BackgroundTriple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems.MethodsTo understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis.ResultsWe isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A).ConclusionsTaken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Stim and Orai proteins comprise the molecular machinery of Ca 2+ release-activated Ca 2+ (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca 2+ -store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca 2+ current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca 2+ -permeable conductance that was relatively resistant to block by Gd 3+. The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.store-operated Ca 2+ entry | gating mechanism | Ca 2+

Background: STIM1-operated Orai1 channels mediate Ca 2ϩ entry for T cell activation, but the gating mechanism remains elusive. Results: The C and N termini of Orai1 have differential roles in STIM1-triggered channel activation. Conclusion:The binding between STIM1 and the Orai1 C terminus docks STIM1 onto the Orai1 N terminus, leading to channel activation. Significance: This stepwise gating mechanism contributes to ER-PM crosstalk for Ca 2ϩ entry, a fundamental process in cell biology.

SummaryViral infections cause plant chlorosis, stunting, necrosis or other symptoms. The down-regulation of chloroplast-related genes (ChRGs) is assumed to be responsible for chlorosis.We identified the differentially expressed genes (DEGs) in Rice stripe virus (RSV)-infected Nicotiana benthamiana, and examined the contribution of 75 down-regulated DEGs to RSV symptoms by silencing them one by one using Tobacco rattle virus (TRV)-induced gene silencing.Silencing of 11 of the 75 down-regulated DEGs caused plant chlorosis, and nine of the 11 were ChRGs. Silencing of a down-regulated DEG encoding the eukaryotic translation initiation factor 4A (eIF4A) caused leaf-twisting and stunting that were visible on RSV-infected N. benthamiana. A region of RSV RNA4 was complementary to part of eIF4A mRNA and virus-derived small interfering (vsiRNAs) from that region were present in infected N. benthamiana. When expressed as artificial microRNAs, those vsiRNAs could target NbeIF4A mRNA for regulation.We provide experimental evidence supporting the association of ChRGs with chlorosis and show that eIF4A is involved in RSV symptom development. This is also the first report demonstrating that siRNA derived directly from a plant virus can target a host gene for regulation.

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive-stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative-stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull-down assays, the N-terminus of RSV RNA-dependent RNA polymerase (RdRp) interacted and co-localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N-terminus of RdRp when expressed alone was not obviously different from when it was co-expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.

In plants, autophagy is involved in responses to viral infection. However, the role of host factors in mediating autophagy to suppress viruses is poorly understood. A previously uncharacterized plant protein, NbP3IP, was shown to interact with p3, an RNA-silencing suppressor protein encoded by Rice stripe virus (RSV), a negative-strand RNA virus. The potential roles of NbP3IP in RSV infection were examined. NbP3IP degraded p3 through the autophagy pathway, thereby affecting the silencing suppression activity of p3. Transgenic overexpression of NbP3IP conferred resistance to RSV infection in Nicotiana benthamiana. RSV infection was promoted in ATG5-or ATG7-silenced plants and was inhibited in GAPC-silenced plants where autophagy was activated, confirming the role of autophagy in suppressing RSV infection. NbP3IP interacted with NbATG8f, indicating a potential selective autophagosomal cargo receptor role for P3IP. Additionally, the rice NbP3IP homolog (OsP3IP) also mediated p3 degradation and interacted with OsATG8b and p3. Through identification of the involvement of P3IP in the autophagy-mediated degradation of RSV p3, we reveal a new mechanism to antagonize the infection of RSV, and thereby provide the first evidence that autophagy can play an antiviral role against negative-strand RNA viruses.

Reduction of osa-miR171b contributes to rice stripe virus symptoms by regulating its targets, while overexpression attenuates symptoms, providing direct experimental evidence that a plant miRNA affects the development of virus symptoms.

MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) libraries of rice infected with Rice stripe virus and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.