Severe pelvic infections following ultrasound-guided transvaginal oocyte retrieval(TVOR) are rare but challenging. Ovarian abscess formation is one of theconsequences and management of such cases as highly debated in pregnant patients. In thiscase report, an early fetal loss following laparoscopic management of ovarian abscess isdescribed and possible etiologies are discussed. Pelvic infections following ultrasound-guided transvaginaloocyte retrieval (TVOR) are rare complicationswith an incidence of 0.6% . The proth hour of ovulation trigger using 250µg of recombinant human chorionic gonadotropin. Retrievalprocess was uneventful without any complications.The patient was prescribed a 5-day course of oral doxycycline  as a part of routine medication after retrievalprocess and vaginal micronized progesterone  for luteal phase support. On the 14th day of transfer,pregnancy was confirmed with a quantitative betahCG(ß-hCG) value of 240 mIU/ml. A subsequentdoubling in serum hCG levels was also observed suggestiveof an early ongoing intrauterine pregnancy.Three weeks after the retrieval procedure, the patientwas admitted to the emergency unit with mild abdominalpain and elevated body temperature (38.3℃). Herphysical examination revealed rebound tenderness inthe lower abdomen and tenderness during bimanualpelvic examination. Pelvic sonography revealed a4.5×4 cm echogenic cystic mass on the left adnexawith mild fluid in the Douglas pouch. Laboratory testswere within the normal range except for leukocytosis (12.500/mm3) and elevated C-reactive protein (8 mg/L) (CRP). An ultrasound-guided drainage of the mass was considered, but due to the anatomic position of the mass and poor cooperation from the patient, the procedure was not successful. Thus an initial empirical antibiotic therapy was started within travenous (IV) ceftriaxone sodium  1gr twice a day and metronidazole 500 mg  three times a day, as we were also unable to obtain satisfactory amount of culture sample from Douglas pouch. Despite full course of antibiotics for 48 hours, mild fever (38℃) and abdominal tenderness persisted and a laparoscopic drainage was decided. On initial evaluation, formations of left ovarian abscess and diffuse pus were confirmed  for male factor subfertility inan assisted reproduction unit, Ankara, Turkey. Hermedical history was unremarkable and physical examinationwas normal. Basal ultrasound examinationon the 3rd day of menstrual cycle revealed a normalpelvic anatomy without appearance of an ovarian cystincluding endometrioma. Following a standard ovarianstimulation with gonadotropins, TVOR was performedat the 36med Figes. AntibioOocyte retrieval process is an invasive procedure with the potential risk of inoculation of the vaginal microorganisms into abdomen. The risk of pelvic infection after TVOR is estimated as 0.6% . It may Topical antiseptic usage for vaginal preparation before TVOR is another controversial issue in the presence of risk factors. However, there has been no such accepted universal approach. Povidone-iodine or chlorhexidine are commonly used to sterilize the vagina, whereas other options include saline irrigation, careful removal with dry swabs, or avoiding them completely by cleansing the vagina with only saline solutions. Tsai et al.  reported2) is highly diffusible and may induce fetal tachycardia and acidosis (2) in the physiologic range. On the other hand, Steinbrook and Bhavani-Shankar (Life threatening complications as a result of assisted reproductive techniques obviously require close surveillance and active management. Ideally, less invasive and conservative approaches should be the first option; nevertheless, severe complications such as pelvic abscess sometimes require more aggressive treatment. Trans-vaginal ultrasound-guided drainage of pelvic abscess is a relatively easy, safe and effective procedure. It has also been proven to be significantly more effective than medical therapy and has been associated with a low surgical morbidity . It is, acidosis . Amos etacidosis  reported-Shankar  reported-Shankar .Management of tubal-ovarian abscess is quite complicated in women of reproductive age, and especially in pregnant patients. Today, in the light of growing evidence, majority of clinicians choose to perform fertility-sparing procedures in management of pelvic abscess. Laparoscopy provides direct visualization of pelvis and allows clinicians to perform additional procedures such as adhesiolysis, salpingotomy, and excision of necrotic tissues, simultaneously.Even though the exact cause of fetal loss is unclear in this case report, pelvic infection can be assumed to be the plausible cause as there was evidence of inflammation reported in the pathological examination of the fetal material. As the duration between surgery and fetal loss was relatively long, it is really hard to say that fetal loss may be linked with the surgical procedure.Long duration of parenteral antibiotic usage is another questionable issue in this case for its toxic consequences on the fetus. However, beyond the etiology of the loss, this report was aimed at raising the issue of alternative management of an abscess when the case is not suitable for ultrasound-guided drainage. In seeking for a more accurate and safe method, especially applicable in early pregnancy, further studies are definitely required.In sum, formation of pelvic abscess following TVOR is a rare, but a serious complication and laparoscopy may be a feasible option when less invasive approaches are unsuccessful during early pregnancy.


Several bacterial pathogens are targeted by macroautophagy (referred to as autophagy hereafter), and autophagy is widely recognized as an important antibacterial defence mechanism A. Actin is a globular, multifunctional protein that forms filaments . Actin  prevents the recruitment of ubiquitin, autophagy receptors, and ATG8 family proteins, thereby inhibiting autophagosome formation Shigella, the polymerization of actin by IcsA/VirG  is strictly required for recognition by ATG5 and the tectonin domain-containing protein TECPR1 to promote autophagosome-lysosome fusion Rickettsia in the spotted fever group, Burkholderia pseudomallei, and Mycobacterium marinum, will help to identify and characterize the diverse roles of actin in autophagy.Some bacterial pathogens, including pathways , 2AIV. IMicrotubules facilitate autophagosome trafficking and are viewed to promote autophagosome biogenesis and the fusion of autophagosomes with lysosomes Listeria ActA can recruit LaXp180/CC1, a binding partner of the microtubule-sequestering protein stathmin Shigella VirA is a type III secretion system (T3SS) effector protein that can destroy surrounding microtubules and help actin-based motility SalmonellaSalmonella-containing vacuole (SCV), and the T3SS effector SifA is essential for this process  interacts with ATG3, a ubiquitin-like conjugating enzyme essential for ATG8 lipidation, and promotes autophagosome biogenesis Shigella, TOCA-1 helps bacteria to avoid recognition by autophagy markers Several pathogenic bacteria can modulate the dynamics of microtubules during infection  process  [25]. WiIntermediate filaments are more stable and exhibit less dynamic behaviour than actin filaments or microtubules. In agreement with this, vimentin intermediate filaments have recently been shown to suppress autophagy dynamics by interacting with Beclin 1/ATG6, a protein crucial for autophagy initiation Chlamydia trachomatis, an obligate intracellular bacterial pathogen, replicates within a large vacuole called an inclusion. 14-3-3β was the first eukaryotic protein found to interact with the chlamydial inclusion Chlamydia can remodel intermediate filaments and actin to provide structural integrity to the inclusion and also to prevent cytosolic detection of inclusion contents  can potentiate an antibacterial effect of autophagy through their ability to stimulate fusion of chlamydial inclusions with autophagosomes The rearrangement of intermediate filaments during bacterial infection is not as well characterized as in the case of actin or microtubules. contents  [34]. InSeptins have been mostly implicated in cytokinesis, a process in which autophagy is tightly regulated, and their role in nondividing cells is poorly understood Shigella infection, septins entrap actin-polymerizing bacteria in cage-like structures that restrict motility and dissemination (Shigella and M. marinum but not for Listeria because ActA masks the bacteria from autophagic recognition (see above). These observations indicate that septin cages assemble in certain pathways of actin polymerization, for example, actin polymerization by WASP family proteins (as occurs in the case of Shigella and M. marinum), and/or that a cytosolic source of membrane is required to facilitate septin assembly surrounding the bacterium. In turn, septins may control the shape of autophagic membranes, as shown for phospholipid-based liposomes in vitro Danio rerio) has suggested that septin cages are an important defence mechanism to clear intracellular bacteria in vivo During mination . ExperimStudies using intracellular bacterial pathogens have shown great potential to significantly advance our understanding of autophagy-cytoskeleton interactions. Despite new insights, many outstanding issues remain. For example, the precise signals targeting bacteria to autophagy and the source of membrane for autophagosome biogenesis  are poorly understood. Work has shown that intracellular bacteria can trigger multiple canonical and noncanonical  autophagy pathways Finally, autophagy dysfunction has been implicated in various human diseases including cancer, neurodegenerative diseases, inflammatory diseases, and infectious diseases. Cytoskeleton dysfunction has also been implicated in many of these diseases. Could autophagy dysfunction underlie the pathogenesis of particular cytoskeleton disorders (and vice versa)? An in-depth understanding of autophagy-cytoskeleton interactions in vivo will be critical to fully appreciate how autophagy and the cytoskeleton can function in immunity and bacterial clearance and if autophagy-cytosketon interactions can be therapeutically manipulated for human health.


Previous studies have shown that the activity of the amygdala is elevated in people experiencing clinical and subclinical levels of anxiety and depression (negative affect). It has been proposed that a reduction in inhibitory input to the amygdala from the prefrontal cortex and resultant over-activity of the amygdala underlies this association. Prior studies have found relationships between negative affect and 1) amygdala over-activity and 2) reduced amygdala-prefrontal connectivity. However, it is not known whether elevated amygdala activity is associated with decreased amygdala-prefrontal connectivity during negative affect states.Here we used resting-state arterial spin labeling (ASL) and blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) in combination to test this model, measuring the activity  and functional connectivity  of one subregion of the amygdala with strong connections with the prefrontal cortex, the basolateral nucleus (BLA), and subsyndromal anxiety levels in 38 healthy subjects.BLA rCBF was strongly correlated with anxiety levels. Moreover, both BLA rCBF and anxiety were inversely correlated with the strength of the functional coupling of the BLA with the caudal ventromedial prefrontal cortex. Lastly, BLA perfusion was found to be a mediator of the relationship between BLA-prefrontal connectivity and anxiety.These results show that both perfusion of the BLA and a measure of its functional coupling with the prefrontal cortex directly index anxiety levels in healthy subjects, and that low BLA-prefrontal connectivity may lead to increased BLA activity and resulting anxiety. Thus, these data provide key evidence for an often-cited circuitry model of negative affect, using a novel, multi-modal imaging approach. One commonly cited model of mental illness is derived from the writings of the neurologist Hughlings Jackson, who observed that frontal lobe lesions were associated with ‘release behaviors’, i.e., disinhibition of suppressed, primitive reflexes that are generated subcortically A novel approach to testing this model is to combine two MRI-based methods that measure “resting-state” brain activity: 1) arterial spin labeling (ASL) functional MRI (fMRI), which uses magnetically-labeled arterial blood water as an endogenous tracer, providing a quantitative measure of regional cerebral blood flow (rCBF), and 2) blood oxygenation level-dependent (BOLD) functional connectivity fMRI, which measures correlations in spontaneous, low frequency fluctuations in the BOLD signal between different brain regions Previous studies using resting-state BOLD data to measure functional connectivity have shown that the basolateral nucleus of the amygdala (BLA) in humans displays a high degree of functional correlation at rest with the ventral medial prefrontal cortex (mPFC), whereas the central nucleus of the human amygdala shows little connectivity with the mPFC Written informed consent was obtained from all subjects prior to enrollment, in accordance with the guidelines of the Partners HealthCare Institutional Review Board, which approved this study.38 healthy adults  using a 12-channel head coil. Subjects underwent two conventional high-resolution three-dimensional structural T1 magnetization prepared rapid acquisition gradient-echo (MPRAGE) scans  = 2530 ms, echo time (TE) = 3.39 ms, flip angle = 7°, resolution 1.3×1×1.3 mm).0 scan  was acquired for resting regional cerebral blood flow (rCBF) quantification purposes One 6-min-2-sec pulsed ASL scan was collected using the “PICORE-Q2TIPS” MRI labeling method One 6-min-12-sec resting BOLD scan  was also acquired.During both the ASL and BOLD scans, subjects were instructed to keep their eyes open, blink normally, and look at a blank screen.Regional CBF levels were extracted from the basolateral nucleus of the amygdala (BLA) using an anatomically-defined BLA region-of-interest (ROI). The BLA ROIs were derived from the Jeulich Histological Atlas www.fmrib.ox.ac.uk/fsl) http://surfer.nmr.mgh.harvard.edu/) tools. The ‘tag’, ‘control’, and M0 scans were first motion-corrected using MCFLIRT X, [(controlX−1+controlX+1)/2−tagX]) to generate perfusion-weighted images. All of the perfusion-weighted maps were then averaged and scaled by a factor proportional to the M0 scan to obtain rCBF maps in absolute values (ml/100 g of tissue/min) ASL data analyses were performed using a combination of FSL  was used as the variable of interest in a linear regression with the time series corresponding to each voxel across the brain. All statistical analyses of correlation data were performed on Fisher z transforms a priori search area for the regression analyses. The search area was confined to a ventral mPFC mask  consisting of voxels showing significant BLA-mPFC functional coupling within arbitrary boundaries containing the ventral mPFC region: x-coordinate ≤|10|, y-coordinate ≥0, and z-coordinate ≤10. Thus this independent, a priori mask was used in order to restrict the subsequent analyses to voxels within the ventral mPFC showing functional connectivity with the BLA, prior to conducting the correlational analyses of interest. Sites showing correlations between BLA-mPFC connectivity and the regression variable (anxiety or rCBF) were deemed significant if they met a cluster-wise correction  within the ventral mPFC. All second-level analyses were conducted using SPM5 (http://www.fil.ion.ucl.ac.uk/spm).Whole-brain random effects multiple regression analyses using STAI state anxiety score or rCBF of the BLA as effects of interest were conducted to identify locations showing correlations between the strength of their connectivity with the seed and the anxiety or BLA rCBF measures. Analyses were restricted to regions of the brain that showed significant connectivity with the seed region (either positive or negative). Based on previous work showing that the BLA is highly anatomically and functionally connected with the ventral mPFC (see Introduction), and a recent meta-analytic factor analysis suggesting that the ventrocaudal mPFC is primarily involved in ‘affect generation’ The location of significant clusters was determined using the Talairach and Tournoux Stereotaxic Atlas A mediation analysis was then conducted to assess the relationships among the results of our regression analyses . The relationship between BLA-mPFC connectivity strength, BLA perfusion levels, and anxiety scores was assessed using the Indirect Mediation Analysis script created by Preacher and Hayes for SPSS The mean (± standard deviation) state anxiety score of this sample  was well below means typically reported in patients with anxiety and other psychiatric disorders −4) and left  amygdala, the majority of which fell within the boundaries of the BLA ROIs  . This fiBLA ROIs . In addiBLA ROIs , as wellBLA ROIs .An average map of the functional connectivity of the BLA confirmed corrected <.02; 3392 mm3; Z = 4.4), orbitofrontal cortex, and superior frontal gyrus  and medial frontal cortex   or SNR measures correlated with anxiety or perfusion .A follow-up, secondary mediation analysis was then conducted to assess the relationship among the findings of the regression analyses conducted above, to distinguish between the following two models also see : 1) BLA To conduct this analysis, BLA-mPFC connectivity levels that had been found to correlate with anxiety or BLA perfusion were extracted for each subject in the following manner: The time course of mPFC resting-state BOLD activity was extracted from 3 mm-radii spheres centered on the locations of the peak correlations with anxiety levels (for the test of Model #1) or BLA perfusion (for the test of Model #2), and then correlated with the time-course extracted from the anatomical BLA seed. These correlations were then transformed into z-scores for each subject, reflecting BLA-mPFC connectivity strength.Testing of Model #1  revealed−4), a significant total effect of perfusion levels on anxiety , but a non-significant direct effect of connectivity on anxiety . Additionally, the direct effect of perfusion levels on anxiety remained significant after taking into account BLA-mPFC connectivity strength , indicating that the relationship between BLA perfusion and anxiety is not mediated by BLA-mPFC connectivity. Furthermore, the bias-corrected bootstrapping test of the indirect effects of BLA perfusion on anxiety through BLA-mPFC connectivity as a mediator (path ab: −.03±.10) did not reach significance, with the 95% confidence intervals  failing to indicate a specific mediated effect. In other words, BLA-mPFC connectivity failed to account for a statistically significant portion of the relationship between BLA perfusion levels and anxiety. In sum, these mediation results support Model #1, that reduced BLA-mPFC connectivity leads to elevated BLA perfusion and subsequent increases in anxiety, rather than the alternative possibility that elevated BLA perfusion leads to poor BLA-mPFC connectivity and increases in anxiety (Model #2).Testing of Model #2  revealedThe goal of this study was two-fold. First, we aimed to determine whether ASL fMRI could detect changes in amygdala activity related to variation in negative affect in healthy subjects. We found strong correlations between the perfusion of the BLA, as well as the hippocampus, putamen, and multiple midline frontal and parietal cortical areas, and anxiety levels, suggesting that ASL is a sensitive tool for measuring this relationship. Second, we sought to test a widely-cited model of psychopathology–the proposal that certain symptoms, including clinical anxiety and depression  is predicted by the strength of amygdala-mPFC connectivity. Pezawas and colleagues first reported that amygdala-mPFC connectivity, as measured by task-elicited BOLD responses, is inversely correlated with temperamental negative affect The present study shows for the first time that amygdala hyper-perfusion is linked to both anxiety and poor connectivity between the BLA and the caudal-ventral mPFC ) in the same subjects. Because conclusions cannot be drawn about the direction of these associations using regression alone, we used a mediation approach to determine whether, in our subjects, reduced connectivity strength within the BLA-mPFC pathway could be linked causally to elevated BLA perfusion and resulting anxiety. These analyses showed that the inverse relationship between BLA-mPFC connectivity and anxiety was significantly mediated by BLA perfusion levels, i.e., that decreased connectivity between the BLA and ventral mPFC contributes to BLA overactivity, resulting in higher anxiety levels. The alternative model, that BLA-mPFC connectivity mediates the relationship between BLA perfusion and anxiety levels, was not significant. Thus, these data provide further support for the Hughlings Jackson, top-down “release” model of negative affect–demonstrating associations among all three components of the model and providing preliminary evidence for a causal relationship between poor mPFC-amygdala connectivity and elevated amygdala activity and anxiety.It should be noted that the mediation analysis in the current study was conducted using data extracted from the regression analyses; thus the findings only apply to this dataset. It will be necessary to repeat these analyses in an independent sample, and in clinically ill samples, to confirm these results. Also, the fact that the direct effect of BLA-mPFC connectivity on anxiety showed a trend towards significance after BLA perfusion was taken into account as a mediator suggests that additional factors, other than amygdala activity, influence the relationship between BLA-mPFC connectivity and anxiety. Follow-up studies can also consider the effects of other potential mediators on these relationships.A model of the function of this pathway has been derived from findings of studies conducted in rodents. The infralimbic (IL) cortex in rodents sends glutamatergic inputs to the GABAergic intercalated cells of the amygdala, which, when stimulated, inhibit the output neurons of the central nucleus of the amygdala In the current study, resting BOLD activity of an extended region of the ventral mPFC was positively coupled with resting BOLD activity of the BLA, consistent with previous findings In the present data, the portion of the amygdala showing the greatest elevation in perfusion in relation to anxiety levels corresponded roughly  to the BLA, rather than the central nucleus of the amygdala, suggesting that disruption of the SGC projection to the BLA  may lead to its disinhibition, resulting in over-stimulation of the central nucleus and elevated arousal and anxiety. Since the GABAergic intercalated cells reside between the basolateral and central nuclei of the amygdala and influence the activity of both nuclei In summary, these results show that ASL is a sensitive tool for objectively measuring the neural effects of subtle variation in anxiety levels. Given that ASL scans are non-invasive, brief and have excellent test-retest reliability, ASL could be used in the future as a clinical procedure which aids in the assessment of risk for future development of psychiatric illnesses, including severe anxiety disorders, such as PTSD, depression or other illnesses associated with impaired functioning of medial temporal lobe structures, such as schizophrenia. Secondly, we also found associations among individual variation in amygdala-mPFC functional coupling, amygdala perfusion, and anxiety levels in the same healthy subjects, with mediation results supporting the model that loss of prefrontal inhibition of the amygdala leads to its over-activity and generation of negative affect. Given that the present findings in healthy subjects are consistent with evidence for similar or related changes in this circuitry in mood and anxiety disorders


Tremendous technological advancements in prostate radiotherapy have decreased treatment toxicity and improved clinical outcomes for men with prostate cancer. While these advances have allowed for significant treatment volume reduction and whole-organ dose escalation, further improvement in prostate radiotherapy has been limited by classic techniques for diagnosis and risk stratification. Developments in prostate imaging, image-guided targeted biopsy, next-generation gene expression profiling, and targeted molecular therapies now provide information to stratify patients and select treatments based on tumor biology. Image-guided targeted biopsy improves detection of clinically significant cases of prostate cancer and provides important information about the biological behavior of intraprostatic lesions which can further guide treatment decisions. We review the evolution of prostate magnetic resonance imaging (MRI) and MRI-ultrasound fusion-guided prostate biopsy. Recent advancements in radiation therapy including dose escalation, moderate and extreme hypofractionation, partial prostate radiation therapy, and finally dose escalation by simultaneous integrated boost are discussed. We also review next-generation sequencing and discuss developments in targeted molecular therapies. Last, we review ongoing clinical trials and future treatment paradigms that integrate targeted biopsy, molecular profiling and therapy, and prostate radiotherapy. Prostate cancer is the most common solid organ malignancy in American men with an estimated 220,800 newly diagnosed cases and projected 27,540 deaths for the year 2015 [The current method of using DRE, PSA, and TRUS biopsy to determine treatment has come under scrutiny. While the incidence of prostate cancer has risen with this screening algorithm, cases of clinically significant disease still go unrecognized and there is concern for overtreatment of more indolent, clinically insignificant cancers as current methods are not able to effectively detect patients who would render a survival benefit from definitive treatment , 4. FurtIn the 1990s, clinicians began using magnetic resonance imaging (MRI) as a tool for staging men diagnosed with prostate cancer. The primary utilization of MRI at that time was identification of extracapsular extension and seminal vesicle invasion because early techniques poorly visualized intraprostatic lesions , 6. The On T2W MRI, hypointense intraprostatic lesions correlate well with cancerous foci found in radical prostatectomy specimens. Similarly, these tumor foci also tend to preferentially enhance dynamic contrast enhanced (DCE) MRI series. The development of magnetic resonance spectroscopic imaging (MRSI), a functional study that detects relative levels of choline and citrate within tumors, adds to the specificity of MRI for intraprostatic lesions . DiffusiThe inclusion of multiple MRI parameters is known as multiparametric MRI (mpMRI). Overlapping modalities in the mpMRI approach corrects for deficiencies inherent in any individual sequence. The use of 2 or more parameters improves the accuracy of detection and localization of prostate cancer –15. CombWhile mpMRI provides valuable anatomic information that often correlates with high-risk histopathology, tissue diagnosis is still essential and remains the gold standard for diagnosing prostate cancer. Recent technological advances have allowed for the integration of mpMRI with ultrasound guided biopsies and this is currently being evaluated as a potential alternative or supplement to the standard TRUS biopsy. Three approaches have emerged that use mpMRI for guiding prostate biopsies including direct “in-bore” MRI biopsies, cognitive fusion, and MRI-TRUS fusion-guided biopsy .p = 0.001) [Initial studies using mpMRI to guide biopsy performed the biopsy under direct visualization in the MRI gantry. The patient first gets a diagnostic mpMRI and returns for biopsy if suspicious lesions are identified. Upon return, biopsies of the lesions are obtained under direct visualization using serial MRI scans to confirm biopsy needle placement. Advantages of this method are that only visualized lesions are biopsied, which decreases the total number of biopsies the patient receives, and this allows for precise documentation of biopsy needle locations –22. The = 0.001) .Cognitive fusion biopsy is the simplest method of combining mpMRI and prostate biopsy. The urologist reviews previously acquired mpMRI images and then biopsies the general location of suspicious MRI lesions using the standard TRUS biopsy technique. The advantage of cognitive fusion biopsy is that it requires no additional equipment or cost, making it most easily adaptable to current practice models. The main disadvantage is strong operator dependency in correlating static MRI findings with dynamic real-time ultrasound findings. Cognitive fusion biopsy also lacks the ability to archive the exact location of the biopsy which could be important for focal therapy or surveillance purposes. Despite these potential shortcomings, the use of cognitive fusion biopsies increases prostate cancer detection and more accurately depicts overall disease burden in high-grade disease , 25. SpeThe newest and most promising form of MRI-targeted prostate biopsy is the fusion of mpMRI with real-time TRUS imaging with postimage processing and software technology. In MRI-TRUS fusion biopsy, a diagnostic mpMRI is used to localize the tumor and a specialized software program fuses these images to a real-time TRUS image seen in the biopsy suite. An important practical advantage of MRI-TRUS fusion biopsy is that the MRI and the TRUS do not have to be physically or temporally linked. MRI data is transferred to one of several models of fusion software enabled 3D-TRUS units that can be located in a standard ultrasound suite. After upload, images of the prostate are remodeled using identification of landmarks  that are present on both the MRI and TRUS platform. Since the prostate on MRI  often differs in shape and contour from the same image on TRUS, the superimposed image must be transformed before successful fusion can occur. This is done through either an elastic or rigid transformation or a combination of both fusion algorithms. These images are shown as either a side-by-side display of the MRI and TRUS images or a single fused image allowing for targeted biopsy of the predelineated regions of interest from the diagnostic mpMRI on the real-time TRUS after fusion. The fusion enabled 3D-TRUS contains a tracking method that fixes the prostate in a 3D coordinate system so that movements of the US probe are mirrored on the fused MRI display. While this method appears to be less operator dependent, there is still need for operator input to assess and adjust altered gland contours or misregistration artifacts.The data supporting the use of MRI-TRUS fusion biopsy is promising. Puech et al. compared the effectiveness of standard 12-core biopsy and MRI-TRUS fusion biopsy and found that fusion biopsy detected 10% more prostate cancer overall and 15% more clinically significant prostate cancer . In the Radiation therapy has been a mainstay in the treatment of prostate cancer since the 1960s with the development of high-energy teletherapy units and linear accelerators. Shortly thereafter, interstitial prostate brachytherapy became a primary treatment modality for organ-confined prostate cancer. Major advances in diagnostic imaging since that time have dramatically improved the ability to accurately target the prostate with smaller and smaller treatment volumes. This, in turn, led to better toxicity profiles, safe dose escalation, and improved disease control –39. MoreThe evolution of external beam radiation techniques and advanced imaging techniques has allowed for increasingly focal radiation therapy with margins around the prostate as small as 5 mm when using standard fractionation schemes . SignifiA second strategy toward dose escalation involves heavy ion-based irradiation such as proton therapy. Proton therapy differs from conventional photon-based radiation therapy in that protons are charged particles that deposit a higher proportion of energy toward the end of their path of travel in a tissue and little to no energy beyond. Therefore, a very steep dose gradient can be created to minimize dose spill into adjacent tissue as compared to photon therapy. Unfortunately, population studies and experiences from large proton centers do not show superiority in disease control or toxicity for proton therapy –43.One of the disadvantages of dose-escalated fractionated radiation to the prostate is the prolonged duration of treatment using standard fractionation schemes of 1.8 to 2 Gy per fraction to total doses of 74 to 80 Gy. In addition, there is a biological rationale for delivering higher radiation dose over a shorter period of time. Thus, multiple phase III trials have been conducted to demonstrate the safety, feasibility, and efficacy of hypofractionated, or shorter than standard, regimens –53. The Cell survival after radiation therapy is modeled by an exponential function that accounts for both direct, called alpha, and indirect, called beta, mechanisms of DNA damage. The ratio, or alpha/beta ratio, of these types of damage can give a general sense of the ability of the tissue to repair that damage. This repair ability is inversely proportional to the alpha/beta ratio. Generally, normal tissues have an alpha/beta ratio around 3 and tumors around 10. Historically, most radiation treatment schedules have been designed to capitalize on these differences in damage repair between tumor and normal tissue by delivering small doses of radiation over a prolonged period of time. This is the case for standard fractionation prostate radiation. More recent data, however, suggests that prostate cancer may actually have a lower alpha/beta ratio than previously suspected. This would mean that there is less benefit to lower dose, fractionated regimens.Using this rationale, recent studies have investigated shorter courses of radiation therapy with higher doses per treatment . Two earExtremely hypofractionated radiation regimens consisting of 5 treatments or less have also been investigated . Three rExternal beam radiation dose escalation and hypofractionation trials increased dose homogeneously to the entire prostate. Despite this, the most common location of recurrence is within the prostate . 80% of More recent evidence supports the idea that dominant intraprostatic lesions, as opposed to multifocal disease, drive the natural course of disease. Furthermore, these dominant lesions are the site of most recurrences –61. UnfoRadiation techniques to deliver focal therapy to prostate lesions involve both external beam radiation and prostate brachytherapy. External beam techniques include IMRT, VMAT, and helical tomotherapy. Multiple dosimetric studies demonstrate the feasibility of escalating dose to an intraprostatic lesion up to 100 Gy with little to no potential for excess toxicity compared to standard whole-gland treatment. A single phase II trial has demonstrated feasibility of escalating dose to an intraprostatic lesion to 80 Gy with toxicity comparable to standard homogeneous dosing .Unfortunately, sacrificing whole-organ dose for focal boost results in inferior biochemical control using both external beam and brachytherapy techniques, especially with intermediate and high-risk disease . In the The approach used in current prostate radiotherapy trials investigating hypofractionation and extreme hypofractionation utilizes a technique called simultaneous integrated boost (SIB) to deliver higher dose to dominant intraprostatic lesions while still delivering an adequate lower dose to the whole prostate. We now know that delivering higher radiation doses benefits men with the most high-risk disease. We also know that only targeting individual lesions within the prostate in these men leads to worse outcomes. Doses as high as 85 Gy have been delivered to the entire prostate with external beam radiation therapy . The preNCT01856855) is investigating the efficacy and toxicity of such an approach [A newer approach to more accurately and appropriately intensify therapy is to combine information obtained by MR-TRUS fusion biopsy with SIB radiation therapy. Fusion biopsy identifies more clinically relevant, high-risk disease, which benefits most from treatment intensification. It also provides direct correlation between imaging and histopathologic findings. Furthermore, other intraprostatic lesions can be biologically risk-stratified to guide treatment planning. A phase II protocol at the University of Alabama at Birmingham NCT018555 is invA big challenge in choosing adequate therapy and determining outcomes from clinical trials in prostate cancer is the significant disease heterogeneity within risk groups. Genomic and molecular analyses of prostate cancer specimens will hopefully help us better characterize disease risk and personalize treatment. Multiple genomic panels have been developed and validated in predicting outcomes based on tissue from radical prostatectomy or biopsy specimens. Prolaris  is a 46-gene expression panel for biopsy and TURP specimens that predicts prostate cancer death. It has been validated in radical prostatectomy specimens to predict biochemical recurrence and distant metastases , 70. DecDespite significant advances in prostate cancer therapy over the last few decades, many men, particularly those with high-risk disease, will have PSA recurrence, develop symptomatic local or distant disease, or die from their prostate cancer. Prostate cancer is a heterogeneous disease and can even manifest heterogeneously within the prostate of a single patient. Prostate cancer therapy is moving rapidly toward personalization, and this approach could significantly improve outcomes for men with high-risk biology. Future clinical trials and standard therapy will biologically risk-stratify patients in order to optimize treatment outcome. Personalized prostate cancer therapy, therefore, will depend on our ability to accurately identify, biopsy, analyze, and treat areas of high-risk disease within the prostate for men with organ-confined disease. The incorporation of MR-TRUS biopsy, molecular testing of biopsy specimens, and focal treatment intensification will make personalized therapy a reality.


Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography–computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate  Constroduction . Reporteoduction ,17,20. SPA.Replication kinetics of the multi-label reporter virus system were characterized in human A549 lung cells B,C. Replper se. Binding to A549 and MDCK cells proceeded rapidly and reached a maxima in ~45 min, paralleling prior analyses [Reporter viruses were used to study early stages in the viral life cycle, beginning with attachment using bioluminescent PASN virions. Hemagglutinin (HA)-mediated attachment to sialic acids on the cell surface is essential during infection and is often disrupted by HA-specific antibodies . Westernanalyses . FurtherFollowing attachment and entry, fluorescent reporter viruses are especially useful in tracking gene expression at the cellular level without the administration of exogenous substrate for reporter activity. Live-cell microscopy detected robust fluorescence in A549 cells infected with PATG and PATR, but not WSN A. Singly50), rely on successful infection and completion of the viral life cycle, our fluorescent and bioluminescent viruses report on gene expression, and not necessarily the capacity to produce infectious particles. It has recently been suggested that a large percentage of influenza virions are capable of initiating infections, but cannot support production of infectious progeny, so-called “semi-infectious” virions [GFP). These experiments routinely detected 3–5 fold more cells expressing virally encoded GFP than would be predicted based on the MOI used to initiate the infection; all of the cells inoculated at an MOI = 0.4 were GFP-positive, when only ~33% are predicted to be infected assuming an ideal Poisson distribution . This suggests that some of the enhanced gene expression for PASTN resulted from complementation during co-infection by two “semi-infectious” particles. Moreover, as WSN enhanced NLuc expression which was only present in the reporter virus genome, these data provide evidence that one mode of multiplicity reactivation occurs by restoring expression from an otherwise inactive gene segment, as opposed to solely supplying a replacement for the defective segment. By exploiting the unique advantages of these reporter viruses, we have established methods to quantitate semi-infectious events and multiplicity reactivation, and probe their potential role during infection and viral evolution.Multiplicity reactivation predicts that semi-infectious events can be complemented during co-infection to restore viral gene expression and potentially virus production . To addrin vivo imaging  and was below the limit of detection by 7 days post infection (dpi), preventing an otherwise lethal infection.A key advantage of using non-invasive reporters is the capacity for longitudinal measures throughout the course of infection and sequential measures using different imaging modalities or spectra. Using longitudinal bioluminescent imaging to measure virus replication and tissue distribution in the same animals, we evaluated the antiviral kinetics of the neuraminidase inhibitor oseltamivir (OSV). Animals were treated beginning 2 days post PASTN inoculated and viral load was quantified by  imaging A. In con18F-FDG, a radiotracer for detecting influenza-mediated inflammation [18F-FDG localized to each of the CT-defined lung volumes (We then paired bioluminescence and PET/CT imaging to measure both virus replication and inflammation. Inflammation was measured in a subset of mice at 5 dpi using ammation  (Figure  volumes C. OSV trin vivo imaging experiments demonstrate the ability to simultaneously measure viral replication, the efficacy of antiviral or immunomodulatory therapy, the host antiviral response, and the close interdependence of these variables.In conclusion, we report the generation of a versatile toolbox of influenza A reporter viruses. These viruses are suited for probing discrete stages of the viral replication cycle at the cellular level—from attachment to the cell, through gene expression, and ultimately the spread of released virus. Moreover, multi-modal


Diabetic cardiomyopathy is defined as ventricular dysfunction initiated by alterations in cardiac energy substrates in the absence of coronary artery disease and hypertension. In addition to the demonstrated burden of cardiovascular events associated with diabetes, diabetic cardiomyopathy partly explains why diabetic patients are subject to a greater risk of heart failure and a worse outcome after myocardial ischemia. The raising prevalence and accumulating costs of cardiovascular disease in diabetic patients underscore the deficiencies of tertiary prevention and call for a shift in medical treatment. It is becoming increasingly clearer that the effective prevention and treatment of diabetic cardiomyopathy require measures to regulate the metabolic derangement occurring in the heart rather than merely restoring suitable systemic parameters. Recent research has provided deeper insight into the metabolic etiology of diabetic cardiomyopathy and numerous heart-specific targets that may substitute or reinforce current strategies. From both experimental and translational perspectives, in this review we first discuss the progress made with conventional therapies, and then focus on the need for prospective metabolic targets that may avert myocardial vulnerability and functional decline in next-generation diabetic care. As of 2013, one in ten individuals living in Western countries was diagnosed with diabetes mellitus (DM), and three in ten were obese .-48.46-48GLP-1R agonists may withstand some of the controversy concerning DPP-4 inhibitors  cofactor . TLRs ardb/db mice administered desacyl ghrelin, the non-octanoylated form of this “hunger hormone” [Genetic engineering has uncovered numerous other key molecules of lipid metabolism with potential use against DCM. This group includes rate-limiting lipases of TAG hydrolysis -65, arachormone” . Remarka2-consumption ratio and reduce hyperglycemia and AGE generation. Insulin resistance and hyperlipidemia may be also controlled by statins, and perhaps more prospectively by incretin-based drugs, which would balance the content of sarcolemmal glucose and FA transporters and exert anti-apoptotic/fibrotic benefits in the cardiomyocyte. In addition, FAT/CD36 interference may provide further protection against myocardial inflammation and stiffness through the attenuation of TLR4-NLRP3 signaling. The design of these cardioprotective plans, however, may lead to difficulties concerning the possibility of provoking other end-organ complications associated with glucotoxicity. To overcome these hurdles, the assessment of differential traits, stages and pathogenic factors in each diabetic patient becomes a clinical priority.As therapeutic resources currently allow diabetic patients to manage glucose homeostasis, reach near-normal life expectancy and delay microvascular complications, it is unacceptable that heart-risk reductions are not yet properly achieved. By targeting DCM we may prevent cardiac functional decline and improve the response to potentially lethal coronary events. Although the fine-tune mechanisms leading to DCM remain partially opaque, restoring cardiac energy metabolism seem to be a cornerstone. In light of the experimental data provided here, we may outline a potential therapeutic strategy focused on correcting the etiological imbalance between lipids and glucose as fuel substrates Figure . Excessi


Obesity and diabetes are increasing at an alarming rate worldwide, but the strategies for the prevention and treatment of these disorders remain inadequate. Brown adipose tissue (BAT) is important for cold protection by producing heat using lipids and glucose as metabolic fuels. This thermogenic action causes increased energy expenditure and significant lipid/glucose disposal. In addition, BAT in white adipose tissue (WAT) or beige cells have been found and they also exhibit the thermogenic action similar to BAT. These data provide evidence indicating BAT/beige cells as a potential target for combating obesity and diabetes. Recent discoveries of active BAT and beige cells in adult humans have further highlighted this potential. Growing studies have also shown the importance of central nervous system in the control of BAT thermogenesis and WAT browning using animal models. This review is focused on central neural thermoregulation, particularly addressing our current understanding of the importance of hypothalamic neural signaling in the regulation of BAT/beige thermogenesis and energy homeostasis. Brown adipose tissue (BAT) is a thermogenic organ that protects the body from cold environment via dissipating chemical energy (lipids and glucose) as heat. For a long period of time, BAT was thought to be only present in certain species of mammals, including rodents, hibernating animals, and newborn humans. Recently, active BAT has been found in adult humans at a cold environment –6. TheseThere are two types of brown adipocytes. The classic brown adipocytes, such as the ones that reside in the interscapular region in rodents, are originated from myf5-positive cells . The othBrown adipose tissue contains a large number of mitochondria that act as a heat generator through uncoupling protein 1 (UCP1). During BAT activation, UCP1 uncouples the oxidation of fatty acids from ATP synthesis to dissipate energy as heat. This function is very important for small mammals to maintain body temperature, especially during cold environment . Fatty aBrown adipose tissue thermogenesis is predominately governed by the sympathetic nervous system (SNS) via the adrenergic receptor signaling pathways. Upon stimulation, sympathetic nerve releases norepinephrine that binds to β3 adrenergic receptors in the membrane of brown adipocytes to activate a cascade of signaling pathways, leading to increases in fatty acid β-oxidation and heat production . Recentlin vivo and in vitro studies have suggested that BAT-associated glucose uptake is regulated by norepinephrine and insulin -induced thermogenesis in iBAT , 70. HowDespite these discrepancies, recent studies at molecular levels provide support for the role of the VMH in thermoregulation. One report has shown that estradiol inhibits AMP-activated protein kinase (AMPK) through the estrogen receptor α (ERα) in the VMH and results in activation of BAT thermogenesis through the SNS . StudiesO-linked β-N-acetylglucosamine (O-GLcNAc) transferase  and orexigenic neurons (NPY and AGRP). Food restriction causes significantly increased expression of NPY and AgRP in the ARC, while food restriction also decreases adaptive thermogenesis , implyinnsferase . O-GLcNA fasting . Since N fasting , ARC AgR fasting . Given tThe neurochemical characterization of the hypothalamic projections to the BAT in rats identifies the presence of melanin-concentrating hormone (MCH) and orexin (hypocretins) neurons in the caudal aspect of LH , implicaMc4r null mice reversed hyperphagia, but did not affect energy expenditure , BAT is activated to produce heat to maintain body temperature in mammals. As discussed above, studies on animal models have shown that the hypothalamus serves as an essential site in this thermoregulation. Particularly, the POA/DMH/rRPa neural system is excited to promote the sympathetic activity in BAT to increase BAT and body temperature in response to cold exposure , 61. UsiN-methyl-d-aspartate receptor blocker MK-801 directly into the caudomedial nucleus of the solitary tract (NTS), indicating that the CCK–NTS signaling also mediates diet-induced BAT thermogenesis. Intriguingly, peripheral administration of CCK activates neurons in the NTS, as well as within the PVN and the DMH using c-Fos as an activation marker  had increased body core and BAT temperature . This efn marker , suggest1A receptor agonist eliminated these thermogenic effects induced by social defeat stress  in adult humans have provided the potential for BAT in combating obesity and associated comorbidities. Using animal models, we now appreciate that the brain regulates sympathetic outflows to BAT to modulate BAT thermogenesis and body temperature. Especially, recent studies have revealed that hypothalamic peptide signaling plays an important role in the control of BAT thermogenesis and WAT browning in rodents Figure . PhysiolThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A 65-year-old patient presented with persistent ulnar-sided pain for years. X-ray revealed no osseous or articular abnormality but mild to moderate basal joint degenerative arthrosis. There was positive ulnar variance bilaterally with cystic changes along the ulnar aspect of the lunate left greater than right, suggestive of “ulnolunate impaction syndrome.”What is the diagnosis?Is there any eponym?What are the etiology and clinical presentation of the condition?What are the treatment options for ulnar impaction syndrome (UIS)?Ulnar impaction syndrome is a degenerative wrist condition caused by the ulnar head impacting upon the ulnar-sided carpal bones. This abutment results in increased load bearing across the ulnar head, triangular fibrocartilage complex (TFCC), and ulnar carpals and subsequent degeneration of the TFCC, chondromalacia of the osseous structures involved , and disruption of the triquetrolunate ligament.1This condition is also known as ulnar abutment or ulnocarpal loading.-3,5The most common predisposing factor is positive ulnar variance, an increased ulnar length relative to the radius. In the positive ulnar variance wrist, the TFCC is stretched and thin, and greater biomechanical forces, specially rotation forces, impact the joint. This positive variance can be congenital1,Conservative methods should be attempted as the first line of treatment and include immobilization for 6 to 12 weeks, nonsteroidal anti-inflammatory drugs, corticosteroid injection and limiting aggravating movements such as pronation, gripping, and ulnar deviation.2Several studies have reported a high percentage of success with ulnar shortening osteotomy. Baek et al


Understanding the signals used by plants to attract seed disperses is a pervasive quest in evolutionary and sensory biology. Fruit size, colour, and odour variation have long been discussed in the controversial context of dispersal syndromes targeting olfactory-oriented versus visually-oriented foragers. Trade-offs in signal investment could impose important physiological constraints on plants, yet have been largely ignored. Here, we measure the reflectance and volatile organic compounds of a community of Malagasy plants and our results indicate that extant plant signals may represent a trade-off between olfactory and chromatic signals. Blue pigments are the most visually-effective – blue is a colour that is visually salient to all known seed dispersing animals within the study system. Additionally, plants with blue-reflecting fruits are less odiferous than plants that reflect primarily in other regions of the colour spectrum. The physical properties of fruits can act as signals of edibility and nutrition to the seed dispersing animals that play a crucial role in moving seeds away from the parent tree and, thus, increasing plant fitness ,2,3,4,5.The fruit syndrome hypothesis posits that suites of fruit signals should be directed at the frugivorous guilds that provide the highest quality seed dispersal service, according to their capacity to receive signals . The fruThe fruit syndrome hypothesis has intuitive appeal and support for the colour-odour trade-off has been shown to exist for bird- versus bat-dispersed species  and in mOur study aims to help clarify this debate by approaching the fruit syndrome hypothesis using a quantitative and multivariate approach. For the fruit syndrome hypothesis to be supported, there should be a negative relationship between chromatically conspicuous fruits and odiferous fruits, i.e., fruits that invest in colour should not invest in odour, due to the predicted trade-off between attracting olfactory-driven versus visually oriented foragers.Here, we evaluate the fruit syndrome hypothesis by testing whether fruits invest in specific signalling strategies at the expense of others. To evaluate potential phylogenetic constraint on these strategies, we first test for the presence of a phylogenetic structure among traits using a species-level phylogeny. We examine chromatic  and odour (olfactory) signals in an analysis of 56 endemic wild fruit species in a tropical dry forest in Madagascar. We predict that fruits that invest in pigment production in non-photosynthetically active regions of the chromatic spectrum will not invest heavily in odour production. Additionally, because plants with a higher surface-area-to-weight ratio  produce more signals relative to overall fruit production  we predict that the relationship between fruit colour and odour will be altered by surface-area-to-weight ratio of fruits.Ethics statement—N/A. Permission for fieldwork granted by Madagascar National Parks and the Government of Madagascar (permit number: 092N_EA07/MG12). Between January and December, 2012, between 5–10 ripe fruits of each species were opportunistically collected in the Ampijoroa region of Ankarafantsika National Park, Madagascar . Ripe fruits of each species were collected directly from trees and all analyses were performed within ~2 hours of fruit collection. Fruit ripeness was determined based on colour, odour, and hardness and confirmed after analysis based on the presence of viable seeds. Seeds were considered viable if they were fully formed, had the approximate mass of seeds known to germinate, and had no evidence of damage. Plants were identified to genus and species using a published tree flora  and an uTo quantify fruit odour, we measured volatile organic compound (VOC) emissions of ripe fruits. Ripe fruits were placed in inert ~1.5 L plastic sampling bags and the atmosphere within each bag was sampled using a vacuum pump  that pulled air through the sample bag  and into two odourant-adsorbent filters . Contamination of the sampling enclosure with ambient VOCs was minimized by passing incoming air through a container of activated carbon. Additionally, blank samples were collected and analysed to identify contamination from the sampling apparatus. Three peaks representing sampling apparatus contamination were detected in each blank sample and these peaks were subtracted from total VOC sums of fruit samples. Trapped VOCs were analysed using the procedure and instrumentation reported in . All VOCReflectance spectra of one ripe fruit of each species were measured relative to a Spectralon white reflectance standard (Labsphere) on-site using a Jaz portable spectrometer and a PX-2 pulsed xenon lamp (Ocean Optics Inc.) emitting a D-65 light source, with a range of 250-720nm Figs  and 2. T10 surface-area-scaled sum of VOC emissions. We calculated the surface area of each fruit using the following equation for the surface area of an ellipsoid: 4π[((ab)p+(bc)p+(ac)p)/3] 1/p where p = 1.6075 [We used a VOC index, calculated as log= 1.6075 . Four re= 1.6075 . ReflectVOC emission, UV, blue, green, and red reflectance were optimized onto a species-level phylogeny as continuous characters, using TNT version 1.1 . The fraTo assess the association between fruit odour production and fruit chromaticity, we calculated the Pearson correlation of UV, blue, green, and red reflectance with VOC index. To account for correlation across normalized spectrum-reflectance values, we developed a multivariate regression model that included a spline transformation of the spectrum-reflectance values. A series of five predictor variables were calculated as weighted sums of spectrum-reflectance values. The weights for the five predictor variables were determined by a natural-cubic spline that had knots at 350, 410, 470, 530 and 590 nm. Because the summed reflectance values between 400 and 700 nm added to 100%, our spline transformation used the 650nm bandwidth as a fixed referent value to protect against multi-collinearity [When mapped onto a model phylogeny, none of the measured fruit traits showed any evidence of being phylogenetically informative. Calculated values of CI  and RI  were much lower than would be sufficient for an informative character . We deteThere was a significant negative relationship between blue reflectance and overall VOC index . We observed no significant relationship between VOC index and any of the other reflectance ranges: UV , green , or red reflectance  and a lack of relationship in the other reflectance bands.To control for the confounding effect of fruit size and correlated reflectance bands, we ran a multivariate model that included a spline transformation of the normalized reflectance values . The figOur prediction that fruit investment in non-photosynthetically active pigments would scale negatively with odour production is partially supported. We find that bluish fruits have significantly lower total VOC emissions than fruits reflecting other hues, which is consistent with the fruit syndrome hypothesis. Specifically our results suggest the existence of a disperser-mediated dichotomy in fruit colour and odour signal production: plants that invest more in blue chromatic signals invest less in odour, yet fruit size is a critical component.The trade-off between blue chromatic reflectance and VOC emission is particularly compelling because reflectance in the blue range of the spectrum (400-500nm) may be doubly costly to a plant as it occupies critical photosynthetic space—both chlorophyll A and B absorb blue wavelengths of light . Thus plThe lack of a relationship between red reflectance (600-700nm) and VOC emission may result from the other benefits to a plant of investment in pigments that reflect at this range of the spectrum . Advantages to red plant pigmentation include anti-fungicidal properties, photoprotection against UV damage, prevention of photoinhibition, and chromatic crypsis against dichromatic (red-green colour blind) herbivores ,46,47. TOur finding that smaller fruits tend to invest heavily in VOC production may reflect the diminutive size of nocturnal, olfactory-driven mammals in Madagascar. Unlike most other tropical systems where the small end of the disperser size spectrum is dominated by avifauna, in Madagascar the smallest seed dispersing animals are mouse and dwarf lemurs of the family Cheirogaleidae . CheirogOur results provide support for the idea that fruit traits may converge to simultaneously attract multiple dispersers with diverse sensory phenotypes by using both colour and odour signals when possible, or by decreasing olfactory signals when producing colours that all dispersers can see well. An important next step will be to record the behaviour of seed dispersers relative to fruit cues. In this forest there are five known seed dispersing mammals, three nocturnal and two cathemeral, ,50 and fS1 Fig(PDF)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.


Zymomonas mobilis ZM481 (ATCC 31823) is an ethanol-tolerant strain that can produce the highest level of ethanol in Z. mobilis from glucose in the shortest time. Here, we report a draft genome sequence of ZM481, which can help us understand the genes related to the ethanol tolerance of this strain. Zymomonas mobilis quickly metabolizes glucose to ethanol via the unique Entner-Doudoroff pathway, which improves some of the most important technoeconomic factors of production, ethanol yield, and productivity, because of low biomass accumulation and high specific surface area for sugar uptake during fermentation  is a high-yielding mutant strain derived from Z. mobilis ATCC 31821 (ZM4), which cannot only produce the highest level of ethanol in the shortest time but also get better ethanol tolerance than the original strain  to accomplish functional annotation by comparing coding gene with nonredundant protein sequences from KEGG rom KEGG . rRNA, trom KEGG , tRNAscarom KEGG , respectBecause ZM481 was derived from chemical mutagenesis of ZM4, whose genome was completely sequenced and well annotated in 2005 , the genLSFP00000000. The version described in this paper is the first version, LSFP01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.


Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events  Ribosome, a dynamic ribonucleoprotein complex (~2.4MDa in prokaryotes and ~4MDa in eukaryotes), is widely known to be the key player of protein synthesizing machinery of every living cell upon which the genetic code within the mRNA is translated into the primary amino acid sequence . Most ofin vitro, confirming Anfinsen’s pioneering proposition that the primary amino acid sequence of a protein determines its folding and given proper conditions, proteins can fold spontaneously either co-translationally or post-translationally  are coloured in navy blue. Entrance of the polypeptide tunnel is marked. (B) Available Domain V structures from different organisms  superimposed showing very less deviation (RMSD < 5Å). Land marks: CP, central protuberance; SB, P-proteins stalk base.(PDF)Click here for additional data file.


The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer.We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. “pre-diagnostic”, we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve.We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis .Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics. A serum test to aid in the diagnosis of cancer has long been the “holy grail” of early cancer detection. Developing a blood based diagnostic assay is ideally suited to a disease such as breast cancer as high risk populations are defined and current screening tools, specifically mammography, are limited in specific populations, i.e. increased breast density in young women. Identification of autoantibodies associated with high risk or pre-invasive disease may facilitate serum-based testing for early detection or risk stratification. Unlike circulating shed tumor proteins or nucleic acids, plasma levels of which are dependent on tumor size, autoantibodies are elicited in significant concentrations even after minimal exposure to the antigen due to cytokine induced production of antibodies by activated B cells . DespiteGenetically engineered mouse models of cancer may provide a model system for identification of diagnostic autoantibodies. As mice develop spontaneous breast cancers, many in middle age, serum can be collected over time and samples for discovery can span both disease-free and disease-bearing states in the same individual. We questioned whether the TgMMTV-neu model could be used to identify autoantibody candidates for the early detection of human breast cancer. Although the TgMMTV-neu expresses the neu proto-oncogene, the genes expressed in the tumors that arise in these animals is similar to the genes expressed in human luminal breast cancer . The micneu)-202Mul) or wild-type FVB mice  were bred under specific pathogen-free conditions at the University of Washington. Animal care and use was in accordance with institutional and national guidelines. The protocol was approved by the University of Washington Institutional Animal Care and Use Committee (Protocol number 2878–01). Mice were enrolled for blood collection and monitoring for spontaneous tumor growth between 4–6 weeks of life  discrete positive plaques were identified from each group of 32,000 clones evaluated. The positive plaques were interrogated with pooled normal FVB serum  was used to identify tumor associated autoantibodies as previously described . BrieflyEvaluation of autoantibodies in mice. Two methods were used for the quantitation of murine antibodies; ELISA and western blot/densitometry. The measurement of serum antibodies to tumor antigen Pdhx was carried out using a lysate ELISA (bacteria expressing the protein of interest) as previously described with the modification that both IgG and IgM responses were evaluated . Experimental serum at a 1:200 dilution and horseradish peroxidase conjugated goat anti-mouse IgG  or IgM  were used. After development, plates were read at an absorbance of 450 nm. The OD of each serum dilution was calculated as the OD of the antigen-coated wells minus the OD of non-antigen encoding lysate-coated wells. The autoantibody concentration (ng/well) was calculated from the 4-parameter fitted standard curve on each plate. All identified antigens were highly homologous between mouse and man, median 95% (range 80–100). Human recombinant proteins were available for Otud6b, Stk39, and Lgals8  and indirect ELISA was performed to measure serum antibodies to tumor antigens as previously described [The assessment of Vps35 and Znf238 autoantibodies was performed by Western blot and densitometry as the recombinant proteins gave insufficient signal in ELISA. 3 pmole recombinant protein of Vps35 or Znf238 (Abnova) were loaded on SDS-PAGE gel and transferred onto nitrocellulose membranes . After blocking with 5% nonfat milk, membranes were incubated with mouse sera (diluted 1:100) overnight. Then the membranes were washed with TBS/0.05% Tween 20 and incubated with peroxidase-labeled goat anti-mouse IgG  or IgM (1:500 dilution) secondary antibody (Invitrogen). After washing, bands were visualized using a peroxidase-linked enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) and the optical density of the specific band was quantified with ImageJ software.Evaluation of antigen expression in human DCIS. To evaluate whether these identified antigens had the potential for being expressed in human pre-invasive lesions, a GEO dataset, GSE26304, was analyzed for expression of the genes encoding the identified pre-diagnostic antigens [Due to the low volume of sample available for analysis, serum autoantibodies to only 3 antigens, Otud6B, Stk39, and Pdhx, were assessed using customized protein microarrays as previously described . We chos2 test. The sensitivity and specificity of a single or combination of antibodies in mice was evaluated using receiver-operating-characteristic (ROC) curve analyses, leading to estimates of the area under the curve (AUC), with 95% confidence intervals. Statistical analysis was carried out in SPSS software, version 15.0. For protein arrays, ROC analysis of marker combinations was performed using a linear regression model based on maximum likelihood estimation. AUC and 95% confidence intervals were calculated using R v2.13.1.For all analysis, significance was assessed using a two-tailed T test or ANOVA with p < 0.05 being considered significant. Differences in the incidence of an antibody response between transgenic mice and controls were evaluated using a xPre-diagnostic autoantibodies directed against six tumor associated antigens were identified in individual TgMMTV-neu mice at significantly higher levels compared to wild-type controls (p < 0.01 for each antigen)  from FVB controls, ROC were generated and AUC calculated Table . The perTo explore the relevance of these antigens to human pre-invasive disease, we evaluated gene expression in an existing data set of normal breast, DCIS, and IDC. 10% of DCIS expressed Pdhx Figure A, Lgals8As human homologues of the murine pre-diagnostic antigens exist and the proteins appear to be expressed in human pre-invasive breast lesions, we explored the potential utility of three of the antigens, Pdhx, Otud6B, and Stk39 in discriminating women who would eventually develop cancer from controls Table A. An evaKinetic studies in mice Figure  demonstrWe questioned whether the use of genetically engineered mouse models of mammary cancer could serve as a tool for developing autoantibody panels useful for the early detection of human breast cancer and/or improved stratification of risk. The identification of cancer predicting autoantibodies, using individuals bearing early stage invasive breast cancers, may not encompass antigens associated with the earliest high risk lesions, thus may limit the utility of any identified panel for screening a general population. Transgenic mouse models of mammary cancer have been shown to have significant genetic similarities to human breast cancer . In addiThere have been several studies that have explored the use of autoantibody panels in the early detection of breast cancer. All have assessed sera from disease bearing individuals and several have focused on well-known tumor associated antibodies such as HER2, p53, NY-ESO, and MUC1 for example ,9. In anThe autoantibodies we found in the pre-diagnostic sera of TgMMTV-neu mice, animals that had not yet developed invasive cancers, were also directed against intracellular proteins. Half of the antigens identified are also associated with inflammation and immunity. Pdhx, a glycolysis protein, is an antigenic component of anti-mitochondrial autoantibodies. The majority of patients with primary billiary cirrhosis have autoantibodies directed against Pdhx . Lgals8 Genes encoding homologues of murine pre-diagnostic antigens are found in human breast, DCIS, and invasive cancers. Our studies demonstrate that women who eventually develop breast cancer have evidence of autoantibodies targeting these pre-diagnostic antigens prior to their first diagnosis of disease. A limited panel of only 3 of the autoantibodies could discern women destined to develop breast cancer from matched controls that did not develop disease with an AUC approaching 0.7. Key to this finding was the use of both IgG and IgM antibodies for detection and assessing sera at a time point more distant from diagnosis. IgM antibodies are the first antibodies secreted by B cells in response to an antigen. B cell proliferation and IgM production are induced simultaneously via cytokine secretion by a number of different immune cells . As T ceThe ideal method to develop an autoantibody panel for the early detection of breast cancer would be to discover autoantigens in women who have not yet developed invasive cancer but are destined to do so. The use of genetically engineered mice that develop spontaneous tumors, allows investigators to model studies such as WHI and collect samples from individuals who have not yet developed cancer but will do so in a defined period of time. Genetic and protein based analyses suggest that there is enough similarity between cancers arising in mice and people that discovery methods in mice might be applicable to human cancer diagnostics.Data presented in this report suggest that, at least for breast cancer, mining the pre-diagnostic autoantibody repertoire of transgenic mice yields viable biomarker candidates for the early detection of the disease.AUC: Area under the curve; DCIS: Ductal carcinoma in situ; IDC: Invasive ductal carcinomas; ROC: Receiver operating curves; SEREX: Serological screening of cDNA expression libraires; WHI: Womens Health Initiative.MLD and SH are inventors on patents held by academic institutions that pertain to data presented in this manuscript.JM and JL executed the experiments as described. EG and LR developed the sera repository for the mice. MMJ, EM, YD, HL, EB, SES contributed laboratory and data analysis support for the experiments described. JSC provided regulatory support. SH and MLD supervised all studies and contributed to data interpretation and manuscript writing and review. All authors read and approved the final manuscript.Serum acquisition and mouse cohorts.Click here for file


Micrometastatic cells in the bone marrow, now usually referred to as “disseminated tumor cells (DTCs)”, can be detected in early stage cancer patients. It has been hypothesized that DTCs represent key intermediates in the metastatic process as possible precursors of bone and visceral metastases, and are indicators of metastatic potential. Indeed, multiple clinical studies have unequivocally demonstrated the prognostic value of these cells in breast and other cancers, as DTCs have been associated with adverse outcomes, including inferior overall and disease-free survival. Despite this established clinical significance, the molecular nature of DTCs remains elusive. The complexity of the bone marrow poses a unique challenge in the isolation and direct characterization of these rare cells. However, recent advances in rare-cell technology along with technical improvements in analyzing limited cell inputs have enabled the molecular profiling of DTCs. In this review, we discuss research featuring the isolation and genomic analysis of DTCs. Emerging work on the molecular characterization of DTCs is now providing new insights into the biology of these cells. The lethal progression of solid tumors usually occurs via hematogenous spread from the primary tumor to distant sites. One of the potential steps in this metastatic process involves tumor cells— termed micrometastases or disseminated tumor cells (DTCs)— transiting from the primary tumor to the bone marrow. Current detection methods for DTCs include immunocytochemical (ICC) assays as well as nucleic acid-based assays using reverse transcriptase-polymerase chain reaction , 2. A nu6-107 bone marrow cells) and the inherent complexity of the bone marrow compartment, major technical challenges have impeded the detection and isolation of DTCs. However, novel strategies for molecular profiling of DTCs have recently emerged, and will potentially shed new light on these rare and elusive cells.Despite the prognostic significance of DTCs, their biology is not well understood. It has been hypothesized that DTCs hold particular significance in cancer metastasis, including: as potential precursors of overt bone or bone marrow metastasis, as a reservoir of cells for further circulation and metastasis to visceral sites, and as a prognostic factor of metastatic potential. Because of their rarity  . ReferenUnfortunately, efforts to implement clinical detection of DTCs have been beset by multiple methodological issues , 12. The® system has allowed for the reliable and reproducible enumeration of CTCs. Clinical studies in metastatic breast, prostate and colon cancer patients have shown that elevated CTC numbers correlate with poor survival [In contrast to DTC research, circulating tumor cell (CTC) detection in peripheral blood illustrates successful translation to commercialization and subsequent clearance by the U.S. Food and Drug Administration. The CellSearchsurvival -16. Othesurvival .Due to the high cellularity of the bone marrow, an enrichment step is most often required to facilitate the detection and isolation of rare DTCs. Methods for enrichment and detection of DTCs have been reviewed in detail , 18, 19.in vitro propagation of DTCs [The potential loss of nucleic acids during isolation has limited the utility of nucleic acid purification from single or small pools of cells prior to downstream molecular analysis. Alternatively, cell(s) can be lysed and the subsequent steps  can be performed within the whole cell lysates . Due to  of DTCs , 23. ExaA wide variety of molecular techniques have helped elucidate novel insights into the molecular biology of DTCs Table . Below, DNA assays. The significance of genomic instability in cancer has encouraged attempts at documenting loss of heterozygosity (LOH) events in DTCs [ in DTCs -26. LOH  in DTCs  or as re in DTCs , which a in DTCs -26.in situ hybridization (M-FISH) has permitted karyotypic analysis in DTCs [A cytogenetic technique known as multiplex fluorescent  in DTCs . This me in DTCs . M-FISH The most widely used genomic profiling technology employed in copy number analysis of DTCs is chromosome comparative genomic hybridization  , 30-34. RNA assays. In addition to genomic DNA characterization, there have been efforts to study the transcriptome of DTCs using a variety of molecular techniques [chniques , 34, 43.chniques . In thischniques . In contchniques , 46. Witchniques . This teIn the following sections, we review studies describing molecular characterization of DTCs. The studies have spanned a decade and half with the first genome-wide analysis reported in 1999 . A majorTP53 tumor suppressor gene and sequencing analysis revealed the inactivation of the remaining allele by a mutation. Further loss-of-heterozygosity (LOH) analysis revealed allelic losses in tumor suppressor genes, APC and CDH1.Klein and colleagues  were theTP53 gene in 19 cells, while the rest did not carry any detectable mutations.In a follow-up study , bone maIn an effort to understand the dynamics of tumor shedding, Schmidt-Kittler et al  analyzedin vitro models.Kraus and colleagues applied an alternative strategy for characterization of DTCs through the establishment of two unique bone marrow cell lines (PC-E1 and PC-R1) derived from 2 M0 prostate cancer patients . AlthougIn vitro cell culturing allowed for the genetic analysis of viable and proliferative DTCs. Examination of the proliferation rates of cytokeratin-positive cells (putative DTCs) revealed no correlation with the proliferative potential of matched primary tumors as determined by Ki-67 staining. In contrast to previous findings by Schmidt-Kittler et al [Using a similar strategy, Gangnus and colleagues  establiser et al , cCGH anCDH1 (E-cadherin) and CTNNB1 (β-catenin), suggesting that they are indeed tumor cells. Comparative analysis with matched primary tumors also revealed shared allelic losses. Of 16 matched cases analyzed, 9 showed at least one area within the matched tumor that shared at least one LOH with its corresponding DTC. Finally, a QPCR-based assay revealed significantly higher incidence of HER2 gene amplification in M1 cells compared to M0 cells, suggesting that this aberration is acquired late in the diseases process. Intriguingly, comparison of the HER2 status between DTCs and matched primary tumors revealed poor congruence with only 1 of the 27 matched cases being concordant.The detection of DTCs with normal karyotypes  promptedHER2 locus. A comparison of genomic profiles between DTCs and single tumor cells from lymph node metastasis of the same patients showed almost no common alterations. For example, tumor cells from the lymph node exhibited gains on 7q and 10q, and losses on 5q; these alterations, however, were absent in DTCs. Despite this genetic divergence, gain of 17q12-21 (HER2) was observed at similar frequencies between the two groups. Quantitative assessment via QPCR analysis confirmed amplification of HER2 in a subset of DTCs with gains on 17q12-21. Interestingly, discordant copy number aberrations including HER2 status were observed between DTCs and matched primary tumors. Survival analysis demonstrated that patients whose DTCs had amplified HER2 had significantly reduced survival compared to those without HER2 amplification. In contrast, HER2 status of matched primary tumors was not associated with increased risk of death.Single DTCs from 30 patients with esophageal cancer were subjected to copy number analysis by cCGH . ResultsAKAP3 and AKAP6.To address the issue of limited resolution of cCGH, a protocol was optimized using a custom 3K BAC array (1Mb resolution) to analyze copy number in single cells . InitialAR) gene. Although fewer and less striking genomic aberrations were found in DTCs from patients with localized disease, similarities with matched primary tumors provided strong evidence that these were truly tumor cells.The inconsistency between the high incidence of DTCs and the low rates of biochemical recurrence and metastasis among prostate cancer patients raised questions of whether DTCs were indeed tumor cells or merely non-malignant epithelial cells residing in the bone marrow . AlthougIn a related study involving non-metastatic prostate cancer patients, DTCs were isolated before surgery (n=14 patients) and during clinical follow-up (n=23 patients) . IsolateA workflow for single cell aCGH analysis was developed using high-resolution oligonucleotide microarrays to allow in-depth assessment of genomic aberrations in single DTCs . InitialMYC oncogene) and loss in 13q (including the RB1 tumor suppressor). Clustering analysis indicated that DTC copy number profiles were more similar to that of the lymph node metastasis than to the primary tumor. Detailed analysis of copy number profiles of single DTCs revealed substantial heterogeneity, but also uncovered the existence of DTC subclones that closely resembled the primary tumor and lymph node metastasis.A two-step assay involving whole genome amplification and oligonucleotide microarray analysis was optimized to analyze copy number changes in single DTCs . PreclinThe first application of NGS in DTC analysis was performed to detect copy number aberrations and copy neutral loss of heterozygosity  . DNA froTWIST1), and the other cluster showing an over-representation of transcripts encoding ribosomal proteins, translational initiation factors, and genes involved in cell cycle and DNA repair. Differential expression analysis of EPCAM-enriched vs. whole bone marrow samples from the same patients revealed a DTC gene expression signature which included TWIST1. To validate the DTC signature, bone marrow samples from an independent set of 30 patients were interrogated via QPCR analysis. Fifteen patients had progressed or developed metastatic disease while the other half had stable disease for a year. Among the transcripts in the DTC signature, only TWIST1 expression was significantly associated with early tumor relapse.To determine associations between gene expression and resistance to chemotherapy, microarray profiling was done on bone marrow samples enriched for EPCAM-expressing cells from 23 early stage breast cancer patients who received neoadjuvant chemotherapy . UnsuperKRT19 and EPCAM were infrequently expressed, detected in 0% and 20% of the samples, respectively. Significant differential expression of SNAIL2 and LAMB1 was observed between patients who experienced a recurrence and those who did not. Candidate therapeutic targets, such as the PTCH1 gene of the Hedgehog pathway, were expressed in patients who developed metastatic disease. In addition, discordance between primary tumor HER2 status and ERBB2 expression in the bone marrow was commonly observed.A gene expression-based approach involving the nCounter (Nanostring) system was developed for detection of DTCs in enriched bone marrow samples . InitialChery and colleagues  performeKlein et al  performeEPCAM and KRT transcripts in single cells from non-metastatic prostate cancer patients than in the controls. Surprisingly, the KLK3 gene, which encodes the prostate specific antigen, was rarely detected in prostate DTCs. In addition, the detection rates for hematopoietic , and erythroid  transcripts were similar between the prostate DTCs and controls cells. Clustering analysis did not clearly separate the three groups from each other (control cells vs. DTCs from non-metastatic vs. DTCs from metastatic patients). Most of the single cells from the same patient did not cluster together suggesting intra-patient heterogeneity. Moreover, parallel CGH analysis showed that a majority of the cells did not possess genomic aberrations, and single cells harboring genomic aberrations were likely to express KLK3 and epithelial transcripts. Notable was the detection of copy number alterations in some cells expressing erythroid and hematopoietic transcripts.Putative EPCAM-positive DTCs from 65 non-metastatic prostate cancer patients were subjected to targeted expression profiling of 17 genes . The panGenomic instability is a well-described hallmark of most cancers characterized by increased levels of genetic alterations involving complete or partial chromosomal loss and/or gain. It is therefore remarkable that numerous studies have shown that a majority of DTCs from non-metastatic patients exhibit very little instability. Those with aberrant profiles possessed fewer alterations in comparison to DTCs from metastatic patients, and displayed a repertoire of genomic aberrations that were infrequent in primary tumors of the same cancer type. Moreover, the common occurrence of aneuploidy relative to chromosome breaks suggested that dissemination occurred early before telomere crisis. In addition, these cells showed high cell-to-cell heterogeneity, whereas those from metastatic patients were more homogeneous, displaying similar patterns of genomic aberrations.The significant genetic differences observed between DTCs and corresponding primary tumors have raised questions on whether the latter could be used as surrogates for the genetic makeup of existing metastatic cancer . DTCs, oAn important question in metastasis research pertains to tumor cell dormancy, including whether and how DTCs can remain dormant over long periods prior to resumption of proliferative activity. Various studies have shown that DTCs can remain quiescent in the bone marrow for extended periods of time (reviewed in ref ). AttempThe cancer stem cell theory proposes that tumors arise from a unique subset of cells capable of self-renewal and multi-lineage differentiation . It is hTWIST1, SNAIL1, and LAMB1 has been observed in DTCs [During dissemination and migration, tumor cells may undergo a process called epithelial-to-mesenchymal transition (EMT), which results in the down-regulation of epithelial markers like cytokeratin and EPCAM . Methods in DTCs , 21. Whe in DTCs . In addiGenomic analyses of DTCs and their corresponding primary tumors from non-metastatic patients have challenged the widely accepted ‘clonal evolution’ model for tumor progression Figure . This moHER2 amplification may be actively selected for during dissemination and tumor progression [An alternative ‘parallel evolution’ model proposes that DTCs disseminate early from the primary tumor, and acquire genomic aberrations independent of the primary tumor. The genomic profiles of DTCs and the primary tumor may therefore show little resemblance. For example, the detection of HER-positive DTCs from HER2-negative primary tumors suggests that gression , 25, 33.gression . Recent gression , 65, 66.gression .Many of the studies in this review support the parallel evolution model , 36, 67.Much progress has been made recently towards genomic characterization of CTCs . DespiteMuch remains unknown about the biology of DTCs. As with CTCs, the field of DTC genomic research is still in its infancy. Initial efforts towards characterization of DTCs have addressed the formidable technical challenges involved in detecting and characterizing these rare cells within the highly complex bone marrow environment. Subsequent improvements in rare-cell detection methods, combined with new techniques for characterization of limited amounts of tumor DNA, have facilitated progress in genome-wide copy number profiling of DTCs. Results from early genomic studies on DTCs have highlighted mechanisms involved in tumor cell dissemination and evolution.Dissecting DTC biology may unlock key riddles about cancer progression. These include the temporal dynamics involved in DTC dissemination as early or late events, and the relationship of DTCs to primary tumors as direct progeny, echoes of shared ancestry, co-evolvers, and/or exemplars of heterogeneity. The relationship of DTCs to CTCs, bone metastasis and other distant metastasis remains unclear as well. DTCs can shed new light on the processes of genomic instability, tumor heterogeneity, dormancy, cancer stem cells, and epithelial-to-mesenchymal transition.Future efforts towards comprehensive genomic analysis of DTCs will facilitate deeper understanding of DTC biology. Although first generation DTC testing did achieve a kind of proof of concept as a prognostic factor in early breast and other cancers, it never gained standard use as a clinical test. Genomic profiling of DTCs will likely lead to novel DTC-based biomarkers with pathogenetic and/or therapeutic relevance.