Lys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC) sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.The primer tRNA for reverse transcription in HIV-1, tRNA Lys isoacceptors in mammalian cells, tRNALys1,2 and tRNALys3, are selectively incorporated into the virus [Lys3 is the primer for initiating minus-strand cDNA synthesis, and its annealing to the 18 nucleotide primer binding site (PBS) region in the 5' part of the viral genome via the 3' 18 nucleotides in tRNALys3 complementary to the PBS, is a key step in viral replication [During assembly of HIV-1, the major tRNAlication .Lys3 and sites of annealing in viral RNA contain double stranded regions which may require denaturation for annealing to proceed efficiently. Nucleocapsid protein (NC) has been shown to facilitate tRNALys3 annealing both in vitro [in vivo [in vitro, and that the protein only has very subtle tertiary structural and helix destabilization effects on tRNALys3 alone [Both tRNAin vitro ,6 and in[in vivo , primaris3 alone -11.Lys3 annealing to genomic RNA in vitro, the annealing of primer tRNA onto the genomic RNA within HIV-1, murine leukemia virus, and avian retrovirus occurs independently of precursor protein processing [Lys3 is annealed efficiently in protease-negative HIV-1 (about 80% that found in wild-type virions), optimal placement on the viral genome to achieve efficient initiation of reverse transcription requires exposure of the viral genome to mature nucleocapsid protein [Lys3 annealing, while mutations in NC sequences within GagPol do not, indicating the importance of Gag NC sequences in the annealing [In vitro, Gag has been reported to facilitate tRNALys3 annealing to viral RNA as efficiently as mature NC [Although processed nucleocapsid proteins have been shown to facilitate tRNAocessing -14. Howe protein . In thesnnealing . In vitrature NC .Lys3 annealing onto the viral RNA, independent of its role in the packaging of tRNALys3 into the virion. We present data herein indicating that the RT connection domain, while non-essential for tRNALys3 incorporation into virions, is required for tRNALys3 annealing to the viral RNA genomeNevertheless, we will present evidence in this report that GagPol still plays an important role in tRNALys incorporation into virions, but is required for the annealing of tRNALys3 to the viral genomeThe RT connection domain within GagPol is not required for tRNA.Lys3, to determine the tRNALys3/genomic RNA in each viral variant. These results are shown graphically in Figure Lys incorporation into virions is not dramatically affected until GagPol sequences including the thumb domain of RT are deleted (Δ581 and Δ715).293T cells were transfected with protease-negative HIV-1 proviral DNA coding for either full length, protease-negative, GagPol (BH10.P-) or C-terminally deleted GagPol species. The different constructs are shown in Figure Lys3 annealed in vivo to the viral RNA genome, total viral RNA was used as the source of primer/template in an in vitro reverse transcription reaction, using exogenous HIV-1 RT, dCTP, dTTP, α-32P-dGTP, and ddATP. This assay measures the amount of extendable tRNALys3 placed onto the viral genome. It is not known if all annealed tRNALys3 is extendable. Since the sequence of the first six dNTP's incorporated is CTGCTA, annealed primer tRNALys3 will be extended by 6 bases, and the extended tRNALys3 can be resolved and detected by one dimensional polyacrylamide gel electrophoresis (1D PAGE). These results are shown in Figure Lys incorporation, but do severely reduce the ability of tRNALys3 to be functionally annealed to the viral RNA genome.To measure the amount of tRNALys3 as BH10P-, but cotransfection of mutant DNA with DNA coding for GagPol gives a small increase in tRNALys3 packaged to over 90% of BH10P- .Attempts were also made to rescue tRNAd to Vpr , but unlIn vitro studies of the interaction between purified RT and tRNALys3 have indicated an interaction between the RT thumb domain and the tRNA [In vivo studies also indicate an important role of the RT thumb domain in GagPol in tRNALys3 viral packaging. tRNALys3 incorporation into HIV-1 is not affected by deletion of the IN domain in GagPol, nor by further deletion of the RNaseH and connection domains in RT, but is severely inhibited by further deletion of the thumb domain as well [Lys3 interacts with the RT thumb domain during incorporation into virions, and Gag nucleocapsid plays a role in promoting tRNALys3 annealing to viral RNA [the tRNA -22. In v as well . Thus tRiral RNA -7, presuLys3 annealing? One possibility, suggested by in vitro studies, is that RT plays a direct role in tRNALys3 annealing. Early work indicated that the in vitro annealing of primer tRNATrp to AMV genomic RNA was promoted by the addition of AMV reverse transcriptase [Lys3, HIV-1 RT was also shown facilitate the in vitro annealing of tRNALys3 to the PBS sequence [in vitro works suggest that RT alone can directly promote tRNALys3 annealing to viral RNA. Whether the RT sequences in GagPol can function similarly in vivo is not known.What then is the role the RT connection domain sequence in GagPol in facilitating tRNAcriptase . In a lasequence . These iLys3 bound to the thumb domain in RT closer to either NC in Gag or to the genomic RNA that is bound to Gag NC. Recent work has indicated that that Pol sequences alone can bind to Gag p6 through the RT sequences in Pol [Lys incorporation into the virus and tRNALys3 annealing to the viral genome at levels approximately 35% those achieved using full-length GagPol. Thus, in addition to the interactions which probably occur between Gag and homologous sequences in the Gag part of GagPol, the interaction of RT sequences in GagPol with Gag p6 could place the RT-bound tRNALys3 closer to Gag NC sequences and viral RNA in the packaging complex. It remains to be determined which sequences within RT bind to Gag p6, but if it were those of the connection domain, this could explain how these sequences could promote tRNALys3 annealing through altering the configuration of GagPol.Alternatively, the RT connection domain may undergo interactions with Gag that may result in placing the tRNAs in Pol . Pol proLys3, and its annealing to HIV-1 RNA. One also finds two separate domains in Gag involved in these same processes. Evidence has been presented supporting the role of lysyl-tRNA synthetase (LysRS) in targeting tRNALys for viral incorporation, through a specific interaction of Gag capsid sequence with LysRS in a tRNALys/LysRS complex [Lys3 annealing [Lys3 annealing, and LysRS may be required to dissociate from tRNALys3 so as to free this tRNA for annealing to the viral RNA.Thus, two separate RT domains (thumb and connection) appear to be involved, respectively, in the viral incorporation of tRNA complex , while onnealing ,16,17. IBH10 and BH10P- are protease-positive and protease-negative strains of HIV-1, respectively . All delTransfection of COS7 cells with wild type and proviral DNA was performed using the calcium phosphate method as previously described . BrieflyViral particles were washed with 1X TNE and cellular or viral proteins were extracted with 1X RIPA buffer . Western analysis was performed using 300 mg cellular protein or 10 μg viral protein, as determined by the Bradford assay [Lys3 into virions, hybridization to dot-blots of viral RNA was carried out with DNA probes complementary to tRNALys3 [Lys3 annealed to genomic RNA, tRNALys3-primed initiation of reverse transcription was measured using total viral RNA as the source of primer tRNA/template in an in vitro HIV-1 reverse transcription reaction, as previously described [Lys3 is extended by 6 bases, and this product can be resolved by 1D PAGE, and quantitated by phosphorimaging, as previously described [Total viral RNA was extracted from viral pellets by the guanidinium isothiocyanate procedure , and distRNALys3  or to getRNALys3 . To measescribed . The seqescribed .SC carried out the molecular genetic studies, assisted by MJ. LK conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter.We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole.-, the bionic Cl- diffusion potentials  were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTRInh-172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways.In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential  of the bronchiole was small , but when gluconate replaced luminal Cl- conductance that is most likely due to CFTR.These results indicate that the small airway of the pig is characterized by a constitutively active Cl Most, if not all, forms of chronic obstruction pulmonary disease (COPD) as well as asthma begin in the small airways. While the pathogenesis of small airway diseases is poorly understood ,2, it is- selective conductance that dominates the electroconductive properties of this epithelium, that is most probably duo to CFTR.At the same time, almost nothing is known with certainty about the transport properties of distal airway epithelia or how fluid movements help maintain hygiene. No doubt, the paucity of understanding is due to the inaccessibility and the fragility of the tissue. Most concepts of the mechanisms and functions at this level have been taken from findings in the upper respiratory tract or from the larger cartilaginous ringed structures of the trachea and bronchi -7. More 2O. The assembly was wrapped in a plastic bag and transported from the abattoir to the laboratory (<60 min) in an insulated box chilled with ice. In the laboratory, small pieces of about 0.5 cm3 were cut from the peripheral lung parenchyma, usually from along the costal diaphragmatic ridge of the lower lobes. In general, the freshest tissue gave the best responses although some tissue responded well after 6–8 hours of storage in a cooled environment.Lungs were excised intact immediately after sacrifice of young pigs (30–60 kg). Lungs were maintained inflated through a ligated plastic tube connected to an aquarium air pump (~1 L/min) to maintain a positive airway pressure of 10–14 cm-HUnder a dissecting microscope, the opening to a small airway was visualized on the proximal cut surface of a small block of lung tissue. The airway opening was then cannulated with a system of two concentric micropipettes -17 with intact and therefore remained embedded in the mass of connective tissue and air filled alveoli that normally surround the bronchi in vivo. The surrounding parenchymal tissue effectively prevented changing the solution in contact with the serosal surfaces of the airway epithelium, which in vivo is the extracellular fluid and in the intact preparation could not be readily removed. NaCl Ringer solution is designed to mimic mammalian extracellular fluid. Therefore, we used NaCl Ringer in the bath to establish electrical continuity with the serosal surface of the bronchiolar epithelium during the entire experimental period. The Ringer solution contained in (mM): Na+ (~155), K+ (4.5), Mg2+(1.2), Ca2+ (1.0), PO43- (3.5), Cl- (152), SO42- (1.2), Glucose (5) buffered to pH 7.4 with NaOH. For ion diffusion studies, 150 mM of Cl- was replaced with an equivalent amount of gluconate (taken as impermeant), HCO3-, NO3-, I-, or Br-. Luminal solutions perfusing the bronchiolar airway were rapidly changed as needed via a manifold distributing stores of the above solutions through a needle tube to the tip of the perfusing pipette  [Inh-172 and GlyH-101 were generous gifts from Dr. A. Verkman, University of California, San Francisco, CA.).The perfused airways were M): Na+ ~55, K+ (4-172 (5) ,20, GlyH-172 (5)   of the system, which includes the summed resistances of the epithelium  plus the core resistance of the lumen plus the extracellular fluid resistance, the resistance of the epithelium relative to Rt was small (even after floating the current passing circuit), and therefore changes in the epithelial resistance were obscured. Consequently, the response of TEP's was taken as the primary indication of the permeability properties of the epithelium.Although we recorded the total resistance . RNA was reversely transcribed using Sensiscript RT Kit (QIAGEN Inc. CA). The resulting first-strand cDNA was directly used for PCR amplification . The conditions for PCR reactions were as follows: 3 min at 94°C ; 35 cycles of 1 min at 94°C, 1 min at 55–60°C, 1 min at 72°C and then 72°C 10 min . For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide.The primers were constructed on the basis of the published cDNA sequence of CFTR, ENaC, NKCC1 and β-Actin from GenBank. Since the pig gene sequence was not complete, primers were obtained from the human accordant gene, in which highly conserved regions were selected. The pairs of primers for CFTR (accession no. NM_000492) were sense 5'-TCCTAAGCCATGGCCACAA-3' and antisense 5'-GCATTCCAGCATTGCTTCTA-3'; sense 5'-GCCTGGCACCATTAAAGAAA-3' and antisense 5'-CTTGCTCGTTGACCTCCACT-3', which generated a 197-bp and 171-bp CFTR PCR product respectively; for α-ENaC (Z92978) were sense 5'-CAACAACACCACCATCCAC-3' and antisense 5'-TAGGGATTGAGGGTGCAGA-3', which generated a 225-bp PCR product; for β-ENaC (NM_000336) were sense 5'-TGCTGTGCCTCATCGAGTTTG-3' and antisense 5'-TGCAGACGCAGGGAGTCATAGTTG-3', which generated a 277-bp PCR product; for γ-ENaC (X87160) were sense 5'-TCAAGAAGAATCTGCCCGTGA-3' and antisense 5'-GGAAGTGGACTTTGATGGAAACTG-3', which generated a 237-bp PCR product; for NKCC1 (U30246) were sense 5'-TCCAGGTAATGAGTATGGTGTCAG-3' and antisense, 5'-GTTAAGATGTAGCCACGAAGAGGT-3', which generated a 205-bp PCR product; and for β-Actin (BC004251) were sense, 5'-TTCAACTCCATCATGAAGAAGTGTGACGTG-3' and antisense, 5'-CTAAGTCATAGTCCGCCTAGAAGCATT-3', which generated a 312-bp PCR product. All primers showed products closely corresponding to the predicted size for expression of RNA transcripts for these genes.The bronchioles were dissected and then fixed in ice-cold 4% formaldehyde buffered in phosphate at 4°C for 3 hours, infiltrated with cryoprotectant (30% sucrose in PBS) overnight, and frozen in OTC medium  at -35°C. Sections of 5 μm thickness were cut on a cryostat microtome (Thermo Electron) and mounted on glass slides (Fisher Superfrost Plus). Antigen retrieval was performed using a pressure cooker . To reduce autofluorescence, sections were treated for 20 min with 1.5% sodium borohydride in PBS. Sections were incubated sequentially with blocking solution (30 min), primary antibody (overnight at 4°C), and secondary antibodies conjugated to Alexa Fluor-488 and/or -546 (Molecular Probes). Confocal images were acquired with a Zeiss LSM-510 microscope and assembled using Adobe Photoshop. CFTR was labeled with rabbit antibody R3194 (courtesy of C. Marino), and ENaC with a rabbit antibody against the β-subunit of human ENaC (kindly courtesy of C. Fuller and D. Benos). Tight junctions were labeled with a mouse antibody against the junction-associated protein zonula occludens-1 (Zymed). Nuclei were stained with TO-PRO-3 (Molecular Probes).Differences in mean measurement were assayed by applying the Student T test to paired or unpaired means as appropriate. A probability  of ≤ 0.05 was taken as significantly different.- gradient. Despite the fact that this small Cl- gradient should render the lumen positive, there was a small spontaneous lumen negative potential of about -3 mV , the TEP decreased slightly, but without statistical significance  to serosa (110 mm) Cl mV Fig. ; Table 145 mM to nce Fig. ; Table 1- with the impermeant anion, gluconate, the TEP hyperpolarized to as much as -90 mV , GlyH-101 (50 μM) and CFTRInh-172 (5 μM) significantly depolarized the TEP by 36.7, 20.0 and 8.7 mV .Under conditions of symmetrical [Cl mV Fig. ; Table 2- in NaCl Ringer. Amiloride (10 μM) was added in order to block Na+ transport. For luminal Cl-, Br-, I-, NO3-, HCO3- and gluconate, the mean estimated Px/PCl were, respectively, 1.0, 0.92, 0.79, 0.65, 0.33 and 0.28 , but in the presence of the Cl- gradient, the TEP hyperpolarized significantly to all agonists; the response appeared to be increased when both ATP and adenosine were added together  was present in all luminal perfusates to block ENaC Na+ conductance.When we added forskolin (5 μM) plus IBMX (100 μM), adenosine (100 μM), ATP (100 μM) or ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gClher Fig. ; Table 4Two sets of different primers for CFTR as well as primers for α-, β-and γ-ENaC, NKCC1, and β-Actin all produced products of predicted sizes for each of the corresponding mRNAs Fig. .Immunoreactive CFTR antibody (gift of C. Marino) was detected in the apical domains of the bronchiolar epithelia in a continuous border of the bronchiole. The tight junctions were labeled simultaneously and appeared as punctate areas within the border staining for CFTR consistent with the expected location for these structures Fig. .The airways are extraordinarily specialized conduits for air to and from the alveoli for gas exchange. They must remain moist in order to remain flexible and to effectively filter air before it enters the delicate tissues of the alveoli. Hence, a principal function of the airway epithelia is to provide and service a continuous layer of aqueous fluid on the airway surfaces. For decades, the upper airways, trachea, and large bronchi have served as models for the entire tracheobronchial epithelial function ,22,23, a- free solution of about -16 mV [- free Ringers [in vivo. We, too, found similar, relatively small TEP voltages in microdissected porcine airways.The functions of the lower peripheral airway epithelium is poorly defined and understood because this tissue is inaccessible and relatively unamenable to standard physiological techniques for study. Nonetheless, a few courageous attempts to unravel these mysteries have been made. About ten years ago, Ballard ,14 and A Ringers . In all - free solutions in the lumen was as much as -90 mV , we found that the bi-ionic TEP with Clin vivo electrical properties in vitro, it is fundamental that the integrity of the epithelial sheet be conserved because TEP measurements depend on separation of charge. Breaks, holes, or tears in the barrier inescapably create electrical shunts, which, by allowing simultaneous back leak of epithelial current, prevent separation of charge. Even though the individual cells or groups of cells of the epithelia function and respond physiologically, the transepithelial voltage signals will be erroneously muted or lost through such shunts. In the present case, it appears that perfusion of the bronchiole without dissection from surrounding parenchyma, minimizes trauma induced shunts and allows detection of a much more complete and robust electrical signal. Unfortunately, the price for this preservation of signal is the inability to control the contra luminal solution. Keeping in mind that the undissected bronchiole is embedded in a mass of air filled alveoli together with numerous other elements of the tissue at this level, it is easy to see that changing the bath solution can have no acute effects on the composition of the contra luminal solution at the basilateral membrane of the epithelium. Consequently, we did not change the bath solution and assumed that normal Ringer solution is sufficiently similar to extracellular fluid in vivo to avoid introducing significant free solution diffusion potentials or altering the physiological composition of the native fluid present in the extracellular compartment of the in tact preparation.In order for an epithelium to reflect its - in the lumen spontaneously created a large lumen negative transmural potential , suggesting that since CFTR is not sensitive to Ca2+ mediated activation either, a.) the conductance is due to CFTR, b.) other Ca2+ activated Cl- conductances must be fully constitutively activated or not present, or c.) luminal application of the ionophore drug does not increase intracellular Ca2+ effectively. Third, CFTR is known to be inhibited by NPPB, CFTRInh-172, and GlyH-101[- conductance in the airway epithelium. However, the fact that none of the inhibitors appeared to completely inhibit the transepithelial potential might argue that another anion channel conductance is present. Two points diminish this argument. First, if the tissue is actively transporting, anion channel inhibitors will not completely ablate the TEP because that component of potential generated by the basilateral K+ emf and the apical Na+ emf should not be blocked and should be reflected across the epithelium in the TEP. Secondly, even when the CFTR Cl- channel has been isolated from active transport components, anion channel blockers do not usually completely block its conductance in native tissue [- conductance of pig nasal airways [Since obstructive airway disease in Cystic Fibrosis arises in the small airways -34 and Cluconate -37. Secoluconate -40. In c GlyH-101,21. These tissue . Moreove airways . It is n- channel that responds to activation of PKA, but both are also apparently insensitive to ionomycin. They both show equally large bionic diffusion potentials for Cl- and are several fold more permeable to Cl- than to Na+, and yet both have very low transmembrane potentials in bilateral isotonic Ringer solutions [Not only does the bronchiole resemble the sweat duct with respect to exhibiting a constitutively active Clolutions . Both arolutions ,41,44, aWe have found that the epithelium of terminal airways of the pig appears to express an anion permeability that constitutively dominates the electroconductive properties of this zone of the airway. Its anion selectivity sequence is similar to that expected for CFTR, and its activity can be enhanced by forskolin/IBMX or decreased by anion channel blockers known to inhibit CFTR. RT-PCR amplification products and specific antibodies identify CFTR in this tissue. The small airway appears to share a number of properties with the human sweat duct and may, by analogy, belong to a class of highly absorptive epithelia.CFTR: Cystic Fibrosis Transmembrane Conductance Regulator+ ChannelENaC: Epithelial Na+-K+-2Cl- CotransporterNKCC: NaTEP: Transepithelial PotentialPKA: Protein Kinase A


We conclude that the contribution of neurons to behavior can depend strongly upon tuning to stimulus dimensions that appear to be irrelevant to the current task, and we suggest that these findings are best explained in terms of the strategy used by animals to perform the task.Cortical neurons are frequently tuned to several stimulus dimensions, and many cortical areas contain intercalated maps of multiple variables. Relatively little is known about how information is “read out” of these multidimensional maps. For example, how does an organism extract information relevant to the task at hand from neurons that are also tuned to other, irrelevant stimulus dimensions? We addressed this question by employing microstimulation techniques to examine the contribution of disparity-tuned neurons in the middle temporal (MT) visual area to performance on a direction discrimination task. Most MT neurons are tuned to both binocular disparity and the direction of stimulus motion, and MT contains topographic maps of both parameters. We assessed the effect of microstimulation on direction judgments after first characterizing the disparity tuning of each stimulation site. Although the disparity of the stimulus was irrelevant to the required task, we found that microstimulation effects were strongly modulated by the disparity tuning of the stimulated neurons. For two of three monkeys, microstimulation of nondisparity-selective sites produced large biases in direction judgments, whereas stimulation of disparity-selective sites had little or no effect. The binocular disparity was optimized for each stimulation site, and our result could not be explained by variations in direction tuning, response strength, or any other tuning property that we examined. When microstimulation of a disparity-tuned site  Greg DeAngelis and Bill Newsome use microstimulation to activate small populations of neurons with different combinations of response properties in monkey visual cortex Determining how information is “read out” of sensory maps in the cerebral cortex is of fundamental importance for understanding how neural activity gives rise to cognitive processes such as perception, planning for action, and working memory. A substantial portion of our knowledge about sensory read-out comes from studies of the middle temporal (MT) visual area, an extrastriate area known to play important roles in processing visual motion information  Do MT columns that possess or lack disparity tuning contribute differentially to direction judgments? We used electrical microstimulation to test the hypothesis that neurons in the nondisparity-selective regions of MT contribute to motion perception, whereas those in the disparity-selective regions are mainly involved in depth perception. Our hypothesis was confirmed for two of the three monkeys in this study: microstimulation of nondisparity-selective sites produced strong direction biases, whereas stimulation of disparity-selective sites had little or no effect. For the third monkey, microstimulation biased direction judgments when it was applied at either disparity-selective or nonselective sites. For disparity-tuned sites that did yield effects on direction judgments, we also asked a second question. (2) Does the influence of a disparity-tuned column on direction judgments vary as a function of the actual disparity of the motion display? We found that stimulation effects were stronger when the disparity of the visual stimulus matched the preferred disparity of the stimulated column.We conclude that tuning for task-irrelevant stimulus dimensions can exert dramatic effects on the contribution of cortical neurons to a particular perceptual judgment. In extreme cases, columns tuned for an irrelevant dimension (disparity) fail to contribute at all to perceptual judgments of the task-relevant dimension (direction). In less extreme cases, the contribution of a column is modulated by tuning along the task-irrelevant dimension, so that microstimulation effects are obtained primarily when the visual stimulus possesses the right conjunction of properties (direction and disparity) to excite the column optimally. We discuss our findings in terms of the strategies employed by animals to solve the task.Microstimulation experiments were performed at 102 recording sites in area MT of three rhesus monkeys  during the performance of the direction discrimination task illustrated in We have previously shown that disparity-selective neurons tend to occur within discrete patches of MT . Given tp << 0.001), with no significant change in the slope of the curve . This effect is qualitatively similar to those obtained previously in our laboratories . Thus, the activity of neurons at this stimulation site did not appear to contribute to direction discrimination.p < 0.05), whereas open symbols indicate nonsignificant effects. The filled and open triangles correspond to the examples shown in r = −0.69, n = 38; monkey T, r = −0.52, n = 36; p << 0.001 for both animals). An analysis of covariance that included monkey identity as a coregressor revealed no significant difference between regression slopes for the two animals . Note that microstimulation almost always produced a significant effect on direction judgments in experiments for which the DTI was less than 0.5. In contrast, significant effects of microstimulation occurred much less frequently when the DTI exceeded 0.5.The result in r = −0.025, p > 0.9, n = 28). We often observed significant effects of microstimulation at sites with strong disparity tuning. It is worth emphasizing that all of the data in We obtained quite different results in a third animal, monkey R B. For thn = 51) was significantly lower than the mean psychophysical thresholds for monkey S  and monkey T  . In contrast, the average slope of the psychometric functions did not differ between the three animals . We shall consider these issues further in the Discussion.The individual differences between monkeys in the data of r = 0.45, p = 0.01, n = 32) as a function of the DTI. The data reveal a clear inverse relationship between the two effects. Columns with low DTIs produce large effects on direction discrimination performance and little or no effect on depth discrimination. In contrast, columns with large DTIs show the converse pattern. In this monkey, therefore, the functional segregation of MT columns according to the strength of disparity tuning is particularly clear.Monkeys T and R were subjects both in the current set of experiments and in a separate study in which we showed that stimulation of disparity-tuned columns influences perceptual judgments of depth . For theIt is important to note that the differences between animals seen in The striking result in n = 162) and in three additional monkeys (n = 409). Across this unbiased sample of 571 recordings, we find no significant correlation between Disparity Tuning Index (DTI) and Direction Tuning Index  between direction-tuning bandwidth and DTI across our sample of 571 MU recordings, indicating that the sharpness of direction tuning also does not covary with disparity selectivity. These observations, combined with the fact that we only performed microstimulation experiments in the portions of MT with the strongest direction tuning , we computed the standard deviation (SD) of directional preferences within a 400-μm region around each microstimulation site. We find no significant correlation between the strength of microstimulation effects and the SD of preferred directions . Correspondingly, there is no significant correlation between the strength of the microstimulation effects and the peak MU response , and all of the microstimulation effects in Systematic variations in responsiveness as a function of disparity tuning (point 3 above) can be excluded as a possible explanation for our findings because there is no correlation between the peak response of MU activity and the DTI , indicating that variations in these parameters are also unlikely to account for the results shown in Finally, using a dataset of 409 MU recordings and a multiple regression analysis, we also tested for correlations between DTI and several other response properties, including preferred speed, Speed Tuning Index, RF eccentricity, optimal stimulus size, and percentage of surround inhibition. None of these variables was significantly correlated with DTI . At these sites, and at many sites in monkey R, we could ask whether the efficacy of microstimulation varied when the random-dot stimulus was presented at different points along the disparity-tuning curve of the stimulated column.The logic of this experiment is illustrated for a disparity-selective site in p < 0.001). In the second block of trials, we presented dots at the nonpreferred disparity (−0.5°), and microstimulation exerted no effect whatsoever on the monkey's choices (p > 0.5). To be certain that this effect did not result from some nonstationarity in electrode position, cell responsiveness, etc. (p < 0.001). At this stimulation site, therefore, we were able to switch the result from a very substantial effect to no effect and back again simply by manipulating the disparity of the random-dot stimuli.ss, etc. , we collp = 0.025). We chose three different disparities at which to perform the direction discrimination task: 0°, 0.6°, and −0.6°. p < 0.0001), with no corresponding change in slope (p > 0.4).p < 0.05) for at least one of the two disparities. We computed a Microstimulation Selectivity Ratio (MSR) as follows:The individual example sites in Figures PE is the effect of microstimulation when dots are presented at the preferred disparity, and NPE is the effect when dots are presented at the nonpreferred disparity. This index is a standard contrast measure, except that the quantities in the denominator are absolute values. This formulation was necessary to keep the index bounded between −1.0 and 1.0.where r = 0.37, F = 9.9, p < 0.005), with no significant differences between the three monkeys  = 0.14, p > 0.8).Thus, as hypothesized see , microstUsing microstimulation to probe the link between neuronal activity and behavior, we have tested whether the contribution of MT neurons to direction discrimination depends on their disparity selectivity. This work addresses the general question of how neurons that are tuned to multiple stimulus dimensions contribute to behavior in situations where one or more of these stimulus dimensions are task-irrelevant. Relatively little is currently known about how the responses of sensory neurons are pooled by decision mechanisms see  and how How can we explain the finding see A that reA second possibility is that the output connections of disparity-selective and nonselective regions of MT have different targets, such that decision mechanisms for motion receive input from nondisparity-selective portions of MT whereas decision mechanisms for depth receive input from disparity-tuned regions. Experiments have not been done to test this hypothesis, so we cannot rule it out. One argument against this idea, however, is that one of the three monkeys (monkey R) did not show a dependence of microstimulation effects on disparity selectivity see B. Thus, A third possibility, which we favor, is that our findings reflect the strategy that each monkey adopted for reading out motion signals from MT during the extended period of training on the task. In this scenario, all regions of MT could project to decision mechanisms for both motion and depth, but the relative weights of the connections would vary with the animal's task strategy. This would allow the read-out strategy to be altered rapidly based on the demands of the task. In our experiments, one strategy for performing the task would be to extract motion signals from all MT columns with the appropriate direction selectivity and spatial RF, regardless of their disparity selectivity. This strategy would entail pooling signals from many columns, including those with unfavorable signal-to-noise ratios due to their poor responsiveness to stimuli of nonoptimal disparity.A second strategy, which could yield better performance, would be to monitor primarily columns that are maximally activated by the stimulus, but this would entail pooling responses from columns with different disparity preferences when the stimulus disparity changed. Thus, some sort of complex “switching” would be required to route information to the decision process from the set of columns optimal for each experiment. A third, and perhaps the simplest, strategy would be to monitor motion signals only from the nondisparity-selective portions of MT; these columns would respond well to all stimulus disparities, providing a good signal-to-noise ratio for all stimulus sets on which the monkey was trained. This strategy offers the further advantage that one can monitor the same set of columns for all stimulus conditions in our task. Given that correlated noise among neurons limits the benefits of pooling across large populations of neurons , this laIf monkeys were to adopt the simple strategy of monitoring only the nondisparity-selective regions of MT, then the microstimulation results shown in r = 0.55, p < 0.01) than for monkey R .We found a statistically significant, but relatively weak, dependence of microstimulation effects on the difference between the preferred disparity of MT neurons and the stimulus disparity see . What acAnother possible source of variability in Considering these likely sources of variability, the fact that we see a significant overall effect in Many of the standard experimental approaches in systems neuroscience  find their utility in exposing correlations between neuronal activity and external stimuli or behavioral states. Of course, finding signals that are correlated with behavior does not prove that those signals underlie the behavior. The value of electrical microstimulation, reversible inactivation, and lesion techniques is that they can establish causal links between neural activity and behavior. In this study, we only microstimulated at sites in MT that had strong directional selectivity; thus, one might assume that all sites would be equally likely to contribute to performance of the direction discrimination task. The central finding of this study is that the contribution of MT direction columns to task performance is modulated by the tuning of the neurons to a stimulus variable that is irrelevant to completion of the task. Thus, even within a single area of the brain, the causal linkage between neurons and behavior may depend on uncontrolled stimulus dimensions, and may be determined by unexpected factors such as task strategy. This result highlights the importance of causal techniques for studying the neural basis of behavior, and suggests that microstimulation studies may be able to reveal how high-level task strategies modulate the read-out of neuronal signals from topographic maps in the brain.(Macaca mulatta) are described elsewhere , all of which had previously been subjects in other studies in the laboratory. Each animal had a scleral search coil implanted in at least one eye (monkey S had coils in both eyes) to allow monitoring of eye position. In addition, each subject was equipped with a head restraint post and a stainless-steel recording chamber that was positioned over the occipital cortex. Electrodes were introduced into the visual cortex through a transdural guide tube that was positioned within a square array of grid holes at 1-mm intervals .All visual stimuli used in this study were dynamic random-dot patterns presented on a standard 21-in. color display . The display subtended 39° × 29° at the viewing distance of 57 cm and was refreshed at a rate of 100Hz. The visual stimuli were generated by a Cambridge Research Systems VSG2/3 board  that was housed in a dedicated PC. Stereoscopic presentation was achieved through the use of ferroelectric shutters  that were switched in antiphase for the two eyes. Left and right half-images were presented on alternate video frames, and the shutters were synchronized to the vertical refresh, thus exposing each eye to the appropriate visual stimulus on alternate frames. With this technique, the quality of stereo separation is limited mainly by phosphor persistence. Thus, random-dot stimuli were always presented using the red gun only, since the red phosphor has a much faster decay than either the green or blue phosphors. We achieved a contrast ratio of approximately 40:1 (“open” eye:“closed” eye) using this approach, and “ghosting” artifacts were barely visible, even under dark-adapted conditions.2-s) for the central patch and 8 dots/(deg2-s) for the background.Monkeys performed two separate tasks in these experiments: a visual fixation task, and a direction discrimination task. In the visual fixation task, a small, yellow fixation point (FP) appeared to begin each trial, and the monkeys were required to maintain fixation within a 2° × 2° or 3° × 3° electronic window, centered on the fixation target, until the fixation target was extinguished. The monkeys received a liquid reward for successful fixation, typically 0.1–0.15 ml of water or juice. If the monkey broke fixation before the end of a trial, the trial was aborted, the data were discarded, and the monkey was not rewarded. During the fixation period, a bipartite random-dot stimulus was presented for 1.5 s. It consisted of a central, circular patch of coherently moving dots that could be presented with variable binocular disparity, and which covered the receptive field of the MT neurons under study. To assist the monkey in maintaining binocular convergence on the FP, we filled the remainder of the visual display with zero-disparity dots that were randomly repositioned every fourth video frame (25 Hz), thus producing a twinkling, zero-disparity background. Each dot was approximately 0.1° in size. Dot density was 32 dots/ operating in constant-current mode. The current was a train of biphasic pulses with a frequency of 200 Hz and an amplitude of 20 μA. Each biphasic event consisted of a 200-μs cathodal pulse followed by a 200-μs anodal pulse, with a 100-μs gap between the two. Microstimulation parameters were chosen to elicit robust perceptual biases but were well below the current and frequency levels at which stimulation has been shown to flatten the slope of the psychometric function . MicrostWe searched for candidate microstimulation sites by examining the tuning properties of MU activity at regular intervals of 100 μm along electrode penetrations through MT. At each recording site, we rated the strength of direction selectivity on a scale from 1 to 3 (3 = strongest tuning), and we carefully estimated the preferred direction of motion (see p > 0.3). Whenever possible  First, we carefully mapped the MU RF of the MT neurons by dragging a small patch of moving dots (100% coherence) through the RF with a pointing device. Spike densities were plotted on a Cartesian map of visual space during this process to facilitate visual mapping of the RF. In addition, we mapped the direction and speed selectivity of the neurons by moving a cursor throughout a polar direction-speed domain while spike densities were again plotted on the screen. From this procedure, we determined the location and size of the MU RF, as well as the preferred direction and speed of motion. We also estimated the range of disparities over which the neurons were selective, and these parameters were then used in subsequent quantitative tests. 2) We next measured a disparity-tuning curve for MU activity at the identified stimulation site, while the monkey performed a block of fixation trials. Nine evenly spaced disparities were typically tested within the disparity range determined from our initial qualitative probing . Neural signals were amplified, filtered (0.5–5.0 kHz), and discriminated using conventional electronics , and event times were stored on magnetic disk with 1 ms resolution. To record MU activity, we simply set the threshold level of our window discriminator to approximately 1–2 SD above the noise level. Thus, a MU event was defined as any deflection of the analog signal that exceeded this threshold. Since the absolute frequency of the MU response depends heavily upon the event threshold, we attempted to achieve a consistent response magnitude from site to site by adjusting our event threshold such that the spontaneous activity level was in the range from 50 to 100 events/s. This setting typically yielded peak MU responses in the range of 300–500 events/s (mean 378.5 ± 78.3 SD).Horizontal and vertical eye-position signals were low-pass filtered with a cutoff frequency of 250Hz, sampled at 1 kHz, and stored to disk at 250 Hz.To construct disparity-tuning curves, we computed the firing rate for each trial during the 1-s stimulus presentation, and we plotted the mean firing rate (± SE) as a function of the horizontal disparity. Smooth curve fits to disparity-tuning curves were achieved using a cubic spline interpolation. To quantify the strength of disparity tuning at each stimulation site, we computed the DTI as follows:Rmax denotes the response to the preferred disparity, Rmin denotes the response to the antipreferred disparity, and S indicates the spontaneous activity level. Values larger than unity can occur if Rmin is less than S. For the quantification of direction-tuning strength  in which we obtained quantitative measurements of both direction tuning and disparity tuning. There is no significant correlation between Direction Tuning Index and Disparity Tuning Index (DTI) across the sample. Note also that the data from monkey S overlap completely with the data from the other animals, indicating that monkey S was not unusual.(358 KB EPS).Click here for additional data file.Figure S2Monkey S is shown in black; monkey T in red.(A) The strength of the microstimulation effect is plotted against the SD of direction preferences within a 400-μm window centered on each stimulation site . There is no significant correlation between these variables, indicating that variability in direction preferences (within the observed range) did not determine the efficacy of microstimulation. Note, however, that all stimulation sites were chosen to have a small range of preferred directions; we did not apply microstimulation at locations in MT where the direction preference changed rapidly over short distances.(B) There is also no significant correlation between the DTI of MU activity at each stimulation site and the SD of direction preferences. This shows that disparity-selective microstimulation sites did not have larger variations in direction preferences.(216 KB PS).Click here for additional data file.Figure S3See (320 KB EPS).Click here for additional data file.


Rural Indian tribes are anthropologically distinct with unique cultures, traditions and practices. Over the years, displacement and rapid acculturation of this population has led to dramatic changes in their socio-cultural and value systems. Due to a poor health infrastructure, high levels of poverty and ignorance, these communities are highly vulnerable to various health problems, especially, communicable diseases including HIV/AIDS. Our study sought to assess knowledge, attitudes and practices regarding sexuality, and the risk factors associated with the spread of HIV/AIDS and STDs among these communities.A nested cross sectional study was undertaken as part of the on going Reproductive and Child Health Survey. A total of 5,690 participants age 18–44 were recruited for this study. Data were obtained through home interviews, and focused on socio-demographics, knowledge, attitudes and behaviors regarding sexuality, HIV/AIDS and other STDs.The study revealed that only 22% of adults had even heard of AIDS, and 18 % knew how it is transmitted. In addition, only 5% knew that STDs and AIDS were related to each other. AIDS awareness among women was lower compared to men (14% vs.30 %). Regarding sexual practices, 35% of the respondents reported having had extramarital sexual encounters, with more males than females reporting extramarital affairs.Lack of awareness, permissiveness of tribal societies for premarital or extra-marital sexual relationships, and sexual mixing patterns predispose these communities to HIV/AIDS and STD infections. There is a dire need for targeted interventions in order to curtail the increasing threat of HIV and other STDs among these vulnerable populations. India is the second most populous nation in the world and has changing sociopolitical and demographic characteristics as well as varied morbidity and mortality patterns . These cAs a direct result of these challenges, India has begun to assess and monitor the impact of HIV/AIDS throughout the country's various states and regions with the assistance of several international health organizations. According to 2003 estimates from UNAIDS, approximately 5.1 million individuals in India are infected with the HIV virus . This trFor effective control of the spread of HIV/AIDS, it is crucial to have data on knowledge, attitudes and behavioral practices for specific population as research has shown that socio-cultural influences, traditional lifestyles, societal norms, and traditions influence HIV/AIDS transmission rates ,6. BecauThe rapid spread of HIV/AIDS in rural Indian communities has been attributed to the country's poor health infrastructure, poverty and lack of awareness ,10. DespThroughout India, approximately 8% of the population lives within rural tribal communities, which are collectively referred to as 'Tribes'. These communities are geographically distinct; with each tribe having its own unique customs, traditions, beliefs and practices. Even within a particular tribal entity, differences in dialect, health practices, unique customs, values, and traditions are apparent. In rural Indian communities indices of reproductive health are typically very poor: maternal mortality rate is about 230 per 100,000 live births and 61.2% of the women suffer from at least one gynecologic pathology . BecauseWe conducted a cross-sectional study nested within an existing enumerative study referred to as the Reproductive and Child Health Survey (RCHS). The RCHS was initially designed to enumerate and ascertain basic demographic and health profiles for all tribal members. Data collection for this particular study was done in two phases. Phase one involved adding additional questions to the original RCHS study to assess risk factors  associated with the transmission of HIV/AIDS and other communicable diseases.The study population comprised of tribal communities living in the southern region of Karnataka Figures ,2. MembeBecause of the enumerative nature of the RCHS, all persons within the age group 18–44 years who participated in the RCHS study were included for phase one of this study. This age group was selected as the focus of the initial study was on reproductive health issues.A semi-structured questionnaire with both open- and close-ended questions was developed to collect information on knowledge, attitudes and behaviors regarding HIV/AIDS, as well as other relevant demographic information not included in the RCHS. The instrument was developed in English, translated to the native language, and subsequently back translated to English for content and language verification. The survey instrument was field-tested for validity purposes and modified accordingly.A team of ten interviewers from the local tribal communities with a minimum of high school education were selected and trained for two weeks to ensure uniform and high-quality data collection. All adults were interviewed separately to ensure confidentiality. Each interviewer read out each of the questions and response choices (where appropriate) to the interviewees and recorded all answers directly on the questionnaire. Verbal informed consent was obtained from each respondent prior to starting the interview.Data were entered into an electronic database using Sybase Central Software . To ensure confidentiality, all respondent identifiers were expunged to create a secondary data set that was used for the final analysis. Frequency tables were generated for selected demographics and health related categorical variables. In addition, univariate analysis was performed on relevant continuous variables. The findings are presented below.Ethical clearance was obtained from ethical boards within each of the tribal communities, and appropriate government agencies were informed about the study objectives. Permission was also obtained from the Institutional Review Board (IRB) at the University of South Florida in Tampa, Florida. Verbal informed consent was obtained from the local leaders, and the individual participants. Because of the high levels of illiteracy, it was not feasible for us to obtain written consent.A total of 11,379 individuals from the 5 tribal communities had been enumerated as part of the RCHS. Of these, 5,690 were within the study age range (18–44 years) and formed the basis for this analysis.Table The findings revealed that these tribal communities did not have a structured marital system; instead members practiced a form of serial monogamy in which they change partners and remarry every four to five years. Regarding sexual practices, 35% of the respondents reported either premarital affairs or extramarital affairs Table . HoweverAmong these communities, there was a low level of knowledge on HIV/AIDS; only 22 % of all study participants  had heard of AIDS Table . Among tIn today's modern world, it is difficult to imagine societies that are still socially and culturally isolated from the rest of civilization; however, they do exist. The tribal societies throughout India have remained socially and culturally alienated from mainstream Indian society until developmental and conservation activities in tribal areas forced interactions between them. Displacement of the tribal people of southern Karnataka has led to a complex process of rapid acculturation and loss of cultural identity; as they struggle to maintain their traditional social structure, they must adopt new skills, beliefs, and practices necessary for success in their new environment. During this acculturation process, they have been faced with a myriad of public health challenges complicated by poverty, ignorance, and reluctance to abandon traditional beliefs and practices that would allow them to assimilate successfully.To date this is the first health related study among the displaced tribal communities of southern Karnataka that has attempted to assess risk threshold for the transmission and spread of HIV/AIDS and other STDs. It is not surprising that knowledge and awareness about HIV/AIDS and STDs was very low among tribal communities compared to the national figures given the degree of isolation, low literacy rates, and minimal access to information.The high level of poverty, inadequate health resources, ignorance and high-risk beliefs and practices among the tribal communities has contributed to the vulnerability of this population. As such it has created a highly susceptible population for the rapid spread of HIV/AIDS and other STDs' as well.Because tribal members are forced to migrate outside of their communities in search for work and increased wages, this may contribute to the spread of HIV/AIDS as many engage in extramarital affairs, seek commercial sex partners, or are under the threat of sexual harassment . While there has been limited scientific research exploring the cultural context of extramarital sexual behaviors, it is generally noted that in these communities, extramarital affairs are condoned and widely practiced especially during periods when women are pregnant or nursing or during period of travel for work . This kiIt is evident from this study that the Indian tribal community is experiencing a latent phase that is potentially a precursor for an HIV/AIDS epidemic. There is a high prevalence of behavioral risk factors, coupled with ignorance, and inadequate health infrastructure thus creating a potential risk for rapid spread of HIV/AIDS, as well as other related diseases. In a country that is struggling to contain the spread of HIV, it is particularly important for concerned parties to pay attention to this population. Currently, virtually no resources are allocated toward the treatment of those infected with HIV/AIDS; the main stay of management is through education and preventive measures to control the spread of the scourge as they represent the most practical and cost effective strategies in this developing nation. It therefore becomes imperative and urgent to address the health concerns revealed in our study in order to formulate effective, culture-sensitive and appropriate intervention programs so that an imminent disaster (i.e. HIV/AIDS epidemic) in this remote and isolated communities can be averted.The author(s) declare that they have no competing interests.All the authors were involved in the design of the study, analysis, interpretation and development of the manuscript.The pre-publication history for this paper can be accessed here:


Dedifferentiated liposarcomas represent heterogeneous tumors with lipomatous and nonlipomatous elements starkly juxtaposed. It is thought that the high grade nonlipomatous elements of the tumor portend a worse prognosis.A 19.8 kg heterogeneous retroperitoneal tumor was successfully and completely resected. Because of its extent, no additional treatment modalities were practicable. The tumor soon recurred. The recurrent tumor differed from the primary tumor in that it was more homogeneous, consisting mainly of nonlipogenic, calcific tissue.Dedifferentiated liposarcomas are known to have a very high recurrence rate. The biological behavior of dedifferentiated liposarcomas is likely dictated by the most aggressive element of these heterogeneous tumors. Sarcomas arising from the retroperitoneum are rare tumors, accounting for 10–15% of all soft tissue sarcomas . These fA 65-year-old male presented with a three-week history of progressively worsening abdominal distension. He denied any abdominal pain but stated that he noticed an increased frequency of bowel movements. His past medical history was unremarkable. On examination, he was afebrile and had a hugely distended abdomen with an immobile, nontender mass occupying all four quadrants of the abdomen. Computed tomographic (CT) scan revealed a large, heterogeneous lobulated mass occupying most of the abdomen Figure . The perWhile neoadjuvant chemotherapy and radiation comprise a frequent approach for retroperitoneal sarcomas at our institution, the extent of the tumor made this approach unfeasible. Resection was therefore planned unless an intraoperative biopsy revealed lymphoma. Resection was accomplished through a T-type incision Figure , and entThe tumor was submitted for histological examination in two parts measuring, 30.0 × 10.0 × 6.5 cm and 32.0 × 20.0 × 10.0 cm, weighing 19.8 kg in total Figure .The patient was discharged from hospital on the seventh postoperative day. He was followed in the surgical oncology outpatient clinic monthly. Four months after resection the patient had a follow-up CT scan, which demonstrated an intra-abdominal recurrence consisting almost completely of a calcified, nonlipomatous tumor Figure . The patde novo, although secondary dedifferentiation can occur with multiple recurrences of a well differentiated liposarcoma [The patient in our case manifested the dedifferentiated variant of liposarcoma. The term "dedifferentiated liposarcoma" refers to the development of a high grade nonlipogenic sarcoma juxtaposed to a well differentiated liposarcoma . The majosarcoma . CT and osarcoma . Calcifiosarcoma ,6. Otherosarcoma ,7. A furosarcoma .Among liposarcomas, the presence of features of the dedifferentiated variant strongly portends a worse prognosis. The overall 5-year survival of dedifferentiated liposarcomas is 20%; the 5-year survival of well differentiated liposarcomas is 83% . DediffeWhile generally the phenotype of the nonlipogenic component does not impact prognosis of dedifferentiated liposarcomas, the presence of calcifications has been identified as an adverse prognostic factor . In the Complete resection of the tumor is perhaps the most important factor determining long-term survival. Unfortunately, the rate of complete respectability is only about 53% . As illuDedifferentiated liposarcomas represent aggressive variants of liposarcomas. Each morphological element of these heterogeneous tumors may manifest completely different biology. The overall biological behavior of dedifferentiated liposarcomas is likely dictated by the most aggressive element, which typically resides in the nonlipomatous portion of the tumor.None declared.SK, HB, WT, and OB made substantial contributions to the intellectual content of the paper, in the interpretation of data, and in drafting the manuscript. All authors read and approved the final manuscript.


This paper compiles data from different sources to get a first comprehensive picture of psychometric and other methodological characteristics of the Menopause Rating Scale (MRS) scale. The scale was designed and standardized as a self-administered scale to (a) to assess symptoms/complaints of aging women under different conditions, (b) to evaluate the severity of symptoms over time, and (c) to measure changes pre- and postmenopause replacement therapy. The scale became widespread used (available in 10 languages).A large multinational survey (9 countries in 4 continents) from 2001/ 2002 is the basis for in depth analyses on reliability and validity of the MRS. Additional small convenience samples were used to get first impressions about test-retest reliability. The data were centrally analyzed. Data from a postmarketing HRT study were used to estimate discriminative validity.Reliability measures (consistency and test-retest stability) were found to be good across countries, although the sample size for test-retest reliability was small.Validity: The internal structure of the MRS across countries was astonishingly similar to conclude that the scale really measures the same phenomenon in symptomatic women. The sub-scores and total score correlations were high (0.7–0.9) but lower among the sub-scales (0.5–0.7). This however suggests that the subscales are not fully independent.Norm values from different populations were presented showing that a direct comparison between Europe and North America is possible, but caution recommended with comparisons of data from Latin America and Indonesia. But this will not affect intra-individual comparisons within clinical trials.The comparison with the Kupperman Index showed sufficiently good correlations, illustrating an adept criterion-oriented validity. The same is true for the comparison with the generic quality-of-life scale SF-36 where also a sufficiently close association has been shown.The currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging women in different regions and over time, it suggests a high reliability and high validity as far as the process of construct validation could be completed yet. The interest of clinical research in aging women and males increased in recent years and thereby the interest to measure health-related quality of life and symptoms. Women, as do men, experience an age-related decline of physical and mental capacity. They observe symptoms such as periodic sweating or hot flushes, impaired memory, lack of concentration, nervousness, depression, insomnia, and bone – joint complaints.women, not by their physician ...4 scoring points [very severe symptoms]). The respondent provides her personal perception by checking one of 5 possible boxes of "severity" for each of the items. This can be seen in the questionnaires in the additional files linked to this publication. The composite scores for each of the dimensions  is based on adding up the scores of each item of the respective dimensions. The composite score  is the sum of the dimension scores. The three dimensions, their corresponding questions and the evaluation are detailed and summarized in an attached file linked to this publication [see Additional file 1].).The MRS scale became internationally well accepted. The first translation was into English . Other tLike in other QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity, and this in addition to the conventional psychometric requirements of test reliability and validity.The aim of this paper is to present additional psychometric data to discuss the methodologically relevant characteristics of the MRS scale.The development of the scale, instrument characteristics , and norms and standardized scores have been published elsewhere -5. This Europe , North America (USA), Latin America , and as example for Asia – Indonesia. Study participants were accrued as a random sample of females aged 40 to 70 years from existing population panels. The sample size in each of the countries was about 1000 females aged 40–70 years, with exception of USA (n = 1500). The participation rates ranged between 46 and 94% across countries. The demographic details of the sample are: On average, about tertiles of the respondents were under 50 years, between 50–59, and over 60 years old in most of the countries, however, about 50% were less than 50 years in Indonesia and in Brazil. The majority of respondents reported a Christian religion in Europe (range: 74% (Germany) to 96% (Spain), 85% in USA, and in Latin America (range: 95% (Argentina) to 97% (Mexico). The use of the MRS was part of this survey, i.e. multinational data became available to reconsider methodological issues more thoroughly such as internal structure of the scale, reliability , and reference values for different population.During the last two years a number of smaller and larger investigations were made from different groups to further check methodological features of the scale. We performed recently a large, multinational survey to represent the situation across nine countries and cultures using existing and for the respective countries representative panels between November 2001 and February 2002 to get information about knowledge, attitudes and behaviour related to hormonal treatment in women aged 40–70 years: For the purposes of reliability assessment we performed a few preliminary studies with a test-retest approach. These small, descriptive studies of community samples of women aged 40–70 were done in summer and fall 2002 by local collaborators in the respective countries, but they were done separately and independent from the main study. These studies were done just for orientation with convenience samples – not representative for the respective population.There is only one intervention study  available to our knowledge. This study has been published  but not With these data available, we were able to scrutinize many methodological characteristics of the MRS scale to review most fundamental psychometric characteristics as well validity parameters.The assessment of scientific measurements depends first of all on the evidence of replicability (consistency) and test-retest reliability. In contrast to systematic and random variation, reliability gives an estimate of method-related measurement error which should be low not to hide or dilute intended systematic changes – due to treatment for example.Table The test-retest correlation coefficients (Pearson's correlation) support the suggestion of a good temporal stability of the total scale and its three sub-scales Table , althougThe test-retest coefficients of the total score range between 0.8 and 0.96 across Europe, North and Latin America, and Asia. When it comes to the subscales with much fewer items, the variation increased and some of the coefficients went down to 0.5 . Altogether, the test-retest stability over a time period of two weeks aggregated at the international level supports the notion of a very acceptable test-retest reliability of the total scale and their three sub-scales.Although there is an impressive set of information currently available concerning the reliability of the MRS scale, there are also limitations: Small sample sizes prevent a final conclusion regarding test-retest reliability in some of the languages the scale has been translated in.Similar to reliability which assesses the consistency of measurement, the validity estimates if a scale measures what it intends to measure. But whereas reliability can be determined straight forward with very few indicators, the validity is almost always a continuous process . It is a process of accumulating evidence for a valid measurement of what is purposed. Therefore, the currently available data are already fairly comprehensive and do pave the way for a focussed and continuing validation process.The first step of validation is usually to multivariately demonstrate a similar internal structure ("dimensions") of a given scale through factor analysis.The first factorial analysis in 1996 was applied to identify the dimensions of the scale. Three dimensions of symptoms/complaints were identified : a psychThe recent large, multinational survey in nine countries of four continents provided data to compare with the initial standardisation sample of the MRS. The question was: Is the internal structure of the MRS results comparable among different countries or cultures. Astonishingly similar factor loadings of the 11 items of the 3 domains of the MRS were observed , and sleep disturbances both 0.5 in somatic and psychological domain (Latin America). These two items had similar problems in Spain, Mexico, and Brazil but not in other countries  will be the main criterion which might not be affected by potential slight differences in the patient reported outcome structure. Therefore the general agreement in the internal structure of the MRS scale across country groups, even accepting the possibility of slight differences in two items  would be closer to 0 than the correlations with the construct of the aggregate total score to which all sub-scales should significantly contribute. But that is theory; Table There are different categories of severity of complaints or problems with QoL. For the comparison of these categories across countries or cultures it is important to understand the prevalence of complaints. Currently, there is only one table with reference values and definitions published – for the German population from the initial standardization of the MRS . Are theThe data of our large multicultural survey permitted such comparisons. The mean values (SD) of the MRS total score and the three domains are depicted in Table However, the total, psychological and somatic scores were systematically higher in Latin America, and systematically (significantly) lower in Asia (Indonesia) than in Europe/North America. The urogenital scores were significantly lower in Latin America/Indonesia than in Europe/US. Obviously the perception of the prevalent symptoms depends on cultural factors – or the symptoms show real differences in prevalence. Thus, direct comparisons of MRS scores between Europe/North America on the one side and regions in Latin America and Asia cannot are not recommended. This does not effect intra-individual comparisons  within these countries and it may also very little affect the comparison of relative changes (pre/post treatment) among different countries. The latter is a hypothesis and needs further evidence form research/experience.Similar mean values could still have a different distribution across the proposed four categories of "severity of complaints": no/little symptoms, mild, moderate, and severe complaints, i.e. for the total scale and the three domains. The prevalence of these categories across the four regions studied is seen in Table In fact, the comparison with other scales of similar purpose is important. It is known from other quality of life scales that comparisons with scales with similar purposes are much more important than comparisons with so-called objective parameters such as exercise tests, physiological or chemical parameters – in our case with hormones.Health related quality of life should be validated against quality of life measured with other generic QoL scales , and against specific instruments to measure symptoms in aging women (e.g. Kupperman index). These data were published elsewhere  but willAlthough the Kupperman Index was not validated according to psychometric standards it is still in use in the medical practice to monitor menopausal symptoms. Therefore a comparison with the fully standardized MRS seems to be reasonable. If one divides the distribution of both scales into quartiles and compares the frequencies, both instruments were found to be closely associated: Kendall's tau-b coefficient 0.75 (95% CI 0.71–0.80) . SimilarTwo sub-scales of the multi-domain quality of life scale SF-36 was compared with the MRS: the somatic sum score (with somatic domain of MRS) and the psychologic sub-scales of both instruments. Both somatic domains were sufficiently well and significantly associated: Kendall's tau-b = 0.43 (95% CI 0.52–0.35); Pearson correlation coefficient r= 0.48 (95% CI 0.58–0.37). That means, the higher the score in the somatic dimension of the MRS, the lower the quality of life according to the somatic sum-score of the SF-36 . Similari.e., the ability of the scale to accurately measure treatment effects and to predict the clinically based assessment of physicians, was not analysed so far. At present, there is one post-marketing study that can be used to preliminary assess discriminative validity. The results will be published soon elsewhere. To this end, many clinicians understand the term "validity" and mean high utility for clinical work or research.The MRS scale is a standardized HRQoL scale with good psychometric characteristics. The use in many countries offered the possibility to compare the test characteristics across countries. Reliability measures (consistency and test-retest stability) were found to be good in all countries where data were obtained – however, some samples were very small and therefore considered as preliminary information.The validity was measured in its various forms: The internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon in women with complaints. The sub-sores and total score correlations showed high coefficients with the total score and less among the sub-scales. This however indicates that the subscales are not fully independent in practice.Comparisons of reference values from different populations showed that the MRS scores can easily be compared between Europe and North America/US. Direct comparisons between Europe/North America and Latin American countries and Asia (Indonesia) should be considered with caution because the severity of reported symptoms seems to differ. The reasons are not clear, further research is needed.The comparison with other scales for menopausal symptoms (Kupperman Index) showed a sufficiently close association and correlation coefficients, i.e. illustrating a good criterion-oriented validity. The same is true for the comparison with the generic QoL scale SF-36 where also high correlation coefficients have been shown.Thus, the currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging males over time or intervention. It suggests a high reliability and high validity as far as the process of construct validation could be completed.KH: responsible for drafting the manuscript and running analyses. AR: responsible for designing and overseeing the multinational survey (2001/2002), contributed to writing and revising of the paper. PP: co-ordination of the field work of the multinational survey, setting up the initial database, and contributed to writing of the paper, responsibility in developing/validating the MRS scale. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript. FS: Provided data of a clinical study, contributed to writing of the manuscript. LAJH: responsible for the collection and evaluation of the data, and involved in writing/revising the paper. DMT: responsible for checking the integrated database, responsible for several analyses regarding validity, and contributed to writing of the paper.


Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus.Vaccinia virus, the prototype member of the family We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells.When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines. Poxviridae, is a large DNA virus whose replication takes place in the cytoplasm of the infected cell [in vitro, little is known about the ability of vaccinia virus to infect different cell types in vivo. Vaccinia virus host range in cell culture is known to be determined by several genes. The importance of host restriction has been highlighted in recent years by the growing use of the Modified Vaccinia Ankara (MVA) virus strain, whose replication is severely impaired in human cells [Vaccinia virus, the prototype of the ted cell . Althougan cells -4. Genesan cells .In addition to host range genes, there are a number of factors that might influence the infection rate of a given cell type, such as the accessibility and amount of receptors, the ability to internalize the virus, and the metabolic state of the cell. In addition to cellular factors, genetic differences in the virus might influence the efficiency and fate of the infection. For instance, cellular nucleotide pools can be one of the factors that, in conjunction with the expression of viral thymidine kinase (TK), may influence the rate of infection.in vivo may be determined by many complex factors that may be dependent on the cell type and metabolic state.The above considerations led us to hypothesize that, although receptors for vaccinia seem to be ubiquitous, and virus replication is relatively independent from the host cell, virus tropism We have focused here on the differences between two widely used strains of vaccinia virus (Western Reserve-WR and MVA), and also to their respective TK(-) mutants, in their ability to infect different cell types in fresh human PBLs.+, up to 20%) and NK cells .Previously, we have shown that GFP expression from a vaccinia virus recombinant can be used to monitor infection by flow cytometry . Where aVaccinia virus MVA and TK-deficient viruses have been proposed as improved recombinant vaccines. In particular, the highly attenuated MVA strain has elicited much interest as a safer vaccine vector. We studied the influence of the virus strain and the TK phenotype in the infection of human PBLs. We thus constructed GFP expressing viruses from vaccinia virus MVA strain, by inserting the GFP cassette downstream of the F13L gene, using an intergenic region for the insertion. Additionally, thymidine kinase-deficient virus recombinants WR-TK(-) and MVA-TK(-) were constructed by inserting the GFP cassette within the viral TK locus. Those viruses grew to high titers and produced, upon infection of cell lines, bright GFP fluorescence (not shown).+ and CD8+ cells were poorly infected, although there was indication of an increased infection of low-CD8 T lymphocytes in comparison with high-CD8 cells. Notably, this experiment confirmed that most of the monocytes (CD14+) was infected in our experimental conditions, and showed a high level of GFP fluorescence. It was of interest to directly compare the ability of vaccinia MVA to infect PBLs with that of the standard laboratory strain WR. A side-by-side comparison of WR-GFP and MVA-GFP showed that both viruses infected a high percentage of CD14+ monocytes , and a low percentage of T lymphocytes . No significant differences were noted in the percentage of CD4 cells infected with both viruses. Although both virus strains were able to infect the majority of monocytes, MVA-GFP produced a lower level of GFP fluorescence than WR-GFP in the infected monocytes. Those differences could be the result of a lower expression level, or a delay in the course of infection, by the MVA strain.The four GFP-expressing viruses were used to infect fresh human PBLs from a different individual, and subjected to flow cytometry analysis at 7 h.p.i. Fig. . The res+ B lymphocytes (7.1% vs 3.5%) and CD56+/CD16+ NK cells(7.6% vs 4%).In addition to increased GFP expression levels, WR-GFP was also more efficient than MVA-GFP for the infection of CD19As stated above, we constructed recombinant viruses from both WR and MVA by insertion of GFP into the TK locus. Infection of different populations in human PBLs with these viruses was again monitored in paralell using specific antibodies Fig. . InfectiDetection of cells infected by GFP-expressing vaccinia viruses provide a fast and sensitive method to measure virus infection . In thisIt is to note that the approach used in this work only allows the detection of viral gene expression derived from the infection, but does not address whether the infection results in the production of progeny virus. Early reports indicated that vaccinia virus cause cythopathic effect in human leukocytes, although only replicated in mitogen-stimulated cell populations, indicating that active cell replication is required for virus replication ,8. In thOur results point to a significant preference of vaccinia virus for certain cell types. In particular, monocytes were the most susceptible cells, followed by B cells and NK cells. In contrast, T cells were infected at very low rates. These observations are in broad agreement with previous studies, where different infection rates have been noted between monocytes and lymphocytes  and betwComparison of the patterns obtained with the two virus strains and their TK(-) mutants indicate that both the virus strain and the TK phenotype may determine the amount of gene expression, as was revealed by the intensity of GFP fluorescence in infected monocytes. In addition, the ability of the virus to infect certain cell types (CD19) seems to be affected to a certain extent by disruption of the TK gene. While this may be derived from our inability to detect those infected cells because of decreased gene expression, we cannot rule out a more direct requirement of TK activity in those cells.MVA vaccinia virus strain has elicited much interest recently because of its safety record. Because clinical complications and side effects of smallpox vaccination are a critical issue in the event of mass vaccination, understanding the basis of MVA attenuation may lead to the development of better vaccine vectors. In this study, a number of differences were noted between the rates of infection obtained with WR and MVA virus strains. While both viruses were able to infect the monocyte population, WR infected B cells and the NK population  more efficiently than MVA. Whether these observations have implications on the pathogenicity or immunogenicity of MVA will require further studies.The fact that both WR and MVA showed a strong preference for certain cell populations indicate that, in addition to host range genes, there are other factors that might influence the infection rate of PBL cells. Those might include a variety of such as the accessibility and amount of receptors, ability to internalize the virus, and the metabolic state of the cell.Monocytes (CD14+ cells) were the cells in the PBL population that showed a greater susceptibility to vaccinia virus infection, as measured by viral gene expression. On the other hand, T lymphocytes (CD3+ cells) were infected with low efficiency. An intermediate susceptibility was detected in B lymphocytes (CD19+ cells) and NK (CD56+ cells). Both the use of a highly attenuated virus strain (MVA) or the disruption of the thymidine kinase gene lead to decreased gene expression in the infected cells. Those observations highlight the existence of a different degree of susceptibility to infection if PBL subpopulations, a fact that may have important implications in understanding virus pathogenicity and immunogenicity.2 atmosphere at 37°C. Plasmid pRB21 [Vaccinia virus strain WR was grown and titrated in BSC-1 or CV-1 cells, grown in minimal essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) and 2 mM L-Glutamine. MVA virus and recombinants were grown in BHK-21 cells (ATCC CCL10) cultured in BHK medium containing 5% FBS, 3 g/ml tryptose phosphate broth and 0.01 M hepes. All cells were maintained in a 5% COid pRB21  containsGCTAGCAAAGGAGAAGAA) and GFPH3 (TTTAAAGCTTTACTAGTGGATCCTCAG), that include NheI and HindIII restriction sites, respectively. After digestion with NheI and HindIII, the gene was inserted into the corresponding sites in plasmid pRB21 [Plasmid pRBrsGFP, designed to mediate the insertion of the gene coding an enhanced version of the green fluorescent protein gene, rsGFP,  was constructed as follows. rsGFP gene in plasmid pQBI25 was amplified using oligonucleotides GFP 5'  after digestion with Viruses WR-GFP and MVA-GFP were obtained by transfection of plasmid pRBrsGFP in cells infected with WR mutant vRB12  or MVA, Recombinant virus VVrsGFPTK, was isolated after transfection of plasmid prsGTK in cells infected with virus WR. Recombinant virus plaques were isolated by plaquing on 143B TK(-) cells in the presence of 25 μg/ml bromodoxyuridine. GFP positive plaques were identified under the microscope, and plaque-purified three times before amplification.Recombinant virus MVA-GFPTK was isolated by transfection of plasmid prsGTK in MVA-infected BHK-21 cells. GFP-positive virus were identified under the microscope, isolated by three consecutive rounds of plaque purification in BHK-21 cells and amplified in BHK-21 cell cultures.Finally, virus recombinants were analyzed by Southern Blot, using digoxigenin-labelled GFP gene sequence as the probe. The analysis demonstrated that the recombinants contained the GFP expression cassette in the desired genome position and that they were stable, double recombinants.Peripheral blood mononuclear cells from healthy subjects were obtained by density gradient centrifugation of heparinized blood on Ficoll-Paque . Cells obtained from the interface were washed three times in saline solution and then resuspended in complete medium (CM) consisting of RPMI 1640  supplemented with 10% FBS (Gibco), 2 mM L-glutamine , 100 U/ml each of penicillin and streptomycin . Viability of the isolated cells always exceeded 95% as determined by trypan blue exclusion.5 PBLs were infected with virus recombinants VV-rsGFP, VVrsGFPTK, MVA-GFP, and MVA-GFPTK, at 10 p.f.u./cell, in 0.7 ml of RPMI medium containing 2% FBS. After 1 h adsorption, cells were pelleted and resuspended in 2 ml of fresh RPMI medium containing 2% FBS. At different infection times, the cells were sedimented by low-speed centrifugation, resuspended in 100 μl FACS-FLOW, and labeled with the appropriate conjugated monoclonal antibodies (mAb) for flow cytometric analysis (FCM) .Infection of human PBLs was performed as follows: 2 × 10Cells were incubated with the antibodies for 30 min at 4°C in the dark, washed twice with saline solution and finally resuspended in 200 μl Cytofix/Cytoperm (BD Pharmingen). Cells were analyzed in a FACSCalibur  and data were processed with Cell Quest software (BD).The author(s) declare that they have no competing interests.JMS carried out the isolation of virus recombinants and performed viral infections and participated in the drafting of the manuscript. LS and GR performed the preparation of PBLs, carried out the flow cytometry and elaborated the data. GR participated in the interpretation of the data and helped in the elaboration of the manuscript. RB conceived the study, designed the virus recombinant constructs, supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript.


Most patients with non-small cell lung cancer who initially respond to gefitinib or erlotinib (tyrosine kinase inhibitors) ultimately develop resistance and disease relapse. What is the mechanism for this resistance? Uncontrolled proliferation of tumor cells is a hallmark of cancer. In many types of cancer, mutations in genes that activate cellular signal transduction pathways contribute to enhanced proliferation and survival of cancer cells. One well-characterized example is mutation in tyrosine kinases, enzymes that regulate the growth and survival of cells. Tyrosine kinase activity is tightly regulated in normal cells, but is dysregulated due to mutation in some cancers, including lung cancer, resulting in enhanced proliferation and survival of cancer cells. The tyrosine kinases are attractive candidates for molecularly targeted therapy in cancer, because cancers become dependent on growth signals from the mutant tyrosine kinases. Tyrosine kinases require ATP for their enzymic activity, and thus small molecules that mimic ATP can bind to mutant kinases and inactivate them.BCR-ABL gene rearrangement [The paradigm for tyrosine kinase inhibition as treatment for cancer using small-molecule inhibitors was first established in the context of chronic myelogenous leukemia (CML) associated with the angement . Imatiniangement , and alsangement .PDGFRB [PDGFRA [KIT [Imatinib is also a potent inhibitor of the ARG, KIT, PDGFRA, and PDGFRB tyrosine kinases. As a consequence, there have been additional dividends from the United States Federal Drug Administration approval of imatinib for treatment of BCR-ABL-positive CML. For example, imatinib is effective in treatment of chronic myelomonocytic leukemia with gene rearrangements that constitutively activate PDGFRB , of hype [PDGFRA , and of FRA [KIT  .epidermal growth factor receptor (EGFR) in patients with NSCLC that are associated with clinical response to the small-molecule EGFR inhibitors gefitinib (Iressa) or erlotinib (Tarceva) [More recently, this paradigm has been extended to treatment of non-small cell lung cancer (NSCLC). Several mutations have been identified in the context of Tarceva) ,8,9, incTarceva) ,8,9. TheBCR-ABL kinase domain itself, including T315I [As might have been anticipated in treatment of cancer with any single agent, resistance to small-molecule tyrosine kinase inhibitors has emerged as a significant clinical problem. This was first appreciated in patients with CML treated with imatinib whose tumors developed resistance, and has been most extensively studied in that context. Although there are many potential mechanisms for development of clinical resistance, most cases of imatinib-resistant CML are due to point mutations in the ng T315I ,11. Siming T315I ,12. Thesng T315I ,14.PLoS Medicine, Pao and colleagues have identified acquired mutations in patients with NSCLC that appear to explain clinical resistance to gefitinib or erlotinib [In an elegant new study in rlotinib . The mecStructural models of EGFR provide structural insights into these biological data. A ribbon structure of erlotinib bound to the EGFR kinase domain  shows thKRAS alleles that have previously been shown by these same authors to confer resistance to these inhibitors [These observations provide convincing evidence that, at least in some patients with NSCLC, resistance to gefitinib or erlotinib can be attributed to acquisition of a T790M mutation in the context of EGFR. However, three additional patients with clinical resistance to gefitinib or erlotinib did not have the T790M mutation, nor did they have mutant It will be important to identify alternative small-molecule inhibitors for the T790M resistance mutation. Structural data suggest that one compound, lapatinib, may subserve this purpose , but it Pao and colleagues' superb study also highlights several important points that may guide development of kinase-targeted therapies in the future. It is clear that, to the extent that small-molecule kinase inhibitors are effective as single agents in treatment of cancer, resistance will develop. Furthermore, based on previous experience, some of these patients are likely to harbor acquired point mutations in the target kinase that confer resistance. Resistance mutations identified via in vitro screens have shown a high degree of correlation with those that develop in vivo, as shown in screens for imatinib-resistant BCR-ABL mutants  and PKC4These findings also emphasize the critical need for re-biopsy of patients with cancer treated with molecularly targeted therapies at time of relapse. Tissue acquisition is more challenging in solid tumors than for hematopoietic malignancies, and may entail risk. Nonetheless, it is clear that data derived from such analyses will be essential to inform approaches to improving therapy for NSCLC and other solid tumors.