The reported isolation of nanobacteria from human kidney stones raises the intriguing possibility that these microorganisms are etiological agents of pathological extraskeletal calcification [Kajander, E. O. & Ç iftçioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274 -8279]. Nanobacteria were previously isolated from FBS after prolonged incubation in DMEM. These bacteria initiated biomineralization of the culture medium and were identified in calcified particles and biofilms by nucleic acid stains, 16S rDNA sequencing, electron microscopy, and the demonstration of a transferable biomineralization activity. We have now identified putative nanobacteria, not only from FBS, but also from human saliva and dental plaque after the incubation of 0.45-m membrane-filtered samples in DMEM. Although biomineralization in our ''cultures'' was transferable to fresh DMEM, molecular examination of decalcified biofilms failed to detect nucleic acid or protein that would be expected from growth of a living entity. In addition, biomineralization was not inhibited by sodium azide. Furthermore, the 16S rDNA sequences previously ascribed to Nanobacterium sanguineum and Nanobacterium sp. were found to be indistinguishable from those of an environmental microorganism, Phyllobacterium mysinacearum, that has been previously detected as a contaminant in PCR. Thus, these data do not provide plausible support for the existence of a previously undiscovered bacterial genus. Instead, we provide evidence that biomineralization previously attributed to nanobacteria may be initiated by nonliving macromolecules and transferred on ''subculture'' by self-propagating microcrystalline apatite.

Background: Pulmonary hypertension (PH) in adults with sickle cell disease (SCD) is associated with early mortality, but no prior studies have evaluated quantitative relationships of mortality to physiological measures of pre-and postcapillary PH. Objectives: To identify risk factors associated with mortality and to estimate the expected survival in a cohort of patients with SCD with PH documented by right heart catheterization. Methods: Nine-year follow-up data (median, 4.7 yr) from the National Institutes of Health SCD PH screening study are reported. A total of 529 adults with SCD were screened by echocardiography between 2001 and 2010 with no exclusion criteria. Hemodynamic data were collected from 84 patients. PH was defined as mean pulmonary artery pressure (PAP) > 25 mm Hg. Survival rates were estimated by the Kaplan-Meier method, and mortality risk factors were analyzed by the Cox proportional hazards regression. Measurements and Main Results: Specific hemodynamic variables were independently related to mortality: mean PAP (hazard ratio [HR], 1.61; 95% confidence interval [CI], 1.05-2.45 per 10 mm Hg increase; P ¼ 0.027), diastolic PAP (HR, 1.83; 95% CI, 1.09-3.08 per 10 mm Hg increase; P ¼ 0.022), diastolic PAP 2 pulmonary capillary wedge pressure (HR, 2.19; 95% CI, 1.23-3.89 per 10 mm Hg increase; P ¼ 0.008), transpulmonary gradient (HR, 1.78; 95% CI, 1.14-2.79 per 10 mm Hg increase; P ¼ 0.011), and pulmonary vascular resistance (HR, 1.44; 95% CI, 1.09-1.89 per Wood unit increase; P ¼ 0.009) as risk factors for mortality.

Purpose: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector^based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. Experimental Design: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector^based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. Results: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an antiapoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. Conclusions:These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths among men in Western countries (1, 2). More men are currently diagnosed at the early stages of prostate cancer and can be effectively treated by surgery or radiation. However, in one third of the