Search

In recent years, surface acoustic wave (SAW) devices have demonstrated great potentials and increasing applications in the manipulation of nano- and micro-particles including biological cells with the advantages of label-free, high sensitivity and accuracy. In this letter, we introduce a novel tilted-angle SAW devices to optimise the acoustic pressure inside a microchannel for cancer-cell manipulation. The SAW generation and acoustic radiation force are improved by seamlessly patterning electrodes in the space surrounding the microchannel. Comparisons between this novel SAW device and a conventional device show a 32% enhanced separation efficiency while the input power, manufacturing cost and fabrication effort remain the same. Effective separation of HeLa cancer cells from peripheral blood mononuclear cells is demonstrated. This novel SAW device has the advantages in minimizing device power consumption, lowering component footprint and increasing device density.

Gallium nitride (GaN) is a compound semiconductor which has advantages to generate new functionalities and applications due to its piezoelectric, pyroelectric, and piezo-resistive properties. Recently, surface acoustic wave (SAW) based acoustic tweezers were developed as an efficient and versatile tool to manipulate nano- and micro-particles aiming for patterning, separating and mixing biological and chemical components. Conventional piezoelectric materials to fabricate SAW devices such as lithium niobate suffers from its low thermal conductivity and incapability of fabricating multiphysical and integrated devices. This work piloted the development of a GaN-based Acoustic Tweezer (GaNAT) and its application in manipulating micro-particles and biological cells. For the first time, the GaN SAW device was integrated with a microfluidic channel to form an acoustofluidic chip for biological applications. The GaNAT demonstrated its ability to work on high power (up to 10W) with minimal cooling requirement while maintaining the device temperature below 32C. Acoustofluidic modelling was successfully applied to numerically study and predict acoustic pressure field and particle trajectories within the GaNAT, which agree well with the experimental results on patterning polystyrene microspheres and two types of biological cells including fibroblast and renal tumour cells. The GaNAT allowed both cell types to maintain high viabilities of 84.5% and 92.1%, respectively.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. Reducing cost and time for manufacturing surface acoustic wave (SAW)-based acoustofluidic devices could facilitate rapid applications of the SAW technology and attract more researchers into this field. We present herein a novel but simple technology to form a cost-effective SAW device by stacking electrodes made from printed circuit board (PCB) and piezoelectric substrate to generate acoustic waves. PCB-based SAW devices (PCB-SAW) have been characterized and compared to the conventional SAW devices made using the cleanroom fabrication processes. The PCB-SAW devices were successfully used to manipulate microparticles within droplets and microchannels, which showed similar performance with those previously achieved using the standard SAW devices. The PCB-SAW was further used as an acoustic tweezer to precisely pattern the human lung cancer cells inside the microchannel. Cell viability tests proved that the PCB-SAW possessed good biocompatibility to be potentially applied as a cost-effective and precise tool for acoustophoresis and beyond.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways. Refereed/Peer-reviewed

In tuberous sclerosis (TSC)–associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2+/− mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2+/− mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.

Gallium nitride (GaN) is a compound semiconductor which shows advantages in new functionalities and applications due to its piezoelectric, optoelectronic, and piezo-resistive properties. This study develops a thin film GaN-based acoustic tweezer (GaNAT) using surface acoustic waves (SAWs) and demonstrates its acoustofluidic ability to pattern and manipulate microparticles. Although the piezoelectric performance of the GaNAT is compromised compared with conventional lithium niobate-based SAW devices, the inherited properties of GaN allow higher input powers and superior thermal stability. This study shows for the first time that thin film GaN is suitable for the fabrication of the acoustofluidic devices to manipulate microparticles with excellent performance. Numerical modelling of the acoustic pressure fields and the trajectories of mixtures of microparticles driven by the GaNAT was performed and the results were verified from the experimental studies using samples of polystyrene microspheres. The work has proved the robustness of thin film GaN as a candidate material to develop high-power acoustic tweezers, with the potential of monolithical integration with electronics to offer diverse microsystem applications.

Correction for ‘Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB’ by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807–1814, DOI: 10.1039/C9LC01192G.

Tuberous sclerosis is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR and development of tumours in multiple organs including the kidneys. The mTOR inhibitors rapamycin and everolimus (rapalogs) have demonstrated clinical efficacy in treating TSC-associated tumours including renal angiomyolipomas. However, tumour responses are usually only partial and regrowth occurs after drug withdrawal. TSC-associated tumours are highly vascular and TSC patients with renal angiomyolipomas have elevated levels of circulating vascular endothelial growth factor A (VEGFA) and VEGFD. Sorafenib inhibits multiple kinases including VEGF receptors and has been used to treat metastatic epithelioid angiomyolipoma in one case but formal trials have not been undertaken. In this study, we investigated tumour angiogenesis and the therapeutic efficacy of everolimus in combination with sorafenib for renal tumours in Tsc2+/- mice. We found that these tumours exhibited remarkably variable angiogenesis despite consistent aberrant activation of mTOR and increased expression of HIF1α and VEGFA. Treatment of 11 month old Tsc2+/- mice for two months with a combination of everolimus and sorafenib significantly reduced the number and size of solid renal tumours, whereas everolimus or sorafenib alone did not. These results suggest that inhibition of mTOR and multiple kinases including VEGF receptors using combination therapy could hold promise for the treatment of TSC-associated tumours that have responded inadequately to a rapalog alone.

Tuberous sclerosis (TSC) is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR. Tumour responses in TSC patients to rapamycin, an allosteric inhibitor of mTOR, or its analogs are partial and reversible probably due to feedback activation of Akt. In this study, we examined the efficacy of GSK2126458, an ATP-competitive dual inhibitor of PI3K/mTOR, in comparison to rapamycin for treatment of renal tumours in genetically engineered Tsc2+/- mice. We found that both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 also significantly reduced the number and size of all lesions (cystic, papillary and solid) although to a lesser extent compared to rapamycin. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Furthermore, GSK2126458 and rapamycin suppressed proliferation of tumour cells. Importantly, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are therefore needed to test whether rapamycin in combination with GSK2126458 could promote apoptosis and thus improve therapy of TSC-associated renal tumours.

Metformin, a substrate of several poly-specific organic cation transporters, is a widely used biguanide for the treatment of type II diabetes. Recent studies suggest that metformin attenuates mTORC1 signalling by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) in the presence or absence of a functional hamartin/tuberin (TSC1/TSC2) complex. Metformin has also been reported to inhibit mTORC1 independent of AMPK through p53-dependent regulated in development and DNA damage responses 1 (REDD1) or by inhibiting Rag GTPases. These observations suggest that metformin could have therapeutic potential for tuberous sclerosis, an inherited disorder characterised by the aberrant activation of mTORC1 and the development of tumours in many organs, including the kidneys. In this study, we investigated the effect of metformin on renal lesions in a Tsc1(+/-) mouse model of tuberous sclerosis. Continuous treatment of metformin for 9 months at doses of up to 600 mg/kg/day had no significant effect on renal lesions in nine treated mice compared to 10 controls. Metformin treatment appeared to attenuate mTORC1 signalling in Tsc1(+/-) kidney tissues but not in renal tumours. Surprisingly, the expression of the organic cation transporters Slc22a1, Slc22a2 and Slc22a3 essential for the cellular uptake of metformin was highly suppressed in renal tumours. Treatment of cultured cells derived from a Tsc1-associated renal tumour with 5-aza-2-deoxycytidine or trichostatin A greatly increased the expression of these genes. These data suggest that the epigenetic suppression of the organic cation transporters in Tsc-associated mouse renal tumours may contribute to the lack of response to metformin treatment.

OBJECTIVE: Transgenic mouse models of tuberous sclerosis (TSC) develop renal cysts, cystadenomas, solid adenomas and carcinomas. Identification and characterisation of these lesions in vivo may help in TSC pre-clinical trials. This study was to evaluate T2 weighted MRI for assessment of renal lesions in two Tsc mouse models. MATERIALS AND METHODS: Tsc1(+/-), Tsc2(+/-) and wild type mice were subjected to a first MRI scan at 12 months of age and a second scan 2 months later. One Tsc2(+/-) mouse was treated with rapamycin for two months after the initial scan. Immediately following the second scan, mice were sacrificed and MRI images were compared to renal histological findings. RESULTS: MRI identified all types of Tsc-associated renal lesions in both Tsc1(+/-) and Tsc2(+/-) mice. The smallest detectable lesions were

ST1 is an artificial mini-chromosome approximately 4.5 Mb in size containing mouse minor and major satellite DNA, human alphoid DNA and sequences derived from interval 5 of the human Y chromosome. Here we have measured the mitotic and meiotic transmission of ST1 and have used the mini-chromosome to define the ability of mice to monitor the presence of unpaired centromeres during meiosis. ST1 is mitotically stable, remaining intact and autonomous in mice for many generations. Female mice efficiently transmit ST1 to their offspring at a frequency approaching 50%. Male mice also reliably transmit the mini-chromosome, though to only 20% of their offspring. Presence of ST1 in males is not associated with any compromise in the output of the seminiferous epithelium nor with histological or immunocytochemical evidence of increased apoptosis, outcomes predicted for a synapsis checkpoint. These data indicate that the presence of an unpaired centromere is not sufficient to arrest male meiosis, implying that univalents are normally eliminated by a mechanism other than a tension-sensitive spindle checkpoint.

We show that the accuracy of mitotic segregation of three engineered, mapped human mini-chromosomes differs between human, mouse and chicken cell lines. We have studied the cause of these differences by analysing the extent of centromere formation on one mini-chromosome immunocytochemically. In human and chicken cell lines the mini-chromosomes segregate accurately and form centromeres but in one mouse cell line centromere formation is undetectable and mitotic segregation is inaccurate. These results indicate that the centromere is maintained by an activity that functions in trans and varies either in amount or specificity between different cells. Structurally defined mini-chromosomes may allow this activity to be studied.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication [1] and have been used to identify genes important for chromosome stability [2] and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells [3,4] but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice [5,6] and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Neurofibromatosis type 1 (NF1), also called von Recklinghausen disease or peripheral neurofibromatosis, is a common autosomal dominant disorder characterised by multiple neurofibromas, café au lait spots, and Lisch nodules of the iris, with a variable clinical expression. The gene responsible for this condition, NF1, has been isolated by positional cloning. It spans over 350 kb of genomic DNA in chromosomal region 17q11.2 and encodes an mRNA of 11-13 kb containing at least 59 exons. NF1 is widely expressed in a variety of human and rat tissues. Four alternatively spliced NF1 transcripts have been identified. Three of these transcript isoforms (each with an extra exon: 9br, 23a, and 48a, respectively) show differential expression to some extent in various tissues, while the fourth isoform (2.9 kb in length) remains to be examined. The protein encoded by NF1, neurofibromin, has a domain homologous to the GTPase activating protein (GAP) family, and downregulates ras activity. The identification of somatic mutations in NF1 from tumour tissues strongly supports the speculation that NF1 is a member of the tumour suppressor gene family. Although the search for mutations in the gene has proved difficult, germline mutation analysis has shown that around 82% of all the fully characterised NF1 specific mutations so far predict severe truncation of neurofibromin. Further extensive studies are required to elucidate the gene function and the mutation spectrum. This should then facilitate the molecular diagnosis and the development of new therapy for the disease.

A panel of 200 unrelated NF1 individuals has been screened for mutations using a panel of specific clones for the entire gene. DNA analysis on conventional Southern blots indicated that (20) 10% of NF1 patients showed aberrant bands. Small lesions involving nucleotide alterations were detected in a further 10 patients; 5 of these alterations have been fully characterised and are the novel mutations in the NF1 gene. A number of mutations were identified in exon 2. Identical mutations in this exon in two unrelated individuals involved an insertion of cytosine into codon 5662 and resulted in an inappropriate stop codon. This mutation also created a new MnlI site. Another novel mutation in exon 2 resulted from the insertion of thymidine at nucleotide 5678, which also created an inappropriate stop codon. We have so far completed the screen of exons 1-9 of the NF1 gene for the identification of mutations and have found no evidence of clustering of such mutations in the gene.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease caused by various types of mutations in the NF1 gene. We have previously developed a locus-specific DNA microarray for detection of copy number changes at the NF1 locus by comparative genomic hybridization (CGH) analysis. The original array contains 183 probes pooled from 444 polymerase chain reaction (PCR) products. In the current work, we have used 493 probes derived from single PCR products (200--998 bp in size) to construct a higher resolution array with a smaller average probe size for molecular diagnosis of NF1. This has improved the average resolution from 12.6 kb in the previous array to 4.5 kb in the current version. The performance of the newly constructed microarray was validated with 14 well-characterized NF1 mutations for CGH analysis. These mutations represent deletions from approximately 7 kb to over 2 Mb in size. Using this array, we examined a total of 55 NF1 patients for copy number changes at the NF1 locus, detecting deletions in four of them. These results demonstrate that a locus-specific microarray constructed from single PCR products can efficiently detect copy number changes at the NF1 locus, providing a simple method for the molecular diagnosis of NF1.

Polyethylene glycol (PEG)-mediated cell fusion is a simple and efficient technique used widely for the production of somatic cell hybrids and for nuclear transfer in mammalian cloning. We describe a basic protocol of PEG-mediated cell fusion for the production of somatic cell hybrids. Fusion can be performed between adherent and suspension cells or between adherent cells or suspension cells. Either whole cells or microcells can be used as donors to fuse with recipient cells. Microcell fusion is particularly useful in transfer of a single or a limited number of chromosomes between various types of cells. Using this method, we have successfully introduced mammalian minichromosomes into a variety of vertebrate cells. The technique described here can be adapted for uses in other cell fusion involved research. This protocol, in principle, provides guidelines for further development of PEG mediated cell fusion technology.

Polyethylene glycol (PEG)-mediated cell fusion is a simple and efficient technique used widely for the production of somatic cell hybrids and for nuclear transfer in mammalian cloning. We describe a basic protocol of PEG-mediated cell fusion for the production of somatic cell hybrids. Fusion can be performed between adherent and suspension cells or between adherent cells or suspension cells. Either whole cells or microcells can be used as donors to fuse with recipient cells. Microcell fusion is particularly useful in transfer of a single or a limited number of chromosomes between various types of cells. Using this method, we have successfully introduced mammalian minichromosomes into a variety of vertebrate cells. The technique described here can be adapted for uses in other cell fusion involved research. This protocol, in principle, provides guidelines for further development of PEG mediated cell fusion technology.

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear how centromere sites are specified on chromosomes in vertebrates. We have previously introduced a mini-chromosome, named ST1, into a variety of cell lines including human HT1080, mouse LA9 and chicken DT40. This mini-chromosome, segregating faithfully in these cells, contains mouse minor and major, and human Y alpha-satellite DNA repeats. In this study, after determining the organisation of the satellite repeats, we investigated the location of the centromere on the mini-chromosome by combined immunocytochemistry and fluorescence in situ hybridisation analysis. Centromeric proteins were consistently co-localised with the minor satellite repeats in all three cell lines. When chromatin fibres were highly stretched, centromeric proteins were only seen on a small portion of the minor satellite repeats. These results indicate that a fraction of the minor satellite repeats is competent in centromere function not only in mouse but also in human and chicken cells.

We have characterized two intragenic polymorphisms in the neurofibromatosis type 1 (NF1) gene by direct sequencing of PCR products. The variants for these polymorphisms were initially detected on Hydrolink gels. One of the polymorphisms involves a G to A transition in intron 41 at the 28th base upstream of exon 42 with an observed {open_quote}G{close_quote}/{open_quote}A{close_quote} heterozygosity of 0.42. The other polymorphism is a T to C transition in intron 16 at the 16th base upstream of exon 17 with an observed {open_quote}T{close_quote}/{open_quote}C{close_quote} heterozygosity of 0.09. In combination with other documented polymorphisms in the NF1 gene, these variants should assist in genetic analysis of NF1 families. 24 refs., 3 figs.

We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.

We have screened a total of 105 unrelated patients with neurofibromatosis type l (NF1) for mutations in exon 28 of the NF1 gene using heteroduplex analysis and single strand conformation polymorphism analysis. One novel mutation has been identified and characterised. This mutation involves a 13-bp deletion (AAACTGGCTGAGC or AACTGGCTGAGCA) from base position 5077 (or 5078) to 5089 (or 5090) of the cDNA coding sequence. This alteration leads to a reading frame shift with a premature amber termination signal (TAG) at codon 1694. In addition, there is a change from lysine to threonine at codon 1693. The truncated gene product is estimated to be 1125 amino acid residues shorter than the predicted normal protein (2818 amino acids).

Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics. Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success. Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications. Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites.

A 4 Mb human mini-chromosome, DeltaDelta2, was transferred from Chinese hamster ovary (CHO) cells into a mouse L cell line. The mini-chromosome could be transferred intact into the L cells, with 112/119 clones maintaining a mini-chromosome of the same size as the original. Ten clones were grown for 30 days in continuous culture. The mini-chromosomes were maintained stably with or without selection at a copy number of 1-2 per cell and none experienced any size alterations, as determined by pulsed-field gel electrophoresis. Thus DeltaDelta2 is structurally and mitotically stable in L cells. This contrasts with results in embryonic stem cells, in which DeltaDelta2 is highly unstable. These findings indicate that established somatic cell lines, such as L cells and CHO cells, have less stringent controls over centromeric function than do normal embryonic cells.

A mammalian artificial chromosome would enable analysis of the cis-acting DNA sequences necessary for mammalian chromosome function and would allow large numbers of genes in a defined sequence environment to be introduced into experimental animals, agricultural livestock or human cells. Recent technical progress suggests that a route to mammalian artificial chromosome construction is now open.

Despite the extensive search for disease causing mutations in exons 28-36 of the neurofibromatosis type 1 (NF1) gene, the NF1 specific mutations so far documented account for only a small proportion of all NF1 cases. In this study, we have used 8 sets of new primers to amplify sequences throughout the NF1 gene, including 10 different exons and their flanking intron sequences. The derived PCR products from 150 independent NF1 patients and 50 normal controls were examined by heteroduplex analysis on Hydrolink gels. Three novel mutations were identified and characterised. Two of these mutations include the same 3-bp deletion (AAT) within exon 17 with a silent codon change from ACA (threonine) to ACG (threonine) and a loss of the codon ATG (methionine). The third mutation is a 10-bp deletion (TTCTCTTGGA) within exon 44 resulting in the formation of an inappropriate stop codon. These results should be useful for the further elucidation of the molecular basis of NF1.