Measurement of luteinizing hormone surge in vaginal discharge: a potential biomarker that enables simple, non-invasive prediction of the periovulatory period.

BACKGROUND: Predicting the periovulatory period is very important for conception. Current approaches to predicting the periovulatory period include monitoring of basal body temperature and urine luteinizing hormone (LH) concentration; however, these methods are time-consuming. Here, we examined the potential of using vaginal discharge (VD) as a non-invasive means of sample collection for determining the LH surge that indicates ovulation. METHODS: Urine and VD samples were collected from 35 healthy women aged 20-39 years. VD samples were collected with panty liners to reduce the burden on participants. Daily first urine samples and used panty liners were collected from the 10th through 19th days of the menstrual cycle. Urine and VD LH (uLH and vLH) levels in the samples were measured by enzyme-linked immunosorbent assay. Measured vLH baseline and first surge values were analyzed using Student's t-test and ROC curves. RESULTS: Samples for a total of 55 menstrual cycles were collected. We used uLH surge to establish the date of ovulation. uLH surges were observed in 49 cycles, 34 of which had corresponding VD samples that qualified for measurement. Five cycles were excluded due to a lack of vLH data. In the remaining 29 cycles, the vLH surge appeared within the fertile window 90% of the time, and the sensitivity and specificity of the test were 86% and 83%, respectively. CONCLUSIONS: VD has potential for use as a sample for predicting the periovulatory period by measuring LH content.

Rationalizing the Influence of the Binding Affinity on the Activity of Phosphoserine Phosphatases.

The Sabatier principle states that catalytic activity can be maximized when the substrate binding affinity is neither too strong nor too weak. Recent studies have shown that the activity of several hydrolases is maximized at intermediate values of the binding affinity (Michaelis-Menten constant: Km ). However, it remains unclear whether this concept of artificial catalysis is applicable to enzymes in general, especially for those which have evolved under different reaction environments. Herein, we show that the activity of phosphoserine phosphatase is also enhanced at an intermediate Km value of approximately 0.5 mM. Within our dataset, the variation of Km by three orders of magnitude accounted for a roughly 18-fold variation in the activity. Owing to the high phylogenetic and physiological diversity of our dataset, our results support the importance of optimizing Km for enzymes in general. On the other hand, a 77-fold variation in the activity was attributed to other physicochemical parameters, such as the Arrhenius prefactor of kcat , and could not be explained by the Sabatier principle. Therefore, while tuning the binding affinity according to the Sabatier principle is an important consideration, the Km value is only one of many physicochemical parameters which must be optimized to maximize enzymatic activity.

[Digital Breast Tomosynthesis Quality Control Manual Overview].

Recently, mammography systems equipped with digital breast tomosynthesis (DBT) have become widely used in Japan. Therefore, it is urgently necessary to establish a quality control method for DBTs. So far, we have been studying acceptance tests for DBTs with reference to EUREF. In 2020, IEC 61223-3-6 was published, which provides not only acceptance tests but also constancy test methods. Therefore, we conducted data collection using DBTs sold in Japan and examined the feasibility of conducting constancy tests. Although there were some items that were difficult to implement in each device, we were able to confirm quality control items that could be implemented in many devices. In addition, we were able to confirm routine tests that enable rapid evaluation. Based on these results, we have developed a "Digital Breast Tomosynthesis Quality Control Manual". In this paper, we report an overview of the manual and the results of routine tests.

Thermodynamic principle to enhance enzymatic activity using the substrate affinity.

Understanding how to tune enzymatic activity is important not only for biotechnological applications, but also to elucidate the basic principles guiding the design and optimization of biological systems in nature. So far, the Michaelis-Menten equation has provided a fundamental framework of enzymatic activity. However, there is still no concrete guideline on how the parameters should be optimized towards higher activity. Here, we demonstrate that tuning the Michaelis-Menten constant ([Formula: see text]) to the substrate concentration ([Formula: see text]) enhances enzymatic activity. This guideline ([Formula: see text]) was obtained mathematically by assuming that thermodynamically favorable reactions have higher rate constants, and that the total driving force is fixed. Due to the generality of these thermodynamic considerations, we propose [Formula: see text] as a general concept to enhance enzymatic activity. Our bioinformatic analysis reveals that the [Formula: see text] and in vivo substrate concentrations are consistent across a dataset of approximately 1000 enzymes, suggesting that even natural selection follows the principle [Formula: see text].

Care provided by midwives and the unmet needs of pregnant and postpartum women: A qualitative study of Japanese mothers.

Objectives: We aimed to clarify the content of care provided by midwives working in hospitals and clinics in Japan and the unmet needs in midwifery care from mothers' perspectives. Design: This study employed a qualitative approach through semi-structured interviews. Setting: Fifteen Japanese women, whose youngest singleton children were aged 12-18 months, were asked to recall their experiences with midwives, from pregnancy through the first postpartum year. Verbatim records were analyzed using thematic analysis. Results: Seven themes regarding the care provided by midwives were generated: confirmation of physical condition, maintenance and promotion of perinatal physiological process, support for better preparation for childbirth, assistance in labour and childbirth, support for a new life with a baby at home, support for the family, and care for comfort and confidence as a mother. Unmet needs were identified in all themes, except for 'confirmation of physical condition' and 'support for the family'. Ten subthemes, under the five themes of unmet needs, were integrated into three categories: midwives' responses to potential concerns, lack of continuity of care, and lack of personalised care. Key conclusions and implications for practice: Midwives in hospitals and clinics in Japan mainly provided care from pregnancy to one-month postpartum, in line with global core competencies. However, they could respond more effectively to the potential concerns of women, and provide continuous, personalised care more sufficiently. Improving working environments for midwives and collaborating with postpartum public health services are key to addressing these unmet needs of women, leading to women-centred care.

Development of University of Tokyo's eating disorders inventory in female athletes.

AIMS: This study aimed to develop a scale to screen for eating disorders in female athletes. METHODS: Preliminary survey: A total of 275 female athletes (mean age: 19.4 ± 1.0 years) and 7 female athletes diagnosed with eating disorders (mean age: 20.1 ± 2.5 years) were administered screening items prepared based on an existing scale, followed by exploratory factor analysis. Main survey: Six items, relating to three factors, were extracted, and 201 female athletes (mean age: 22.3 ± 4.8 years) and 6 female athletes diagnosed with current or a history of eating disorders (mean age: 18.8 ± 2.9 years) were queried. The diagnostic validity of the scale was then evaluated. RESULTS: Preliminary survey: Questions (α = 0.71) were extracted from six items, relating to three factors, and collectively termed the University of Tokyo's eating disorders inventory in female athletes (TEDIFA). To determine the scale cut-off score, ROC analysis was performed with the total score, and the cut-off and gray zone scores were set at 13 and 11, respectively. Main survey: At the cut-off score of 13, AUC, sensitivity, and specificity were 0.83 (p < 0.05), 75%, and 90%, respectively. CONCLUSIONS: The scale that was developed, TEDIFA, consisted of six items. The cut-off scores were set at 11 for the gray zone (sensitivity: 75%; specificity: 56%; accurate diagnosis rate: 60%), and 13 for positivity (sensitivity: 75%; specificity: 90%; accurate diagnosis rate: 87%), demonstrating the reliability and validity of the scale.

Urinary Biopyrrin Levels and Their Relationship with the Menstrual Cycle and Concomitant Symptoms Among Healthy Nonpregnant Women of Reproductive Age: A Cohort Study.

Background: Urinary biopyrrin (UBP) is an oxidative metabolite formed from the reaction of bilirubin with reactive oxygen species. Previous studies have explored the relationship between UBP levels and certain diseases or pregnancy. However, UBP levels in healthy nonpregnant women have not been well examined. We aimed to clarify the representative value of UBP in healthy nonpregnant women and explore its relationship with menstrual cycles and concomitant symptoms. Methods: We included healthy, nonpregnant Japanese women aged 20-39 years with normal body mass index and menstrual cycle. In total, 1260 urine samples collected during 43 menstrual cycles of 36 women were analyzed to determine the representative values and reference intervals of UBP levels. The correlation between daily UBP levels and the order of the day was explored, and median UBP levels of 5-day clusters were compared using Friedman and Mann-Whitney U tests. These analyses were also conducted in women with concomitant symptoms during the menstrual cycle. Results: The median UBP level in all samples was 0.2291 (reference: 0.0102-2.9335) µmol/gCr. There was no significant relationship between the median UBP level and menstrual cycle, regardless of the presence of self-manageable symptoms during or before menstruation. Conclusions: The representative UBP value and its reference interval can serve as standards for comparison with other populations. Our findings suggest that the UBP level may be an objective oxidative stress indicator that is less sensitive to menstrual cycle and concomitant symptoms. UBP levels in healthy nonpregnant women could be assessed regardless of the menstrual cycle and concomitant symptoms.

[Examination of the Quality Control Items for Digital Breast Tomosynthesis System in Japan].

Mammography equipment attached to the digital breast tomosynthesis (DBT) system is widespread in Japan. However, there are no guidelines for quality control methods for DBT in Japan. Therefore, it is necessary to rapidly establish a performance evaluation procedure and a quality control procedure for DBT. In this study, we conducted basic experiments using DBTs of five companies (Canon Medical, Fujifilm Medical, GE Healthcare, Hologic, Siemens) already sold in Japan and examined feasible common items. We aimed to establish a quality control method for DBT in Japan. The measurement was performed based on the European Reference Organisation for Quality Assured Breast Screening and Diagnostic Services (EUREF) breast tomosynthesis quality control protocol, version 1.03. In this study, we tried to measure 18 items in DBT. We examined whether the 18 items could be measured using each device; it is not an evaluation of device performance based on the measured values. There were some management items that were difficult to implement due to the specifications of DBT, such as devices that required pressure on DBT operation, problems due to the shape of bucky, and devices that did not have stationary mode. There were also problems with measurement data; for example, devices could not retrieve projection data and reconstruction data. This study clarified points to be considered for establishing common quality control items. In the future, we will carefully refer to the recently published IEC 61223-3-6, consider international harmonization, and establish DBT guidelines customized for the Japanese market.

Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences.

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.

Fragmentation of acetate-CoA ligase gives a clue to understand domain rearrangement history of NDP-forming acyl-CoA synthetase superfamily proteins.

NDP-forming type acyl-CoA synthetase superfamily proteins are known to have six essential subdomains (1, 2, 3, a, b, c) of which partition and order are varied, suggesting yet-to-be-defined subdomain rearrangement happened in its evolution. Comparison in physicochemical and biochemical characteristics between the recombinant proteins which we made from fragmented subdomains and wild-type protein, acetate-CoA ligase in a hyperthermophilic archaeon, consisting of two distinct subunits (α1-2-3 and ßa-b-c) provided a clue to the mystery of its molecular evolutionary passage. Although solubility and thermostability of each fragmented subdomain turned out to be lower than that of wild-type, mixture of the three synthetic subunits of α1-2, α3, and ßa-b-c had quaternary structure, thermostability, and enzymatic activity comparable to those of the wild-type. This suggests that substantial independence and mobility of subdomain 3 have enabled rearrangement of the subdomains; and thermostability of the subdomains has constrained the composition of the subunits.

Structural comparisons of phosphoenolpyruvate carboxykinases reveal the evolutionary trajectories of these phosphodiester energy conversion enzymes.

Inorganic pyrophosphate (PPi) consists of two phosphate molecules and can act as an energy and phosphate donor in cellular reactions, similar to ATP. Several kinases use PPi as a substrate, and these kinases have recently been suggested to have evolved from ATP-dependent functional homologs, which have significant amino acid sequence similarity to PPi-utilizing enzymes. In contrast, phosphoenolpyruvate carboxykinase (PEPCK) can be divided into three types according to the phosphate donor (ATP, GTP, or PPi), and the amino acid sequence similarity of these PEPCKs is too low to confirm that they share a common ancestor. Here we solved the crystal structure of a PPi-PEPCK homolog from the bacterium Actinomyces israelii at 2.6 Å resolution and compared it with previously reported structures from ATP- and GTP-specific PEPCKs to assess the degrees of similarities and divergences among these PEPCKs. These comparisons revealed that they share a tertiary structure with significant value and that amino acid residues directly contributing to substrate recognition, except for those that recognize purine moieties, are conserved. Furthermore, the order of secondary structural elements between PPi-, ATP-, and GTP-specific PEPCKs was strictly conserved. The structure-based comparisons of the three PEPCK types provide key insights into the structural basis of PPi specificity and suggest that all of these PEPCKs are derived from a common ancestor.

Discovery and analysis of a novel type of the serine biosynthetic enzyme phosphoserine phosphatase in Thermus thermophilus.

Studying the diversity of extant metabolisms and enzymes, especially those involved in the biosynthesis of primary metabolites including amino acids, is important to shed light on the evolution of life. Many organisms synthesize serine from phosphoserine via a reaction catalyzed by phosphoserine phosphatase (PSP). Two types of PSP, belonging to distinct protein superfamilies, have been reported. Genomic analyses have revealed that the thermophilic bacterium Thermus thermophilus lacks both homologs while still having the ability to synthesize serine. Here, we purified a protein from T. thermophilus which we biochemically identified as a PSP. A knockout mutant of the responsible gene (TT_C1695) was constructed, which showed serine auxotrophy. These results indicated the involvement of this gene in serine biosynthesis in T. thermophilus. TT_C1695 was originally annotated as a protein with unknown function belonging to the haloacid dehalogenase-like hydrolase (HAD) superfamily. The HAD superfamily, which comprises phosphatases against a variety of substrates, includes also the classical PSP as a member. However, the amino acid sequence of the TT_C1695 was more similar to phosphatases acting on non-phosphoserine substrates than classical PSP; therefore, a BLASTP search and phylogenetic analysis failed to predict TT_C1695 as a PSP. Our results strongly suggest that the T. thermophilus PSP and classical PSP evolved specificity for phosphoserine independently. ENZYMES: Phosphoserine phosphatase (PSP; EC 3.1.3.3); serine hydroxymethyltransferase (EC 2.1.2.1); 3-phosphoglycerate dehydrogenase (EC 1.1.1.95); 3-phosphoserine aminotransferase (EC 2.6.1.52).

Phosphoserine Phosphatase Is Required for Serine and One-Carbon Unit Synthesis in Hydrogenobacter thermophilus.

Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilusIMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.

Heterogeneity of the serine synthetic pathway in Entamoeba species.

Phosphoserine phosphatase (PSP) catalyzes the third step of the phosphorylated serine biosynthetic pathway, and occurred multiple times in evolution, while enzymes catalyzing the first and second steps in the pathway have single respective origins. In the present study, we examined the existence of PSP among genus Entamoeba including a human enteric parasite, Entamoeba histolytica. E. histolytica as well as majority of Entamoeba species have the first and second enzymes, but lacks PSP. In contrast, a reptilian enteric parasite, Entamoeba invadens possesses canonical PSP. Thus, there are variations in the existence of the serine biosynthetic ability among Entamoeba species.

Cultural immersion through international experiences among Japanese nurses: Present status, future intentions, and perceived barriers.

AIM: Given limited exposure to various ethnicities, languages, and cultures, providing health care to an increasing foreign population in Japan will likely be challenging for Japanese nurses. This study aimed to examine past and intended future international experiences of Japanese nurses to assess their cultural immersion level. METHODS: A cross-sectional electronic survey was conducted among 2029 nurses in 2010. Participants were categorized by travel purpose, and the frequency of non-holiday travel was analyzed. To examine participants' desire for and perceived feasibility of future non-holiday international experiences by background characteristics, logistic regression analyses were performed. RESULTS: Of 1039 participants, 10.1% had past non-holiday international experiences, with 80% having traveled to high-income, English-speaking countries. The median value for travel frequency was once, and the median duration of travel was less than 1 month. The most common purpose of travel was participation in short-term programs (e.g. professional training, language study). Fifty-one percent of female nurses reported a desire for future non-holiday international experiences. Of these, 37.2% considered such experiences feasible. Age of the youngest child, having nursing specialization, English proficiency, and past international experience were significant predictors for feasibility. CONCLUSION: Japanese nurses with foreign experience were considered valuable human resources for culturally appropriate care. Efforts should be made to integrate them into the Japanese healthcare setting. The present study revealed room for improvement in foreign language proficiency and cross-cultural training with a focus on non-English-speaking and developing countries. A supportive workplace environment should be created that allows nurses to pursue the international experiences they desire.

Discovery of an intermolecular disulfide bond required for the thermostability of a heterodimeric protein from the thermophile Hydrogenobacter thermophilus.

Factors that increase protein thermostability are of considerable interest in both scientific and industrial fields. Disulfide bonds are one of such factors that increase thermostability, but are rarely found in intracellular proteins because of the reducing environment of the cytosol. Here, we report the first example of an intermolecular disulfide bond between heteromeric subunits of a novel-type phosphoserine phosphatase from a thermophilic bacterium Hydrogenobacter thermophilus, which contributes to the protein thermostability at the physiological temperature. Comparison of remaining soluble proteins between wild-type and cysteine-deleted mutant using SDS-PAGE revealed that the disulfide bond increases the thermostability of the whole protein by tightly connecting a subunit with low solubility to the partner with higher solubility. Furthermore, it was strongly suggested that the disulfide bond is formed and contributes to the stability in vivo. This finding will open new avenues for the design of proteins with increased thermostability.

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria.

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.

Elevated serum levels of neutrophil elastase in patients with influenza virus-associated encephalopathy.

We examined serum levels of various cytokines, chemokines, growth factors, and adhesion molecules in patients with uncomplicated influenza (n=20) and influenza virus-associated encephalopathy (IE) (n=18) to understand the underlying mechanism of IE. We found that IL-1ß, IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, G-CSF, GM-CSF, TNF-α, TIMP-1, MMP-9, sE-selectin, and neutrophil elastase were elevated significantly in sera from patients with uncomplicated influenza and those with IE, compared with normal controls (n=20). Of note, neutrophil elastase, sE-selectin, IL-8, and IL-13 were elevated significantly in IE as compared with uncomplicated influenza. In the present study, for the first time, we found that serum levels of neutrophil elastase were increased in patients with IE compared with uncomplicated influenza, which suggested that cerebral endothelial damage in the development of IE was mediated by neutrophil elastase. The present study implied that anti-elastase agents are possibly an effective therapeutic protocol for IE, but this needs further elucidation.

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2-GST-sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses. BIOLOGICAL SIGNIFICANCE: Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities. Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite. We have found numerous new splicing-related factors which could have therapeutic benefit. We also detected all the DExH/A RNA helicases involved in splicing and splicing proofreading control. Still, Entamoeba is very inefficient in splicing fidelity, thus we may have found a possible model system to study these processes.