Improved Detection of preclinical Colorectal Liver Metastases by High Resolution Ultrasound including Molecular Ultrasound Imaging using the targeted Contrast Agent BR55.

PURPOSE: Aim of the present study was to investigate the sensitivity of high resolution ultrasound (HRU), standard contrast-enhanced ultrasound (CEUS) and CEUS using a novel vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agent for the detection of hepatic metastases in a mouse model of colorectal cancer using clinical standard technology. MATERIALS AND METHODS: The human colon cancer cell line HT29, transfected with luciferase cDNA for in vivo bioluminescence monitoring, was injected intrasplenically into CB17.SCID mice. Mice were monitored weekly by bioluminescence and after 2 and 4.5 weeks by HRU and CEUS. Contrast media (untargeted BR1, targeted BR55) was applied and digital cine loops from the arterial phase (15 - 45 sec), portal venous phase (50 - 120 s) and late phases (3 - 5 min, 1hour) of the whole liver were analyzed. Data were correlated with postmortem histopathology. RESULTS: Without contrast enhancement, lesions > 4 mm were reliably detected. After use of untargeted CEUS, lesions > 2 mm were reliably detected and enhanced rim vascularization and late-phase wash-out was shown. With BR55, lesions > 0.8 mm were reliably detected with excellent documentation of vascularization. A persistent contrast enhancement was seen > 30 min after injection. Contrast-enhancement patterns with BR55 significantly correlated with CD31 (R2 = 0.74) and VEGFR2-immunohistochemistry (R2 = 0.66). CONCLUSION: Detection of metastases by HRU and CEUS was earlier and more accurate than monitoring via bioluminescence. In vivo monitoring of hepatic micrometastases can thus be performed without prior modification of cancer cells using standard technology.

Efficacy and sustainability of natural products in COVID-19 treatment development: opportunities and challenges in using agro-industrial waste from Citrus and apple.

Natural products have been used in the treatment of illnesses throughout the history of humankind. Exploitation of bioactive compounds from natural sources can aid in the discovery of new drugs, provide the scaffold of new medicines. In the face of challenging diseases, such as the COVID-19 pandemic, for which there was no effective treatment, nature could offer insights as to novel therapeutic options for control measures. However, the environmental impact and supply chain of bioactive production must be carefully evaluated to ensure the detrimental effects will not outweigh the potential benefits gained. History has already proven that highly bioactive compounds can be rare and not suitable for medicinal exploitation; therefore, the sustainability must be accessed before expensive, time-demanding, and large trials can be initialized. A sustainable option to readily produce a phytotherapy with minimal environmental stress is the use of agro-industry wastes, a by-product produced in high quantities. In this review we evaluate the sustainability issues associated with the production of phytotherapy as a readily available tool for pandemic control.

The genus Capsicum: a phytochemical review of bioactive secondary metabolites.

The Capsicum genus is one of the most popular plants consumed and cultivated worldwide, containing approximately 50 000 varieties of pepper. Due to its wide biodiversity, the chemical composition within the genus also presents a great variability. Its major applications are in food and pharmacological industry, as pepper presents a chemical composition rich in capsaicinoids, carotenoids, flavonoids and volatile compounds which is attributed to the ability of the fruit to remove insipidity, produce aromas and act against oxidative diseases. Due the existence of several cultivars there is a huge intraspecific chemical variability within each species, which can be considered as an obstacle when selecting and cultivating a species to be applied as a natural product source for a specific objective. The usage of pepper-based products in different industrial areas requires pre-established ranges of chemical compounds, such as capsaicinoids, which in high concentration are toxic when consumed by humans. Applying a pepper with a chemical profile closely related to the concentration that is required after industrial processing can improve efficacy and effectiveness of the process. An insight into the chemical characteristics of major secondary bioactive compounds within Capsicum, the factors that affect their concentration and their chemosystematic implication are reported and discussed.

Neuromuscular characterisation in Cerebral Palsy using hybrid Hill-type models on isometric contractions.

BACKGROUND: Muscles of individuals with Cerebral Palsy (CP) undergo structural changes over their lifespan including an increase in muscle stiffness, decreased strength and coordination. Being able to identify these changes non-invasively would be beneficial to improve understanding of CP and assess therapy effectiveness over time. This study aims to adapt an existing EMG-driven Hill-type muscle model for neuromuscular characterisation during isometric contractions of the elbow joint. METHODS: Participants with (n = 2) and without CP (n = 8) performed isometric force ramps with contraction levels ranging between 15 and 70% of their maximum torque. During these contractions, high-density EMG data were collected from the M. Biceps and Triceps brachii with 64 electrodes on each muscle. The EMG-driven Hill-type muscle model was used to predict torques around the elbow joint, and muscle characterisation was performed by applying a genetic algorithm that tuned individuals' parameters to reduce the RMS error between observed and predicted torque data. RESULTS: Observed torques could be predicted accurately with an overall mean error of 1.24Nm ± 0.53Nm when modelling individual force ramps. The first four parameters of the model could be identified relatively reliably across different experimental protocols with a full-scale variation of below 20%. CONCLUSION: An HD-EMG muscle modelling approach to evaluating neuromuscular properties in participants with and without CP has been presented. This pilot study confirms the feasibility of the experimental protocol and demonstrates some parameters can be identified robustly using the isometric contraction force ramps.

Characterization of the expressed gene and several processed pseudogenes for the mouse ribosomal protein L30 gene family.

Five cloned genes encoding the mouse ribosomal protein L30 were isolated from a recombinant DNA library and characterized by restriction mapping and nucleotide sequence analysis. Only one of these genes has introns and is expressed; the others are inactive processed pseudogenes. The expressed gene consists of five exons and four introns spanning 2,723 nucleotides. Transcripts of this gene are processed into the mature L30 mRNA by pathways that exhibit both constraints and flexibility with regard to the order of intron excision. The L30 mRNA which is 457 to 468 nucleotides in length excluding the polyadenylic acid tail, exhibits some microheterogeneity at its 3' end and encodes a basic protein of 115 amino acids. The 5' portion of the rpL30 gene has some novel features which are remarkably similar to the previously characterized mouse rpL32 gene. These include homologous sequences in the -60 to -340 region, the absence of a good TATA consensus sequence, and the presence of a palindromic pyrimidine sequence that spans the cap site.

Nonproductive kappa immunoglobulin genes: recombinational abnormalities and other lesions affecting transcription, RNA processing, turnover, and translation.

Six nonproductive kappa immunoglobulin genes (kappa- alleles) were cloned and sequenced. The structural abnormalities discerned from sequence analysis were correlated with functional lesions at the level of transcription, RNA processing, turnover, and translation. Four kappa- alleles, three containing V kappa genes and one not, are transcribed at normal or even greater than normal rates, the defects in these genes being expressed at various posttranscriptional levels. The other two kappa- alleles, both of which lacked V genes, exhibited greatly depressed yet clearly detectable transcriptional activity. These results are consistent with a hierarchical relationship between enhancer and promoter elements in which the enhancer establishes transcriptional competence at the kappa locus and the promoter (or pseudopromoter) determines the relative level of transcriptional activity. One of the structural abnormalities discovered in this study, a large deletion which removes the entire J kappa region, also provides new insight into the mechanism of VJ and VDJ recombination.

Robotic assessment of neuromuscular characteristics using musculoskeletal models: A pilot study.

OBJECTIVE: Non-invasive neuromuscular characterization aims to provide greater insight into the effectiveness of existing and emerging rehabilitation therapies by quantifying neuromuscular characteristics relating to force production, muscle viscoelasticity and voluntary neural activation. In this paper, we propose a novel approach to evaluate neuromuscular characteristics, such as muscle fiber stiffness and viscosity, by combining robotic and HD-sEMG measurements with computational musculoskeletal modeling. This pilot study investigates the efficacy of this approach on a healthy population and provides new insight on potential limitations of conventional musculoskeletal models for this application. METHODS: Subject-specific neuromuscular characteristics of the biceps and triceps brachii were evaluated using robot-measured kinetics, kinematics and EMG activity as inputs to a musculoskeletal model. RESULTS: Repeatability experiments in five participants revealed large variability within each subjects evaluated characteristics, with almost all experiencing variation greater than 50% of full scale when repeating the same task. CONCLUSION: The use of robotics and HD-sEMG, in conjunction with musculoskeletal modeling, to quantify neuromuscular characteristics has been explored. Despite the ability to predict joint kinematics with relatively high accuracy, parameter characterization was inconsistent i.e. many parameter combinations gave rise to minimal kinematic error. SIGNIFICANCE: The proposed technique is a novel approach for in vivo neuromuscular characterization and is a step towards the realization of objective in-home robot-assisted rehabilitation. Importantly, the results have confirmed the technical (robot and HD-sEMG) feasibility while highlighting the need to develop new musculoskeletal models and optimization techniques capable of achieving consistent results across a range of dynamic tasks.

The novel activation of ABL by fusion to an ets-related gene, TEL.

In human leukemia, activation of the ABL proto-oncogene locus on chromosome 9 most commonly occurs as a result of its fusion to the BCR locus on chromosome 22. The resulting chimeric protein displays an elevated tyrosine kinase activity. We have identified a novel activation of ABL which involves a gene located on chromosome 12, designated TEL. Like BCR, TEL is fused in-frame with ABL and produces a fusion protein with an elevated tyrosine kinase activity when assayed in an immune complex. The amino-terminal sequences of TEL encode a helix-loop-helix motif which may mediate dimerization.

MLL self fusion mediated by Alu repeat homologous recombination and prognosis of AML-M4/M5 subtypes.

Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9 in a major breakpoint cluster region within a centromeric 8.3-kb BamHI fragment, whereas rearrangement in one patient was the result of a direct tandem duplication of exons 2-6 of MLL. Analysis of sequences at the duplication junction revealed that the points of MLL fusion within introns 6 and 1 both lie within Alu elements. This suggests the involvement of Alu repeat mediated homologous recombination in MLL self fusion. For the 10 rearranged samples, cytogenetics analysis revealed a normal karyotype in 3, and 3 had abnormalities other than 11q23. Survival analysis of patients revealed no difference between those with rearrangement of MLL and those showing the germ-line configuration.

Isolation and characterization of a pufferfish MLL (mixed lineage leukemia)-like gene (fMll) reveals evolutionary conservation in vertebrate genes related to Drosophila trithorax.

The MLL gene is interrupted and fused to a number of partner genes as a result of chromosomal translocations in human leukemias. MLL is a very large protein with a unique domain structure and large regions of homology to Drosophila trx. To define the key structural and functional domains of the MLL protein in vertebrates, we have cloned the genomic region encoding an MLL-like gene in the compact model vertebrate genome of Fugu rubripes. While the similarity between the mouse and human MLL proteins is very high, a lower overall similarity is present between the Fugu and mammalian proteins. Several new highly conserved regions were identified in the portion of the protein included in the MLL leukemia-associated fusion proteins. The conserved nature of regions of similarity between vertebrate forms of MLL and the Drosophila TRX proteins, as well as other domains previously suggested to have a functional role in MLL (including the AT hooks and the DNA methyltransferase domain), was also observed. Therefore, strong evolutionary constraints limited sequence divergence within these domains. The information derived from this comparative analysis will form the basis for the functional study of the MLL protein, particularly as it relates to human leukemogenesis.

Characterization of the translocation breakpoint in a patient with Philadelphia positive, bcr negative acute lymphoblastic leukaemia.

Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22) translocation was first recognised in CML patients by its 22q- or Philadelphia (Ph) chromosome. While all Ph positive CML patients so far described have a chromosome 22 breakpoint within the breakpoint cluster region (bcr) located in the 3' part of the phl gene, only some Ph positive ALL patients have breakpoints in bcr. We have cloned the breakpoint of the 9q+ chromosome from the DNA of a Ph positive ALL patient in whom there is no breakpoint in the bcr. The non-chromosome 9 sequences of the breakpoint region are shown to be derived from chromosome 22. The breakpoint in chromosome 22 is shown to be the first intron of the phl gene about 66kb upstream of the bcr. Using probes from this intron, rearrangements were detected in the DNA of two out of twelve additional Ph positive, bcr negative ALL patients.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

MLL2, the second human homolog of the Drosophila trithorax gene, maps to 19q13.1 and is amplified in solid tumor cell lines.

The Mixed Lineage Leukemia (MLL) gene is commonly involved in translocations in infantile leukemia and is amplified in some cases of adult myeloid leukemia. A homolog of MLL denoted MLL2, which represents the second human homolog of the Drosophila trithorax gene, was characterized by assembling ESTs, the KIAA0304 cDNA clone, RT - PCR fragments and a new clone isolated from a cDNA phage library and compared to the available genomic sequence. The MLL2 gene maps to 19q13.1, a region of frequent rearrangement or amplification in solid tumors. MLL2 consists of an 8.5 - 9 kb transcript and spans 20 kb of genomic DNA. The predicted MLL2 protein possesses all of the major domains defined in MLL and the two genes have a similar genomic structure. We find that MLL2 is amplified in two of 14 pancreatic carcinoma cell lines and one of five glioblastoma cell lines and is a likely critical gene in 19q13.1 amplifications. It is also a candidate for chromosomal rearrangements involving this chromosome locus. MLL2 is one additional mammalian trithorax-group gene with involvement in human cancer.

ts BCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.

Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming properties of BCR-ABL and are relevant to the clinical intransigence of Ph-positive leukaemias.

An improved method for detection of B-lymphoid clonality by polymerase chain reaction.

Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporated in a single reaction. By using this method clonal IgH gene rearrangement is detected in 15 of 16 cases of B-lineage malignancy. Significantly, this series included four cases of B-lymphoma in which previous attempts to detect PCR clonal IgH gene rearrangements using a VH FR3 primer were unsuccessful. In two of these cases, nucleotide sequence analysis of the amplified DNA showed that failure to prime with the VH FR3 primer was likely to be attributable to insufficient homology with the target sequence. The use of the approach described in this paper should significantly improve the reliability of detection of B-lymphoid clonality by PCR.

Molecular analysis of a CML patient with a long duration of chronic phase before and after lymphoid blast crisis.

A patient who was diagnosed with chronic myeloid leukemia remained in chronic phase for 14 years before progressing into a lymphoid blast crisis in 1983. The acute phase was successfully treated, and the patient has remained in an indolent chronic phase to date. Cytogenetic and molecular analysis during this second chronic phase confirm the presence of the Philadelphia chromosome and its transcribed BCR-ABL mRNA. The breakpoint within M-bcr occurred in the 3' portion of the region and expressed a hybrid joining the b3 exon of BCR to the a2 exon of ABL.

bcr-abl oncogene activation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to CML, do not exhibit chromosomal breakpoints in the major cluster region or mcr (Ph+ mcr- ALL). These cases may have a novel bcr-abl fusion gene instead. We tested this hypothesis in eight Ph+ mcr- ALL patients by amplifying the putative hybrid part of the bcr-abl cDNA, using the polymerase chain reaction method. All cases examined showed the same joining of the first exon of the bcr gene to the c-abl oncogene. Thus, the novel bcr-abl fusion in Ph+ mcr- ALL is the result of a molecularly distinct Ph chromosome. This allows the definition of Ph+ leukemias by their respective bcr-abl oncogene activation. Moreover, the cDNA amplification method we use is a clinically useful tool to screen for bcr-abl oncogene activations in leukemia patients.

Differentiation stage-specific expression of a gene during granulopoiesis.

Differential screening of a recombinant cDNA library using cDNAs transcribed from poly(A)+ RNA of normal or leukemic leukocytes revealed a number of recombinants homologous to mRNAs characteristic of particular leukemias. The occurrence of one of these (pCG14) in high abundance was shown to be sufficiently characteristic of the circulating leukocyte population of chronic granulocytic leukemia (CGL) patients to distinguish them from all other populations of leukocytes. We have now characterized the gene encoding this mRNA and shown that its expression is specific to the granulocyte lineage in hemopoietic cells and is, moreover, limited to a narrow stage of differentiation during granulopoiesis. Our results explain why high levels of pCG14 RNA are characteristic of chronic granulocytic leukemia peripheral blood leukocytes.