Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria.Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic  Hyper-oxaluria, either as a result of inherited metabolic disorders or many other intrinsic and extrinsic factors, is one of the major risk factors for developing kidney stones In 1994, we were the first group to note that oxalate renal cell interactions involved alterations in gene expression Oxalate a metabolic end product is freely filtered at glomerulous and undergoes bi-directional transport in the renal tubules. We demonstrated for the first time that oxalate exposure to the renal cells results in plethora of changes, including changes in gene expression, re-initiation of DNA synthesis, and cell death Several recent studies have suggested the usefulness of Kidney Injury Molecule -1 (KIM-1) as an early indicator for renal injury resulting from chemical toxicity or ischemic injury in vitro and in vivo methods that are well documented as models for experimental hyperoxaluria for these studies. For in vitro studies, treating cells in tissue culture with different concentrations of Sodium Oxalate or COM-Crystals has been established as an efficient model system to test the effects of hyper-oxaluria et alIn the present study, we determined the effect of hyper-oxaluria on the expression status and shedding of KIM-1. We used both in vivo and in vitro studies indicate that hyperoxalria increases the expression of KIM-1 mRNA and protein in renal cells and tissues. Moreover, the ectodomain of KIM-1 was also identified in the urine collected from hyper-oxaluric rats by Western blot analysis. This study, thus, suggests that KIM-1 may be a potential urinary biomarker for oxalate mediated renal injury.Results from both the Oxalate exposure has been shown to induce cellular injury in epithelial cells and one of the markers for tubular injury is the over-expression and shedding of Kidney Injury Molecule (KIM-1) in vitro using proximal tubular epithelial cells, we extended the study to an in vivo model for hyper-oxaluria. Wistar rats were given 0.75% Ethylene Glycol in drinking water to develop hyper-oxaluria. Immuno-histochemical staining using the antibody that binds to the C-terminal region of KIM-1 suggested that the expression of the protein is significantly higher in hyper-oxaluric rat kidney in as little as 1 week . Kidney tissue dissected out of rats fed EG for 3 weeks showed evidence of crystal deposition in the lumens of the tubules. Bi-refringent crystals were observed under polarized light  and therTo determine the nature of the chemical composition of the crystals, tissue sections were stained with Von Kossa's staining for Calcium. Tissue sections collected from rats after 3 week of EG diet, showed positive von Kossa's staining, suggesting that the observed crystal deposits were primarily composed of Calcium Oxalate . Tissue Tubular injury in rodents has been shown to cause shedding of the ectodomain of KIM-1 protein, which can be detected in the urine. Urine was collected both from control and hyper-oxaluric rats for a period of 24 hours and western blot analysis was used to identify KIM-1 in the urine. Hyper-oxaluric rat urine showed shed KIM-1 protein in the urine, where as the control rats did not have any protein in the urine . This suOur observations also did not suggest any changes in the water or feed intake of the treatment group compared to the control rats. Results presented in in vivo and in vitro models of hyper-oxaluria.Hyper-oxaluria either as a result of increased Oxalate intake or metabolic disorders afflicts different age groups and is a primary source of discomfort. Persistent and prolonged exposure of the kidneys and urinary tract to higher Oxalate loads leads to increased renal injury and is usually considered to be an initiator for developing kidney stones In the present study, exposure of HK-2 cells to increased Oxalate concentrations over-expresses KIM-1 mRNA and protein. KIM-1 mRNA levels are significantly increased in as little as 30 min after exposure to oxalate . Controlin vivo model, Wistar rats were given drinking water with 0.75% Ethylene Glycol for a period of 3 weeks. Rat models of hyper-oxaluria were based on Ethylene Glycol in various concentrations ranging from 0.75% to 2.5% in drinking water For developing an Immuno-histochemical staining using antibodies against the C-terminal region of KIM-1 showed up-regulation of KIM-1 in tissue sections collected from hyper-oxaluric rats compared to control . The incKIM-1 has been shown to be localized to proximal tubular cells and may play an important role in the phagocytosis of apoptotic and dead cells We observed a reduction in the Urinary Oxalate on week 3 in the hyper-oxaluric rats compared to the amount of urinary oxalate on weeks 1 and 2. This may be a reflection of the observed deposition on birefringent crystals in the lumen of the tubules . In addiIn summary, our study shows that KIM-1 is over-expressed in human kidney epithelial cells upon exposure to higher oxalate concentrations. Since, increased KIM-1 expression is observed over a sustained period of time after hyper-oxaluric exposure, over-expression and shedding of KIM-1 may be potentially used as an early marker in hyper-oxaluria mediated tubular injury.All chemicals used in this study were procured from Sigma . Microcentrifuge concentrators Microcon YM-10 for concentrating rat urine were procured from Millipore Corporation .Male Wistar rats  3 to 5 week old with an average weight of 150 gm were according to the Center for Laboratory Animal Care (CLAC) guidelines at the University of Colorado Vivarium. Randomized groups were given either control water or water with 0.75% Ethylene Glycol. For metabolic studies, the rats were housed in metabolic cages for a period of 24 hours for measuring water intake and collecting urine for chemical analysis. The rats were acclimatized for a period of 24 hours to ensure continuity. Urine was collected for a period of 24 h at the end of each week and tissue samples from sacrificed animals was asceptically collected and processed. For histological studies, part of the tissue was fixed in formalin and embedded in paraffin wax; or stored in RNAlater solution for extracting RNA.Human Kidney Epithelial Cells, HK-2 were procured from ATCC and maintained in DMEM medium supplemented with 10% Fetal Bovine Serum and antibiotics. Before Oxalate treatments, cells were serum starved for 16 to 20 hours. Media components were procured form Invitrogen Corporation  as described previously Urine analysis for concentrations of different metabolites was performed with the assistance of University of Colorado Hospital Clinical laboratory using standard procedures. Oxalate concentration in the urine was estimated using a AS10 ion chromatography column Kidney tissue dissected from rats was fixed in 10% Formaldehyde and embedded in Paraffin wax. 5 uM thin sections were used for histochemical staining; sections were stained for assessing fine structures by Hematoxylin and Eosin Detection of Calcium Oxalate crystal deposits was by VonKossa's staining and was done by the Pathology Laboratory of the University of Colorado Denver using standard procedures RNA was isolated from tissue stored in RNAlater using the RNEasy Kit according to manufacturer's recommendations . 1 ug total RNA was used to synthesize cDNA using iScript cDNA synthesis Kit . PCR was performed with gene specific primers using Platinum Taq Polymerase  and separating the products on a 1% agarose gel. Primers were procured from Integrated DNA Technologies  and primer sequences for rat and human Kidney Injury Molecule (KIM-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are described in Kidney tissue from rats and HK-2 cells were washed with Ice cold PBS and then lysed immediately with boiling hot 2X SDS gel loading buffer with 2- Mercaptoethanol. Cellular proteins were resolved using SDS-Polyacrylamide gels and then transferred onto Polyvinylidene difluoride membrane (Millipore Corporation). Two different antibodies that represent the N- terminal region and the C-terminal region of KIM-1 protein were procured from Abcam Inc. , while GAPDH antibody was procured from Cell Signaling Technology Inc. Quantification of the band intensities was performed using the densitometry analysis function of Quantity One 1-D gel analysis software .Unless otherwise mentioned, two dimensional two sample equal variance student's T-test was used for statistical analysis. A p<0.05 was considered significant.


Spike-timing-dependent plasticity (STDP) modifies the weight (or strength) of synaptic connections between neurons and is considered to be crucial for generating network structure. It has been observed in physiology that, in addition to spike timing, the weight update also depends on the current value of the weight. The functional implications of this feature are still largely unclear. Additive STDP gives rise to strong competition among synapses, but due to the absence of weight dependence, it requires hard boundaries to secure the stability of weight dynamics. Multiplicative STDP with linear weight dependence for depression ensures stability, but it lacks sufficiently strong competition required to obtain a clear synaptic specialization. A solution to this stability-versus-function dilemma can be found with an intermediate parametrization between additive and multiplicative STDP. Here we propose a novel solution to the dilemma, named log-STDP, whose key feature is a sublinear weight dependence for depression. Due to its specific weight dependence, this new model can produce significantly broad weight distributions with no hard upper bound, similar to those recently observed in experiments. Log-STDP induces graded competition between synapses, such that synapses receiving stronger input correlations are pushed further in the tail of (very) large weights. Strong weights are functionally important to enhance the neuronal response to synchronous spike volleys. Depending on the input configuration, multiple groups of correlated synaptic inputs exhibit either winner-share-all or winner-take-all behavior. When the configuration of input correlations changes, individual synapses quickly and robustly readapt to represent the new configuration. We also demonstrate the advantages of log-STDP for generating a stable structure of strong weights in a recurrently connected network. These properties of log-STDP are compared with those of previous models. Through long-tail weight distributions, log-STDP achieves both stable dynamics for and robust competition of synapses, which are crucial for spike-based information processing. Modifications of the strength (or weight) of synaptic connections between neurons that occur in an activity-dependent manner are hypothesized to play an active role in generating the structure of neuronal networks In this paper, we propose a novel STDP rule, termed log-STDP, that can produce long-tail distributions of synaptic strengths similar to those reported in recent experiments. Pyramidal cells in the rat visual cortex exhibit lognormal-like distributions for the amplitudes of excitatory postsynaptic potentials (EPSPs) Moreover, we study the functional implications of log-STDP in terms of synaptic specialization. We focus on how STDP can achieve both a stable weight distribution and effective selection of synaptic input pathways, which we refer to as the stability-versus-function “dilemma”. Additive STDP (add-STDP) can rapidly and efficiently select synaptic pathways by splitting synaptic weights into a bimodal distribution of weak and strong synapses We first explain how we derived the novel model of log-STDP. Then, we study the synaptic dynamics for a single neuron whose plastic synapses are stimulated by an arbitrary number of input spike trains, as illustrated in Following previous studies Here we present the mathematical description of ‘log-STDP’. In this phenomenological model, the change in the synaptic weight induced by pre- and postsynaptic spikes at respective times The shape of the weight distribution produced by STDP can be adjusted via the scaling functions in proportion as compared to a weaker weight; see Before studying the dynamics induced by log-STDP, we discuss the role of noise in our model in the light of previous models. Our model involves two sources of noise in the STDP dynamics, via the white noise Compared to the study by van Rossum et al. A last point concerns spike-pair restrictions: all pairs of pre- and postsynaptic spikes contribute to STDP in the present study, which implies more updates and thus more noise in the synaptic dynamics. Consequently, even though individual updates in our version of mlt-STP are less noisy than in the original model of van Rossum et al. Our theoretical framework allows us to evaluate the weight distribution produced by an arbitrary weight-dependent STDP model, by combining (1) and (5). In this section, we focus on the case of uncorrelated input spike trains, for which (5) is valid. However, the theoretical prediction may not be reliable when the synaptic dynamics does not have a stable fixed point. For example, add-STDP requires taking into account the effect of input-output spike-time correlations to obtain a bimodal distribution of As a first control, we verify that the stationary distributions in The toy model is sufficiently simple to obtain an analytical expression for the spread of the resulting distribution, see (24) in Rapid adaptation to the external world is enhanced when weights experience a certain degree of noise. With log-STDP, synapses are shuffled because of the plasticity-intrinsic noise rate  Hz . The outand 8 Hz . The evoThe temporal “antisymmetry”  of the learning window has been shown to favor correlated inputs, therefore generating weight specialization When the inputs are only weakly correlated  of 50 inputs. As illustrated in After the correlation switch at 500 s, the potentiated weights from the first correlated group return to their baseline equilibrium value, close to the fixed point In order to assess whether the interesting dynamics produced by log-STDP for a single neuron also holds in the case of a recurrent network, we first reproduce a previous result of network self-organization Following the results in e neuron . Even wiHowever, these input correlations do affect the fine structure of recurrent connections for log-STDP. To show this, we firstly examine the “survival” of the potentiated synapses in the top of the distribution, as in tructure , which gCompared to log-STDP, the weights are shuffled more quickly with mlt-STDP and no structure develops. This is illustrated in Finally, we assess the persistence of weights in the strong tail in another manner. Because input correlations are sustained here, it makes sense to check how many times each weight appears in the strong tail. The repeated presence of weights in the tail implies some consistency for an emerged weight structure, even though some weights get repressed and pushed out at some times. We thus calculate for each weight The present paper proposes a novel STDP model called log-STDP that combines a number of interesting properties. Log-STDP inherently produces long-tail  distributions of synaptic strengths that agree with physiological observations A first important feature of log-STDP is its log-like saturating LTD, an intermediate variation between constant and linear functions. The scaling functions in (6) have been designed to coincide with mlt-STDP model in the range of “small” weights . Similar to mlt-STDP and in contrast to add-STDP and nlta-STDP, log-STDP requires neither “hard” or “soft” upper bound on the weights to secure the stability of their distribution.Another property of log-STDP that supports its functional capabilities is the noise in the STDP update. Due to the sublinear LTD (and quasi-constant LTP),  weights . Here weThe present analysis only considers all-to-all spike contributions to STDP. For low (input and output) firing rates, as was used here, typical interspike intervals are larger than the temporal range of STDP. This means that the synaptic dynamics for models with restricted interactions, where not all pairs of spikes contribute to STDP Because of its sublinearly increasing LTD, log-STDP alone produces a long-tail weight distribution, even for uncorrelated inputs. The change of curvature around the fixed point of the dynamics , assuming that the following conditions are satisfied:The pre- and postsynaptic spike trains are (quasi) probabilistically independent for all pairs input/output; this is a good approximation in the case of many uncorrelated Poisson-generated inputs.The neuronal output firing rate is not too high  leads to approximated expressions that do not take the neuron model into account, but describe satisfactorily the asymptotic weight distribution when the learning dynamics has a stable fixed point Under assumption (i), the pre- and postsynaptic spike trains behave as two Poisson processes. This means that (12) can be rewritten asUsing (13), the drift Likewise, we can evaluate the noise term The results in (14) and (15) are reproduced in (5) in the main text. There, we have dropped the input firing rate To obtain a group of spike trains with a given correlation strength Now we extend the result in (14) and (15) to incorporate input-output correlations. We do not aim to develop the full theory here for integrate-and-fire neuron. Rather, we aim for a simpler result using the Poisson neuron. This provides insight on sensitivity of STDP rules to input spike-time correlations for integrate-and-fire neuron . The firWhen a correlated event occurs, the synchronous incoming spike volley causes the firing probability We take input spikes as references now to evaluate In particular, the equilibrium weight ts e.g.,  is givenUsing the expressions in (20), In order to get analytical insight about a suitable STDP model that generates long-tail distribution of synaptic weights, we consider the following functions:This toy model of “plasticity” inspired us to formulate the weight dependence for log-STDP in (6). Despite the discrepancies between the functions The STDP model is detailed in the main text in (3), (4) and (6). The baseline simulation uses A previous study Similar results were obtained with the following log-like LTD scaling function In our analysis, we compare log-STDP with other previous reference models, namely add-STDP Add-STDP Mlt-STDP has a linear weight dependence for LTD and constant LTP Nlta-STDP The simulation results presented in this paper use the usual leaky integrate-and-fire neuron model with conductance-based synapses. The evolution of the membrane potential Figure S1Comparison of the weight dependence schemes and resulting weight distributions for different models of STDP. Each row corresponds to the model whose name is written on the left: log-STDP for our novel model; mlt-STDP Column A:Column B: The drift Column C: Resulting weight distribution (same color coding as B) with linear axes (similar to (EPS)Click here for additional data file.Figure S2Simulated weight distribution obtained for various choices of parameters for log-STDP. Similar plots to (EPS)Click here for additional data file.Figure S3Simulation with a single neuron similar toin the main text with two correlated input pools.A: Schematic diagram of the neuron (top blue filled circle) stimulated an uncorrelated pool (bottom open circles) and two correlated pools (bottom red filled circles). Darker gray indicates a stronger correlation strength. B: Evolution of the synaptic weights for the configuration in A. The correlation strengths are C: Evolution of the neuronal output firing rate for the configurations: (EPS)Click here for additional data file.Figure S4Weigth potentiation and firing rate as a function of the input correlation with the Poisson neuron.A: The single Poisson neuron is stimulated by 1000 inputs of which 100 have correlations (strength on x-axis). The simulated weights from the correlated pool (circles) are taken from 10 simulations of duration 500 s and their mean is indicated by the thick black curve (with error bars for the standard deviation). The predicted equilibrium weights using Eq 21 in the main text is represented by the blue curve. The predictions neglect the noise in the STDP update, as well as the synaptic delays, but it is satisfactory up to correlation strengths equal to 0.6. Discrepancies come from neglecting the noise, which become non-negligible for large weights. B Similar to A with the output firing rate.(EPS)Click here for additional data file.Figure S5Time histogram of the neuronal spiking response to correlated events for a Poisson neuron. Comparison between log-STDP (red) and mlt-STDP (purple). The parameters given in Sec ‘Parameters used in numerical simulation with the Poisson neuron model’ above. Similar to (EPS)Click here for additional data file.Figure S6Weight emerged structure in a network stimulated by two independent correlated pools.A: Schematic representation of the network before (left) and after (right) the learning epoch. The recurrent network of 500 neurons was stimulated by a pool of 2800 uncorrelated input spike trains (not shown) and two identical correlated pools of 100 spike trains each (bottom red circles), which exhibited delta correlations  at the end of the learning epoch, where darker pixels indicate stronger weights. Among recurrent connections, the within-group connections were potentiated while the between-group connections remained weak.(EPS)Click here for additional data file.Figure S7Distribution of the firing rates for the network neurons corresponding to  in the main text. The same color coding applies: solid curve for log-STDP and dashed for mlt-STDP; red for correlated inputs and purple for uncorrelated inputs.(EPS)Click here for additional data file.


Statement of problem: Techniques and recommendations for the restoration of endodontically treated teeth have changed from the use of custom cast metal post and core system to glass fiber-reinforced (GFRC) post and composite core system. Has this latest prefabricated glass fiber reinforced post and composite core system increased the fracture resistance of teeth and reduced the incidence of unrestorable root fractures. Purpose: The purpose of this study was to evaluate the incidence of root fracture and mode of failure of endodontically treated teeth restored with two different post and core systems.Material and Methods: Forty maxillary central incisors were randomly divided into two groups. (n=20). All teeth received endodontic treatment. First group was restored with custom cast post and core system. Second group was restored with glass fiber post and composite core system. In Both the groups posts were cemented with adhesive resin cement. Compressive load was applied at an angle of 130 to the long axis of teeth at a cross head speed of 1 mm/min until fracture occurred. Data were analyzed with student “t” test P<.001.Results: The mean value for fracture resistance was (331.4025) N in Group -I Custom cast Ni-Cr post and core and (237.0625) N in Group -II Glass fiber reinforced post and composite core system. Students “t” test shows the significant difference in fracture resistance of two groups. Conclusion: This study showed that the incidence of root fracture was significantly higher in custom cast Ni-Cr post and core system than glass fiber post and composite core system. A more favourable mode of failure was observed in teeth restored with Group II glass fiber post system. Key words:Post-and-core technique, glass fiber post, cast post and-core system, fracture resistance, endodontically treated teeth.  The restoration of endodontically treated teeth has been studied extensively . Posts aEndodontically treated anterior teeth have traditionally been restored with cast or wrought metal posts and cores. These metallic posts have a much higher modulus of elasticity than the supporting dentine; this mismatch in modulus could lead to stress concentration and leads to failure. This has lead to search for a plastic based material that has modulus closer to that of dentine .Tooth-colour posts have increased in popularity since they were introduced in 1997 . PrefabrThe cement used for cementation with glass fiber post is resin adhesive cement. This cement provides stronger union between post and core and tooth structure using adhesive bonding technique. This adhesive restorative cement transmits and distributes functional stresses across the bonding interface to the tooth more properly . IntegraThe purpose of this study was to evaluate the incidence of root fracture and mode of failure of endodontically treated teeth restored with two different types of posts custom cast Ni-Cr post and core system; glass fiber reinforced post and composite core system.Forty, freshly extracted human maxillary central incisors were selected for the study. The teeth were collected irrespective of the age, sex, and side of the arch. All the teeth were without root caries, root fillings, root cracks, and minimum of 10mm of root length. The selected teeth were stored in artificial saliva (wet mouth ICPA C09002) at room temperature until used for study, to avoid their dehydration. All the teeth were decoronated horizontally at cemento-enamel junction, perpendicular to long axis of teeth. Endodontic treatment of selected teeth was completed as: A#15-50 using step back technique. After endodntic treatment all the prepared teeth were randomly divided into two groups of twenty teeth each,Group I- In this group twenty samples of endodontically treated teeth is restored with Custom cast Ni-Cr alloy post and core (CP) system. Group II- In this group twenty samples of endodontically treated teeth is restored with Glass fiber-reinforced post and composite core (GFRP) system. Group I (CP) Peeso reamers  were used to prepare the post space. For Group II (GFRP), special drills supplied with the kit, were used to prepare post space, leaving 3 mm apical seal in the root canal. Prepared post space was evaluated with the radiograph.For Group I (CP) - Wax pattern for cast post and core was prepared. Paper pin was roughened and dipped in molten blue inlay wax and inserted into the canal. Incremental addition of wax was done to make a post pattern. The core was built up to achieve a desired core height of 4 mm, and bucco-lingual and mesio-distal dimensions 1.5 mm less than to the corresponding total bucco-lingual and mesio-distal dimensions of the specimen. The post and core wax pattern was sprued and invested. Casting was done with Ni-Cr alloy  and casting of post and core were obtained and finished.For Group II (GFRP), the prefabricated glass fiber posts were cemented with adhesive resin cement (3M) ESPE (N120174). Then it was light cured for 40 sec to achieve complete polymerization. Core build up was done with Tetric N-cerem, Ivoclar Vivadent (N-12096). The preformed polyester matrix was filled with the core build up material placed on the specimen. Each increment was light cured for 60 sec. It was finished to the final core height of 6 mm and bucco-lingual and mesio-distal dimensions corresponding to that of the tooth, with the help of composite finishing kit.Cementation of cast post in Group I (CP) were done with adhesive resin cement. The procedure of cementation was same as done for group II (GFRP), post system.With free hand all preparations were finished with a diamond bur  at high speed with water spray . All finish lines were placed at level of cemento-enamel junction. Single coat of spacer was applied to the core part of the specimen. The core was dipped into the molten blue inlay wax, to give a uniform layer of wax over the entire surface. Crown patterns were prepared for two groups of post and core system by duplicating polycarbonate crown forms (size 10).Investing and casting was done with the Ni-Cr alloy . Castings were retrieved, finished and polished.Cementation of crown was done with the adhesive resin cement. All specimens roots were dipped into molten wax to a depth of 2mm below the CEJ to give a uniform layer 0.2-0.3 mm of wax over the entire root surface.40 Acrylic resins blocks were fabricated by mixing the self- cure acrylic resin DPI-RR cold cure P-13103, L-13102 and pouring it into block – holder part of metal jig. Specimens were held perpendicular into the center of block– holder part of metal jig. After setting of acrylic resin, dewaxing was done to achieve a space for simulated periodontal ligament. Type I- regular viscosity regular body, polyvinyl siloxane impression material (Reprosil-Dentsply) 100313 was applied over the root surfaces and also into the mold surface in acrylic resin blocks. Each specimen was reinserted into acrylic blocks up to 2mm below CEJ and held under digital pressure until material was set. Excess material was removed with the help of B. P. knife. All specimens were prepared and stored in artificial saliva until tested. Stainless steel metal attachment tool were custom made of the desired dimensions according to the testing machine. Metal jig had the provision for holding acrylic resin blocks that orient the specimen at an angle of 130 degree to the load application tip of the attachment tool. The whole as-sembly was fitted in the universal testing machine  and load was applied on the palatal surface of cast crown, 2mm from incisal edge, at an angle of 130 degrees to the long axis of the root, at a cross head speed of 1mm\min until failure occurred as seen in Fig. . The meaThe mean fracture resistance of Custom Cast Ni-Cr post and core group and Glass fiber reinforced composite post and core group was 331.4025) N, (237.0625) N respectively.  system was as follow- 90% of the specimens had root fracture, 10% of the specimens had cement failure. Post fracture, core fracture and post-core junction fractures were not observed in this group  had shown the more favourable mode of failure as only 10% of the specimens had shown the root fracture as compared to the Group-I (CP) in which 90% of the specimens had shown the root fracture. The cast restorations had highest modulus of elasticity (220 GPa) as compared glass fiber post 13-40 GPa) whereas the modulus of elasticity of dentine is 15-25 GPa). In glass fiber post and composite core system modulus of elasticity is similar to dentin had resulted in improved stress distribution between the post and dentin thus resulting in improved flexibility of teeth under applied loads. Fiber posts contribute to minimizes the risks of unrestorable root fractures -40 GPa w. It has -25 GPa. Insua et al (Sidoli GE et al (Sirimai S et al (While in case of cast post and core system due to high difference in modulus of elasticity resulting in stress concentration at the apical region of root leads to catastrophic root fractures. These findings are shown in other studies conducted by ua et al , Sidoli GE et al , Sirimai S et al .The cement used in this study was resin adhesive cement. Adhesive system for post cementation improves marginal adaptation with improved seal -23. ReliMohhamad N (Cormier CJ (Tay FR (This cement provides stronger union between post and core and tooth structure using adhesive bonding technique. hhamad N  claimed rmier CJ , Tay FR  (Tay FR .A few Incidence of post fracture are observed in glass fiber post and core system. This can be due to the weak bond between the internal glass fibers and resin matrix . In caseWithin the limitation of this study, it was concluded that a more favourable mode of failure was observed in teeth restored with Group II glass fiber post and composite core system. Teeth restored with Group I custom cast post system showed catastrophic vertical root fractures which are non repairable. So glass fiber post and composite core system have been recommended because they improve teeth flexibility under applied loads as well as stress distribution between post and dentin to reduce the risk of root fracture, the most serious type of failure.


Cdc20-anaphase promoting complex/cyclosome (Cdc20-APC/C) E3 ubiquitin ligase activity is essential for orderly mitotic progression. The deubiqituinase USP44 was identified as a key regulator of APC/C and has been proposed to suppress Cdc20-APC/C activity by maintaining its association with the inhibitory protein Mad2 until all chromosomes are properly attached to the mitotic spindle. However, this notion has been challenged by data in which a lysine-less mutant of Cdc20 leads to premature anaphase, suggesting that it's ubiquitination is not required for APC/C activation. To further evaluate its role in checkpoint function and chromosome instability, we studied the consequences of over-expression of mouse Usp44 in non-transformed murine embryonic fibroblasts. Here we show that cells with high Usp44 are prone to chromosome segregation errors and aneuploidization. We find that high Usp44 promotes association of Mad2 with Cdc20 and reinforces the mitotic checkpoint. Surprisingly, the APC/C-Cdc20 substrate cyclin B1 is stabilized in G2 when Usp44 is over-expressed, but is degraded with normal kinetics once cells enter mitosis. Furthermore, we show that USP44 expression is elevated in subset of T-cell leukemias. These data are consistent with an important role for USP44 in regulating Cdc20-APC/C activity and suggest that high levels of this enzyme may contribute to the pathogenesis of T-ALL. Mitotic Checkpoint Complex, or MCC) to the APC/C co-activator molecule Cdc20 Cdc20 and the APC becomes active.The cells of a majority of human cancers carry abnormal numbers of chromosomes, a condition known as aneuploidy Recently, two RNAi-based functional genetic screens were performed in order to identify novel gene products involved in the mitotic checkpoint To address issues raised by previous RNAi studies, we have studied the consequences of over-expression of Usp44 in non-transformed mouse fibroblasts. As was observed in cells depleted of USP44, we observe increased levels to grossly disrupt normal chromosome segregation, leading to aneuploidy. These changes are accompanied by functional and biochemical evidence of reduced Cdc20-APC/C activity, with the substrate cyclin B1 stabilized in G2, and early mitosis. These observations suggest that USP44 is an inhibitor of APC/C activity. Lastly, we show that levels of USP44 are highly elevated in human T-cell acute lymphoblastic leukemia, suggesting a role for these molecular defects in the pathogenesis of this disease.To examine the impact of excess Usp44 on mitotic chromosome segregation, we used live-cell microscopy to follow MEFs transduced with lentivirus encoding either empty, or Usp44-HA, as they progress through mitosis. Chromosomes were visualized through the expression of histone H2B-YFP. When compared with control cells, we observed a significant increase in chromosome missegregation in MEFs expressing Usp44-HA .Cherry). We conducted live-cell imaging experiments and grouped cells by the level of expression – with those having dim expression (Usp44CherryLO) and others with bright expression (Usp44CherryHI). The rate of chromosome missegregation was increased in both groups compared with the Usp44-HA pool, with 42% abnormalities in the Usp44CherryLO group, and 48% in the Usp44CherryHI group, with the predominant abnormality being chromosome bridging. This suggests that the data from cells expressing Usp44-HA may underestimate the mitotic defects resulting from excess Usp44.As these data come from a polyclonal pool of drug-resistant MEFs, there is likely to be significant variation in the level of Usp44 over-expression. To determine if mitotic errors vary with the level of expression, we transduced MEFs with a fusion between Usp44 and the fluorescent protein mCherry  on primary MEFs. Expression of wild-type Usp44 was associated with a substantial increase in whole chromosome losses or gains compared with cells transduced with empty virus . An averDespite several attempts (five different immunogens in ten rabbits), we were unsuccessful in raising a polyclonal antibody suitable for the detection of endogenous mouse or human USP44 by western blot, nor did any of the commercially available antibodies recognize over-expressed human or mouse Usp44. Thus, it impossible to determine the degree of protein over-expression. We therefore determined the abundance of Usp44 mRNA by quantitative real-time (qRT)-PCR in a panel of 4 MEF lines used in the experiments reported here. In these cells, we detected an average 95-fold . In early mitosis, we observed a significant increase in cyclin B1 levels compared with control , cells began to enter mitosis within 24 hours following release. Compared with control, Usp44-HA expression led to an increase in the levels of cyclin B at all time points, suggesting reduced APC/C activity . Notably control . In subs control .Cerulean) into MEFs stably transduced with either empty virus or Usp44Cherry  and then followed cells by live-cell microscopy. We marked cells in G2, either by centrosome association of cyclin B1, or by recognizing the early stages of chromosome condensation, and followed them through mitosis. As there is no cycloheximide present, the amount of cyclin-B1Cerulean present is the balance of new synthesis and degradation. As has been reported by others using mouse cells, we observe that cyclin B1 levels gradually decline following nuclear envelope breakdown ; Cherry fusion protein localized predominantly to the nucleus, with scattered signal in the cytoplasm in a punctate or vesicular pattern near the nucleus . Comparing cancer to normal tissues, eight cancer groups have significant alteration in the level of USP44 . Furthermore, non-degradative ubiquitination may affect the Cdc20 functioning.USP44 has been identified in two recent functional genomic screens as an important protein in the mitotic checkpoint The observation that Usp44 overexpression specifically leads to anaphase bridges argues against a non-specific disruption of normal mitotic functions. We do not observe significant increases in the rates of lagging or misaligned chromosomes in cells expressing excess Usp44. The latter two defects are commonly seen in cells experiencing a bypass of the mitotic checkpoint and in the presence of defects in the microtubule –kinetochore “error correction machinery” that requires aurora B. An association has been proposed between defects in the DNA repair or replication processes and the formation of anaphase bridges The idea that prolonged checkpoint signaling may lead to chromosome missegregation and aneuploidy was recently explored in the work by the Benezra group who demonstrated aneuploidy and enhanced tumorigenesis in a mouse model with an inducible Mad2 transgene We were particularly surprised to find that the highest levels of exogenous Usp44 occurred in interphase, and that levels were relatively low in mitosis. As we clearly observe mitotic phenotypes resulting from excess Usp44, this suggests that either the levels of exogenous Usp44 are still above normal in mitosis, or that the impact of high levels in interphase persists into mitosis, leading to mitotic errors. While we clearly demonstrate that levels of cyclin B are increased in G2, consistent with the latter explanation, we cannot determine whether the levels of Usp44 are in fact elevated in mitosis due to the lack of a suitable antibody. Our findings in this regard are at odds with a recent report where high levels of Usp44-HA were seen in mitosis, with degradation coinciding with mitotic exit. While we initially thought this was due to inter-species variation, our results in HeLa cells were similar to the mouse data. More detailed work regarding the regulated degradation of USP44 is needed to clarify these discrepancies. Our work is consistent with a recent study using MEFs with low levels of BubR1 that demonstrate a requirement for APC/C inhibition in G2. While low levels of cyclin B1/Cdk activity in mitosis had previously been shown in these BubR1 hypomorphic cells Mutations in several CIN genes have been described in a variety of human cancers. While the majority of cases of most types of tumors have obvious aneuploidy, it is becoming more clear that, with the emerging recognition of acquired uniparental disomy, the true number of cases where aneuploidy contributes to cancer pathogenesis is likely even larger. The role of aneuploidy in T-ALL was highlighted in recent studies of the TLX1 oncogene. This gene is involved in translocations in up to 30% of adult cases of T-ALL. Recently, its role in the pathogenesis of this disease was shown to involve chromosome instability, as TLX transgenic mice developed a high incidence of T-ALL characterized by increased aneuploidy It is important to note that UbcH10 over-expression has been observed in many tumors of a variety of types. Recently, transgenic over-expression of UbcH10 was shown to drive the development of spontaneous tumors in mice, providing conclusive evidence of its oncogenic potential The use of human disease samples was performed following the approval of the Mayo Clinic Institutional Review Board (IRB). Clinical samples were obtained following an additional review by the Children's Oncology Group (COG), Acute Lymphoblastic Leukemia Biology Subcommittee. Samples were acquired, following informed consent, by the COG in the course of several multi-institutional clinical trials, and were completely de-identified prior to their being provided to the authors. As the consent used by the COG included the use of these samples in future biologic research, the Mayo Clinic IRB waived the need for further consent prior to the use of these samples in our studies. All studies involving animals were reviewed and approved by the Mayo Clinic Institutional Animal Use and Care Committee (Protocol # A25510).http://www.ebi.ac.uk/Tools/msa/clustalw2/)). Using the human USP44 cDNA as a benchmark, we performed a BLAT search (http://genome.ucsc.edu/) against the murine genome and identified an open reading frame with a high degree of homology to the human sequence  including a highly conserved catalytic cysteine. This sequence (herein referred to as Usp44) was cloned by PCR from cDNA generated from mouse kidney RNA using the RNeasy kit (Qiagen) according to manufacturer instructions. This cDNA is expressed in all tested tissues in the mouse using quantitative real-time PCR. The RNA was converted to cDNA using the superscript III kit (Invitrogen) according to manufacturer instructions. The cDNA was cloned with an N- or C-terminal HA tag, or mCherry C-terminal fusion, into the lentiviral vector pTSIN-IRES-Puro in which we inserted the IRES-puromycin resistance cassette cloned from pIRES-Puro (Clontech) into an engineered multiple cloning site that was inserted between the BamH1 and KpnI sites of pTSIN. Cells used in this study include murine embryonic fibroblasts (MEFs) that were generated from day 13.5 embryos resulting from an intercross of C57BL/6 mice Upon review of the NCBI data for mouse Usp44, we noted that its sequence and genomic structure differ from all other known alleles for this gene regardless of species. The predicted amino acid sequence from this locus would lack a catalytic cysteine and would have relatively poor alignment with the human sequence .Live cell imaging experiments were performed as previously described in detail Following approval by the Mayo Clinic Institutional Review Board (IRB), a series of 25 de-identified samples with a diagnosis of T-cell acute lymphoblastic leukemia (ALL) were obtained from Mayo Clinic sample banks, and from the Children's Oncology Group , and were used for analysis of USP44 mRNA. Controls consisted of peripheral T-cells isolated from discarded filter-cones from volunteer donors to the Mayo Clinic blood donor center


Endomorphins are endogenous opioid peptides that cause potent antinociception in rodent models of acute and neuropathic pain with less undesirable side effects than opioid alkaloids. However, endomorphins are poorly suited to clinical applications because of low membrane permeability and a susceptibility to enzymatic degradation. Glycosylation and lipidation have proven to be two of the most robust approaches for the generation of new therapeutic endomorphin derivatives. Conjugation with lipoamino acids (LAA) confers an amphipathic character to the peptide, which improved interaction between the peptide and the lipid bilayer of the cell membranes, increasing permeability. Glycosylation can also improve peptide stability and blood brain barrier (BBB) transport. It is believed that an endocytotic mechanism (transcytosis) is responsible for the systemic delivery of water-soluble glycopeptides. This review discusses the application of glycosylation and lipidation strategies to improve the drug-like properties of endomorphins. Pharmacologically active endomorphin analogs with less adverse effects are also discussed. Opioid analgesics such as morphine are among the most commonly used for the treatment of severe pain. Although opioid analgesics are useful for the relief of nociceptive pain, their efficacy against neuropathic pain is limited. Furthermore, they are associated with a range of undesirable side effects such as constipation, respiratory depression, tolerance, and physical dependence, particularly with long-term use. Investigations into new effective treatments for neuropathic pain that could replace opioid alkaloids have predominantly focused on the development of peptide analogs with selectivity for μ-opioid (MOP) receptors  which had high MOP receptor selectivity, but low affinity , possess affinity for the three opioid receptors: μ (MOP), δ (DOP), and κ (KOP), with low to moderate specificity. β-endorphins have comparable affinity for both MOP and DOP receptors. Met- and leu-enkephalins are DOP receptor endogenous ligands and dynorphins are ligands of KOP receptors. Mammalian peptides with high selectivity and affinity for MOP receptors were not known until the discovery of endomorphins. In Different strategies have been investigated for the effective delivery of endomorphin.Use of peptidase inhibitors such as tripeptides diprotin A and B, Tyr-Pro-Ala-NH2 (EMDB-2), and Tyr-Pro-Ala-OH (EMDB-3), enhanced and prolonged the antinociceptive effects of endomorphins  of glucose-C8LAA derivative, this was not as pronounced as the Papp of the compounds modified only with C8LAA compared to endomorphin-1. Due to a significant reduction in the receptor binding affinity of the C8LAA analog, a further modification was performed on the backbone structure of the parent peptide. The unnatural amino acid 2′,6′-dimethyltyrosine (Dmt) the more hydrophobic and conformationally restricted residue compared to Tyr  relative to the parent peptide. This analog exhibited enhanced stability and permeability  with all compounds bearing an overlapping Phe in the sequence. It was proposed that the addition of substance P fragments to lipo-endomorphin-1 may decrease the development of tolerance and physical dependence. Two C10-modified endomorphin-1/SP hybrid peptides showed significantly improved half-life and membrane permeability. However, of the two promising LAA-modified derivatives with high stability and permeability, only the one that contained the SP7−11 fragment produced potent activity at MOP receptors with significant binding affinity and retained selectivity. The docking scores obtained from conformational studies were also in agreement with the Kiμ values obtained in the receptor binding affinity experiments  and fractional absorption values (Fa) in humans. This makes the results from Caco-2 cell studies a reliable predictor of the oral absorption of compounds in human  model of pain in rats. This analgesia was opioid receptor-mediated and was produced following intravenous (i.v.) administration. The two C10LAA-modified endomorphin-1 peptides produced higher potency than C8LAA analogs and even morphine in this model of rats with EDet al. synthesized glycosylated endomorphin-2 analogs by conjugating glucose or 2,3,4,6-tetra-O-acetyl glucose with the hydroxyproline (Hyp) residue. The MOP receptor binding affinity and agonist activity was abolished in all of these analogs therefore the compounds were not further investigated for their in vivo pain relieving activity (Biondi et al., To date, many glycosylated analogs of various neuropeptides such as deltorphin (Negri et al., N-terminal conjugation of endomorphin-1 with lactose succinamic acid resulted in a significant antinociception following oral administration to CCI rats. This effect was comparable with that of morphine, for which various oral dosage forms are currently in clinical use (Varamini et al., in vivo and in vitro biological activity of endomorphin analogs. Over fifteen years since the discovery of endomorphins, investigations have led to the production of derivatives with acceptable selectivity and MOP receptor-binding affinity. However, only limited progress has been made in the production of compounds with outstanding metabolic stability and membrane permeability, while retaining their pharmacodynamic properties. These are critical criteria in the field of peptide drug delivery to overcome the obstacles and succeed in the development of peripherally active and BBB-permeable analgesics suitable for clinical applications. Thus far, two of the most successful strategies have been shown to be glycosylation and lipid modification. Two examples are the development of an orally active lactose-modified and systemically effective LAA-conjugated derivative of endomorphin-1 with high potential for the treatment of neuropathic pain.Endomorphins have been shown to elicit a potent pain relieving effect in different acute and neuropathic pain models in animals after central administration. More importantly this effect is concomitant with little to no undesirable side effects associated with the application of opioids like morphine. These promising effects have been strong motivation for investigators to design and synthesize a substantial number of endomorphin derivatives. Numerous studies report developments in our understanding of the structure activity relationship properties, bioactive conformation, physiological characterizations, This review highlights the two modifications that have made the most improvements to the therapeutic and side effect profile of endomorphins. Presently potent and promising lead compounds have been developed which are prospective to proceed from research to the pharmaceutical industry. These achievements are the outcome of extensive research having been made to develop opioid peptide-based analgesics like endomorphins for the effective management of neuropathic pain.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012  in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged .Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond. Light-Emitting Diode Fluorescence Microscopy (LED-FM) for sputum smear examination is recommended by the World Health Organization (WHO) for detection of acid-fast bacilli in high tuberculosis (TB) burden countries In India (high TB burden country), The performance of LED-FM in improving yield of sputum smear positive TB cases has not been evaluated under routine programmatic conditions in India and elsewhere in the world. In this study, we assessed whether implementation of LED-FM services in these settings was operationally feasible and led to increased yield of smear positive TB among presumptive TB patients  who underwent sputum smear examination for diagnosis of pulmonary TB.This was a before-and-after study designed to evaluate the impact of introduction and implementation of LED-FM services in detecting sputum smear positive TB cases in medical college DMCs under RNTCP, India.There are over 350 medical colleges across India and each of these is associated with tertiary care teaching hospitals. Most of these medical colleges collaborate with RNTCP and have a designated microscopy centre (DMC) for the diagnosis of sputum smear positive pulmonary TB cases and a facility for treatment of TB patients In 2012, ZN SSM was replaced by Auramine O based LED-FM in 200 medical colleges RNTCP DMCs. The training module of RNTCP for fluorescence microscopy was revised by a technical working group consisting members from National TB Reference Laboratories (NRL) and Intermediate TB Reference Laboratories (IRL), Central TB Division (CTD), WHO, FIND, and The Union and was approved by CTD. Based on this module LTs and STLSs were trained for 3 days and 4 days respectively at NRLs and IRL. The training consisted of modular readings, interactive lectures, discussions and hands-on training. The trainees were only certified after they successfully read the panel slides of different grades and scored 70% in the post test based on MCQ’s.)In these DMCs, each PTP undergoes two sputum smear examinations including one ‘spot’ and a second ‘early-morning’ sputum specimens External quality assurance (EQA) protocol for sputum smear microscopy as specified by the RNTCP for ZN microscopy and LED-FM microscopy was in place in all these DMCs. The EQA guidelines related to LED-FM was circulated to the districts implementing LED-FM in medical colleges RNTCP DMC. Onsite evaluation of microscopy (ZN SSM and LED-FM) is routinely carried by STLS on monthly basis. Five positive and five negative slides are checked between two visits of the STLS by a systematic random un-blinded sampling. They are also advised to read the doubtful slides. The LTs were also advised to read the doubtful slides in higher magnification i.e. 1000X for confirmation. The random blinded rechecking (RBRC) is also carried at district level on monthly basis for ZN SSM The target population included persons with presumptive TB referred for diagnostic sputum smear examination at the above described DMCs. The results of sputum examinations of persons with presumptive TB attending these DMCs between July to December 2011 using ZN SSM (2011/ZN) were compared with those results between July to December 2012 from LED FM (2012/FM).In order to assess whether the increase in the proportion of sputum smear positive TB cases were at-least 10% from the baseline (assuming it to be 10% before the introduction of LED-FM services) with 80% power and 95% confidence interval, a design effect of 2, a sample size of 14,950 persons with presumptive TB were required from the 2011 and 2012 periods. As ∼2000 patients with presumptive TB undergo sputum smear examination per DMC per year, in order to achieve the sample size a minimum of 15 DMCs were required. The 15 DMCs were selected randomly by population proportion to size sampling. For each of these 15 DMCs, another DMC within the same district, with comparable workload and implementing ZNSSM services throughout the period was chosen to act as a control group. For this group, ZN SSM results during intervention period (2012/ZN) were compared with ZN SSM results collected during baseline period (2011/ZN).The study variables included age, sex and results of each of the sputum samples. Data was extracted from the RNTCP Laboratory Registers maintained at the selected facilities.The data was double-entered into electronic format created in EpiData  which were further validated and analysed.The data was cross-tabulated, comparing the period prior and after introduction of LED-FM. Statistical significance of the result was obtained from Pearson’s Chi Square test with Yates correction and P≤0.05 was considered statistically significant.Ethics approval for the study was obtained from Ethics Advisory Group of International Union Against TB and Lung Disease. Administrative approval to access the records was obtained from Central TB Division, Ministry of Health and Family Welfare, Government of India. This study was record review of laboratory registers and data was analyzed anonymously. The informed consent of the patients was also waived by EAG for this study.A total of 25,685 and 27,030 TB symptomatics underwent sputum smear examination in the selected 15 medical colleges during the baseline period (2011/ZN) and intervention period (2012/FM) respectively. Interestingly nearly two-thirds were males. The median age (interquartile range) was 45 (30–60) years for 2011/ZN and median age (interquartile range) was 45 (29–59) years for 2012/FM. The patient population before and after LED-FM implementation was similar by age and sex. Total of 25,159 (2011/ZN) and 26,426 (2012/FM)patients with presumptive TB were available for analysis after excluding the patient with presumptive TB who did not submit either spot or early morning sputum in 2011/ZN  and 2012/FM  Table 2.The proportion of sputum smear positive increased from 13.6% (2011/ZN) to 17.8% (2012/FM)  after implementation of LED-FM microscopy. The percentage change in the positivity rate was 30% Table 3.The increase in proportion of sputum smear positive in the sites that implemented LED-FM services was observed across both sexes and all age sub-groups Table 3.Our study is the first to show the successful implementation of LED-FM AO staining services in routine programmatic conditions in India. We found that the introduction of LED-FM services in medical college DMCs resulted in an increase in the yield of pulmonary sputum smear positive pulmonary TB patients. Similar findings have been reported from peripheral laboratories of China Previous studies, which were specifically-designed in research settings, showed an increase of 8–10% in the proportion sputum positive patients with LED fluorescence microscopy. These studies had shown that the increase in yield in positivity was predominantly among those with scanty gradeThere has been a concern expressed in the past about higher chance of false-positive smears  with LED-FM and the suggestion that all scanty and doubtful cases be confirmed by ZN SSM Sputum smear fluorescence microscopy in Senegal revealed that 5.8% of sputum smear positive follow up cases had scanty grades in comparison to new cases (2.5%) We did not collect information on the number of smear positive TB cases initiated on treatment. This would have been helpful to ascertain if increased number of cases detected actually translated into increased number of cases initiated on treatment. Further, our study evaluated the performance LED-FM services in tertiary care teaching hospitals. There is need for further assessment of performance in other more peripheral microscopy centres to assess the overall expected impact of roll-out of LED-FM services on case notification rates.This study was limited by the fact that the data were extracted from routine records and we were unable to control the quality of data recording. As an operational research study, it lacked the rigour and internal validity of an intervention trial. The patient populations compared might have had important differences which might be partially responsible for the difference in results. Measuring all the patient characteristics was beyond the scope of the current study and while we are cognizant of this limitation, we note the groups were similar by age and sex and feel the random selection of sites would have mitigated any differences. We evaluated routine practice and have no information concerning skills and knowledge of those undertaking the sputum smear examinations and if they varied between the two time periods. Further studies assessing cost-effectiveness of LED-FM in comparison to ZN microscopy are in progress and will be reported separately.The implementation of LED-FM in high work load medical college DMCs increased the detection of smear positive cases. Our study has confirmed the advantage of LED fluorescence microscopy in routine practice in busy laboratories, previously shown in controlled research environments. This provides valuable operational evidence needed for scale-up of this technology in similar settings in India and beyond.


Investigating the steadiness of the phase-coupling between the time-course of the reader's voice and brain signals of subjects with autism spectrum disorder (ASD) passively listening to connected speech using magnetoencephalography (MEG). In typically developed subjects, such coupling occurs at the right posterior temporal sulcus (pSTS) for frequencies below 1 Hz, and reflects the neural processing of sentence-level rhythmic prosody at the prelexical level.Live) or recorded (Recorded) voices continuously reading a text in French for five minutes. Coherence was computed between the reader's voice time-course and ASD subjects' MEG signals. Coherent neural sources were subsequently reconstructed using a beamformer.Cortical neuromagnetic signals were recorded with MEG  while seven right-handed and native French-speaking ASD subjects  listened to live  included in a previous study.Significant coupling was found at 0.5 Hz in all ASD subjects in This study discloses a preserved coupling between the reader's voice and ASD subjects' cortical activity at the right pSTS. These findings support the existence of preserved neural processing of sentence-level rhythmic prosody in ASD. The preservation of early cortical processing of prosodic elements in verbal language might be exploited in therapeutic interventions in ASD. Autism spectrum disorders (ASD) represent an heterogeneous group of neurodevelopmental disorders of variable severity characterized by clinical features that can be classified into two main groups: social/communication disorders and narrow interest/stereotyped-repetitive behaviors , so that the spectrotemporal information is concurrently extracted within separate neural streams for speech perception and then integrated in brain regions involved in subsequent lexical computations for speech comprehension  and the CVC approach. We predict that pSTS dysfunction in ASD might be reflected in a weaker coupling in ASD subjects, or that the location of coherent brain areas will differ from that observed in typically developed subjects. Conversely, if coupling is unaffected in ASD, it might represent a theoretical support for therapeutic interventions based on low-frequency prosodic content of language to foster social communication in this disorder.ASD subjects' clinical details are summarized in Test de Langage Oral Complexe pour Collégiens (TLOCC) Seven right-handed and native French-speaking subjects  diagnosed with Asperger Syndrome (2 patients) or autistic disorder (5 patients) were included in the study. The handedness was assessed with the Edinburgh handedness Inventory The study had the prior approval by the ULB-Hôpital Erasme Ethics Committee. The ASD subjects and their parents gave written informed consent before participating to the study.Data obtained in the ten right-handed and native French-speaking typically developed healthy adult subjects  included in the seminal CVC study Live) or recorded (Recorded) voices continuously reading a text in French during 5 min. Both texts were chosen for their emotional neutrality in order to reduce the effect of affective prosody, and for the absence of any dialog in order to minimize prosody modulations. As in the seminal study, the first condition (Live) consisted in the passive listening of a text (http://www.eodi.org/la_revolution_francaise.html) read by a native French-speaking male . Subjects were asked to fixate the gaze at a point in the MSR to avoid any gaze contact with the reader. In a second condition (Recorded), subjects passively listened to a recording of a native French-speaking female reading a French text (http://www.litteratureaudio.com/livre-audio-gratuit-mp3/zola-emile-la-terre.html) during 5 min. This condition was designed to test the influence of human interactional factors  on the CVC phenomenon. A loudspeaker  was positioned 2 m in front of the subjects inside the MSR. Signals of the recorded voice were recorded time-locked to MEG signals. In a third 5-min session (Rest), subjects were asked, as in the two previous conditions, to relax, fixate the gaze at a point in the MSR and not to move. The three experimental conditions  were randomized across subjects.The experimental paradigms used in this study were adapted from The methods used for data acquisition, preprocessing, and analyses have been extensively detailed in MEG signals were recorded at the ULB-Hôpital Erasme with a whole-scalp-covering neuromagnetometer . Head position inside the MEG helmet was continuously monitored using four head-tracking coils. The locations of the coils and at least 150 head-surface  points with respect to anatomical fiducials were determined with an electromagnetic tracker . The recording passband was 0.1–330 Hz for MEG signals and 0–330 Hz for accelerometer and recorded voice signals; all signals were sampled at 1 kHz.Live were preprocessed off-line using the spatio-temporal signal-space-separation  to subtract system artefacts. Then, for frequency and coherence analyses, 2048 ms epochs were extracted with 1638 ms overlap, leading to a frequency resolution of 0.5 Hz. Epochs where MEG signals exceeded 3 pT (magnetometers) or 0.7 pT/cm (gradiometers) were rejected to avoid contamination of the data by eye movements, muscle activity, or artefacts of the MEG sensors. This yielded more than 590 artifact-free epochs for each subject and condition.Continuous MEG data were preprocessed off-line using the signal-space-separation method to suppress external interferences and to correct for head movements Live; 130–300 Hz, Recorded) and rectified in order to compute for each epoch the coherence between the f0 time-course and MEG signals. In both conditions, the analyses were based on the voice fundamental frequency (f0) as this parameter is a highly relevant feature for speech processing  template. Notwithstanding the risk of decrease in spatial accuracy, the MNI template was used rather than individual structural magnetic resonance imaging (MRI) in order to facilitate ASD subjects' recruitment as some of them refused to undergo MRI. For each subject, MEG and segmented MNI coordinate systems were coregistered using the three anatomical fiducial points for initial estimation and the head-surface points to manually refine the surface coregistration. The MEG forward model was then computed using the MNE suite . Cortical sources coherent with the f0 time-course were identified using dynamic imaging of coherent sources (DICS) f-mean across subjects of normalized maps, according to Finally, to produce coherence maps at the group level, we computed the generalized The statistical significance of sensor-space individual-level coherence was assessed using surrogate data-based statistics, based on coherence computed between surrogate f0 time-courses and original MEG data The statistical significance of source-space group-level coherence was assessed using a non-parametric permutation test based on coherence between vocal signals and rest condition MEG data Non-parametric Wilcoxon and Mann-Whitney test were used to assess the difference between conditions and between ASD and typically developped subjects from the seminal CVC study Live , nor between ASD subjects and the typically developed healthy adult subjects investigated in the seminal CVC study .In line with the seminal CVC study carried out in a group of healthy adult subjects No other peak in coherence was found at the sensor level, and particularly at 4–6 Hz. The 0.5-Hz coherent frequency was therefore considered as the only frequency of interest to compute individual and group-level coherence maps.p<0.05) in Live and at the middle temporal gyrus (MTG)  in Recorded. Considering the limited spatial resolution afforded by the coregistration procedure used in this study, these coherence local maxima can be considered very close to the one reported in the seminal CVC study  Group-level analysis revealed maximum coherence at the right STS .Correlation analyses did not disclose any association between sensor-space coherence levels and age or behavioural scores . Indeed, it has been proposed that the temporality of the sensory stimulations, apart from their multimodal character, plays a key role in the pathophysiological basis of some behavioural impairments characterizing ASD Finally, a functional MRI study investigating the neural basis of prosodic speech deficits  in subjects with high functioning autism failed to find any clear deficit in pSTS recruitment compared with typically developed healthy subjects Importantly, the preservation of early cortical processing of prosodic elements in verbal language might potentially be exploited in therapeutic interventions in ASD. An anecdotic report already suggests in 1979 that the stress put on the melodic content of language has been helpful in the language therapy of an autistic boy Recorded) obtained significant sensor-space coherence levels with values similar to those observed in the seminal CVC study, we consider that the conclusions of this study are not affected by this limitation.First, ASD subjects were compared with the typically developed healthy subjects investigated in the seminal CVC study Second, the use of the MNI template for the source space coherence analyses in ASD subjects limits the accuracy of the coherent source localization  Finally, we did not test behaviourally the rhythmic prosody perception skills of our ASD subjects, which limits the interpretation of the functional relevance of the preserved CVC phenomenon in this population. Indeed, even if our result highly suggest a normal neural processing of the suprasegmental speech rhythmic prosody at the prelexical level, we cannot determine if this preserved coupling gives rise to normal prosody perception at the behavioural level. The ASD subjects included in this study had weak to clear expressive prosodic disorder. This highlights the importance to investigate in future studies the correlation between CVC levels and prosody perception skills.This study discloses a preserved coupling between the reader's voice and the listener's cortical activity in ASD subjects at the right pSTS. These findings support the existence of preserved neural processing of sentence-level rhythmic prosody in ASD. The preservation of early cortical processing of prosodic elements in verbal language might be exploited in therapeutic interventions in ASD.


We describe a scar-related reentrant ventricular tachycardia circuit with a proximal segment in an endocardial basal septal scar and an exit in a region of slow conduction in a non-overlapping region of epicardial basal lateral scar. The 12-lead EKG demonstrates criteria for a basal lateral epicardial VT, however the same morphology could be produced with a longer stim-latency with pace mapping or VT induction from the endocardial septal region of scar. A significant segment of myocardium demonstrated no endocardial or epicardial scar on electroanatomic mapping, suggesting the presence of a mid-myocardial isthmus. Further evidence was provided by assessment of unipolar settings. The epicardial VT that initially appeared to originate from the basal lateral epicardial region, was successfully treated with radiofrequency ablation of the lateral aspect of the endocardial septal scar. We present a 46 year-old man with a history of non-ischemic cardiomyopathy (CM), likely of familial origin, status-post implantable cardioverter-defibrillator (ICD) implantation who was transferred to our center after presenting with ventricular tachycardia (VT) storm and recurrent ICD shocks for monomorphic VT, with a rate between 165-175 beats-per-minute, despite therapy with antiarrhythmic medications.The patient underwent endocardial and epicardial mapping as is the protocol at our institution for any patient with a non-ischemic cause of their CM. Endocardial and epicardial electroanatomic maps (EAM) were obtained with a Carto 3 system . Monomorphic VT was induced with double extrastimuli testing. Standard 12-lead EKG morphology demonstrated a VT with a right bundle branch block pattern, V5 transition and inferior axis at a rate of 170 beats-per-minute . The EKGAn excellent pace-map could be produced from the basal lateral epicardial surface  with a sInterestingly, at a late potential site  within tRadiofrequency ablation of the lateral aspect of the septal endocardial scar at sites of late and fractionated potentials was undertaken. After ablation of this region the clinical tachycardia was non-inducible. The patient was free of VT at greater than 9 months follow-up off of antiarrhythmic medications.The above findings demonstrate that the VT exit site was the basal lateral epicardium. The findings of no corresponding endocardial basal lateral scar with endocardial septal pace-maps that were matches to the epicardial pace-maps at a lateral site, in combination with a VT that could be induced from the endocardial septal region with a PMI suggests a mid-myocardial connection between the two scar regions that was not adequately visualized with standard EAM system settings.This is the first description in the literature we are aware of demonstrating a VT circuit that likely required a bridging mid-myocardial segment for non-overlapping regions of scar on the endocardium and epicardium. While current practice and available mapping techniques lead electrophysiologists to label VT as having an endocardial or epicardial origin, myocardial scar formation does not follow an either/or pattern. Rather many, if not most, myocardial scars that predispose to reentry likely involve a complex three-dimensional substrate that may include the endocardium, mid-myocadium and epicardium. This finding has been reported in a canine model . HoweverAnter and colleages  reportedThese early studies and this case raises the importance of a complete electroanatomic map with pacemapping used as an adjunct technique for scar-related reentry, especially in a situation where the initially proposed exit site of the tachycardia demonstrates no late potential activity in a hemodynamically untolerated VT.Lastly, this case illustrates that 12-lead morphology is only predictive of the exit site of a reentrant circuit. There are likely many VT morphologies that indicate the need for epicardial mapping and ablation with subendocardial or intramural isthmuses and vice versa.The use of pace-mapping and not entrainment mapping is a limitation of this manuscript. Stim-QRS latency during pacing is not necessarily synonymous with Stim-QRS during entrainment. However, the authors interpret the longer stim-QRS latency to be similarly indicative of progressive distance from the exit site and evidence that in this case there was a single preferential exit site toward the lateral epicardium.


Bacillus thuringiensis (Bt) has evolved in several insect species through the modification of insect midgut binding sites for Bt toxins, and reports of costs associated with Bt resistance are common. Studies on the costs of Bt-resistance restrict the insect to a single artificial diet or host-plant. However, it is well documented that insects can self-select appropriate proportions of multiple nutritionally unbalanced foods to optimize life-history traits. Therefore, we examined whether Bt-resistant and susceptible cabbage loopers Trichoplusia ni differed in their nutrient intake and fitness costs when they were allowed to compose their own protein:carbohydrate diet. We found that Bt-resistant T. ni composed a higher ratio of protein to carbohydrate than susceptible T. ni. Bt-resistant males exhibited no fitness cost, while the fitness cost  was present in resistant females. The absence of the fitness cost in resistant males was associated with increased carbohydrate consumption compared to females. We demonstrate a sex difference in a fitness cost and a new behavioural outcome associated with Bt resistance.Evolved resistance to xenobiotics and parasites is often associated with fitness costs when the selection pressure is absent. Resistance to the widely used microbial insecticide  Bacillus thuringiensis (or its Cry toxins expressed in plants) has resulted in the evolution of resistance Bt are well-documented and primarily relate to reductions in survival, fecundity, and mass and increases in development time in comparison to unselected susceptible insects The evolution of resistance to pathogens, parasites and chemical insecticides is often accompanied by negative pleiotropic effects in the absence of the selection pressure Bt Cry toxins, necessary for the toxins to cause death Bt resistance is likely to be influenced by diet. This has been shown to be the case, with the costs of Bt resistance being exacerbated on lower quality or better-defended diets or host plants The most common genetic resistance mechanism is the modification or loss of midgut binding proteins (cadherin or aminopeptidase N) for Bt relies on the use of non-Bt refuges Bt resistance and their underlying mechanisms were better understood, this knowledge could potentially be used to enhance resistance management strategies. Most studies of fitness costs associated with Bt resistance restrict the insect to a single artificial diet or host-plant. However, in nature, most herbivorous insects have access to foods that vary in nutritional quality, and they can self-compose their diet from multiple, nutritionally unbalanced foods to optimize life-history traits Bt was likely to alter nutrient intake and that resistant insects may be able to compensate for fitness costs when given a choice of diets. We used the Geometric Framework for nutrition to test our hypotheses by comparing Bt-resistant and Bt-susceptible lines of cabbage loopers, Trichoplusia ni (Hübner). In this approach it is possible to quantify how an organism regulates the intake of two or more food components at the same time using a graphical model T. ni has developed resistance to Bt as a result of the frequent applications in greenhouses Bt-resistant T. ni have well-documented, context-dependent costs, including reduced survival and pupal weight, and increased development time A key tactic in managing the evolution of resistance to Bt-resistant T.ni colony was originally collected from a commercial tomato greenhouse in British Columbia, Canada in 2001 Bt-resistant strain used in the present study was collected during the same year and from a greenhouse close to the source of the Cry1Ac resistant strain used to determine alterations in aminopeptidase Ns  every generation to maintain resistance. The Bt-susceptible colony is a revertant line obtained by not exposing the resistant line to BtBt-resistant insects used here were not selected with Bt for one generation (Bt-RU) to avoid possible transgenerational effects Bt than Bt-S at the time of the experiment.The Experimental larvae were reared on the wheat-germ based diet (colony diet) from egg-hatch until exposure to the wheat-germ-free, nutritionally-defined treatment diets. ‘Treatment diets’ consisted of altering the ratios of protein (casein) and digestible carbohydrate (sucrose) that made up 60% of the dry weight in the following proportions (% protein: % carbohydrate): 50∶10, 30∶30, 20∶40, 10∶50. Other ingredients include Wesson’s salt (5%), cholesterol (1.5%), ascorbic acid (1%), sorbic acid (0.5%), sodium alginate (2.5%), sucrose-free Vanderzant vitamin mix (3.5%), wheat-germ oil (1%) and cellulose (25%) . Diets were suspended in 1.35% agar solution in a 5∶1 agar solution:dry diet ratio. The wheat-germ based ‘colony diet’ also consisted of approximately 60% protein and carbohydrate, and had an approximate protein to digestible carbohydrate ratio of 1 to 1.1.Freshly moulted final (fifth) instar Bt-S and Bt-RU larvae were weighed and individually provided with two pre-weighed nutritionally suboptimal but complementary diet blocks, and were allowed to self-compose their diet. Three choice experiments were performed to examine the consistency of self-selected macronutrient ratios, with a choice between the highest P:C ratio diet block (50∶10) and one of the carbohydrate-biased or equal ratio diet blocks (i) 50∶10 & 30∶30, ii) 50∶10 & 20∶40, and iii) 50∶10 & 10∶50). A total of 72, 75, and 79 larvae were used for each choice experiment, respectively. The diet was replaced each day until pupation; any diet remaining was collected, dried at 50°C for 24 hrs until constant mass, and then weighed. Daily consumption was estimated by calculating the difference between the dry initial and final remaining mass of the diet blocks. The dry initial mass of the diet blocks was estimated by constructing a regression equation with pre-weighed diet blocks without larvae. All larvae successfully pupated, and the date of pupation was recorded. Three days after pupal initiation, pupae were sexed and then dried at 50°C for 48 hrs until constant mass and then weighed.Differences in the cumulative bivariate consumption of protein and carbohydrate by day between Bt-RU and Bt-S were analysed by multiple analysis of covariance (MANCOVA) using Pillai’s trace statistic. Sex and choice experiment were included as fixed effects and initial larval weight was included as a covariate. Univariate analyses of protein and carbohydrate intake were obtained from post hoc analyses as part of the bivariate MANCOVA. Analysis of covariance (ANCOVA) for pupal weight was performed separately. Pupal weight was squared before analysis to normalize the data. Tukey HSD comparison was used to compare means among treatments. Time to pupation was analyzed using accelerated failure-time analysis using a Weibull distribution. For all analyses, all factors and their interactions were fitted initially in the model and non-significant interactions were removed sequentially to produce the final minimal model. SAS 9.3 was used for all analyses . The dataset has been made available as supporting information (Dataset S1).When provided with two nutritionally suboptimal but complementary diet blocks, the intake target of Bt-S larvae was more carbohydrate-rich than Bt-RU larvae ; Table 1This prompted an examination of the univariate ANCOVA results for protein and carbohydrate intake separately. Protein consumption showed a significant three-way interaction that is likely due to differences in the direction of the effects among choice experiments. However, there were no significant differences when compared by Tukey HSD . There wHigher carbohydrate consumption by Bt-RU males was associated with heavier pupal mass, such that they weighed the same as Bt-S pupae . Broadly, this is in line with other Lepidoptera, in that protein intake is either equal to or greater than carbohydrate ingestion Allowing susceptible and resistant Bt susceptibility in Bt-resistant T. niT. ni males mate multiple times and it is hypothesized that increased weight improves mating success and frequency Bt susceptibility has been found in T. niBt-challenge, and thus ingest more protein, whereas males are selected for mating success and frequency. Virally-infected caterpillars have shown similar behaviour to improve their likelihood of surviving infection by reducing carbohydrate consumption while maintaining protein consumption Bt tolerance has been shown to be transferred to the next generation by maternal effects Bt-RU females weighed less than the susceptible pupae of both sexes, whereas resistant males weighed the same. However, male Bt-RU achieved a larger pupal weight at the cost of longer development time. Male pupal weight is weakly heritable and positively correlated with Bt. Studies with more lines of Bt-resistant T.ni and other species are needed to confirm the generality of this finding. Investigation of precise mechanisms are beyond the scope of this paper but altered nutrient intake might result from changes in the feedback mechanism that provides information about the nutrient content of food and chemical composition of the haemolymph T. ni. Whether the results from this study hold in situations where other protein and carbohydrate sources are available or on plants differing in nutritional quality warrant further investigation.Given that Bt-RU and Bt-S are back-crossed to maintain genetic similarity and have been continuously reared on the same diet, these differences in feeding behaviour are consistent with an association with resistance to T. ni larvae will self-compose a specific balance of nutrients, and that the evolution of pathogen resistance can change feeding behaviour to alter the nutrient intake target. Consumption of a higher ratio of protein by Bt-RU suggests that they may seek out plant parts higher in protein, adding to their destructive potential as pests by preferentially consuming the reproductive and younger parts of plants that are protein rich. Alternatively, such behaviour might enhance refuge-based resistance management strategies if these plant parts contain higher levels of defensive phytochemicals. Understanding the behavioural and physiological outcomes of resistance selection is important in predicting the destructive potential of resistant insects and the success of resistance management strategies.Herbivorous insects have access to a variety of plants and plant parts that vary in nutritional content. Our experiments demonstrate that Figure S1Variability of protein intake. Least squares means of the three-way interaction between colony, sex, and choice experiment on (DOCX)Click here for additional data file.Dataset S1Data of nutrient intake and development measures.(XLS)Click here for additional data file.