Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

[Manufacturing classification system for oral solid dosage forms of traditional Chinese medicines(â ¢): classification of granule dissolving behaviors].

Solubility is an important sensory quality attribute of traditional Chinese medicine(TCM) granules. In this paper, 90 batches of granules(30 batches of TCM formula granules, 30 batches of Chinese patent medicine granules and 30 batches of Japanese Kampo granules) were used as the research objects. The turbidity sensor was used to characterize the turbidity curve of the granule dissolution process. The classification system of granule dissolution behaviors was constructed from three dimensions: dissolution degree, equilibrium time, and dissolution mechanism. According to the equilibrium time, the granule dissolution rates were divided into three categories : faster(<100 s), general(101-300 s) and slow(>301 s). According to the turbidity curve profile, the granule dissolution mechanisms were classified into dissolution-controlled type(α-type), dispersion-controlled type(ß-type), and dispersion-controlled type followed by dissolution-controlled type(γ-type). The proportion of TCM formula granules, Chinese patent medicine granules and Japanese Kampo granules with complete dissolution or slight turbidity at the end of dissolution was 46.7%, 96.7%, and 10.0%. The proportion of TCM formula granules, Chinese patent medicine granules, and Japanese Kampo granules with faster dissolution rates(<100 s) was 23.3%, 26.7%, and 40.0%. The average dissolution rate of Japanese Kampo granules was faster than that of TCM formula granules, and it was slightly faster than the average dissolution rate of Chinese patent medicine granules. The dissolution mechanism of Chinese patent medicine granules was mainly α-type, while that of Japanese Kampo granules was mainly ß-type, and the three types of dissolution mechanisms of TCM formula granules accounted for a relatively average. The purpose of improving the solubility and dispersion of granules can be achieved by combining the comprehensive application of various functional excipients with the small dosage of Japanese Kampo granules and the wide addition scope of excipients. In the process of transforming TCM compound prescriptions into formulas, there is still much room for innovation in formula excipients and process optimization.

Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked ß-N-Acetylglucosamine Strategy.

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked ß-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

The first report of Cryptosporidium testudinis in Chinese alligators (Alligator sinensis) in China.

Several Cryptosporidium species that infect reptiles, especially squamates, are well described, but there is limited data about Cryptosporidium species infecting crocodilians. In this study, we assess the occurrence of intestinal parasites using traditional microscopic examination and describe the prevalence and Cryptosporidium species in the captive-bred Chinese alligators (Alligator sinensis) in eastern China using molecular methods. The results of microscopic examination showed that no intestinal parasites were detected among the 491 fecal samples examined from the Chinese alligators. The overall prevalence for Cryptosporidium was 0.41% (2/491) by PCR detection using the SSU rRNA locus. Sequence and phylogenetic analysis of the SSU rRNA, COWP, and actin genes revealed the presence of Cryptosporidium testudinis, which has been isolated primarily from chelonians. This is the first detection of the specific DNA of C. testudinis in the feces of the Chinese alligator. This study expands our knowledge of the Cryptosporidium species involved in crocodiles, and more extensive studies are necessary to confirm the validity of C. testudinis in crocodiles.

Does liver resection/transplantation affect respiratory induced liver motion in patients with hepatocellular carcinoma?

The purpose of this study was to evaluate the changes in magnitude of three-dimensional (3D) liver motion after liver resection/transplantation in patients with hepatocellular carcinoma (HCC) using four-dimensional (4D)-computed tomography (CT) images. From January 2012 to April 2016, 74 HCC patients underwent 4D-CT scans under a free-breathing state to assess respiratory liver motion. Of the 74 patients, 40 did not have a liver resection/transplantation (Group A), 34 with liver resection/transplantation. 15 underwent major or minor resection in the right liver lobe (Group B), 14 underwent major or minor resection in the left liver lobe (Group C), and five underwent liver transplantation (Group D). The 4D-CT images were sorted into 10 image series according to the respiratory phase from the end inspiration to the end expiration, and then transferred to treatment planning software. All liver contours were drawn by a single physician and confirmed by a second. Liver relative coordinates were automatically generated to calculate liver respiratory motion in different axial directions and compiled into a single composite image. Differences in respiratory liver motion were assessed using one-way ANOVA. The average liver respiratory motion in the cranial-caudal direction and 3D magnitude were 10.46 ± 2.78 mm (range, 5.60-18.80 mm) and 11.74 ± 2.65 mm (range, 7.45-20.79 mm) for patients without liver resection/transplantation, and 7.74 ± 2.79 mm (range, 2.20-12.90 mm) and 9.07 ± 2.38 mm (range, 4.79-14.08 mm) for posthepatectomy/post-transplant patients respectively. There were significant differences between Group A and B, Group A and C, Group A and D. However, there were no significant differences among Group B, C, and D. Liver resection/transplantation greatly affected respiratory-induced liver motion in patients with HCC. We, therefore, recommend discriminatory internal target volume (ITV) determination for patients with or without liver resection/transplantation undergoing external radiotherapy for hepatic tumors while respiratory motion management is unavailable.

Characterization of the new GII.17 norovirus variant that emerged recently as the predominant strain in China.

Human noroviruses are the most important viral pathogens causing epidemic acute gastroenteritis, in which the GII.4 viruses have been predominant worldwide for the past decades. During 2014-2015 winter season, a new GII.17 variant emerged as the predominant virus in China surpassing the GII.4 virus in causing significantly increased acute gastroenteritis outbreaks. Genome sequences of the new GII.17 variant was determined and compared with other GII.17 noroviruses, revealing residue substitutions at specific locations, including the histo-blood group antigen-binding site and the putative antigenic epitopes. Further study of GII.17 outbreaks focusing on host susceptibility showed that the new GII.17 variant infected secretor individuals of A, B, O and Lewis types. Accordingly, the P particles of the new GII.17 variant bound secretor saliva samples of A, B, O and Lewis types with significantly higher binding signals than those of the P particles of the previous GII.17 variants. In addition, human sera collected from the outbreaks exhibited stronger blockade against the binding of the new GII.17 P particles to saliva samples than those against the binding between the P particles of previous GII.17 variants and saliva samples. Taken together, our data strongly suggested that the new GII.17 variant gained new histo-blood group antigen-binding ability and antigenic features, which may contribute to its predominance in causing human norovirus epidemics.

Emergence of human caliciviruses among diarrhea cases in southwest China.

BACKGROUND: Acute diarrhea is one of the most serious problems in global public health that causes considerable morbidity and mortality worldwide. Human caliciviruses (HuCV) including norovirus (NoV, genogroup GI and GII) and sapovirus (SaV), is a leading cause of acute sporadic diarrhea in individuals across all age groups. However, few studies had been conducted clarifying the characteristics of HuCV in diarrhea cases across all age groups in China. Our study was aimed at assessing the HuCV-related diarrhea burden and NoV genotypes distribution in southwest China. METHODS: The study was conducted in four hospitals in Kunming city, Yunnan province, from June 2014 to July 2015. Stool specimens were collected from 1,121 diarrhea cases and 319 healthy controls in outpatient departments. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect NoV (GI, GII) and SaV. Sequencing was applied to confirm the three viral infections and phylogenetic analysis was performed to determine their genotypes. A structured questionnaire was used to record the demographic information and clinical symptoms of subjects. RESULTS: HuCV was detected at an 11.0 % infection rate in 1,121 diarrhea cases and at 3.4 % rate in 319 non-diarrhea subjects (p < 0.0001, OR = 3.5, 95 % CI 1.8-6.5). The prevalence of the NoV genogroup GII and genotype GII.4 in diarrhea cases was significantly higher than that found in healthy controls (p < 0.0001, p = 0.018, respectively). NoV GII (n = 118, 10.5 %) was the most common HuCV subtype in diarrhea cases, followed by SaV (n = 3, 0.3 %) and NoV GI (n = 2, 0.2 %). Of 118 NoV GII strains isolated from diarrhea patients. GII.4 (n = 55, 46.6 %) was the predominant strain, followed by GII.3 (n = 28, 23.7 %), GII.12 (n = 25, 21.2 %), GII.17 (n = 8, 6.8 %), and GII.5 (n = 2, 1.7 %). Of the 55 GII.4 strains, the GII.4 Sydney 2012 variant had absolutely predominant prevalence (n = 52, 94.5 %), followed by the NoV GII.4-2006b variant (n = 3, 5.5 %). The GII.4 Orleans 2009 variant was not found in diarrhea cases of the study. CONCLUSIONS: NoV GII was the major genogroup and GII.4 was the most predominant strain detected in diarrhea patients. The GII.17 is an emergent variant in sporadic diarrhea and might become the predominant strain in diarrhea cases in the near future. Rapid, accurate detection kits need to be developed to help us find and treat NoV-associated diarrhea in clinical settings in a timely manner.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China.

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.

Thermoregulatory behavior is widespread in the embryos of reptiles and birds.

Recent studies have demonstrated that thermoregulatory behavior occurs not only in posthatching turtles but also in turtles prior to hatching. Does thermoregulatory behavior also occur in the embryos of other reptile and bird species? Our experiments show that such behavior is widespread but not universal in reptile and bird embryos. We recorded repositioning within the egg, in response to thermal gradients, in the embryos of three species of snakes (Xenochrophis piscator, Elaphe bimaculata, and Zaocys dhumnades), two turtles (Chelydra serpentina and Ocadia sinensis), one crocodile (Alligator sinensis), and four birds (Coturnix coturnix, Gallus gallus domesticus, Columba livia domestica, and Anas platyrhynchos domestica). However, we detected no significant thermoregulation by the embryos of two lizard species (Takydromus septentrionalis and Phrynocephalus frontalis). Overall, embryonic thermoregulatory behavior is widespread in reptile as well as bird species but may be unimportant in the small eggs laid by most lizards.

Enhanced native chemical ligation by peptide conjugation in trifluoroacetic acid.

Chemical ligation of peptides is increasingly used to generate proteins not readily accessible by recombinant approaches. However, a robust method to ligate "difficult" peptides remains to be developed. Here, we report an enhanced native chemical ligation strategy mediated by peptide conjugation in trifluoroacetic acid (TFA). The conjugation between a carboxyl-terminal peptide thiosalicylaldehyde thioester and a 1,3-dithiol-containing peptide in TFA proceeds rapidly to form a thioacetal-linked intermediate, which is readily converted into the desired native amide bond product through simple postligation treatment. The effectiveness and practicality of the method was demonstrated by the successful synthesis of several challenging proteins, including the SARS-CoV-2 transmembrane Envelope (E) protein and nanobodies. Because of the ability of TFA to dissolve virtually all peptides and prevent the formation of unreactive peptide structures, the method is expected to open new opportunities for synthesizing all families of proteins, particularly those with aggregable or colloidal peptide segments.

Real-world outcomes of anti-vascular endothelial growth factor therapy for retinal vascular vein occlusion in Tibet, China.

AIM: To evaluate the outcomes of intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents for patients with retinal vein occlusion (RVO) related-macular edema (ME) in Tibetan. METHODS: A retrospective, observational, single-center study. The demographic and clinical data of 90 RVO Tibetan patients (93 eyes) treated with either ranibizumab or conbercept in Tibet Autonomous Region People's Hospital from Jan 2018 to December 2019 were collected. RESULTS: The mean patient age was 56.8±10.6y, 45 (50%) of them were female. The mean living altitude was 3867.8±567.9 m. At the last visit, the best-corrected visual acuity (BCVA) significantly increased (52.2±21.8 letters) in comparison with the baseline (38.2±24.1 letters, P<0.001); while the central retinal thickness (CRT) significantly reduced (245.5±147.6 µm) in comparison with the baseline (504.1±165.2 µm, P<0.001). The 43.0% of the eyes gained ≥15 letters, 60.2% of the eyes gained ≥10 letters, and 78.5% of the eyes gained ≥5 letters. No vision loss was noted in 92.5% of the eyes, 4 eyes lost more than 10 letters during follow-up period. The mean number of injections was 2.4±1.8. No severe ocular or systemic adverse events related to either the drug or injection were noted. CONCLUSION: Anti-VEGF therapy is effective and safe in Tibetan patients for the treatment of RVO related ME.

Magnitude and influencing factors of respiration-induced liver motion during abdominal compression in patients with intrahepatic tumors.

PURPOSE: The purpose of this study was to use 4-dimensional-computed tomography (4D-CT) to evaluate respiration-induced liver motion magnitude and influencing factors in patients with intrahepatic tumors undergoing abdominal compression. METHODS: From January 2012 to April 2016, 99 patients with intrahepatic tumors were included in this study. They all underwent 4D-CT to assess respiratory liver motion. This was performed during abdominal compression in 53 patients and during free-breathing (no abdominal compression) in 46 patients. We defined abdominal compression as being effective in managing the breath amplitude if respiration-induced liver motion in the cranial-caudal (CC) direction during compression was ≤5 mm and as being ineffective if >5 mm of motion was observed. Gender, age, body mass index (BMI), transarterial chemoembolization history, liver resection history, tumor area, tumor number, and tumor size (diameter) were determined. Multivariate logistic regression analysis was used to analyze influencing factors associated with a breath amplitude ≤5 mm in the CC direction. RESULTS: The mean respiration-induced liver motion during abdominal compression in the left-right (LR), CC, anterior-posterior (AP), and 3-dimensional vector directions was 2.9 ± 1.2 mm, 5.3 ± 2.2 mm, 2.3 ± 1.1 mm and 6.7 ± 2.1 mm, respectively. Univariate analysis indicated that gender and BMI significantly affected abdominal compression effectiveness (both p < 0.05). Multivariate analysis confirmed these two factors as significant predictors of effective abdominal compression: gender (p = 0.030) and BMI (p = 0.006). There was a strong correlation between gender and compression effectiveness (odds ratio [OR] = 7.450) and an even stronger correlation between BMI and compression effectiveness (OR = 10.842). CONCLUSIONS: The magnitude of respiration-induced liver motion of patients with intrahepatic carcinoma undergoing abdominal compression is affected by gender and BMI, with abdominal compression being less effective in men and overweight patients.

Molecular characterization and tissue expression profiles of prepro-vasoactive intestinal peptide in the Chinese alligator (Alligator sinensis) during the active and hibernating periods.

The Chinese alligator (Alligator sinensis), a freshwater crocodilian endemic to China, is one of the most endangered crocodilian species; up to this date, very little is known about the endocrine regulation of its metabolic activities during different physiological states. In this study, we characterized the structure of the prepro-vasoactive intestinal peptide in Chinese alligator (prepro-caVIP) for the first time and examined its expression profiles in various tissues during the active and hibernating periods. The prepro-caVIP cDNA consists of a 221-bp 5'-untranslated region (UTR), a 606-bp complete coding region (CDS), and a 312-bp 3'-UTR, which encodes the 201-amino acid prepro-caVIP containing a 28-amino acid vasoactive intestinal peptide (VIP) and a 27-amino acid PHI (peptide histidine isoleucine). Multiple alignment analysis showed that VIP shares 100% identity with the given birds, reptiles, and African clawed frog, and 89% identity with mammals, 96% with fishes. Real-time quantitative PCR showed that the prepro-caVIP is widely expressed in all the examined tissues, and the expression level is significantly higher in small intestine, stomach, pancreas, lung, and skeletal muscle, whereas lower in heart, liver, spleen, kidney, ovary, and oviduct. During hibernation, the expression level of caVIP was significantly decreased in small intestine (P < 0.01), pancreas, and skeletal muscle (P < 0.05), whereas significantly increased in liver, spleen, and lung (P < 0.01). The wide distribution of caVIP and its differential expression changes in various tissues during hibernation implicated that it might play multiple effects in Chinese alligator and participate in the physiological adaptation of various organs in a paracrine and/or neurocrine manner.

Clinical benefits of new immobilization system for hypofractionated radiotherapy of intrahepatic hepatocellular carcinoma by helical tomotherapy.

OBJECTIVE: A comprehensive clinical evaluation was conducted, assessing the Body Pro-Lok immobilization and positioning system to facilitate hypofractionated radiotherapy of intrahepatic hepatocellular carcinoma (HCC), using helical tomotherapy to improve treatment precision. METHODS: Clinical applications of the Body Pro-Lok system were investigated (as above) in terms of interfractional and intrafractional setup errors and compressive abdominal breath control. To assess interfractional setup errors, a total of 42 patients who were given 5 to 20 fractions of helical tomotherapy for intrahepatic HCC were analyzed. Overall, 15 patients were immobilized using simple vacuum cushion (group A), and the Body Pro-Lok system was used in 27 patients (group B), performing megavoltage computed tomography (MVCT) scans 196 times and 435 times, respectively. Pretreatment MVCT scans were registered to the planning kilovoltage computed tomography (KVCT) for error determination, and group comparisons were made. To establish intrafractional setup errors, 17 patients with intrahepatic HCC were selected at random for immobilization by Body Pro-Lok system, undergoing MVCT scans after helical tomotherapy every week. A total of 46 MVCT re-scans were analyzed for this purpose. In researching breath control, 12 patients, randomly selected, were immobilized by Body Pro-Lok system and subjected to 2-phase 4-dimensional CT (4DCT) scans, with compressive abdominal control or in freely breathing states, respectively. Respiratory-induced liver motion was then compared. RESULTS: Mean interfractional setup errors were as follows: (1) group A: X, 2.97 ± 2.47mm; Y, 4.85 ± 4.04mm; and Z, 3.77 ± 3.21mm; pitch, 0.66 ± 0.62°; roll, 1.09 ± 1.06°; and yaw, 0.85 ± 0.82°; and (2) group B: X, 2.23 ± 1.79mm; Y, 4.10 ± 3.36mm; and Z, 1.67 ± 1.91mm; pitch, 0.45 ± 0.38°; roll, 0.77 ± 0.63°; and yaw, 0.52 ± 0.49°. Between-group differences were statistically significant in 6 directions (p < 0.05). Mean intrafractional setup errors with use of the Body Pro-Lok system were as follows: X, 0.41 ± 0.46mm; Y, 0.86 ± 0.80mm; Z, 0.33 ± 0.44mm; and roll, 0.12 ± 0.19°. Mean liver-induced respiratory motion determinations were as follows: (1) abdominal compression: X, 2.33 ± 1.22mm; Y, 5.11 ± 2.05mm; Z, 2.13 ± 1.05mm; and 3D vector, 6.22 ± 1.94mm; and (2) free breathing: X, 3.48 ± 1.14mm; Y, 9.83 ± 3.00mm; Z, 3.38 ± 1.59mm; and 3D vector, 11.07 ± 3.16mm. Between-group differences were statistically different in 4 directions (p < 0.05). CONCLUSIONS: The Body Pro-Lok system is capable of improving interfractional and intrafractional setup accuracy and minimizing tumor movement owing to respirations in patients with intrahepatic HCC during hypofractionated helical tomotherapy.

4D-CT scans reveal reduced magnitude of respiratory liver motion achieved by different abdominal compression plate positions in patients with intrahepatic tumors undergoing helical tomotherapy.

PURPOSE: While abdominal compression (AC) can be used to reduce respiratory liver motion in patients receiving helical tomotherapy for hepatocellular carcinoma, the nature and extent of this effect is not well described. The purpose of this study was to evaluate the changes in magnitude of three-dimensional liver motion with abdominal compression using four-dimensional (4D) computed tomography (CT) images of several plate positions. METHODS: From January 2012 to October 2015, 72 patients with intrahepatic carcinoma and divided into four groups underwent 4D-CT scans to assess respiratory liver motion. Of the 72 patients, 19 underwent abdominal compression of the cephalic area between the subxiphoid and umbilicus (group A), 16 underwent abdominal compression of the caudal region between the subxiphoid area and the umbilicus (group B), 11 patients underwent abdominal compression of the caudal umbilicus (group C), and 26 patients remained free breathing (group D). 4D-CT images were sorted into ten-image series, according to the respiratory phase from the end inspiration to the end expiration, and then transferred to treatment planning software. All liver contours were drawn by a single physician and confirmed by a second physician. Liver relative coordinates were automatically generated to calculate the liver respiratory motion in different axial directions to compile the 10 ten contours into a single composite image. Differences in respiratory liver motion were assessed with a one-way analysis of variance test of significance. RESULTS: The average respiratory liver motion in the Y axial direction was 4.53 ± 1.16, 7.56 ± 1.30, 9.95 ± 2.32, and 9.53 ± 2.62 mm in groups A, B, C, and D, respectively, with a significant change among the four groups (p < 0.001). Abdominal compression was most effective in group A (compression plate on the subxiphoid area), with liver displacement being 2.53 ± 0.93, 4.53 ± 1.16, and 2.14 ± 0.92 mm on the X-, Y-, and Z-axes, respectively. There was no significant difference in respiratory liver motion between group C (displacement: 3.23 ± 1.47, 9.95 ± 2.32, and 2.92 ± 1.10 mm on the X-, Y-, and Z-axes, respectively) and group D (displacement: 3.35 ± 1.55, 9.53 ± 2.62, and 3.35 ± 1.73 mm on the X-, Y-, and Z-axes, respectively). Abdominal compression was least effective in group C (compression on caudal umbilicus), with liver motion in this group similar to that of free-breathing patients (group D). CONCLUSIONS: 4D-CT scans revealed significant liver motion control via abdominal compression of the subxiphoid area; however, this control of liver motion was not observed with compression of the caudal umbilicus. The authors, therefore, recommend compression of the subxiphoid area in patients undergoing external radiotherapy for intrahepatic carcinoma.