The Effect of Histopathological Growth Patterns of Colorectal Liver Metastases on the Survival Benefit of Adjuvant Hepatic Arterial Infusion Pump Chemotherapy.

BACKGROUND: Histopathological growth patterns (HGPs) are a prognostic biomarker in colorectal liver metastases (CRLM). Desmoplastic HGP (dHGP) is associated with liver-only recurrence and superior overall survival (OS), while non-dHGP is associated with multi-organ recurrence and inferior OS. This study investigated the predictive value of HGPs for adjuvant hepatic arterial infusion pump (HAIP) chemotherapy in CRLM. METHODS: Patients undergoing resection of CRLM and perioperative systemic chemotherapy in two centers were included. Survival outcomes and the predictive value of HAIP versus no HAIP per HGP group were evaluated through Kaplan-Meier and Cox regression methods, respectively. RESULTS: We included 1233 patients. In the dHGP group (n = 291, 24%), HAIP chemotherapy was administered in 75 patients (26%). In the non-dHGP group (n = 942, 76%), HAIP chemotherapy was administered in 247 patients (26%). dHGP was associated with improved overall survival (OS, HR 0.49, 95% CI 0.32-0.73, p < 0.001). HAIP chemotherapy was associated with improved OS (HR 0.61, 95% CI 0.45-0.82, p < 0.001). No interaction could be demonstrated between HGP and HAIP on OS (HR 1.29, 95% CI 0.72-2.32, p = 0.40). CONCLUSIONS: There is no evidence that HGPs of CRLM modify the survival benefit of adjuvant HAIP chemotherapy in patients with resected CRLM.

Histopathological growth patterns of neuroendocrine tumor liver metastases.

Histopathological growth patterns (HGPs) of liver metastases represent a potential biomarker for prognosis after resection. They have never been studied in neuroendocrine tumor liver metastases (NETLM). This study evaluated if distinct HGPs can be observed in resected NETLM and if they have prognostic value. Sixty-three patients who underwent resection of NETLM between 01-01-2001 and 31-12-2021 were retrospectively included. HGPs were scored on Haematoxylin&Eosin slides using light microscopy, distinguishing desmoplastic- (dHGP), pushing- (pHGP) and replacement HGP (rHGP). Average HGP scores were calculated per patient. Each patient was classified according to predominant HGP. Overall and Disease-Free Survival (OS and DFS) were evaluated through Kaplan-Meier analysis and Cox regression. Eighteen patients had predominant dHGP (29%), 33 had predominant pHGP (52%) and 11 had predominant rHGP (17%). One patient had mixed HGP (2%). Five-year OS was 76% (95%CI: 66-87%) for the overall cohort. Five-year OS was 92% (95%CI: 77-100%) for dHGP, was 73% (95%CI: 59-91%) for pHGP, 50% (95%CI: 25-100%) for rHGP. Five-year DFS was 39% (95%CI: 19-83%) for dHGP, 44% (95%CI: 27-71%) for rHGP and 50% (95%CI: 23-100%) for pHGP. There was no significant association between HGP and OS or DFS in multivariable analysis. Distinct HGPs could be identified in NETLM. In patients who underwent resection of NETLM, no association was found between HGPs and postoperative survival. Half of the patients with NETLM have a predominant pushing growth pattern, which is a rare growth pattern in liver metastases from breast and colorectal cancer.

Circulating tumour cells are associated with histopathological growth patterns of colorectal cancer liver metastases.

Histopathological Growth Patterns (HGPs) have prognostic and predictive value in patients with Colorectal Liver Metastases (CRLM). This study examined whether preoperative measurement of Circulating Tumour Cells (CTCs) is associated with HGP. CTCs were prospectively enumerated in 7.5 ml of blood using the FDA-approved CellSearch system in patients who underwent local treatment of CRLM with curative intent between 2008 and 2021. All CTC samples were collected on the day of local treatment. Patients treated with neoadjuvant chemotherapy for CRLM or with extrahepatic disease at the time of CTC sampling were excluded. HGP was scored retrospectively following the current consensus guidelines. The association between CTCs and HGP was investigated through multivariable logistic regression. Data were available for 177 patients, desmoplastic HGP (dHGP) was observed in 34 patients (19%). There were no statistically significant differences in patient and tumour characteristics between dHGP and non-dHGP at baseline. Patients with dHGP had longer overall - and disease-free survival (logrank p = 0.003 and 0.003, respectively) compared to patients with non-dHGP. CTCs were not detected in 25(74%) of dHGP patients and in 68(48%) of non-dHGP patients (chi-squared p = 0.006). Preoperative absence of CTCs was the only significant predictor for dHGP in multivariable logistic regression (Odds Ratio 2.7, 95%CI 1.1-6.8, p = 0.028), Table 3. Preoperative absence of CTCs is associated with dHGP in chemo naive CRLM patients without extrahepatic disease. Based on our results, CTC count alone is not sufficient to preoperatively identify HGPs, but integration of CTC count in multivariable prediction models may aid the preoperative identification of HGPs of CRLM.

Treatment of metachronous colorectal cancer metastases in the Netherlands: A population-based study.

BACKGROUND: This study aimed to describe the treatment of metachronous colorectal cancer metastases in a recent population-based cohort. METHOD: Patients with stage I-III colorectal cancer (CRC), diagnosed between January 1st and June 30th, 2015 who were surgically treated with curative intent were selected from the Netherlands Cancer Registry. Follow-up was at least 3 years after diagnosis of the primary tumour. Treatment of metachronous metastases was categorized into local treatment, systemic treatment, and best supportive care. Overall survival was estimated using Kaplan-Meier method. RESULTS: Out of 5412 patients, 782 (14%) developed metachronous metastases, of whom 393 (50%) underwent local treatment (LT) with or without systemic therapy, 30% of patients underwent only systemic therapy (ST) and 19% only best supportive care (BSC). The most common metastatic site was the liver (51%) followed by lungs (33%) and peritoneum (22%). LT rates were 69%, 66%, and 44% for liver-only, lung-only and, peritoneal-only metastases respectively. Patients receiving LT and ST were significantly younger than patients receiving LT alone, while patients receiving BSC were significantly older than the other groups (p < 0.001). Patients with liver-only or lung-only metastases had a 3-year OS of 50.2% (43.3-56.7 95% CI) and 61.5% (50.7-70.6 95% CI) respectively. Patients with peritoneal-only disease had a lower 3-year OS, 18.1% (10.1-28.0 95% CI). CONCLUSION: Patients with metastases confined to the liver and lung have the highest rates of local treatment for metachronous metastatic colorectal cancer. The number of patients who underwent local treatment is higher than reported in previous Dutch and international studies.

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.

Laparoscopic surgery in a patient with ventriculoperitoneal shunt: monitoring of shunt function with transcranial Doppler.

We describe the use of transcranial Doppler (TCD) monitoring during laparoscopic resection of an ovarian cyst in a young woman who previously underwent ventriculoperitoneal shunting for hydrocephalus. Shunt function was not altered by pneumoperitoneum, except during transient episodes of high intra-abdominal pressure. The role of TCD monitoring during laparoscopic procedures in patients with cerebrospinal fluid shunt is discussed.

Identification and characterization of a mitochondrial thioredoxin system in plants.

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor.

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.

Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes.

The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.

Characterization of a germin-like protein gene expressed in somatic and zygotic embryos of pine (Pinus caribaea Morelet).

Germin-like proteins (GLPs) ionically bound to the walls of preglobular somatic embryos of Pinus caribaea Morelet are markers of this early developmental stage. In order to reveal the physiological implications of such markers during early embryo development, we isolated a cDNA clone from somatic embryos predicted to encode a protein with sequence similarity to GLPs. PcGER1 has an open reading frame corresponding to a 220 amino acid polypeptide with a putative N-glycosylation site on Asn-69. The presence of a 24 amino acid putative signal peptide supports the hypothesis of an apoplastic location. The N-terminal 20 amino acid sequence of the predicted mature protein is identical to the amino terminal sequence of GP111, one of the extracellular pine GLPs previously identified. Southern blot hybridizations indicate that PcGER1 is probably unique in the pine genome. Transcripts homologous to PcGER1 are abundant in all embryogenic lines, absent from nonembryogenic lines, and present in quiescent zygotic embryos but not in the female gametophyte, the haploid storage tissue of conifers. Their abundance sharply decreases during germination. Isolation of gf-0.8, a genomic fragment identical to PcGER1 cDNA sequence, confirms that no introns disrupt the coding region as it has been already described for wheat gf-2.8 and gf-3.8 genomic clones. Recombinant PcGER1, produced in Escherichia coli, is recognized by antibodies raised against the GP111 N-terminal nonapeptide and the unglycosylated wheat germin monomer. The implications of GLPs in pine embryogenesis are discussed.

Intron position as an evolutionary marker of thioredoxins and thioredoxin domains.

In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequenced Arabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence of Chlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.

Three glycosylated polypeptides secreted by several embryogenic cell cultures of pine show highly specific serological affinity to antibodies directed against the wheat germin apoprotein monomer.

Embryogenic tissues of Pinus caribaea Morelet var hondurensis produce extracellular proteins; among them germins have been identified. Two-dimensional electrophoresis followed by electroblotting onto a polyvinylidene difluoride membrane allowed isolation and N-terminal amino acid sequencing of extracellular GP111, which is present within the five embryogenic cell lines studied. The amino acid sequence showed strong homologies with the sequences of germins deduced from cDNA sequencing, starting at the same amino acid position but one, compared with other sequences of mature germins deduced from protein sequencing. Immunoblots of embryogenic and nonembryogenic extracellular proteins indicated that the polypeptide GP111 plus two others with similar relative molecular mass values are present in embryogenic cell lines but not in nonembryogenic ones. They were recognized by an antiserum raised against the nonglycosylated monomer of wheat germin. The cross-reaction between pine and wheat apoproteins was highly specific. An antiserum against the glycosylated pentameric germin-like protein (an oxalate oxidase) of barley cross-reacted with all three, as well as with several other glycosylated polypeptides.

A clinical study of the parameters and effects of temporary arterial occlusion in the management of intracranial aneurysms.

Temporary occlusion of intracranial arteries has emerged as a valuable technical adjunct in the management of intracranial aneurysms. The current study considered 121 patients (from a group of 234 consecutive aneurysm patients treated during a 2-yr period) who underwent elective temporary arterial occlusion. Twenty-one patients were excluded from further study because of an intraoperative rupture of an aneurysm, the elective sacrifice of afferent or efferent vessels, or the performance of an extracranial-intracranial arterial bypass graft; the remaining 100 patients underwent elective temporary occlusion under a standard neuroanesthetic regimen, including etomidate-induced burst suppression, normotension, normovolemia, and normothermia. In the postoperative period, radiographic evidence of ischemic brain injury in the distribution of the arteries occluded was selected as the end point for the failure of occlusion tolerance. The parameters evaluated with respect to this end point included the duration and nature of the temporary arterial occlusion, the number of the occlusive episodes, the specific vascular territory occluded, patient age, neurological status, presence of subarachnoid hemorrhage, vasospasm, and aneurysm size. Several parameters were found to be related to the postoperative development of ischemic injury. Patients more than 61 years of age and those in poor neurological condition (Hunt and Hess Grades III to IV) did not tolerate temporary occlusion as well as patients who were younger and in better condition. Patients occluded for less than 14 minutes routinely tolerated the iatrogenic ischemia; the 95% confidence level for the toleration of occlusion without the development of infarction occurred at 19 minutes. All patients occluded for more than 31 minutes had both clinical and radiographic evidence of cerebral infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli.

Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated. Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45%. In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein. Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E. coli cells. It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture). The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group. Recombinant plant type NTR is as effective as E. coli NTR in the DTNB (5,5'-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different. These results are discussed in relation to the primary structures of NADPH thioredoxin reductases.

A new concept of photogeneration of cations: Evidence for photoejection of Ca(2+) and Li (+) from complexes with a crown-ether-linked merocyanine by picosecond spectroscopy.

Time-resolved transient absorption and gain spectra of DCM-crown and its complexes with Li(+) and Ca(2+) in acetonitrile are measured in the wavelength range 370-670 nm after subpicosecond excitation at 425 nm. The results give evidence for the fast formation of free DCM-crown from both excited complexes, with a faster rate for the Li(+) complex. A two-step mechanism is found for the initial decay (within 30 ps) of the free DCM-crown.

Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts.

Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated. The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds. Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks. These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h). Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes). Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H4 gene. They seem to be induced in protoplasts by a synergistic effect of wounding and maceration. Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium.

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed.

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h2) was isolated using thioredoxin h1 cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 bp respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h1 (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h1 and h2, do not have any signal peptides and are most probably cytoplasmic. Using the 3' regions of the mRNAs, two probes specific for thioredoxins h1 and h2 have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h1 and a 4.5 kb fragment encodes h2. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h2 is present in N. sylvestris and N. tomentosiformis but that thioredoxin h1 is absent from both putative ancestors. Thus, the thioredoxin h1 gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA nucleotide sequence and expression of a tobacco cytoplasmic ribosomal protein L2 gene.

The ribosomal protein L2 is an essential component of the ribosomal large subunit by its relation to the peptidyl transferase reaction, subunit association and elongation factor G-GTP binding. We have isolated a 937 nucleotide long cDNA encoding a cytoplasmic ribosomal L2 protein. Its deduced protein contains 260 amino acid residues and shows 65% identity with eucaryotic RL2 but only 32% identity with the chloroplast homologue. In addition, the protein presents the PROSITE signature which matches all the 50S and 60S L2 proteins and the two residues involved in the peptidyl transferase activity. The corresponding mRNA is accumulated in young plant tissues, in growing cell suspension and in germinating seeds but is not detectable in mature plant tissues, stationary cell suspension and in dry seeds. The mRNA accumulation is correlated with the growth process. Southern blot hybridization shows that cytoplasmic ribosomal protein L2 is encoded by two types of gene which could originate from each parent. highly homologous L2 genes were also detected by Southern blots in the genomes of several monocot and dicot plant species.