Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System.

Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii.

Construction of Fe Nanoparticles Interfacial Layer on Micron Al Surface: Boosting the Efficient Energy Release of High-Energy DAP-4 as a Gradient Catalyst.

The high-energy (H2dabco)[NH4(ClO4)3] (DAP-4) with excellent energetic performance attracts wide attention from researchers. The investigation of its interaction with the Aluminum (Al) is of great importance. However, the higher ignition threshold of DAP-4 and the dense oxide layer (Al2O3) of Al severely limit the energy release efficiency of Al/DAP-4. In this study, a new idea to is first proposed to improve and adjust the thermal decomposition and combustion performance of Al/DAP-4 by constructing a highly dispersed iron (Fe) nanoparticle interfacial layer. It acts as a gradient catalyst to promote the thermal decomposition and combustion of DAP-4 and Al, and it also act as an oxygen transport channel to promote the contact and reaction of oxidizing gases with the internal reactive Al powder. It reduces the thermal decomposition temperature of Al@Fe-3/DAP-4 from 386.30 °C (Al/DAP-4) to 349.48 °C and leads to the vigorous combustion. Theoretical calculations show that Fe nanoparticle interfacial layer can facilitate the transport of oxygen through the established oxygen transport channels, and it can also significantly improve the energetic properties of Al@Fe-3/DAP-4 composites. In conclusion, the new approach is proposed to improve the performance of metal fuel/oxidizer composites by constructing interfacial layers, which is expected to promote their practical applications.

Differential responses of soil bacteria, fungi and protists to root exudates and temperature.

The impact of climate warming on soil microbes has been well documented, with studies revealing its effects on diversity, community structure and network dynamics. However, the consistency of soil microbial community assembly, particularly in response to diverse plant root exudates under varying temperature conditions, remains an unresolved issue. To address this issue, we employed a growth chamber to integrate temperature and root exudates in a controlled experiment to examine the response of soil bacteria, fungi, and protists. Our findings revealed that temperature independently regulated microbial diversity, with distinct patterns observed among bacteria, fungi, and protists. Both root exudates and temperature significantly influenced microbial community composition, yet interpretations of these factors varied among prokaryotes and eukaryotes. In addition to phototrophic bacteria and protists, as well as protistan consumers, root exudates determined to varying degrees the enrichment of other microbial functional guilds at specific temperatures. The effects of temperature and root exudates on microbial co-occurrence patterns were interdependent; root exudates primarily simplified the network at low and high temperatures, while responses to temperature varied between single and mixed exudate treatments. Moreover, temperature altered the composition of keystone species within the microbial network, while root exudates led to a decrease in their number. These results emphasize the substantial impact of plant root exudates on soil microbial community responses to temperature, underscoring the necessity for future climate change research to incorporate additional environmental variables.

The splicing factor SR2 is an important virulence factor of Toxoplasma gondii.

Serine/arginine-rich (SR) proteins are key factors with important roles in constitutive and alternative splicing (AS) of pre-mRNAs. However, the role of SR splicing factors in the pathogenicity of T. gondii remains largely unexplored. Here, we investigated the role of splicing factor SR2, a homolog of Plasmodium falciparum SR1, in the pathogenicity of T. gondii. We functionally characterized the predicted SR2 in T. gondii by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The results showed that SR2 was localized in the nucleus and expressed in the tachyzoite and bradyzoite stages. In vitro studies including plaque formation, invasion, intracellular replication, egress and bradyzoite differentiation assays showed that deletion of SR2 in type I RH strain and type II Pru strains had no significant effect on the parasite growth and bradyzoite differentiation (p > 0.05). Interestingly, the disruption of SR2 in RH type I (p < 0.0001) and Pru type II (p < 0.05) strains resulted in varying degrees of attenuated virulence. In addition, disruption of SR2 in type II Pru strain significantly reduced brain cyst burden by ~80% (p < 0.0001). Collectively, these results suggest that splicing factor SR2 is important for the pathogenicity of T. gondii, providing a new target for the control and treatment of toxoplasmosis.

Correlation of Efficient Dispersion and Catalytic Performance of Nanocombustion Catalysts in Energetic Materials: A Case Study of the Nanocopper Oxide Catalyst with Superfine Ammonium Perchlorate.

Obviously, the dispersion of nanocatalytic materials has significant influence on their catalytic performance. In this study, an evaluation method for the dispersion of nanomaterials was established according to the different solid UV absorptions of different substances by taking the dispersion of nanocopper oxide (nano-CuO) in superfine ammonium perchlorate (AP) as an example. The nano-CuO/superfine AP composites with different nano-CuO dispersions can be obtained by changing the process parameters, such as varying the grinding method, the grinding strength, and the grinding time. Three replicate experiments were carried out for different composites to derive the average values of absorbance at 212 nm, and the dispersion of nano-CuO in superfine AP was calculated using the difference equation, as the solid UV curves at 210-214 nm were almost identical for each sample, especially at 212 nm. The properties of different samples were tested by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (IR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), differential scanning calorimetry (DSC), and thermogravimetry-mass spectrometry (TG-MS). The results show that the particle size and structure of superfine AP in the composites prepared by different methods were not changed. The XRD and IR techniques in this study were unable to characterize the dispersion of nano-CuO in the composites due to its low content. The dispersion of nano-CuO in the nano-CuO/superfine AP composites was significantly enhanced with the increase of grinding strength and grinding time, and the dispersion of nano-CuO was positively correlated with its catalytic performance, which means that the thermal decomposition performance of different composites improved with the increasing dispersion of nano-CuO. Highly dispersed nano-CuO exhibited a significant catalytic effect on superfine AP in TG-MS. The above conclusions demonstrate the accuracy of the difference equation for evaluating the dispersion of nanomaterials based on solid UV curves, which is expected to be used extensively in evaluating the dispersion of nanocatalytic materials in energetic materials.

Modeling the dynamic behavior of manipulator joint based on giant magnetostrictive material.

The existing space manipulator joints with permanent magnet synchronous motors (PMSMs) have many deficiencies, such as large size, low output torque, and long settling time. In this paper, the dynamic behavior of space manipulators with smart giant magnetostrictive material (GMM) joints is studied. A nonlinear dynamic model considering the magneto-thermal-mechanical coupling for the manipulator is established, and an experiment is conducted to evaluate the performance of the GMM joint. After verifying the accuracy and availability of the model, we find that comparing with the PMSM joint, manipulators with the GMM joint have better performance, which has large output torque, fast settling time, and wide temperature adaptation range. These essential investigations will be of significant benefit to the applications in space manipulators.

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System.

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.

The positive correlation between the dispersion and catalytic performance of Fe2O3 nanoparticles in nano-Fe2O3-ultrafine AP energetic composites supported by solid UV-vis spectroscopy.

Recently, the widespread use of nanocatalytic materials has contributed to an enormous improvement in the performance of energetic materials, especially, highly dispersed nanomaterials. However, the lack of quantitative methods for analyzing the dispersion of nanomaterials limits their further widespread use. Although various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), etc. are used to analyze the relative dispersion of nanomaterials, it is not possible to quantitatively analyze their dispersion. Therefore, there has been an effort to develop new methods for the quantitative analysis of nanocatalytic materials. Fortunately, we were able to analyze the dispersion of nanocatalytic materials using the difference in their UV absorbance. More importantly, we established the corresponding difference equation to quantify the dispersion of nanocatalytic materials, which is capable of quantifying the dispersion of nano-Fe2O3 in nano-Fe2O3-ultrafine AP composites. The accuracy of the difference equation was verified using a variety of techniques and the desired results were obtained. Based on the above conclusions, the quantitative analysis method for the dispersion of nanomaterials that we established is expected to be widely used and promote the development of energetic materials.

Synthesis of Covalently Modified Energetic Graphene Oxide/CuO Composites with Enhanced Catalytic Performance for Thermal Decomposition of Ammonium Perchlorate.

In this study, a new covalently modified energetic graphene oxide (CMGO) was synthesized by introducing the energetic component 4-amino-1,2,4-triazole on GO sheets through valence bond bonding. The morphology and structure of CMGO were studied by scanning electron microscopy, energy-dispersive spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffractometry, and X-ray photoelectron spectroscopy, and the results showed that CMGO was successfully synthesized. Then, CMGO/CuO was prepared by loading nano-CuO onto CMGO sheets using an ultrasonic dispersion method. Furthermore, the catalytic effect of CMGO/CuO on the thermal decomposition of ammonium perchlorate (AP) was investigated using differential scanning calorimetric technique and thermogravimetric analysis. The results revealed that the high decomposition temperature TH and Gibbs free energy ΔG⧧ of the CMGO/CuO/AP composite decreased by 93.9 °C and 15.3 kJ/mol compared with those of raw AP, respectively. The CMGO/CuO composite exhibited more significant catalytic effect on the thermal decomposition of AP than GO/CuO, and the heat release Q of CMGO/CuO/AP was greatly increased from 132.9 to 1428.5 J/g with 5 wt % CMGO/CuO. The above results demonstrated that CMGO/CuO is an excellent composite energetic combustion catalyst, which is expected to be widely used in composite propellants.

Synthesis of α-CF3 Amides via Palladium-Catalyzed Carbonylation of 2-Bromo-3,3,3-trifluoropropene.

Amide compounds are important organic compounds, which play an important role in biomedical chemistry, materials science, life science, and other fields. The synthesis of α-CF3 amides, especially compounds containing 3-(trifluoromethyl)-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepine-2-one, has long been a challenge due to the tensile properties and instability of the rings. Here, we report an example of palladium-catalyzed carbonylation of CF3-containing olefin to form α-CF3 acrylamide. By controlling the ligands, we can get different amide compounds as products. This method has good substrate adaptability and functional group tolerance.

[Changes in the structure and function of soil prokaryotic communities in subalpine Picea asperata plantations]. / äºšé«˜å±±ç²—æžäº‘æ‰äººå·¥æž—åœŸå£¤åŽŸæ ¸å¾®ç”Ÿç‰©ç¾¤è½ç»“æž„ä¸ŽåŠŸèƒ½å˜åŒ–.

The structural and functional characteristics of soil prokaryotic community are important for maintaining ecosystem functions. In this study, we examined the diversity and compositions, the key drivers, as well as functional characteristics of prokaryotic communities in the rhizosphere and non-rhizosphere soils of Picea asperata with different stand ages using high-throughput sequencing technique and bioinformatics methods. The results showed that ß-diversity of soil prokaryotic communities in both rhizosphere and non-rhizosphere showed significant differences among different stand ages, but no significant difference between rhizosphere and non-rhizosphere in the same stand age. In terms of community composition at the phylum level, the relative abundances of Proteobacteria and Rokubacteria showed an increasing trend with the increases of stand age, while the relative abundance of Actinobacteria showed a decreasing trend, but no significant difference was observed between 75 year-old planted forests (PF75) and natural forests (NF). The relative abundances of Firmicutes and Thaumarchaeota in the soil of the 25 year-old planted forests (PF25) were significantly higher than in other planted forests and NF. At the genus level, the relative abundances of RB41, Terrimonas and Acidibacter showed an increasing trend with the increases of stand age, and RB41 and Terrimonas in rhizosphere soil of PF75 were significantly higher than those in NF. Soil properties and vegetation characteristics jointly influenced the structure of soil prokaryotic communities, with herb layer coverage, soil pH, total phosphorus, and total nitrogen as major drivers. The functional characteristics of soil prokaryotic communities were significantly different among different stand ages. The relative abundances of functions involved in carbon and nitrogen cycle, e.g., cellulolysis and nitrification, decreased with the increases of stand age, whereas that of sulfate respiration involved in the sulfur cycle increased. We proposed that the structure and functional characteristics of soil prokaryotic communities could serve as important indicators of the development stages of P. asperata forests. In the later stages of plantation forest development, soil nutrient availability could be improved by mediating phosphorus-dissolving and nitrogen-enhancing microorganisms to maintain the stability of the plantation ecosystem.

[Diversity and network features of fungal community in the soils of planted and natural Picea asperata forests.] / äººå·¥ä¸Žå¤©ç„¶äº‘æ‰æž—åœŸå£¤çœŸèŒç¾¤è½å¤šæ ·æ€§åŠèŒç¾¤ç½‘ç»œå ³ç³»ç‰¹å¾.

The diversity and interactions of soil fungal community are the key to maintain the diversity and stability of ecosystem. In this study, we examined the structure, diversity and co-occurrence networks of fungal community in rhizosphere and non-rhizosphere soils of planted and natural Picea asperata forests using high-throughput sequencing technique and bioinformatic methods. The results showed that Inocybaceae and Sebacinaceae were dominant family in soils of planted and natural forests, respectively. At the genus level, Inocybe was dominant one in soils of planted and natural forests. There were significant differences in ß-diversity of fungal communities between rhizosphere and non-rhizosphere soils in both planted and natural forests. There were no significant correlations between environmental variables and the relative abundance and α-diversity of fungal communities. Herb layer coverage, soil water content, total organic carbon concentration, and plant species richness played important roles in explaining the variations of ß-diversity of fungal communities. Results of the network analysis showed that the negative correlations were dominant among soil fungal communities in natural forest, suggesting that the competition of different groups in natural forest. Moreover, there were more negative correlations in non-rhizosphere soils than in rhizosphere soils, which indicated that fungal communities in non-rhizosphere soils comprised more competitive network structure than in the rhizosphere soils. Biomarker species were identified based on differential abundance analysis. Sebacinaceae was the single shared keystone species in the fungal network which had significant differences among rhizosphere and non-rhizosphere soils of planted and natural forests. Therefore, it is suggested that the variation of differential species in the soil fungal communities between the planted and natural forest might had limited influence on the stability of the community of planted and natural forests.

[Major Ionic Features and Their Possible Controls in the Surface Water and Groundwater of the Jinghe River].

To study the hydrochemical characteristics of tributaries of the Jinghe River (Heihe River Basin) and their controlling factors, from 2014 to 2015, 242 samples of surface water and groundwater were collected during the dry and flood seasons from the Jinghe River. After determining the main water chemical ion content by comprehensively using correlation analysis, Piper three-line graphs, and Gibbs graph, the spatiotemporal changes in the hydrochemical characteristics of the Heihe River Basin were analyzed. The results showed that all the water samples in the study area were weakly alkaline. In the dry season of 2014, the cations in the surface water and groundwater were mostly Na+, accounting for 56% and 58% of the total cations, respectively. The anions were mainly SO42-, accounting for 33% and 39% of the total anions, respectively. In the other three periods, the main cation and anion components were HCO3- and Na+, accounting for 44%-46% and 42%-56% of the corresponding totals, respectively. In the dry season, the TDS of surface water and groundwater gradually increases along the river from upstream to downstream. From the dry season to the flood season, the hydrochemical types of the surface water changed from Na-Mg-Cl-SO4 to Ca-Mg-HCO3 and of the groundwater from Mg-Cl-SO4 to Ca-Na-HCO3. Most of the water chemistry samples were distributed in the middle and upper parts of the Gibbs diagram, indicating that the formation of hydrated chemical ions in the basin was mainly affected by rock weathering and evaporation-enrichment. However, the effects of human activities on water chemistry were more significant in the dry season than in the flood season.

Enzymatic changes in myosin regulatory proteins may explain vasoplegia in terminally ill patients with sepsis.

The current study was conducted with the hypothesis that failure of maintenance of the vascular tone may be central to failure of the peripheral circulation and spiralling down of blood pressure in sepsis. Namely, we examined the balance between expression of myosin light chain (MLC) phosphatase and kinase, enzymes that regulate MLCs dephosphorylation and phosphorylation with a direct effect on pharmacomechanical coupling for smooth muscle relaxation and contraction respectively. Mechanical recordings and enzyme immunoassays of vascular smooth muscle lysates were used as the major methods to examine arterial biopsy samples from terminally ill sepsis patients. The results of the present study provide evidence that genomic alteration of expression of key regulatory proteins in vascular smooth muscles may be responsible for the relentless downhill course in sepsis. Down-regulation of myosin light chain kinase (MLCK) and up-regulation of MLCK may explain the loss of tone and failure to mount contractile response in vivo during circulation. The mechanical studies demonstrated the inability of the arteries to develop tone when stimulated by phenylephrine in vitro. The results of our study provide indirect hint that control of inflammation is a major therapeutic approach in sepsis, and may facilitate to ameliorate the progressive cardiovascular collapse.

Reproductive isolation and pollination success of rewarding Galearis diantha and non-rewarding Ponerorchis chusua (Orchidaceae).

BACKGROUND AND AIMS: Increasing evidence challenges the conventional perception that orchids are the most distinct example of floral diversification due to floral or prezygotic isolation. Regarding the relationship between co-flowering plants, rewarding and non-rewarding orchids in particular, few studies have investigated whether non-rewarding plants affect the pollination success of rewarding plants. Here, floral isolation and mutual effects between the rewarding orchid Galearis diantha and the non-rewarding orchid Ponerorchis chusua were investigated. METHODS: Flowering phenological traits were monitored by noting the opening and wilting dates of the chosen individual plants. The pollinator pool and pollinator behaviour were assessed from field observations. Key morphological traits of the flowers and pollinators were measured directly in the field. Pollinator limitation and interspecific compatibility were evaluated by hand-pollination experiments. Fruit set was surveyed in monospecific and heterospecific plots. KEY RESULTS: The species had overlapping peak flowering periods. Pollinators of both species displayed a certain degree of constancy in visiting each species, but they also visited other flowers before landing on the focal orchids. A substantial difference in spur size between the species resulted in the deposition of pollen on different regions of the body of the shared pollinator. Hand-pollination experiments revealed that fruit set was strongly pollinator-limited in both species. No significant difference in fruit set was found between monospecific plots and heterospecific plots. CONCLUSIONS: A combination of mechanical isolation and incomplete ethological isolation eliminates the possibility of pollen transfer between the species. These results do not support either the facilitation or competition hypothesis regarding the effect of nearby rewarding flowers on non-rewarding plants. The absence of a significant effect of non-rewarding P. chusua on rewarding G. diantha can be ascribed to low levels of overlap between the pollinator pools of two species.

Clinical Evaluation of Catheter-induced Urinary Tract Infection and Role of the Systemic Antibiotics Treatment / 대한비뇨기과학회지

The lymphatic system is of considerable importance in the spread of malignant tumors. Lymphography provides a simple and direct radiological method of demonstrating clinically unsuspected metastases in lymph nodes. Our study was intended to assess the role of lymphography in the management of the genitourinary tract tumors. The following results were obtained 1) It is helpful in the assessment of plaints with carcinoma of the penis, who have inguinal metastasis and are under consideration of inguinal lymphadenectomy. 2) It is helpful in determining the radiation portals. 3) It is helpful in deciding the stage of disease and in expecting the prognosis. 4) It is superior to IVP in detecting retroperitoneal metastasis. 5) It is valuable in confirming the completeness of lymphadenectomy. 6) It is helpful in reviewing the response to radiotherapy and chemotherapy, and also in the evaluation of recurrence within one year.

Clinical Study to the Problem of Intestinal Gas for Intravenous Urography / 대한비뇨기과학회지

Intravenous urography is very important in the detection of pathological conditions of the urinary tract, but the presence of intestinal gas shadows may be very confusing in the interpretation of urograms. There is considerable difference of opinion regarding the desirability of preparing patients for excretory urography. we obtained clinical impression and statistical results that intestinal gas was increased with prolonged duration after last diarrhea, also with sleeping after last diarrhea. So we attempted one new method of preparation in which employed 30cc, of castor oil for catharsis two times at night before examination and next morning 6:00 a. m. and result was compared with other methods. A total of 300 cases, for the most in-patients, consisted of 6 groups, were studied in this report. All patients had nothing by mouth after midnight until the X-rays were completed and pyelograms usually were taken in the morning. Following methods of preparation were studied. Group 1 : 60 cc. castor oil at bed time. Group 2 : 60 cc. castor oil at 4:00 a. m. Group 3 : 30 cc. castor oil at bed time and 30 cc. at 6:00 a. m. Group 4 : 60 cc. castor oil at bed time and enema in the morning. Group 5 : No cathartics and enema. Group 6 : Dulcolax 2 tablets at bed time. The X-ray films were graded as A) excellent B) good C) fair D) poor by three or more examiners. Group 3. afforded best result. We believed that result was due to prevention of intestinal fermentation by ingestion of castor oil at bed time and passage of swallowed air before X-ray examination by repeated ingestion at 6:00 a. m. A larger series of cases over a longer period of time, for more accurate statistical study, is warranted.