Impact of imaging magnification on colorectal surgery: a matched analysis of a single tertiary center.

BACKGROUND: Laparoscopy has been increasingly applied in colorectal surgery, and imaging systems have been improving concurrently. The present study aims to compare outcomes following colorectal surgery with the 4K and traditional high-definition (HD) video systems. METHODS: All consecutive patients undergoing laparoscopic colorectal surgery between April 2016 and June 2020 were retrospectively retrieved from a prospective institutional database. The study population was matched according to the imaging system (4K versus HD groups) through a propensity score matching (PSM) based on perioperative characteristics of 15 patients. A stratified analysis according to surgical procedures (right, left colectomy, and low anterior resection) was also performed. Primary endpoints were intraoperative blood loss and perioperative transfusions. Also, intra- and postoperative morbidity, operative time, lymph node harvest, and length of hospital stay (LOS) were investigated as secondary outcomes. RESULTS: After PSM, 225 patients were included in both 4K and HD groups. The intraoperative blood loss was significantly lower in the 4K group (p = 0.008), although no different volumes of blood transfusion were required. Postoperative complications presented in similar proportions, while significantly higher rates of abdominal collection (p = 0.045), reoperation (p = 0.005), and postoperative urinary disorders occurred in the HD group. After stratification, the right colectomy subgroup shared similar associations with the study population. LOS did not change between groups, although readmissions were significantly lower in the 4K group (p < 0.001). CONCLUSIONS: The 4K imaging system represents a technological advance providing better surgical outcomes, such as the minimization of intraoperative blood loss and postoperative morbidity.

Actual malignancy risk of either operated or non-operated presumed mucinous cystic neoplasms of the pancreas under surveillance.

BACKGROUND: Despite the low malignant potential of pancreatic mucinous cystic neoplasms (MCNs), surgery is still performed. The aim of this pragmatic study was to assess the outcome of surgery and surveillance for patients presenting with a presumed MCN at the first evaluation. METHODS: Data for patients with a presumed MCN observed from 2000 to 2016 at the Verona Pancreas Institute and San Raffaele Hospital were extracted from prospective databases. The endpoints were risk of malignancy at pathology and rate of misdiagnosis for the surgical series, expressed as an odds ratio (OR) with 95 per cent confidence interval, and disease-specific survival (DSS) for the surveillance cohort investigated by the Kaplan-Meier method. RESULTS: A total of 424 patients were identified. In the surgical series (229 patients), the rate of misdiagnosis was 19.2 per cent. The rate of malignant MCNs was 10.9 per cent (25 patients). The overall rate of malignancy, including misdiagnoses, was 11.3 per cent (26 patients). Predictors of malignancy were mural nodules (OR 27.75, 95 per cent c.i. 4.44-173.61; P < 0.001), size at least 50 mm (OR 13.39, 2.01 to 89.47; P = 0.007), and carbohydrate antigen 19.9 level (OR 3.98, 1.19 to 13.30; P = 0.025). In the absence of mural nodules and enhancing walls, none of the resected presumed MCNs smaller than 50 mm were malignant. Only patients with high-risk stigmata undergoing surgery experienced a significantly reduced 5-year DSS compared with all other patients (88 versus 100 per cent; P = 0.031). CONCLUSION: Presumed MCNs with mural nodules, enhancing walls or cysts of 50 mm or larger should be considered for upfront surgical resection owing to the high risk of malignancy. In the absence of these features, the incidence of malignancy is negligible, favouring surveillance in selected patients given the low risk of malignancy and the high rate of misdiagnosis. LAY SUMMARY: Malignant degeneration of presumed pancreatic mucinous cystic neoplasms takes several years, if it occurs at all. Mural nodules, enhancing walls or cysts of 50 mm or larger call for surgical resection owing to an increased risk of malignancy; otherwise, surveillance seems a good option.

Malignant degeneration of presumed pancreatic mucinous cystic neoplasms takes several years, if it occurs at all. Mural nodules, enhancing walls or cysts of 50 mm or larger call for surgical resection owing to an increased risk of malignancy; otherwise, surveillance seems a good option.

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques.

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO-d6 using two-dimensional homo- and 1H-13C hetero-correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO-d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193-199] are discussed.

Vasoactive intestinal polypeptide carboxy-terminal fragment, VIP(22-28), and other fragments of VIP, in the central nervous system of the rat.

The possible existence in rat brain tissues of shorter peptides related to VIP has been examined. VIP and PHI both contain paired basic amino acid residues at which posttranslational cleavage of these peptides might occur. Antiserum to VIP(22-28) was raised in rabbits. The antiserum was carboxy-terminus directed, showing cross-reactivity with all tested peptides containing the VIP carboxy-terminus sequences. Chromatographic analysis of rat brain extracts demonstrated that recovered VIP(22-28) immunoreactivity [VIP(22-28)-ir] was heterogeneous, consisting of a major fraction [60-70% of total VIP(22-28)-ir] which eluted as authentic VIP(1-28) on gel filtration and on reversed phase high performance liquid chromatography (HPLC) columns. A second fraction (30-35% of total VIP(22-28)-ir] eluted from gel filtration columns in the position of VIP(22-28). HPLC analysis of this fraction from extracts of rat cortex, hippocampus, and midbrain indicated that it was heterogeneous. One component corresponded to authentic VIP(22-28). The other two components have not been identified; one appears to be a VIP fragment intermediate in size between VIP(1-28) and VIP(22-28).

Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin.

Three human Lactobacillus strains, coded B21060, B21070 and B21190, have recently been isolated. The strains show a series of features (acid and bile resistance, adhesion to various types of mucosal cell) which make them particularly promising for the preparation of probiotic products. In the present study, the ability of the strains to inhibit the growth of pathogens in coculture was investigated. Lactobacilli were incubated simultaneously or after one overnight growth with enterotoxigenic Escherichia coli, Salmonella enteritidis or Vibrio cholerae. After 24 and 48 h, bacterial counts of the pathogens and of the lactobacilli were performed. The results showed that these Lactobacillus strains inhibited the in vitro growth of E. coli and S. enteritidis under both conditions. Moreover, a cumulative effect was observed for mixtures of lactobacilli. In contrast, no significant inhibition of Vibrio cholerae growth was observed, provided that the pH of the medium was kept constant. The presence of the pathogens did not affect the growth of the Lactobacillus strains. Moreover, each of the Lactobacillus strains showed coaggregation ability with two pathogenic E. coli strains, namely ATCC 25922 and ATCC 35401.

In vitro and in vivo activity of alkylating bombesin receptor antagonists on small cell lung carcinoma.

Bombesin (BN) and bombesin-like peptides are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. Six peptides belonging to a new class of alkylating BN analogues have been selected according to their characteristics evidenced on Swiss 3T3 fibroblasts: high binding affinity to BN receptor, relevant inhibition (> 60%) of the proliferative stimulus induced by BN, long-lasting effect and specificity for BN receptor. The six peptides were able to bind BN receptors on SCLC cells and to inhibit the growth of two SCLC cell lines: NCI-H69 and NCI-N592. Conversely, they did not inhibit the growth of tumor cell lines devoid of BN receptors. Two of them were tested in vivo on N592 cells transplanted into nude mice. The peptide carrying a Cab [p-bis(2 chloroethyl)aminobenzoyl] moiety proved to be completely inactive. The second peptide, with a Melphalan moiety (Mel), showed a moderate activity (33-45% of tumor growth inhibition) without any toxicity. The low solubility of this compound prevented the use of the higher doses in vivo.

Bombesin receptor antagonists. 4. Chloromethylketone and chloroethylnitrosourea derivatives.

Four alkylating bombesin (BN) analogues (two C-terminal and one N-terminal chloromethylketone derivatives, and one chloroethylnitrosourea derivative) were synthesized and tested in Swiss 3T3 fibroblasts for receptor binding and mitogenic activity. Although they bound to the BN receptor with no or very weak mitogenic activity, no one analogue inhibited BN-induced thymidine incorporation in the contemporaneous treatment; only one behaved as a weak receptor antagonist when given 24 h before BN stimulation.

Bombesin receptor antagonists. 5 new irreversible alkylating analogues.

New alkylating bombesin analogues were synthesized in order to increase their solubility and stability in aqueous solutions. The best compromise between these parameters and the biological properties (receptor binding and antagonistic activity) was achieved with 4-[bis(2-chloro-ethylamino)]benzoyl derivatives of the BN (7-14) octapeptide carrying a (13-14) reduced peptide bond independently of the presence of a His12 residue, either free or protected.

Bombesin receptor antagonists. 3. Irreversible alkylating analogues: melphalan derivatives.

Bombesin (BN)-like peptides (such as GRP, gastrin-releasing peptide) are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. The present paper deals with a new class of bombesin analogues carrying a nitrogen mustard at their N-terminus. Due to the irreversible binding to the BN receptor(s), these peptides eventually block the mitogenic effects of the natural ligand(s), regardless of their intrinsic "agonistic" or "antagonistic" structures. In Swiss 3T3 fibroblasts they inhibit [125I]GRP binding in the nanomolar/micromolar range. According to their "agonistic" or "antagonistic" structural features, they do or do not induce [3H]thymidine incorporation and p 115 phosphorylation. In competition experiments, alkylating "antagonists" selectively inhibit BN-induced thymidine incorporation either when given simultaneously with or 24 hours before the BN challenge. Alkylating "agonists" display antagonistic effects only in the sequential treatment.

Bombesin receptor antagonists. 2. Analogues based on substance P antagonists.

Although bombesin (BN) and substance P share only the C-terminal dipeptide amide, some substance P receptor antagonists are also weak bombesin receptor antagonists. In order to increase the selectivity of the antagonism for the BN receptor, a series of hybrid peptides were synthesized by the solid-phase methodology, and screened on 3T3 fibroblasts for binding and mitogenic activity. The analogues inhibiting BN-induced thymidine incorporation were further tested for peripheral (amylase release and urinary bladder contraction) and central activity (grooming behaviour).

Bombesin receptor antagonists [corrected]. 1. Analogues with deleted, inverted or substituted amino acid residues.

Bombesin is a 14-residue peptide hormone which serves a variety of biological functions, including cell growth control in Swiss 3T3 cultured fibroblasts. It has been also involved in an autocrine system regulating the growth of small cell lung carcinoma. A series of bombesin analogues were developed by amino acid deletion, inversion or substitution in the carboxy-terminal region, identified by the target cell receptor. Their properties were examined for: i) competitive binding assays; ii) mitogenic induction and inhibition assays; iii) effects on early cellular events (inositol phosphates production and protein tyrosine phosphorylation). Inversion of the Trp residue, or deletion of the C-terminal tripeptide amide, induced complete loss of receptor affinity and biological effects. Deletion of the His residue, or its derivatization as His (Dnp) in conjunction with Met deletion or modification, gave rise to compounds with reduced receptor affinity and weak antagonistic properties. Other modifications in the C-terminal tripeptide affected the potency but not the biological profile of the parent peptide. These results indicate the basis for the eventual design of improved, specific bombesin receptor antagonists and stimulate further studies on their possible application in the therapy of human small cell lung cancer.

Total synthesis of horse heart cytochrome c. Preparation and characterization of the (1-66)apofragment.

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

Secondary structure prediction of sauvagine, a novel biologically active polypeptide from a frog.

The secondary structure of sauvagine has been predicted by the statistical method of Chou & Fasman. The results are compared with those obtained by the analysis of the secondary structure of the corticotropin releasing factor. The pattern of helices and beta-turns within these polypeptides is similar. Thus there is an evolutionary relationship in both sequence and secondary structure between these polypeptides, in accordance with their pharmacological properties.

Complexation which facilitates rejoining of horse cytochrome c apofragment [Homoser-lactone65](1-65) or [Homoser-lactone65] (23-65) to apofragment (66-104).

We have shown that two CNBr fragments of horse apocytochrome c, [Homoser-lactone65](1-65) and (66-104), bind to the ferric heme fragment (1-25)H to form a non-productive three-fragment complex, and that when the heme of this complex has been kept reduced for 48 h at 25 degrees, the peptide bond between residues 65 and 66 is restored with a yield of 24% or more. We have also shown that another CNBr fragment [Homoser-lactone65](23-65), but not [Homoser-lactone65](39-65), similarly rejoins to fragment (66-104) in the presence of the ferrous heme fragment with 25% or more yield. For complex of ferro-heme fragment [Hse-lacton65](1-65)H and apofragment (66-104) of horse cytochrome c, which restores the peptide bond between residues 65 and 66 (located on the left side of the heme) (cf. Harbury, H.A. (1978) in Semisynthetic Peptides and Proteins (Offord, R.E. & DiBello, C., eds.), pp. 73-89, Academic Press, New York). Corradin & Harbury have suggested that axial ligation of methionine 80 to the heme (on the left side) is important. Consistent with their idea, fragment [Hse80](66-104) was found not to link to [Hse-lactone65](1-65) in the presence of ferro(1-25)H. Furthermore, the present studies indicate that the interaction involving residues 26 to 38 (on the right side) is also important for such a conformation which assists in the rejoining of the two apofragments. Combining these two ideas, we suggest that restoration of the peptide bond between residues 65 and 66 reflects the structural integrity of these complexes in the reduced form. Thus, the present reaction system can be used not only for chemical synthesis of [Homoser65] apocytochrome c but also to extend amino acid substitution studies of cytochrome c to residues 1 to 64.

Coupling of DTPA to proteins: a critical analysis of the cyclic dianhydride method in the case of insulin modification.

The reaction between the cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA), a bifunctional reagent, and proteins under various conditions was studied using porcine insulin as a model protein. The reaction was compared with that between citraconic anhydride, a monofunctional reagent, and insulin. Products were characterized chromatographically and electrophoretically before and after deesterification by hydroxylamine. A DTPA-conjugated product was further characterized by proteolytic fragmentation. The reaction with citraconic anhydride yielded the expected number of products exclusively acylated on amino groups. In contrast, the reaction with the cyclic dianhydride of DTPA under all conditions examined yielded a much higher number of products than expected. Among the products formed were O-acylated ones and products of intermolecular cross-linking. It is concluded that the use of the cyclic dianhydride of DTPA does not allow the reliable preparation of proteins or other macromolecules conjugated with a high number of DTPA molecules in which each molecule of DTPA is linked to one amino group of the macromolecule through a single amide bond.

Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.

A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.

Total synthesis of horse heart cytochrome C.

A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis of an entire protein.

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids.

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.