Elucidating the functional role of nitric-oxide in Bacillus subtilis by proteomic analysis

A Nitric-oxide synthase-like (NOS) protein has been found to be present in several Gram-positive bacteria, including Bacillus subtilis. NOS generates nitric-oxide from the amino acid L-arginine via the stable intermediate N-hydroxy-L-arginine. The function of NO production in Gram-positive bacteria has not yet been elucidated, but studies indicate a function in signal transduction. In this study, a proteomic approach is used to examine the physiological role of NO in the Gram-positive model bacteria, B. subtilis. Protein profiles obtained from two-dimensional gel electrophoresis of cells grown in the presence of aminoguanidine, a known NOS inhibitor, revealed the downregulation of three proteins via NO inhibition over a pI range of 4 – 7 when compared to an uninhibited sample. These three proteins were identified by LC-MS/MS as a nucleoside diphosphate kinase, an MreB-like protein, and a phage shock A protein homolog. The down-regulation of these proteins via NOS inhibition provides preliminary evidence that NO plays a signal transduction role in Gram-positive bacteria

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

<p>Abstract</p> <p>Background</p> <p><it>Penicillium marneffei </it>is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in <it>P. marneffei </it>that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of <it>P. marneffei</it>.</p> <p>Results</p> <p>Whole cell proteins from the early stages of mould and yeast development in <it>P. marneffei </it>were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated <it>RanA</it>, was subsequently cloned and characterized. The <it>P. marneffei </it>RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous <it>Aspergillus </it>proteins.</p> <p>Conclusion</p> <p>This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in <it>P. marneffei</it>. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in <it>P. marneffei</it>.</p

Genomic Impact of Neoadjuvant Therapy on Breast Cancer: Incomplete Response is Associated with Altered Diagnostic Gene Signatures.

PURPOSE: Neoadjuvant therapy (NAT) has been shown to clinically downstage locally advanced breast cancers. This study aimed to determine whether a meaningful change in gene signatures occurs between pre- and post-NAT breast cancers for patients who do not achieve a pathologic complete response. METHODS: The current analysis included women from the prospective Neoadjuvant Breast Registry Symphony Trial who had breast cancer and awaited NAT. MammaPrint and BluePrint (Agendia, Inc., Irvine, CA) assays were performed on pre- and post-NAT breast tumor samples. RESULTS: At the completion of NAT, 93 patients with residual disease had their remaining tumor analyzed for MammaPrint and BluePrint. Of 93 patients, 21 switched tumor classification: 16 from high risk (HR) to low risk (LR) and 1 from LR to HR (p \u3c 0.001). Four additional patients switched molecular subtype but remained HR. Although only 17 patients switched in their MammaPrint risk classification, the underlying MPIndex was significantly altered after treatment across all patients (p \u3c 0.001). Additionally, the three BluePrint indices for luminal, human epidermal growth factor receptor 2 (HER2), and basal type also were significantly altered after treatment, in a subtype-dependent manner. CONCLUSION: This substudy showed that NAT significantly altered the genomic signature of the patient\u27s breast cancer compared with the patient\u27s pretreatment genomic profile. These alterations occurred in a subtype-dependent manner, suggesting that NAT may have either eliminated the most susceptible tumor subclone, leaving the treatment resistant clone with a different genetic signature, or altered molecular characteristics of the original cancer

Estrogen receptor variants in ER-positive basal-type breast cancers responding to therapy like ER-negative breast cancers

AbstractImmunohistochemically ER-positive HER2-negative (ER+HER2−) breast cancers are classified clinically as Luminal-type. We showed previously that molecular subtyping using the 80-gene signature (80-GS) reclassified a subset of ER+HER2− tumors to molecular Basal-type. We report here that molecular reclassification is associated with expression of dominant-negative ER variants and evaluate response to neoadjuvant therapy and outcome in the prospective neoadjuvant NBRST study (NCT01479101). The 80-GS reclassified 91 of 694 (13.1%) immunohistochemically Luminal-type tumors to molecular Basal-type. Importantly, all 91 discordant tumors were classified as high-risk, whereas only 66.9% of ER+/Luminal-type tumors were classified at high-risk for disease recurrence (i.e., Luminal B) (P &lt; 0.001). ER variant mRNA (ER∆3, ER∆7, and ERα-36) analysis performed on 84 ER+/Basal tumors and 48 ER+/Luminal B control tumors revealed that total ER mRNA was significantly lower in ER+/Basal tumors. The relative expression of ER∆7/total ER was significantly higher in ER+/Basal tumors compared to ER+/Luminal B tumors (P &lt; 0.001). ER+/Basal patients had similar pathological complete response (pCR) rates following neoadjuvant chemotherapy as ER−/Basal patients (34.3 vs. 37.6%), and much higher than ER+/Luminal A or B patients (2.3 and 5.8%, respectively). Furthermore, 3-year distant metastasis-free interval (DMFI) for ER+/Basal patients was 65.8%, significantly lower than 96.3 and 88.9% for ER+/Luminal A and B patients, respectively, (log-rank P &lt; 0.001). Significantly lower total ER mRNA and increased relative ER∆7 dominant-negative variant expression provides a rationale why ER+/Basal breast cancers are molecularly ER-negative. Identification of this substantial subset of patients is clinically relevant because of the higher pCR rate to neoadjuvant chemotherapy and correlation with clinical outcome.</jats:p

Clinical and expression analysis data files supporting study into molecular reclassification from Luminal to Basal-type breast cancer and links with expression of dominant-negative ER variants

Data files supporting the related publication: Estrogen Receptor Variants in ER-positive Basal-type Breast Cancers Responding to Therapy like ER-negative Breast CancersStudy design and methodsThe related study reports the link between molecular reclassification from Luminal to Basal type tumors and the expression of dominant-negative ER variants. The study examines the expression of total ER and ER variants mRNA in ER+/Basal breast cancer patients enrolled in the prospective neoadjuvant NBRST registry trial (NCT01479101) and compare these expression values to patients with ER+/Luminal B breast cancer. The study also investigates pathological complete response rates to neoadjuvant therapy and 3-year follow-up data for both patient groups within the NBRST trial and compares those to ER-/Basal breast cancer patients.The MammaPrint 70-gene expression profile (risk of recurrence signature) and BluePrint 80 gene expression profile (molecular subtyping signature) were employed to reclassify the luminal and basal types in the study.Sample size and wider cohort size:694 ER positive tumours (based on IHC) of 1072 from the wider cohort NBRST cohort.Clinical trial registration number: NCT01479101Software needed to access data:All files in this repository are in .xlsx spreadsheet format, accessible via MS Excel or open office software.Data access:All files available in this repository are openly accessible under the licence stated. MammaPrint and BluePrint analyses were run in a central laboratory involving collaboration with diagnostic company Agendia, which included the sharing of RNA samples and relevant clinical information and outcome data of this special subgroup of breast cancers, but does not cover making raw array data generally available. Files from the NBRST clinical database are held in institutional file storage and are available on request from Dr Groenendijk. Only requests under CC BY-NC-ND terms will be accepted. The NBRST clinical database is not publicly available because data were used under license for the current study. Data are however available upon reasonable request and with permission of Agendia. Access requirements includes application submission and signing of a confidentiality agreement.Please apply to Dr Groenendijk with data access requests: Department of Pathology, Erasmus MC Cancer Institute P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands email: [email protected] Raw data files (listed by figure/table in related article)IHC = immunohistochemistryFF = fresh frozen samplesFFPE = formalin-fixed paraffin embedded samples.pCR = pathological complete responseRD = residual diseaseData supporting Figure 1.xlsx - normalised total ERα mRNA expression levels by sample number for the subgroups: Normal breast, ER+/Basal and ER+/Luminal-BData supporting Figure 2.xlsx - total ERα mRNA expression and the percentage of ER positivity by IHC. Sample versus subgroups: ER IHC %, ER+/Basal FFPE, ER+/Basal FF,ER+/Luminal-B FF, ER+/Luminal-B FFPE. Data supporting Figure 3.xlsx - ratio of ERd7 mRNA expression and total ERalpha mRNA expression for ER+/Basal tumors, ER+/Luminal B tumors and normal breast tissues. Sample versus ER+/Basal FF, ER+/Luminal-B FF, ER+/Basal FFPE, ER+/Luminal-B FFPE, Normal breast FF.Data supporting Figure 4.xlsx - Statistical data supporting Kaplan Meier curves for distant metastasis free interval (DMFI) in HER2-negative patients in NBRST study with follow-up (N=538). A, DMFI for all patients stratified by ER status and molecular subtyping. B, DMFI of Basal patients (N=252) stratified by ER status and response to neoadjuvant therapy. 3 tabs consisting of raw statistics: time (months), n.risk, n.event, survival, standard error, and lower and upper 95 confidence intervals. Data supporting Table 1.xlsx - classification of ER-positive tumors (IHC ≥1%) in NBRST (N=694). Tumor sample (by ID) by MammaPrint and BluePrint in terms of risk, HER2 and ER statuses, ER%, PR statuses and PR%, all via IHC, as well as ER+ borderline classification.Data supporting Table 2.xlsx - clinical characteristics of subset of HER-2 negative patients in NBRST study with follow-up (N=538). Tumor sample (by ID), ER status via IHC and BluePrint, Age, Ethnicity, Menopausal.status, cT.stage, cN.stage, Histopathologic tumor type, Histological grade, classification via MammaPrint and BluePrint, Neoadjuvant.therapy, Adjuvant endocrine therapy, Event DMFI (1 or 0), Days to DMFI.Data supporting supplementary figures 1-3 - holding for basal, luminal and normal subgroups (FF and FFPE): mRNA expression values for Total ER, ERa-36, ERΔ3, ERΔ5, ERΔ7 and the relative expression of ERΔ3, as well as statistical data supporting Kaplan Meier curves by PR status.</div