A Pathway to Personalizing Therapy for Metastases Using Liver-on-a-Chip Platforms.

Metastasis accounts for most cancer-related deaths. The majority of solid cancers, including those of the breast, colorectum, prostate and skin, metastasize at significant levels to the liver due to its hemodynamic as well as tumor permissive microenvironmental properties. As this occurs prior to detection and treatment of the primary tumor, we need to target liver metastases to improve patients' outcomes. Animal models, while proven to be useful in mechanistic studies, do not represent the heterogeneity of human population especially in drug metabolism lack proper human cell-cell interactions, and this gap between animals and humans results in costly and inefficient drug discovery. This underscores the need to accurately model the human liver for disease studies and drug development. Further, the occurrence of liver metastases is influenced by the primary tumor type, sex and race; thus, modeling these specific settings will facilitate the development of personalized/targeted medicine for each specific group. We have adapted such all-human 3D ex vivo hepatic microphysiological system (MPS) (a.k.a. liver-on-a-chip) to investigate human micrometastases. This review focuses on the sources of liver resident cells, especially the iPS cell-derived hepatocytes, and examines some of the advantages and disadvantages of these sources. In addition, this review also examines other potential challenges and limitations in modeling human liver.

A liver microphysiological system of tumor cell dormancy and inflammatory responsiveness is affected by scaffold properties.

Distant metastasis is the major cause of breast cancer-related mortality, commonly emerging clinically after 5 or more years of seeming 'cure' of the primary tumor, indicating a quiescent dormancy. The lack of relevant accessible model systems for metastasis that recreate this latent stage has hindered our understanding of the molecular basis and the development of therapies against these lethal outgrowths. We previously reported on the development of an all-human 3D ex vivo hepatic microphysiological system that reproduces several features of liver physiology and enables spontaneous dormancy in a subpopulation of breast cancer cells. However, we observed that the dormant cells were localized primarily within the 3D tissue, while the proliferative cells were in contact with the polystyrene scaffold. As matrix stiffness is known to drive inflammatory and malignant behaviors, we explored the occurrence of spontaneous tumor dormancy and inflammatory phenotype. The microphysiological system was retrofitted with PEGDa-SynKRGD hydrogel scaffolding, which is softer and differs in the interface with the tissue. The microphysiological system incorporated donor-matched primary human hepatocytes and non-parenchymal cells (NPCs), with MDA-MB-231 breast cancer cells. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes, and was more responsive to inflammatory stimuli. The proportion of tumor cells entering dormancy was markedly increased in the hydrogel-supported tissue compared to polystyrene. Interestingly, an unexpected differential response of dormant cells to varying chemotherapeutic doses was identified, which if reflective of patient pathophysiology, has important implications for patient dosing regimens. These findings highlight the metastatic microphysiological system fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic breast cancer.

How to research the mechanisms of non-pharmacological cardiac interventions.

OBJECTIVE: To discuss research into the mechanisms of non-pharmacological interventions for cardiac populations. METHODS: Overview of past research and theory. RESULTS: Non-pharmacological interventions for cardiac patients (including: cardiac rehabilitation, heart failure disease management programs and psychosocial interventions) have never been so common or diverse, but also have never been subject to so much scrutiny and skepticism. Better understanding of outcomes of these interventions is an urgent global priority. Mechanisms are the "underlying entities, processes, or structures which operate in particular contexts to generate outcomes of interest." PRACTICE: Research into the mechanisms of non-pharmacological interventions offers useful and robust knowledge of how and why cardiac interventions work that can be vital to explaining outcomes from interventions and inconsistencies in results. CONCLUSIONS: Research into intervention mechanisms can inform the design and optimization of interventions. IMPLICATIONS: We recommend that future research into the mechanisms of non-pharmacological interventions for cardiac population 1) view effectiveness as 'somewhat' patterned, 2) conceptualize mechanisms adequately, 3) assume they are hidden, 4) examine how context affects mechanisms, and 6) address what works for whom, when, and why.

Spontaneous dormancy of metastatic breast cancer cells in an all human liver microphysiologic system.

BACKGROUND: Metastatic outgrowth in breast cancer can occur years after a seeming cure. Existing model systems of dormancy are limited as they do not recapitulate human metastatic dormancy without exogenous manipulations and are unable to query early events of micrometastases. METHODS: Here, we describe a human ex vivo hepatic microphysiologic system. The system is established with fresh human hepatocytes and non-parenchymal cells (NPCs) creating a microenvironment into which breast cancer cells (MCF7 and MDA-MB-231) are added. RESULTS: The hepatic tissue maintains function through 15 days as verified by liver-specific protein production and drug metabolism assays. The NPCs form an integral part of the hepatic niche, demonstrated within the system through their participation in differential signalling cascades and cancer cell outcomes. Breast cancer cells intercalate into the hepatic niche without interfering with hepatocyte function. Examination of cancer cells demonstrated that a significant subset enter a quiescent state of dormancy as shown by lack of cell cycling (EdU(-) or Ki67(-)). The presence of NPCs altered the cancer cell fraction entering quiescence, and lead to differential cytokine profiles in the microenvironment effluent. CONCLUSIONS: These findings establish the liver microphysiologic system as a relevant model for the study of breast cancer metastases and entry into dormancy.

Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824.

In this report, a novel zymogram assay and coupled phosphoketolase assay were employed to demonstrate that Clostridium acetobutylicum gene CAC1343 encodes a bi-functional xylulose-5-P/fructose-6-P phosphoketolase (XFP). The specific activity of purified recombinant XFP was 6.9 U/mg on xylulose-5-P and 21 U/mg on fructose-6-P, while the specific activity of XFP in concentrated C. acetobutylicum whole-cell extract was 0.094 and 0.52 U/mg, respectively. Analysis of crude cell extracts indicated that XFP activity was present in cells grown on arabinose but not glucose and quantitative PCR was used to show that CAC1343 mRNA expression was induced 185-fold during growth on arabinose when compared to growth on glucose. HPLC analysis of metabolites revealed that during growth on xylose and glucose more butyrate than acetate was formed with final acetate:butyrate ratios of 0.72 and 0.83, respectively. Growth on arabinose caused a metabolic shift to more oxidized products with a final acetate:butyrate ratio of 1.95. The shift towards more oxidized products is consistent with the presence of an XFP, suggesting that arabinose is metabolized via a phosphoketolase pathway while xylose is probably metabolized via the pentose phosphate pathway.

Cardiac rehabilitation: into the future.

Cardiac rehabilitation is increasingly recognised as an integral component of comprehensive cardiac care. The evidence supporting its effectiveness in reducing morbidity and mortality and improving quality of life is compelling. Yet, despite this recognition and exhortations that its implementation should be a key priority, most cardiac patients do not receive rehabilitation. Service provision varies markedly and many programmes are focused on select populations, often operate in an inflexible manner and fail to add potential value. Issues of suboptimal referral, enrolment and completion are poorly addressed and the potential for embracing novel methods and the latest technology are rarely exploited. This paper reviews the current status of cardiac rehabilitation and proposes ways to improve access and uptake and reduce inequity to ensure that those who are likely to benefit from this complex intervention do so.

Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.

BACKGROUND: Whereas studies have revealed that the cryopreservation of human semen increases sperm DNA fragmentation, the mechanisms involved in this type of cryo-injury are largely unknown. Elucidation of these mechanisms may provide insight into preventing such injury. METHODS: We obtained 60 semen samples from 60 men and conducted experiments to determine the cause of cryopreservation-induced DNA fragmentation using 8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative stress, percentage caspase positive cells as an indicator of apoptosis, the potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK. RESULTS: Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P < 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r = 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P < 0.001) although the caspase inhibitor demonstrated no difference to the control. CONCLUSIONS: Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.

Genomic and genetic approaches for the identification of antifungal drug targets.

Understanding how novel antifungal compounds work in target cells is useful not only in facilitating the discovery of new drugs but also new tools that can be used for further exploration of the targeted biological pathways and their regulation. Various genomic and genetic technologies have been developed in the model yeast Saccharomyces cerevisiae, and have been successfully used to identify drug target pathways. This review discusses the methods developed for some of these technologies, and how they have been used to evaluate the cellular pathways affected by a variety of therapeutic drugs and inhibitors. The advantages and disadvantages of each method are considered, and new advances are highlighted where applicable. The investigation of the mechanism of action of new antifungal compounds will undoubtedly lead to the development of new antifungal therapies targeting new fungal pathways that are more specific and less toxic than currently available antifungal drugs.

Results of a postal survey of scrapie in the Shetland Islands in 2003.

In February 2003, a postal survey of 1279 sheep farmers in the Shetland Islands yielded 586 responses (46 per cent response rate). The principal aim of the survey was to gather information on the history and control of scrapie. Overall, 28.5 per cent of the respondents thought they had had a case of scrapie in their flock at some time. There was a slow increase in the proportion of affected flocks during the 1970s, followed by a more rapid increase during the 1980s and early 1990s, and a decline from the mid-1990s onwards. The peak proportion of affected flocks was approximately 6 per cent in 1994. Of the farmers who had ever had scrapie in their flock, 97.1 per cent had attempted to control the disease. The most common method of control was breeding from non-susceptible tups, used by 90.6 per cent of the affected flocks and 75.1 per cent of the flocks that had never been affected. A comparison of the characteristics of the affected and unaffected flocks indicated that an increased risk of scrapie was associated with the larger flocks, the open flocks and the flocks that bought in lambs. The basic reproduction ratio for the spread of scrapie between flocks was estimated to be 1.47, and the mean duration of an outbreak within a flock was estimated to be approximately two years.

The Shetland Islands scrapie monitoring and control programme: analysis of the clinical data collected from 772 scrapie suspects 1985-1997.

There were 574 scrapie positive suspects (histopathological scrapie lesions present) and 198 scrapie negative suspects (histopathological scrapie lesions absent). The greatest number of scrapie cases were recorded in sheep of 2, 3 and 4 years of age which represented 17%, 36% and 23% of the scrapie positive suspects, respectively. The sign sensitivities and specificities for the ten recorded signs were, respectively: pruritus (62%, 42%), ataxia (23%, 74%), hyperaesthesia (32%, 74%), wool loss (25%, 73%), fleece discolouration (29%, 85%), bruxism (23%, 69%), nibbling reflex (17%, 58%), head rubbing (47%, 78%), poll rubbing (25%, 83%). These single signs had poor discriminatory values with likelihood ratios close to one (range 0.89-1.21); combinations of the four signs, pruritus, wool loss, ataxia, hyperaesthesia and emaciation were more discriminatory (range 0.30-4.3). This study covered a time period when bovine spongiform encephalopathy (BSE) might have been introduced into the sheep population on the Shetland Islands via contaminated feed. No temporal changes could be detected in the age structure of the affected animals.

Improving evidence based cardiac care and policy implementation over the patient journey: the potential of coronary heart disease registers.

Coronary heart disease registers offer considerable potential for providing increased support for practitioners, facilitating improvements in patient care, and allowing efficient monitoring of care provision and outcomes.

Biochemical mode of action and differential activity of new ecdysone agonists against mosquitoes and moths.

THQ (1-aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline) compounds were identified by FMC Corporation in cell-based assays that used ecdysone receptors from Drosophila melanogaster, Heliothis virescens, or Plodia interpunctata. THQ compounds showed weak insecticidal activity against H. virescens and, therefore, were not developed further. Several ecdysone agonists based on THQ chemotype have been synthesized and tested for their activity against a number of EcRs in transactivation assays. The THQ compound, RG-120768, activated AaEcR (EcR from A. aegypti) but did not activate EcRs cloned from other insects. In transactivation assays, all six THQ ligands tested functioned through AaEcR but not through CfEcR (EcR from Choristoneura fumiferana). Three THQ compounds that showed higher activity in transactivation assays were tested in tobacco bud moth, H. virescens, and yellow fever mosquito, A. aegypti. These compounds showed higher activity in A. aegypti when compared to their activity in H. virescens. These data show that the THQ ligands are a new class of non-steroidal ecdysone agonists with preferential activity against mosquitoes.

Hepatozoon sauritus: a polytopic hemogregarine of three genera and four species of snakes in north Florida, with specific identity verified from genome analysis.

Hemogregarines from Thamnophis s. sirtalis, Coluber constrictor priapus, Elaphe obsoleta quadrivittata, and E. g. guttara in northern Florida appeared to be conspecific on the basis of similar gamonts from all the hosts and sporogonic stages obtained from 3 hosts. The resemblance of gamonts to those of Hepatozoon sauritus, described from T. sauritus sackenii in southern Florida, justified comparison of DNA isolates from the type infection of H. sauritus with samples from each of the northern Florida hosts and with a morphologically distinct species, H. sirtalis, from northern Florida. A nucleotide sequence (530 bp) alignment of the 18S ribosomal RNA gene revealed 2 hemogregarine haplotypes that varied at 15 sites (p distance = 2.8%), which included 10 transitions and 5 transversions. Two well-supported clusters (100% bootstrap support) were revealed by a neighbor-joining tree topology. One cluster included the type infection of H. sauritus and all 4 of the other samples from the northern Florida hosts, with samples of H. sirtalis comprising a second cluster. Hepatozoon sauritus, therefore, is a polytopic species in contrast to the 8 other Hepatozoon species thus far described from snakes in Florida, each of which appears to parasitize a single host species.

Transfectant mosaic spheroids: a new model for evaluation of tumour cell killing in targeted radiotherapy and experimental gene therapy.

BACKGROUND: We describe an in vitro tumour model for targeted radiotherapy and gene therapy that incorporates cell population heterogeneity. MATERIALS AND METHODS: Transfectant mosaic spheroids (TMS) and transfected mosaic monolayers (TMM) are composed of two cell populations derived from a single cell line. The cells of one population were transfected with the noradrenaline transporter gene (NAT), allowing active uptake of a radiolabelled targeting agent meta-[131I]iodobenzylguanidine ([131I]MIBG); the other population of cells was derived from the same parent line and transfected with a marker gene - green fluorescent protein (GFP). After treatment with [131I]MIBG, cell kill was determined in TMM by clonogenic assay and in TMS by clonogenic assay and spheroid growth delay. RESULTS: We have used the TMS model to assess the 'radiological bystander effect' (radiation cross-fire) conferred by the beta-emitting radiopharmaceutical [131I] MIBG whose cellular uptake is facilitated by the transfected gene encoding NAT. We show that cell killing by [131I]MIBG in both TMS and TMM cultures increased in direct proportion to the fraction of NAT-transfected cells and that the degree of cell killing against fraction transfected was greater in TMS, suggestive of a greater bystander effect in the three-dimensional culture system. CONCLUSIONS: TMS provide a useful model for assessment of the effectiveness of targeted radiotherapy in combination with gene therapy when less than 100% of the target cell population is expressing the NAT transgene. Further, this novel model offers the unique opportunity to investigate radiation-induced bystander effects and their contribution to cell cytotoxicity in radiotherapy and other gene therapy applications.

Intron/exon organization and polymorphisms of the PLK3/PRK gene in human lung carcinoma cell lines.

PLK3/PRK, a conserved polo family protein serine/threonine kinase, plays a significant role at the onset of mitosis and mitotic progression. Recently, PLK3/PRK has been shown to induce apoptosis when overexpressed in cell lines and is also implicated in cell proliferation and tumor development. Forty lung tumor cell lines were used for single-strand confirmation polymorphism (SSCP) analysis and DNA sequencing to examine the mutational status of PLK3/PRK. No missense or nonsense mutations were revealed in the lung carcinoma cell lines examined. However, three polymorphisms were identified as: a G to A at position 720, an A to G at 1053, and a G to C at 1275. Intron/exon boundaries were determined by amplification of genomic DNA with PLK3/PRK exon-specific primers. The amplification products with increased size relative to the cDNA were sequenced. Fifteen exons throughout the open reading frame were characterized. None of the introns were exceptionally large, typically ranging from 100-300 basepairs in length. These results suggest that although PLK3/PRK expression is downregulated in a majority of lung carcinoma samples, mutational inactivation of the coding sequence of the PLK3/PRK gene appears to be a rare event in lung cancer.

Multisite pooling study using ligase chain reaction in screening for genital Chlamydia trachomatis infections.

BACKGROUND: Ligase chain reaction (LCR), a nucleic acid amplification assay, is a highly specific and sensitive test for detecting Chlamydia trachomatis in cervical and urethral swabs as well as first-void urine specimens. GOAL: To examine the suitability of using the LCR test to detect C trachomatis in pooled cervical specimens. STUDY DESIGN: The performance of LCR in pooled specimens was compared with individual specimen testing at six laboratories using 3,170 cervical swab specimens randomly selected from specimens received for routine testing in the participating laboratories. These samples then were combined consecutively into 634 pools of 5 specimens and 317 pools of 10 specimens. A reduced sample to cutoff ratio of 0.2 or more was used for the pooled specimens. RESULTS: Of the 188 positive specimens (98.9%), 186 were identified when single specimens were analyzed. When pools of 5 or 10 specimens were evaluated, 99.5% and 98.9% of the positive swabs, respectively, were identified correctly. Two positive specimens were detected only through pooling. CONCLUSIONS: Pooling samples for detection of C trachomatis by LCR is sensitive and specific. Depending on the prevalence of infection (positivity), LCR testing may result in cost savings, as compared with individual testing of specimens.

Phenolic compounds from Miconia myriantha inhibiting Candida aspartic proteases.

Assay-guided fractionation of the ethanol extract of the twigs and leaves of Miconia myriantha yielded two new compounds, mattucinol-7-O-[4' ',6' '-O-(S)-hexahydroxydiphenoyl]-beta-D-glucopyranoside (1) and mattucinol-7-O-[4' ',6' '-di-O-galloyl]-beta-D-glucopyranoside (2), along with mattucinol-7-O-beta-D-glucopyranoside (3), ellagic acid (4), 3,3'-di-O-methyl ellagic acid-4-O-beta-D-xylopyranoside, and gallic acid. Complete (1)H and (13)C NMR assignments of compound 1, which possesses a hexahydroxydiphenoyl unit, were achieved using the HMBC technique optimized for small couplings to enhance the four-bond and two-bond H/C correlations. Compounds 1 and 4 showed inhibitory effects against Candida albicans secreted aspartic proteases, with IC(50) of 8.4 and 10.5 microM, respectively.

A developmental study of the Desert hedgehog-null mouse testis.

Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in Drosophila. A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5-12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.