respondents declared their education as secondary - 135 people (59%), then 48 people (21% chose the answer higher, basic - 39 people (17%), primary - 6 people (2%). 2 people (1%) completed their education at lower secondary school. Due to the length of service, the respondents can be divided into 6 groups. The most numerous group of 88 people (38%) are drivers who have been working in the profession for one to four years. Among the respondents there were 60 people (26%) with 5-10 years of work experience, then 43 people (19%) working 11-20 years, 24 people (10%) working less than a year, while 11 people (5%) were employed with transporting loads from 21 to 30 years. Only 4 people (2%) of the respondents can boast over 30 years of professional experience. The total length of the employment periods should indicate the level of knowledge and experience gained. The graph shows that drivers with various years of service took part in the survey, which has a positive impact on the accuracy of the results. In conclusion, the largest group of people was respondents who answered 1-4 years, which means that the road transport department has developed, and this resulted in an increase in the demand for people authorized to drive heavy goods vehicles. Methodology: Before the actual study, a pilot study was performed. It was aimed at acquiring preliminary knowledge about the studied community and testing the correctness of the questionnaire. A questionnaire survey was used to conduct the research. There were 15 single-choice closed questions and 10 open-ended questions in the survey. Some questions included photos. The complete questionnaire was made available through the Facebook social network on transport thematic groups gathering professional drivers and delivered to the transport companies previously discussed. Results: The survey was aimed at extracting the knowledge of professional drivers traveling both in Poland and other European countries about the securing of loads in road transport. The questionnaires included a properly prepared pool of questions and research techniques, which are also important for the accuracy and reliability of the research. The questions were arranged and edited in a clear and legible manner for the respondent, both in electronic and paper form. The tools used are closed-ended questions and open-ended questions with photos. The main part of the study was conducted on 230 respondents. It turned out that drivers do not have sufficient knowledge in the area in which they operate on a daily basis. Some drivers refused to answer the questionnaires, even knowing that their answers would be anonymous or they had no knowledge about it. As the survey shows, more than half of the respondents did not participate in any educational form related to cargo securing. The problem may result from the savings of the people managing the enterprise, as well as the reluctance of drivers to improve their own qualifications. Most of the respondents do not use the aids and measuring instruments available on the market to check the necessary amount of fixing means to ensure the safety of other road users. The tabular or template method of determining the number of lashings eliminates the need to use mathematical formulas, which could be problematic for some professional drivers. These are measures that improve the work and make it easier without the need to perform complex calculations. In the course of the research, difficulties arose in the form of the apparent reluctance of professional drivers to fill out an anonymous questionnaire. Unfortunately, some companies also refused to take part in the survey. It can be assumed that this was caused by the fear of the consequences that could be drawn by the legislator or the fear of the overall result of the study in the case of a specific company. Conclusion: It is required from transport to be effective, and thus profitable and safe, and it is conditioned by the proper securing of the cargo. To sum up, it can be stated that the essence of securing loads in road transport is the appropriate selection of securing means, securing methods and available security systems so that the cargo with a specific weight, shape, plasticity and other own characteristics is immobilized on a properly selected loading surface and prevents its movement. The material collected in the course of the research and the analyzes carried out show that professional drivers have insufficient knowledge in the field of load securing. The problem of poor knowledge of the subject, externalized among drivers, is a significant problem that may lead to dangerous road situations and may generate unnecessary costs in the enterprise. In this situation, its managers should react. An example of a response to such signals may be the employment of a person providing consultancy services in the field of cargo securing, training or audits in transport companies. Retraining professional drivers or organizing training for those who are just taking their first steps in the profession is crucial. A person who becomes aware of the scale of the risk and shows the visual effects of road accidents with greater diligence and accuracy will secure the load that has been assigned to him and make sure that he drives to the road with a vehicle that does not pose a threat. Lashing loads is a field of transport in which an individual approach should be taken to the load taken for transport. Standard mathematical formulas are helpful for this. But not all charges can be formulated and act schematically. Various loads of different shapes, sizes and other own characteristics are a challenge for people responsible for their safe transport. The driver carrying the load can make sure that it has been properly secured, without the need to use complicated mathematical formulas. It may turn out to be problematic for him, which is why publications have been made to facilitate these issues. Each type of trailer has a different security method. The driver, acquiring the license, is obliged to have knowledge in this topic and the translation of the lack of competence is groundless. On the current labor market, entrepreneurs dynamically develop their fleet following the prospect of a potential customer, which results in the transport of various goods. They are looking for specialized drivers with skills, failure to improve qualifications may result in job loss. Load securing is a field of transport that is constantly expanding its range. Demanding from the people involved to create innovative  solutions to emerging problems posed by loads during transport. The guarantee of the  search for the most effective methods of fastening and auxiliary devices is the growing transport and the willingness to transport various loads. To conclude the considerations undertaken in this paper, it should be stated that the common goal of people involved in the transport of goods should be to improve the safety of all road users and to comply with applicable road regulations.