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.

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.

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.

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.

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.

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.

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.