Prevalence and intervention of preoperative anemia in Chinese adults: A retrospective cross-sectional study based on national preoperative anemia database.

BACKGROUND: Preoperative anemia is an important pillar of perioperative patient blood management. However, there was no literature comprehensively described the current situation of preoperative anemia in China. METHODS: We conducted a national retrospective cross-sectional study to assess the prevalence and intervention of preoperative anemia in Chinese adults. Data were from the National Preoperative Anemia Database based on hospital administration data from January 1, 2013 to December 31, 2018. FINDINGS: A total of 797,002 patients were included for analysis. Overall, 27.57% (95% CI 27.47-27.67) of patients had preoperative anemia, which varied by gender, age, regions, and type of operation. Patients who were female, age over 60 years old, from South China, from provinces with lower per capita GDP, underwent operations on the lymphatic and hematopoietic system, with laboratory abnormalities were more likely to have a high risk of preoperative anemia. Among patients with preoperative anemia, 5.16% (95% CI 5.07-5.26) received red blood cell transfusion, 7.79% (95% CI 7.67-7.91) received anemia-related medications such as iron, erythropoietin, folic acid or vitamin B12, and 12.25% (95% CI 12.10-12.40) received anemia-related therapy (red blood cell transfusion or anemia-related medications) before operation. The probability of preoperative RBC transfusion decreased by 54.92% (OR 0.46, 95% CI 0.46-0.47) as each 10-g/L increase in preoperative hemoglobin. Patients with preoperative hemoglobin less than 130 g/L was associated with longer hospital stay and more hospital costs. Patients with severe preoperative anemia given iron preoperatively had lower intra/post-operative RBC transfusion rate, shorter length of stay and less hospitalization costs, but no similar correlation was found in patients with mild and moderate preoperative anemia and patients given erythropoietin preoperatively. INTERPRETATION: Our present study shows that preoperative anemia is currently a relatively prevalent problem that has not been fully appreciated in China. More researches will be required to optimize the treatment of preoperative anemia. FUNDING: National Natural Science Foundation of China and the Logistics Support Department of the Central Military Commission.

[Accumulation of 9α-hydroxy-4-androstene-3,17-dione by co-expressing kshA and kshB encoding component of 3-ketosteroid-9α-hydroxylase in Mycobacterium sp. NRRL B-3805].

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.

The unique evolution of the programmed cell death 4 protein in plants.

BACKGROUND: The programmed cell death 4 (PDCD4) protein is induced in animals during apoptosis and functions to inhibit translation and tumor promoter-induced neoplastic transformation. PDCD4 is composed of two MA3 domains that share similarity with the single MA3 domain present in the eukaryotic translation initiation factor (eIF) 4G, which serves as a scaffold protein to assemble several initiation factors needed for the recruitment of the 40S ribosomal subunit to an mRNA. Although eIF4A is an ATP-dependent RNA helicase that binds the MA3 domain of eIF4G to promote translation initiation, binding of eIF4A to the MA3 domains of PDCD4 inhibits protein synthesis. Genes encoding PDCD4 are present in many lower eukaryotes and in plants, but PDCD4 in higher plants is unique in that it contains four MA3 domains and has been implicated in ethylene signaling and abiotic stress responses. Here, we examine the evolution of PDCD4 in plants. RESULTS: In older algal lineages, PDCD4 contains two MA3 domains similar to the homolog in animals. By the appearance of early land plants, however, PDCD4 is composed of four MA3 domains which likely is the result of a duplication of the two MA3 domain form of the protein. Evidence from fresh water algae, from which land plants evolved, suggests that the duplication event occurred prior to the colonization of land. PDCD4 in more recently evolved chlorophytes also contains four MA3 domains but this may have resulted from an independent duplication event. Expansion and divergence of the PDCD4 gene family occurred during land plant evolution with the appearance of a distinct gene member following the evolution of basal angiosperms. CONCLUSIONS: The appearance of a unique form of PDCD4 in plants correlates with the appearance of components of the ethylene signaling pathway, suggesting that it may represent the adaptation of an existing protein involved in programmed cell death to one that functions in abiotic stress responses through hormone signaling.

Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively.

The eukaryotic translation initiation factor (eIF) 4G functions as a scaffold protein that assembles components of the translation initiation complex required to recruit the 40S ribosomal subunit to an mRNA. Although many eukaryotes express two highly similar eIF4G isoforms, those in plants are highly divergent in size and sequence from one another and are referred to as eIF4G and eIFiso4G. Although the domain organization of eIFiso4G differs substantially from eIF4G orthologs in other species, the domain organization of plant eIF4G is largely unknown despite the fact that it is more similar in size and sequence to eIF4G of other eukaryotes. In this study, we show that eIF4G differs from eIFiso4G in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively.

Competitive and noncompetitive binding of eIF4B, eIF4A, and the poly(A) binding protein to wheat translation initiation factor eIFiso4G.

Eukaryotic translation initiation factor 4G (eIF4G) functions to organize the assembly of initiation factors required for the recruitment of a 40S ribosomal subunit to an mRNA and for interacting with the poly(A) binding protein (PABP). Many eukaryotes express two highly similar eIF4G isoforms. eIFiso4G, one of two isoforms in plants, is highly divergent and unusually small in size. Unlike animal and yeast eIF4G, the domain organization of plant eIF4G proteins is largely unknown. Consequently, little is known about the conservation of plant eIF4G with those in other eukaryotes. In this study, we show that eIFiso4G is similar to other eIF4G proteins in that there are interaction domains for eIF4A and PABP and we identify, for the first time, the interaction domain for eIF4B. In contrast to previous reports, two eIF4A binding domains in eIFiso4G were identified, similar in number and organization to those of animal eIF4G. The eIFiso4G domain organization does differ, however, in that the N-terminal eIF4A binding domain overlaps with the eIF4B and PABP binding domains. Moreover, the eIF4B and PABP binding domains overlap. PABP and eIF4B compete with eIF4A for binding eIFiso4G in the absence of the C-terminal eIF4A binding domain but not when both eIF4A binding domains are present, suggesting that the C-terminal eIF4A interaction domain functions to stabilize the association of eIF4A with eIFiso4G in the presence of eIF4B or PABP. Competitive binding to eIFiso4G was also observed between eIF4B and PABP. These observations reveal an important function of the C-terminal eIF4A binding domain in maintaining the interaction of multiple partner proteins with eIFiso4G despite the substantial divergence in its size and domain organization.

Translation initiation factor 4B homodimerization, RNA binding, and interaction with Poly(A)-binding protein are enhanced by zinc.

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. eIF4B also helps to organize the assembly of the translational machinery through its interactions with eIF4A, eIF4G, eIF3, the poly(A)-binding protein (PABP), and RNA. Although the function of eIF4B is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. Mammalian eIF4B is a constitutive dimer; however, conflicting reports have suggested that plant eIF4B may exist as a monomer or a dimer. In this study, we show that eIF4B from wheat can form a dimer and we identify the region responsible for its dimerization. Zinc stimulated homodimerization of eIF4B and bound eIF4B with a Kd of 19.7 nM. Zinc increased the activity of the eIF4B C-terminal RNA-binding domain specifically. Zinc promoted the interaction between eIF4B and PABP but not the interaction between eIF4B and eIF4A or eIFiso4G, demonstrating that the effect of zinc was highly specific. The interaction between PABP and eIFiso4G was also stimulated by zinc but required significantly higher levels of zinc. Interestingly zinc abolished the ability of eIFiso4G to compete with eIF4B in binding to their overlapping binding sites in PABP by preferentially promoting the interaction between eIF4B and PABP. Our observations suggest that wheat eIF4B can dimerize but requires zinc. Moreover zinc controls the partner protein selection of PABP such that the interaction with eIF4B is preferred over eIFiso4G.

Exploring the mechanism of selective noncovalent adduct protein probing mass spectrometry utilizing site-directed mutagenesis to examine ubiquitin.

Mass spectrometry (MS) is emerging as an additional tool for examining protein structure by way of experiments where structurally related mass changes induced in solution are subsequently detected in the gas phase. Selective noncovalent adduct protein probing (SNAPP) is a recent addition to this type of experiment. SNAPP utilizes noncovalent recognition of lysine residues with 18-crown-6 (18C6) to monitor changes in protein structure. It has been observed that the number of 18C6 adducts that attach to a protein is a function of the structure of the protein. The present work seeks to examine the underlying chemistry which controls the differential attachment of 18C6 to lysine by using ubiquitin as a model system. Ubiquitin is a small protein with a structure that has been well characterized by multiple techniques. Site-directed mutagenesis was used to create a series of ubiquitin mutants where the lysine residues were exchanged for asparagine one at a time. These mutants were then evaluated by SNAPP-MS to determine the relative contribution of each lysine as a binding site for 18C6. It was found that attachment of 18C6 is largely controlled by the strength of intramolecular interactions involving lysine residues. Salt bridges provide the greatest interference, followed by hydrogen bonds. In addition to determining the mechanism for SNAPP, insights are provided about the structure of ubiquitin including confirmation of the existence of two dynamic states for the native structure. These results are discussed in relation to the biological functions of ubiquitin.

eIF4G, eIFiso4G, and eIF4B bind the poly(A)-binding protein through overlapping sites within the RNA recognition motif domains.

The poly(A)-binding protein (PABP), a protein that contains four conserved RNA recognition motifs (RRM1-4) and a C-terminal domain, is expressed throughout the eukaryotic kingdom and promotes translation through physical and functional interactions with eukaryotic initiation factor (eIF) 4G and eIF4B. Two highly divergent isoforms of eIF4G, known as eIF4G and eIFiso4G, are expressed in plants. As little is known about how PABP can interact with RNA and three distinct translation initiation factors in plants, the RNA binding specificity and organization of the protein interaction domains in wheat PABP was investigated. Wheat PABP differs from animal PABP in that its RRM1 does not bind RNA as an individual domain and that RRM 2, 3, and 4 exhibit different RNA binding specificities to non-poly(A) sequences. The PABP interaction domains for eIF4G and eIFiso4G were distinct despite the functional similarity between the eIF4G proteins. A single interaction domain for eIF4G is present in the RRM1 of PABP, whereas eIFiso4G interacts at two sites, i.e. one within RRM1-2 and the second within RRM3-4. The eIFiso4G binding site in RRM1-2 mapped to a 36-amino acid region encompassing the C-terminal end of RRM1, the linker region, and the N-terminal end of RRM2, whereas the second site in RRM3-4 was more complex. A single interaction domain for eIF4B is present within a 32-amino acid region representing the C-terminal end of RRM1 of PABP that overlaps with the N-proximal eIFiso4G interaction domain. eIF4B and eIFiso4G exhibited competitive binding to PABP, supporting the overlapping nature of their interaction domains. These results support the notion that eIF4G, eIFiso4G, and eIF4B interact with distinct molecules of PABP to increase the stability of the interaction between the termini of an mRNA.

Wheat eukaryotic initiation factor 4B organizes assembly of RNA and eIFiso4G, eIF4A, and poly(A)-binding protein.

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. Although this function is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. To gain insight into its functional conservation, the organization of the functional domains of eIF4B from wheat has been investigated. Plant eIF4B contains three RNA binding domains, one more than reported for mammalian or yeast eIF4B, and each domain exhibits a preference for purine-rich RNA. In addition to a conserved RNA recognition motif and a C-terminal RNA binding domain, wheat eIF4B contains a novel N-terminal RNA binding domain that requires a short, lysine-rich containing sequence. Both the lysine-rich motif and an adjacent, C-proximal motif are conserved with an N-proximal sequence in human and yeast eIF4B. The C-proximal motif within the N-terminal RNA binding domain in wheat eIF4B is required for interaction with eIFiso4G, an interaction not reported for other eIF4B proteins. Moreover, each RNA binding domain requires dimerization for binding activity. Two binding sites for the poly(A)-binding protein were mapped to a region within each of two conserved 41-amino acid repeat domains on either side of the C-terminal RNA binding domain. eIF4A bound to an adjacent region within each repeat, supporting a central role for these conserved eIF4B domains in facilitating interaction with other components of the translational machinery. These results support the notion that eIF4B functions by organizing multiple components of the translation initiation machinery and RNA.

Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene.

A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5' upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of alpha-globulin.