Super-resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high-performance computing (HPC) cluster and quantify the speed advantage for a four-node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD-enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software. LAY DESCRIPTION: Optical microscopy is now able to provide images with a resolution far beyond the diffraction limit thanks to relatively new super-resolved microscopy (SRM) techniques, which have revolutionized the ability to study biological structures. One approach to SRM is to randomly switch on and off the emission of fluorescent molecules in an otherwise conventional fluorescence microscope. If only a sparse subset of the fluorescent molecules labelling a sample can be switched on at a time, then each emitter will be, on average, spaced further apart than the diffraction-limited resolution of the conventional microscope and the separate bright spots in the image corresponding to each emitter can be localised to high precision by finding the centre of each feature using a computer program. Thus, a precise map of the emitter positions can be recorded by sequentially mapping the localisation of different subsets of emitters as they are switched on and others switched off. Typically, this approach, described as single molecule localisation microscopy (SMLM), results in large image data sets that can take many minutes to hours to process, depending on the size of the field of view and whether the SMLM analysis employs a computationally-intensive iterative algorithm. Such a slow workflow makes it difficult to optimise experiments and to analyse large numbers of samples. Faster SMLM experiments would be generally useful and automated high throughput SMLM studies of arrays of samples, such as cells, could be applied to drug discovery and other applications. However, the time required to process the resulting data would be prohibitive on a normal computer. To address this, we have developed a method to run standard SMLM data analysis software tools in parallel on a high-performance computing cluster (HPC). This can be used to accelerate the analysis of individual SMLM experiments or it can be scaled to analyse high throughput SMLM data by extending it to run on an arbitrary number of HPC processors in parallel. In this paper we outline the design of our parallelised SMLM software for HPC and quantify the speed advantage when implementing it on four HPC nodes compared to a powerful desktop computer.

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein acute myeloid leukemia (AML)1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via downregulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML.

Balling defect of the additively manufactured titanium lattice implants easily leads to muscle tissue rejection, which might cause failure of implantation. Electropolishing is widely used in surface polishing of complex components and has potential to deal with the balling defect. However, a clad layer could be formed on the surface of titanium alloy after electropolishing, which may affect the biocompatibility of the metal implants. To manufacture lattice structured β-type Ti-Ni-Ta-Zr (TNTZ) for bio-medical applications, it is necessary to investigate the impact of electropolishing on material biocompatibility. In this study, animal experiments were conducted to investigate the in vivo biocompatibility of the as-printed TNTZ alloy with or without electropolishing; and proteomics technology was used to elaborate the results. The following conclusions were drawn: (a) a 30% oxalic acid electropolishing treatment was effective in solving balling defects, and ~21 nm amorphous clad layer would be formed on the surface of the material after polishing; (b) the electropolished TNTZ suggested decreased cell cytotoxicity and improved blood biocompatibility as compared to as-printed TNTZ; (c) the amorphous clad layer could make a barrier to prevent Ta and Zr ions from penetrating into the muscle tissue, and could form a good tissue regeneration at the implantation site during 4 weeks, indicating that the electropolished TNTZ has the potential as implants; and (d) the cells attached to the electropolished TNTZ showed higher antioxidant capacity but less proliferation than attached to as-printed TNTZ.

1. Cell-specific reverse transcriptase-polymerase chain reaction (RT-PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+-H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2 receptors in the duct. 2. The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM). 3. Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct. 4. Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1-like activity in the BLM and NHE2-like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present. 5. Activation of P2 receptors resulted in activation of an NHE-independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+. 6. Stimulation of P2U receptors expressed in the BLM activated largely NHE1-like activity, and stimulation of P2Z receptors expressed in the LM activated largely NHE2-like activity. 7. The interrelation between basolateral and luminal NHE activities and their respective regulation by P2U and P2Z receptors can be used to co-ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.

Cancer is a disease of genome instability and genomic alterations; now, genomic heterogeneity is rapidly emerging as a defining feature of cancer, both within and between tumors. Motivation for our pilot study of tumor heterogeneity in esophageal squamous cell carcinoma (ESCC) is that it is not well studied, but the highest incidences of esophageal cancers are found in China and ESCC is the most common type. We profiled the mutations and changes in copy number that were identified by whole-exome sequencing and array-based comparative genomic hybridization in multiple regions within an ESCC from two patients. The average mutational heterogeneity rate was 90% in all regions of the individual tumors in each patient; most somatic point mutations were nonsynonymous substitutions, small Indels occurred in untranslated regions of genes, and copy number alterations varied among multiple regions of a tumor. Independent Sanger sequencing technology confirmed selected gene mutations with more than 88% concordance. Phylogenetic analysis of the somatic mutation frequency demonstrated that multiple, genomically heterogeneous divergent clones evolve and co-exist within a primary ESCC and metastatic subclones result from the dispersal and adaptation of an initially non-metastatic parental clone. Therefore, a single-region sampling will not reflect the evolving architecture of a genomically heterogeneous landscape of mutations in ESCC tumors and the divergent complexity of this genomic heterogeneity among patients will complicate any promise of a simple genetic or epigenetic diagnostic signature in ESCC. We conclude that any potential for informative biomarker discovery in ESCC and targeted personalized therapies will require a deeper understanding of the functional biology of the ontogeny and phylogeny of the tumor heterogeneity.

Castration-resistant prostate cancer (CRPC) threatens the health of men in general and no effective therapeutics currently exists for the treatment of CRPC. It is therefore of great importance to find a novel molecule that can be a biomarker and a therapeutic target for CRPC. First, we found that the serum fibrinogen gamma (FGG) levels in patients with CRPC were significantly higher than those with localized prostate cancer (PCa) through iTRAQ proteomics and ELISA experiments. Immunohistochemistry, quantitative real-time polymerase chain reaction and western blot also showed an increase of FGG expression in CRPC tissues and cells. Then we proved the proliferation, invasion and migration ability of CRPC cells were significantly reduced after FGG knockdown. The number of apoptotic cells increased at least sixfold after FGG silencing, and was observed in conjunction with an upregulation of p53, caspase 3, clea-caspase 3, and Bax, and a downregulation of Bcl2 and survivin. FGG knockdown in DU145 cells resulted in smaller xenografts than control cells in a mouse model. and we established that FGG is modulated by IL-6 which was increased in CRPC patients via phosphorylation of STAT3. The data suggests that FGG may be a potential therapeutic target and prognostic marker for CRPC.

During an immune response naive T helper (Th) cells differentiate into two functionally distinct subsets, Th1 and Th2, based on their cytokine secretion profile and immunomodulatory function. c-Jun amino terminal kinase (JNK) regulates Th cell differentiation by activating a transcriptional program required for cytokine production. We have recently identified a TNFR superfamily death domain-containing molecule, death receptor (DR)6, which potently activates JNK. T cells from DR6-deficient mice are substantially impaired in JNK activation. When DR6(-/-) mice were challenged with protein antigen, their T cells hyperproliferate and display a profound polarization toward a Th2 response whereas Th1 differentiation is not equivalently affected. In addition, DR6(-/)- T cells showed preference toward Th2 differentiation in vitro. The phenotype seen in the DR6(-/)- mice is not due to the apoptotic pathway. Therefore, DR6, working through JNK, rather than apoptosis, functions to attenuate the Th2 response. This is the first demonstration of a role in the activation and differentiation of Th cells by DR6 in particular and DRs in general.

Dezocine is considered to be an alternative medication for managing postoperative pain. The aim of this study was to assess the efficacy and safety of this drug in this regard.

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1alpha, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.

Permanganate probing and abortive initiation assays were used to investigate the role of ATP in several successive stages of transcription initiation at the activated adeno E4 and mouse DHFR promoters. Removal of ATP at several points along the multi-step pathway blocked further progress towards its completion. Most strikingly, even if the DNA transcription start site is opened using ATP, the subsequent removal of ATP disallows formation of the first phosphodiester bond of the RNA. After ATP-dependent formation of a short RNA, a new transcription complex forms, which is more stable and has a longer open region. Both RNA and ATP appear to play roles in the formation of this complex. The need for ATP throughout this multi-step initiation pathway leads to new and unexpected possibilities for the use of energy and ATPases in transcription initiation.

One of the major unresolved issues in treating pain is the paradoxical hyperalgesia produced by opiates, and accumulating evidence implicate that EphBs receptors and ephrinBs ligands are involved in mediation of spinal nociceptive information and central sensitization, but the manner in which ephrinB/EphB signalling acts on spinal nociceptive information networks to produce hyperalgesia remains enigmatic. The objective of this research was to investigate the role of ephrinB/EphB signalling in remifentanil-induced hyperalgesia (RIH) and its downstream effector.

Sle3 is a NZM2410/NZW-derived lupus-susceptibility interval on murine chromosome 7, which is associated with spontaneous lupus nephritis (SLN), and also anti-GBM-induced glomerulonephritis (GN). The tissue kallikrein gene cluster is located within the Sle3 interval and constitutes potential candidate genes for this locus. We have recently reported that renal kallikrein expression was upregulated by anti-GBM antibody challenge in a strain-specific manner and that it was significantly underexpressed in the anti-GBM-sensitive strains, including B6.Sle3. Further sequencing and functional studies reported earlier provided evidence that kallikreins could constitute disease genes in lupus. In this report, we have used an adenoviral vector to deliver the klk1 gene to B6.Sle3 congenics to directly test if kallikreins might have a protective effect against anti-GBM-induced nephritis. Our data show that klk1 gene delivery ameliorated anti-GBM-induced nephritis in B6.Sle3 congenics. Taken together with earlier studies, these findings indicate that kallikreins play an important protective role in autoantibody-initiated GN and could constitute potential candidate genes for anti-GBM-induced GN and SLN.

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The "window of susceptibility" to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this "window". We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD(R) IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD(R) IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.

Substantial evidence suggests that breast cancer initiation, recurrence and drug resistance is supported by breast cancer stem cells (BCSCs). Recently, we reported a novel role of Aurora kinase A (AURKA) in BCSCs, as a transactivating co-factor in the induction of the c-Myc oncoprotein. However, the mode of action and transcriptional network of nuclear AURKA in BCSCs remain unknown. Here, we report that nuclear AURKA can be recruited by Forkhead box subclass M1 (FOXM1) as a co-factor to transactivate FOXM1 target genes in a kinase-independent manner. In addition, we show that AURKA and FOXM1 participate in a tightly coupled positive feedback loop to enhance BCSC phenotype. Indeed, kinase-dead AURKA can effectively transactivate the FOXM1 promoter through a Forkhead response element, whereas FOXM1 can activate AURKA expression at the transcriptional level in a similar manner. Consistently, breast cancer patient samples portrayed a strong and significant correlation between the expression levels of FOXM1 and AURKA. Moreover, both FOXM1 and AURKA were essential for maintaining the BCSC population. Finally, we demonstrated that the AURKA inhibitor AKI603 and FOXM1 inhibitor thiostrepton acted synergistically to inhibit cytoplasmic AURKA activity and disrupt the nuclear AURKA/FOXM1-positive feedback loop, respectively, resulting in a more effective inhibition of the tumorigenicity and self-renewal ability of BCSCs. Collectively, our study uncovers a previously unknown tightly coupled positive feedback signalling loop between AURKA and FOXM1, crucial for BCSC self-renewal. Remarkably, our data reveal a novel potential therapeutic strategy for targeting both the cytoplasmic and nuclear AURKA function to effectively eliminate BCSCs, so as to overcome both breast cancer and drug resistance.