Bacilli directly target response regulators of bacterial two-component systems and modulate their activity. Their effects are controlled by binding of signaling peptides to an allosteric site. Hence Raps exemplify a class of monomeric signaling receptors, which we call switchable allosteric modulator proteins (SAMPs). These proteins have potential applications in diverse biomedical and biotechnical settings, but a quantitative understanding of the impact of molecular and cellular factors on signal transduction is lacking. Here we introduce mathematical models that elucidate how signals are propagated though the network upon receptor stimulation and control the level of active response regulator.Rap proteins from 50 are controlled in addition by the relative ligand affinities. By tuning receptor properties, either graded or more switch-like (memory-less) response functions can be fashioned. Furthermore, we show that other contextual factors (e.g. relative concentrations of network components and kinase activity) have a substantial impact on the response, and we predict that there exists a modulator concentration which is optimal for response amplitude.Based on a systematic parameter analysis of the models, we show that key features of the dose-response behavior at steady state are controlled either by the molecular properties of the modulator or the signaling context. In particular, we find that the biochemical activity (i.e. non-enzymatic vs. enzymatic) and allosteric properties of the modulator control the response amplitude. The Hill coefficient and the ECB. subtilis to show how our models can contribute to an integrated view of SAMP signaling by combining biochemical, structural and physiological insights. Our results also suggest that SAMPs could be evolved or engineered to implement diverse response behaviors. However—without additional regulatory controls—they can generate rather variable cellular outputs.We discuss data on Rap-Phr systems in The online version of this article (doi:10.1186/s12918-016-0274-3) contains supplementary material, which is available to authorized users. For almost all forms of life on Earth, the ability to process environmental information is vital for adaptation and survival. For cellular organisms such as bacteria, this function is carried out by biochemical signaling systems. Two-component systems, each consisting of a sensor histidine kinase and a response regulator, are a common form of bacterial signal transduction. The histidine kinase responds to the presence of specific signals that modulate its auto-phosphorylation activity and transfers the phosphoryl group to the response regulator, which in most cases activates or represses gene expression in accordance with its phosphorylation status . In addiBacillus are one of the best characterized classes of modulator proteins, and are encoded not only in the chromosomal genome  and modulator [MT]. Class II parameters may be actively controlled by the cell by altering gene expression depending on environmental conditions, and they might also show considerable variability from cell to cell—due to gene expression noise.To quantitatively analyze the system response upon receptor stimulation it is useful to classify the model’s parameters into two categories. Class I comprises parameters that describe the molecular properties of the modulator, while Class II comprises all other parameters which determine the “signaling context” or the “state” of the signaling system, such as the total concentrations of regulator :Finally, the Class I and Class II parameters introduced above are evaluated relative to the absolute dissociation constant Like any binding modulator, an enzymatic modulator first binds the active regulator and enzymatically inactivates it in a second, separate step. In this case, there are two allosteric mechanisms that derive from the molecular properties of the modulator. As with the binding modulator there is a mutual allosteric influence of the binding affinities. In addition, the enzymatic activity of the modulator may be subject to allosteric regulation, i.e. the signal and the regulator can in principle stimulate or inhibit the enzymatic activity. As a result, the allosteric properties of the modulator protein may be described by two parameters, one describing the sign and strength of binding cooperativity and the other capturing the sign and strength of enzymatic cooperativity Fig. .Fig. 3Alγ , [ST] and [MT] to be constant.Here we also assume the total concentrations of  of the free regulator. Therefore, for systems with positive cooperativity, the signal acts as a repressor, i.e. the signal is an allosteric agonist.As seen from Eq.μ. If the modulator concentration is too low the regulator will be free, irrespective of the presence of the signal. On the other hand, if the modulator concentration is too high, the regulator will always be associated with the modulator. In the intermediate concentration regime the signal can free the regulator from the regulator-modulator complex when bound to a modulator with negative cooperativity or, conversely, can trap the regulator in the SMR complex by interacting with a modulator with positive cooperativity. In order to maximize the output dynamic range the  modulator and the regulator concentration must fulfil the following relationship:Notably, for a given modulator protein, the response amplitude Eq., as plotK1* K5), the more modulator is required. Thus the signaling context, as quantified by μ, is a key parameter that will affect the ability of the signaling system to transfer information. Moreover, the Hill coefficient h can also be altered by varying μ. For Rap-like modulator proteins with negative cooperativity, the Hill coefficient increases as a function of μ and, at the same time, the EC50 shifts to higher input levels. Indeed, μ can even shift the response from a graded (h ≤ 1) to an ultrasensitive induction (h > 1). For modulators with positive α, the model predicts even more complex dependencies on μ . Hence the system always operates under conditions which are favorable for information transfer, which allows us to focus on the effects of Class I parameters.To systematically analyze how the physicochemical properties of a SAMP can affect signal transduction, we assumed that the concentration of each modulator is set at the level that optimizes the response amplitude , i.e. a high dissociation constant K1 or a small total regulator concentrations [RT] will tend to broaden output dynamic range.and is plotted in Fig. h as a function of α and β to gain insight into how the properties of the modulator protein shape the response function  exhibit ultrasensitive response curves, while SAMPs that display strong cooperativity but preferentially bind to the response regulator (large β) tend to show graded responses. Note that, since our model does not support bistable outputs, switching always occurs without memory.In general, there are several ways in which a cellular response can be induced as a function of the signaling input. For example, for certain two-component systems the output can be modulated in a graded fashion while others display switch-like properties, either with or without hysteresis, depending on their exact architecture –46. We tion Fig. . We find. For Rap-like binding modulators with a more switch-like response characteristic, strong cooperative interactions not only increase the response amplitude and the Hill coefficient of the response curve, but also shift the threshold concentration required to trigger the switch to higher signal levels. This is evidenced by rising EC50 levels with increasing α. On the other hand, for modulators that implement graded responses, the EC50 can be tuned by varying β as well as α , we were able to derive the fairly compact mathematical expression:A again reflects the output dynamic range, which is therefore a complicated function of various Class I and Class II parameters.A derivation of Eq.  is givenA > as a function of the cooperativity parameters 4 different parameter sets for the Class II parameters  as a measure for the sensitivity of the qualitative response to changes in the signaling context. The results clearly demonstrate that coherent SAMPs show a robust qualitative response (H = 0), signals act as agonist or inverse agonist, respectively and the response amplitude tends to increase as the strength of the cooperative effects increases. In contrast, incoherent SAMPs may switch their qualitative behavior depending on the signaling context (H > 0), and show smaller average signal amplitudes  results in more switch-like behavior. Moreover, the EC50 shifts with increasing β to higher levels  use the canonical start codon, translation of many Rap phosphatases  is attenuated by the use of a non-optimal start codon. In addition, in order to retain sensitivity to the signal, enzymatic SAMPs would have to adjust their levels as the kinase activity changes. This would also shift the EC50 levels. This might at least partially explain why the transcription of Rap phosphatase genes  is so tightly regulated  with the otherwise indicated parameters using the ode15s solver. To verify the steady-state condition the fsolve function was used. The response amplitude was compared to the explicit mathematical expressions given by Eq. . To derive the steady state dose-response curve the models were numerically integrated to steady state at a fixed level of the total amount of signal  that results in 50 % of the normalized response. At this concentration the elasticity ε of the normalized curve was calculated numerically from Eq. (The escribed . In briefrom Eq. . FinallyNot applicable.Not applicable.All data supporting our findings is contained within this article.


Despite various strategies that aim at persuading relatives to permit donation, the family veto of donation still constitutes a significant impediment to respecting potential donors’ wishes to have their deaths benefit others . PersonaPODDs offer several advantages to those wishing to donate their organs after death. The most important facet is their personalised nature. Rather than being shown a tattered organ donor card by a nurse, or simply being told that their relative was on the register, or faintly half-remembering a decades-old conversation, family members will vividly remember a specific video statement that they received; if they have forgotten it, they can be shown it again. Furthermore, PODDs are much more contemporaneous than alternative methods of stating organ intentions: if relatives question whether any stated intentions are current, they can be shown the annual reconfirmations and any updates to the PODD. Additionally, if any relatives are still reluctant, other relatives and friends who are perhaps more up-to-date with the donor’s intentions will have their resolve to prevent use of a veto strengthened by the PODD.Ideally, access to PODDs could be integrated into national donor registers, with a link from the register to the PODD document and video(s), so that doctors can facilitate family access to PODDs for those who do not use email or when disputes arise. Another advantage of PODDs is that they can be used to stop any family disagreements about what the deceased would have wanted. Essentially, PODDs are extremely powerful persuasive tools that virtually guarantee that families will not veto donation by addressing the key reasons given by families for refusing donation . Widespr1. Go to PODD website.2. Fill in organ directive form.3. Record short video.4. Enter email addresses of recipients.5. Choose reconfirmation frequency.6. Sign and submit.PODD: Personalized organ donation directive.The author declares that he has no competing interests.


Automatic summarization generation of sports video content has been object of great interest for many years. Although semantic descriptions techniques have been proposed, many of the approaches still rely on low-level video descriptors that render quite limited results due to the complexity of the problem and to the low capability of the descriptors to represent semantic content. In this paper, a new approach for automatic highlights summarization generation of soccer videos using audio-visual descriptors is presented. The approach is based on the segmentation of the video sequence into shots that will be further analyzed to determine its relevance and interest. Of special interest in the approach is the use of the audio information that provides additional robustness to the overall performance of the summarization system. For every video shot a set of low and mid level audio-visual descriptors are computed and lately adequately combined in order to obtain different relevance measures based on empirical knowledge rules. The final summary is generated by selecting those shots with highest interest according to the specifications of the user and the results of relevance measures. A variety of results are presented with real soccer video sequences that prove the validity of the approach. This tremendous explosion in visual content and the myriad of applications that have arisen, has spurred the need to develop advanced tools for content-based image and video indexing and retrieval  that represent the shot. An example of a particular analysis tool is the person’s detector.Low-level audio MPEG-7 descriptors for every audio frame.The motion descriptors (Motion Activity and Camera Motion) associated to the shot.Detection of Zoom-in and Zoom-outs in the analyzed video shot.A list of key-frames that represent the video shot.Color Layout, Color Structure and Dominant Color MPEG-7 descriptors associated to every key-frame in the shot.Faces detected in the key-frames. Percentage of skin pixels in a key-frame.Long shots detected in the key-frames.Replays detected in the broadcasted video feed time codes.Referee’s whistle time codes.The information collected from every shot include:The details of the algorithms used in each module are explained in “The final stage of the system is the highlights summary generator module, that is composed of a filter bank that scores each shot with a set of elementary and advanced filters and the shot selection stage that generates the final summary by assessing the previous scores and a set of user parameters. The elementary filters find events of interest in the video shot. For example, one elementary filter can be the audio power increase inside the shot or the detection of the referee’s whistle. Then, elementary filters are combined together in order to create a set of advanced filters that will give a final score to every shot. The objective is to assign positive or negative weights to each elementary filter in order to obtain the final score assigned to every shot. These filters are flexible enough to let the user interact with the system, specifying the relative importance of each elementary filter or, alternatively, to be statistically trained by a linear classifier. The user may define the number of advanced filters, their structure, the weighting of elementary filters, the percentage of every advanced filter included in the summary and the summary duration. The highlights summary generator module is presented in detail in “Groundtruth A: Three matches from 1st division and two matches from 2nd division.Groundtruth B: Three matches from 2nd division and three matches from 3rd division.Groundtruth C: One match from 1st division, one match from 2nd division and one match from 3rd division.In order to evaluate the performance of all algorithms of the proposed approach and the overall automatic highlights summary generator, three different video groundtruths have been generated. Each groundtruth contains the following soccer matches:These video sequences have been produced by the Catalan television (TVC) and they belong to the Spanish soccer league. Each match lasts approximately 2 h and is in MPEG-4 Simple Profile format, has a resolution of 360 × 288 and a GOP structure IPPPPPPP. All the results presented in the sequel have been obtained using these video groundtruths.This section explains the techniques used to analyze the video sequences. Each approach is introduced briefly and the interested reader is addressed to the references for further details. It has to be noted that a complete study and comparison of the state of the art of each technique presented below is out of the scope of the paper. However, when possible, particular results for each approach are provided to validate the selected technique.In order to generate a video summary, the original video sequence is usually split into a minor temporal unit. Depending on the type of summary this unit can range from a video scene up to a single frame .The general overview of the system is shown in Figure http://mozart.dis.ulpgc.es/Gias/modesto.html), to increase the detection rate. The skin filter is derived from the method proposed in  than first division since the TV production for these categories have a different production process. The good results found in these adverse and demanding scenarios proof the validity of the approach to be used in easier 1st division matches.The most interesting events of interest in a soccer game are usually presented through replays from different scene viewpoints or in slow motion. Hence, replay detection approaches can offer reliable descriptors in the highlights summarization process.The TV production style in soccer media usually identifies the beginning and ending of a replay using two identical logos. Therefore, the strategy to detect a video sequence soccer replay can be based on logo detection and is generally implemented in four stages  samples in the band of interest for every audio frame. That band is selected between 3.5 and 4.5 kHz as broadly include the frequencies produced by professional whistles. The reduced set of samples of the DFT is computed using the Goertzel algorithm  followed in the near shots by the soccer player’s celebration (Persons) and/or a replay detection (Replay). Table The proposed system is able to detect satisfactorily over the 70% of the total amount of goals of the five soccer matches analyzed. However its performance presents some unpredictable limitations due to changes in TV production style or in external uncontrolled factors as for instance the public that influences the audio power producing peaks and/or significant increments. It is important to highlight that soccer match 4 and 5 present a higher number of goals not detected and the main reason stems from the fact that these sequences do not contain replays.This paper presents a novel framework for generating automatic video highlights of soccer broadcasting video sequences. The proposed approach consists on segmenting the video sequence into shots and scoring them using a set of low-level and mid-level audio-visual descriptors. Of special interest in the approach is the use of the audio information that provides additional robustness to the overall performance of the summarization system. Low-level descriptors describe audio, color and motion through MPEG-7 standards and mid-level descriptors annotate persons, whistles, zooms, long-shots and replays. The scoring process linearly combines and weights each descriptor by taking into account end-user preferences and custom soccer highlights are then generated. The proposed framework has been tested in a specific scenario and satisfactory results have been achieved.


Entodinium are responsible for much of the bacterial protein turnover. As yet no method to control protozoa in the rumen that is safe and practically applicable has been developed, however a range of plant extract capable of controlling if not completely eliminating rumen protozoa have been described.First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear. Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult to obtain absolute quantification. Despite repeated attempts it has proven impossible to maintain rumen protozoa in axenic culture. Thus it has been difficult to establish conclusively a role of ciliate protozoa in rumen fiber degradation. The development of techniques to clone and express ciliate genes in λ phage, together with bioinformatic indices to confirm the ciliate origin of the genes has allowed the isolation and characterization of fibrolytic genes from rumen protozoa. Elimination of the ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11%. Our recent findings suggest that holotrich protozoa play a disproportionate role in supporting methanogenesis whilst the small  First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite the fact that protozoa can contribute up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear . Here weSince they were first discovered by Dasytricha ruminantium and Polyplastron multivesiculatum based on 18S rRNA genes and Diplodinium dentatum, Entodinium caudatum, Epidinium caudatum, Eudiplodinium maggii, Isotricha intestinalis, Ophryoscolex purkynjei, and Polyplastron multivesiculatum.Early molecular studies on rumen ciliate protozoa focussed on sequencing of 18S rRNA genes to clarify the internal phylogeny of the class Litostomatea . Embley Numerous qualitative and quantitative studies focused on the diversity of ciliates have been performed since then; these studies have used a variety of techniques, including restriction fragment length polymorphism RFLP; , denaturMicroscopy holds several advantages over PCR-based molecular methods for studying ciliate protozoa. First, while the vast majority of intestinal ciliates have been characterized morphologically, there is a lack of 18S rRNA gene reference sequences for many of the observed genera and species. Second, as discussed below, copy number variation of ribosomal RNA genes across the different genera or under different growth conditions may skew the observed proportions of these genera in a sample . StudiesEntodinium, tended to be under-represented, larger protozoa such as Epidinium or Polyplastron tended to be over-represented by a pyrosequencing approach compared to microscopic enumeration  and protozoa-free animals have been used to study the role of ciliate protozoa in the rumen function without been affected by dietary interventions (in vivo studies comprising 48 comparisons (6 (sulfur hexafluoride) tracer method (25% of studies).Despite the fact that that protozoa make up a large portion of the rumen biomass, their role in ruminal fermentation and their contribution to the metabolism and nutrition of the host is still an area of substantial controversy . In the ventions . This paparisons . Most ofn) in each comparison. The meta-analysis was computed fitting a random-effect model with a DerSimonian-Laird estimator (2) in the Metafor package of R for each category separately as follow:Multiple comparisons were included from an individual publication with multiple studies. For each comparison included in the analysis, the effect size was calculated as the natural logarithm of the response ratio  and variance of the ratio calculated based on the reported standard deviation or standard error of the mean for each comparison . All defstimator  for assei = true effect size in the ith study, μ = overall true effect size and ie = random deviation from the overall effect size [iu ∼ N ] ] . For evaP = 0.07). Elimination of protozoa from the rumen significantly decreased rumen OM digestibility  and particularly NDF  and ADF digestibility , probably as a result of the loss of protozoal fibrolytic activity. This activity seems however to differ across the different protozoal groups: Large Ophyroscolecidae such as Epidinium, Polyplastron and Eudiplodinium have greater endoglucanase and xylanase activity (Entodinium spp. have only weak activity (Dasytricha has glucosidase and cellobiosidase activity but negligible fibrolytic activity (P = 0.089), CP , NDF , and ADF  is probably the most consistent of the observed effects of protozoal elimination and seems to be due to decreased bacterial protein breakdown and feed protein degradability in the absence of rumen protozoa  seems to highlight the role of protozoa in the synthesis of VFA and feed degradation. The ability of protozoa to engulf exogenous fatty acids (P < 0.001), slightly increased acetate  and had no effect on propionate molar proportions. In a previous meta-analysis (r = 0.95) but negative with propionate (r = –0.94) and butyrate (r = –0.91), therefore our observations might indicate that the shift in the molar proportions of VFA induced by defaunation seems to be mainly driven by a decrease in the fiber digestion. On the basis of stoichiometry, such a shift in rumen VFA production should result in a decrease in methane production as less metabolic H2 will be available as a substrate for methanogenesis (in vivo and in vitro studies (in vivo (P = 0.074). In line with these observations, in a meta-analysis containing 28 experiments and 91 treatments, it has been reported a significant linear relationship between methane emissions and protozoal concentration (r = 0.96) with a decrease in methane yield averaged 8.14 g/kg DMI per each log unit reduction in the protozoal concentration (2 in their hydrogenosomes (a mitochondria-like organelle), their ability to host epi- and endo-symbiotic methanogens and to protect them from oxygen toxicity (2 transfer that favors both the methanogens and the protozoa (P = 0.48). An alternative hypothesis suggests that defaunation results in the elimination of protozoa-associated methanogens, which could be considered as one of the most active methanogens communities in the rumen  than control animals. The reason for this seems to be based on the availability of an ecological niche for the bacteria when protozoa are not present in the rumen combined with the removal of protozoal predation (P < 0.001), Ruminococcus albus  and Ruminococcus flavefaciens  in the absence of protozoa. This observation suggests that fiber digestion in the rumen is a complicated task which requires the symbiotic collaboration of several fibrolytic microbes, including rumen protozoa, to carry out the initial stages of fiber colonization and digestion; therefore the absence of rumen protozoa seems to have a detrimental effect on this fibrolytic consortium and ultimately in fiber digestion. However, The effect of defaunation on the rumen microbial ecosystem is not limited to only the methanogen population and it has been demonstrated that defaunated animals had significantly greater ruminal bacterial populations  and greater average daily gain . These positive effects of defaunation are particularly obvious with poor quality diets in which the average daily gain was low, possibly as a result of the lower availability of fermentable energy and rumen digestible protein for the rumen microbes (P = 0.001), possibly as a result of a lower heat production . Other hypothesis to explain the better performance of defaunated animals rely on a more efficient use of dietary protein. There is clear evidence that holotrich protozoa leave the rumen more slowly than bacteria (15N and 35S and purine bases), microbial N minus specific bacterial markers  yielded variable estimates of protozoal outflow and sometimes even negative values due to methodological limitations for the markers used  and total non-ammonia N flow . Defaunation also increased the efficiency of microbial protein synthesis  as a result of both a better microbial proteosynthesis and a lower OM digestion. Protozoal generation time is far higher than that of bacteria, thus the energetic requirements for maintenance are higher when expressed as a ratio of protein leaving the rumen  in different sequential orders  could be beneficial to animals fed with fibrous diets. However, these fibrolytic protozoa and Entodinium spp. have a substantial potential to engulf and degrade bacteria (P < 0.001) due to a combination of lower dietary CP degradation in the rumen and lower rumen bacterial breakdown. However, the great variability observed in terms of N balance highlights the fact that defaunation may have different effects on the overall efficiency of N utilization depending on the diet consumed by the animal in particular the level of intake and particle passage rate through the tract  and a numerical decrease in mono and polyunsaturated fatty acids  which possibly did not reached significance as a result of diet-dependent effects.The effects of defaunation on ruminal lipid metabolism are less defined. The protozoa contribute to the total microbial lipolytic activity but their role in bio-hydrogenation is less well-understood  althoughThe findings reported in our meta-analysis need to be carefully interpreted since most of the studies had methodological limitations due to the intrinsic difficulty of the defaunation process leading to:(1)Confusion between the effect of defaunation and period when using the same experimental animals in time,(2)vice versa, moreover protozoal flow is often underestimated using traditional microbial markers Studies assessing methane production have not accounted for other variable effects besides protozoal abundance ,(4)Animals are often fed at levels far below the production levels of intake,(5)In some studies the adaptation period after defaunation/refaunation is too short for the microbial ecosystem to fully adapt,(6)Elimination of rumen protozoa modifies the ecological structure in the rumen and ultimately alternative microbial groups can take over ecological niche previously filled by protozoa.Thus, more studies are needed using state-of-the-art technologies to quantify protozoal activity  as well as the ruminal protozoal pool size relative to protozoal N outflow for better understanding of the role of protozoa on the ruminant’s metabolism. These studies should be done at production levels of intake in order to truly assess the effect of defaunation per unit of product (milk or meat) produced.Current concerns regarding the role of livestock in global warning has driven researchers to search for strategies to manipulate rumen protozoa to decrease methane production. As has been previously mentioned, there is a linear relationship between protozoal concentration and methane emissions  and it hMost approaches to defaunation rarely result in the total removal of protozoa from the rumen with their effectiveness largely dependent on diet composition . Treatme2 sink to methanogenesis  and tannins (r = 0.55), but this relationship was weaker (r = 0.45) with respect to essential oils. Methane inhibition by organosulfur compounds was not associated with changes in the protozoal population , and Quillaja saponaria which contains triterpene saponins. Lately, other sources of saponins such as tea saponins have being explored (in vitro studies (in vivo studies have shown that the antiprotozoal effect of saponins tends to disappears after 7–14 days of administration (in vitro studies (The literature suggests that saponins mitigate methanogenesis mainly by reducing the numbers of protozoa, whilst condensed tannins act by both reducing the number of protozoa and by a direct toxic effects on methanogens, whereas essential oils act mostly by a direct toxic effect on methanogens . In agrepulation  as such pulation . Regardipulation . This inexplored . The antexplored . It has explored . This efexplored . The sup studies . Howeverstration . It has stration . Thus, i studies  using 2, studies .in vivo effect on rumen protozoa has been observed. It has been suggested that the reasons for the lack of effect may be related to insufficient amount of specific Ig delivered in saliva, need of an adjuvant to optimize the production of salivary antibodies, target other antigens of protozoa to generate a greater immune response.Australian researchers have demonstrated the potential of vaccination against methanogens as a method for mitigating methane emissions . AlthougSince the landmark publication of the Rumen Protozoa by All authors have equally contributed to the preparation of this publication.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.


In this paper, a novel camera tamper detection algorithm is proposed to detect three types of tamper attacks: covered, moved and defocused. The edge disappearance rate is defined in order to measure the amount of edge pixels that disappear in the current frame from the background frame while excluding edges in the foreground. Tamper attacks are detected if the difference between the edge disappearance rate and its temporal average is larger than an adaptive threshold reflecting the environmental conditions of the cameras. The performance of the proposed algorithm is evaluated for short video sequences with three types of tamper attacks and for 24-h video sequences without tamper attacks; the algorithm is shown to achieve acceptable levels of detection and false alarm rates for all types of tamper attacks in real environments. EspecThe variety of camera tamper attacks can be classified into three types: defocused, covered and moved camera events –10. A deMost previous studies on tamper detection have compared features of the current frame with those of the background frames ,10–12. Fetc. Furthermore, there is no measure that can be used for fair comparison of the false alarm rate among tamper detection algorithms. Therefore, it is required to design a unified tamper detection algorithm with a small number of features that is robust against scene dynamics. It is also required to provide a way to measure the false alarm rate and to allow performance evaluation for sufficiently long video sequences.These studies have used different features and algorithms to detect each tamper event. Although a unified tamper detection criterion was proposed in our previous study , based oIn this paper, a novel unified tamper detection algorithm is proposed to detect all types of camera tamper attacks from video sequences with scene dynamics based on object and edge information in video sensor networks or IP camera-based video surveillance environments. Because most video sensor nodes and IP cameras act as input sources for video analytics modules residing on themselves or for remote servers that may be the video data sinks, taking advantage of video analytics information, such as information of objects and the background, can greatly reduce the computational complexity of tamper detection. In this study, to tackle the false alarm problem of scene dynamics, edge information and object information extracted from video analytics modules are used to detect camera tamper attacks without using the computationally-intensive transforms and filters used in conventional studies. Object removal for edge extraction and adaptive thresholds for different scene and illumination characteristics are proposed to reduce the false alarm rate and to enhance the accuracy of tamper detection, respectively. Furthermore, in order to maintain the tamper attack alerts until an operator's investigation and cancellation, background absorption of tamper events after the occurrence of tamper events is prevented.The organization of this paper is as follows. In Section 2, the architecture of the video analytics algorithms is proposed; this architecture is suitable for the proposed camera tamper detection algorithm. In Section 3, we propose a unified camera tamper detection algorithm based on background frame and object information from the video analytics algorithms. In Section 4, we present experimental results for the proposed algorithm with real daytime and nighttime video sequences, including camera tamper events. In Section 5, we present our conclusion and suggestions for further work.2.Most conventional studies have used a background frame as a reference for comparison with a current frame ,10–12. AA conventional video analytics algorithm based on background subtraction is shown in In this study, we attempt to detect all types of camera tamper events with a unified criterion that can be embedded into video analytics algorithms based on background subtraction. As a common and unified feature to detect covered, defocused and moved events, edge change information between current and background frames is used. To decrease the false alarm rate that results from scene dynamics, object information from the video analytics algorithm is used to prevent foreground edges from being used for detection. 3.3.1.For camera tamper detection, it is necessary to find common features and signal characteristics in covered, defocused and moved events. For covered and defocused events, captured image frames get simpler and finally lose their high frequency components. For moved events, due to the changing or changed camera angles, captured image frames may lose a large number of high-frequency components due to the motion blurring effect during a camera moving period or the number of high frequency components at the final moved angle may be quite different from that in the initial angle.Conventional studies of tamper detection have focused on the decreasing tendency of high-frequency components in input image frames after tamper attacks –13,16,18In tamper detection, scene dynamics, such as the moving of foreground objects or time-varying illumination conditions, may affect the accuracy and false alarm rate of tamper detection. Conventional studies have suffered from performance degradation due to scene dynamics –11,14,163.2.The proposed tamper detection algorithm is shown in Therefore, the authors propose to use object information to exclude regions of objects in the background and current frames from the calculation of the EDR for tamper detection. The background frame is generated using GMM; foreground objects can be extracted by conventional background subtraction-based video analytics algorithms, as can be seen in In our previous study, a Canny edge detector was used to detect the edges for the current and background frames . However3.3.i.e., the high-frequency component of the current frame, tends to decrease. However, using only the edge information for detection may cause a false alarm for scene dynamics due to foreground objects. Therefore, the authors propose to use a measure that can exclude the regions of foreground objects; in this method, the object information can be taken from the video analytics algorithm.During a camera tamper attack, the number of edge pixels, n, the ratio of the number of edge pixels that have disappeared in the current frame to the number of edge pixels in the background frame, is defined as follows.p in the current frame n and the background frame updated right before the frame n, respectively; values are one for an edge pixel and zero for a non-edge pixel. CR represents the region in the current frame without any foreground object; this region can be obtained from the object information provided by the video analytics algorithms.To measure the amount of edge change when excluding foreground objects, the EDR for the current frame a and b represent the edge of an object or noise existing only in the current image. For the 4 × 4-sized background and current images, nEDR = 0.5.EDR represents the decreased number of edge pixels in the current frame, compared to the number of pixels in the background frame, due to camera tamper attacks. If the foreground region is quite complex, with many edges, only removing the edges in the current frame may greatly affect the EDR for non-tamper attacks. Therefore, for a fair comparison, an object removal mask should be applied to both the current and the background frames.If camera tamper attacks occur, an entire image frame is sometimes detected as a single foreground object, because object detection is based on background subtraction. Such a situation may cause wrong EDR calculation by excluding entire edges of the image frame. Therefore, in our study, only objects less than a certain threshold in size are subjected to object removal. The threshold should be determined by considering the statistics of object information in the video sequences.3.4.Although the background is updated by GMM gradually, if some changes in scene content are maintained in specific regions in the image frame or in the entire frame for a long period, the changes will be gradually absorbed into the background. i.e., something sprayed on the camera keeps the camera from capturing a normal video sequence until the replacement or cleaning of the camera lens. Therefore, if the tamper attack is not handled quickly enough by the operator, the tampered region on the image frame may be absorbed into the background, and the tamper attack will not be detected.General camera tamper attacks last long; To solve this problem, if the detected tamper attack lasts longer than a certain period, a tamper flag is fixed in such a way that it cannot be unfixed by the tampered background, as shown in 3.5.After object removal and EDR calculation for incoming image frames, a tamper decision should be made. Using an adaptive threshold, the proposed algorithm decides on the presence of a tamper attack by comparing the difference between the EDR and the average EDR.initT frames. After this period, the AEDR is updated if no tamper attack is found to have occurred, as follows.n and l represent the number of frames processed and the number of frames with tamper attacks until the current frame, respectively. thm frame, with values of zero for no tamper and one for tamper events. If any tamper attack occurs, the AEDR keeps the current value.During the initial period of tamper detection, the average EDR (AEDR) is calculated using the EDRs of the first The threshold should be adaptive to the time- or location-dependent illumination conditions or plain background. Because the level of EDR depends on the characteristics of the scene contents, the threshold for tamper detection should be adaptive to scene characteristics. Scene characteristics can be classified into four cases: normal, low illumination, large foreground region and plain background.For the low illumination case, especially for nighttime sequences, although artificial lights or infrared (IR) capture capability are used to enhance the quality of the video sequences, extracted features for tamper detection have low reliability due to noise and low signal intensity. However, the proposed algorithm can exclude most noise from the EDR calculation without any anti-noise processing, because background frames generated by GMM include persistent signal changes that gradually exclude highly-variant noises. Because the number of edges in low illuminated frames tends to decrease due to low signal intensity, the level of EDR is generally lower than that in daytime video sequences. Edge changes due to instant changes of illumination are occasionally detected as objects and are excluded for EDR calculation by object removal.For the case of large foreground region, there exist many foreground objects that can cover quite a large portion of the image frame; the region left for edge comparison provides a smaller number of edge pixels for EDR calculation. For the case of a plain or uniform background, the regions for EDR calculation also have quite a small number of edge pixels, and the EDR is at a lower level.thn frame is given as follows.nE, W and H represent the number of edge pixels in the n-th background frame, the width and the height of a frame. The constants for the adaptive threshold are set empirically after the analysis of edge characteristics for various types of scene contents.An adaptive threshold is proposed to detect tamper attacks without parameter adjustment for time-varying scene characteristics due to illumination or environmental changes, especially for nighttime video sequences with a lower number of edge pixels. The adaptive threshold for tamper detection in the 3.6.As illustrated in W represents the size of the tamper validation window in frame interval units.To prevent wrong false alarms due to noise or temporary scene characteristics changes, a tamper validation count is defined as follows.n-th frame is set after validation as follows.setT is a pre-configured interval for tamper validation. There exists a trade-off between setT and the false alarm rate. This value was empirically set at three frame intervals in our experiments.Finally, a tamper flag for the fixT. Once the tamper flag is fixed, only operators can clear it.If tamper attacks last longer than the learning period of the background generation by GMM, the tamper attacks get absorbed into the background. To prevent the release of the tamper flag due to long lasting tamper attacks, the tamper flag is fixed to a set state for tamper attacks lasting longer than the pre-configured period 4.4.1.initT and fixT were all set at 600 frame intervals. Since no commonly-used test sequences for camera tamper detection are available, we captured the test video sequences in the vicinity of our work place. A detailed description of the captured test sequences is given in To evaluate the performance of the proposed tamper detection algorithm, various surveillance video sequences in QVGA (320 × 240) resolution at 30 Hz were used in our experiments. The pre-configured periods The test sequences include 48 daytime and 45 nighttime video sequences with camera tamper attacks and six video sequences without any attack. There are 10 covered, 23 moved and 15 defocused video sequences in the daytime sequences and 17 covered, 14 moved and 14 defocused video sequences in the nighttime sequences. Each sequence with camera tamper attacks includes at least one covered, moved or defocused event. Each test sequence is edited such that tamper attacks occur after Frame 600, which is the minimum time for stable background generation. In general, the background monitored by a surveillance camera is not so plain as the walls of a building or floors with a only few edges, as is shown in etc.The video sequences for false alarm performance evaluation, shown in 4.2.The accuracy of the proposed tamper detection algorithm is measured by the following detection rate, precision rate and daily false alarm rate with the terms defined in T represents the overall length of video sequences in the unit of days.Additionally, the daily false alarm rate (DFAR) is defined as follows.4.3.In 4.4.The performance of the conventional and proposed camera tamper detection algorithms are summarized in For covered events in all daytime and nighttime sequences, the proposed algorithm shows a 95.8% detection rate and the same precision rate. For moved events, it shows a 97.3% detection rate and the same precision rate. The proposed algorithm detected defocused camera events with a detection rate of 96.7% and a precision rate of 93.5%. For all kinds of tamper attacks, the proposed algorithm shows a 96.5% detection rate and a precision rate of 95.7%. Compared with the detection accuracies of the conventional algorithms, shown in 4.5.Most conventional studies have evaluated false alarm performance with very short video sequences with or without tamper attacks –10,12,14Therefore, the authors propose using DFAR in 5.A unified camera tamper detection algorithm that can detect all types of tamper attacks based on edge and object information is proposed. The edge disappearance rate of the background frame is defined as a unified measure of the deviation of the current frame from the background frame, excluding scene dynamics due to foreground objects. Tamper attacks are detected by comparing the difference between the EDR and the AEDR, with the adaptive threshold reflecting environmental conditions. The proposed algorithm outperforms most conventional studies in the detection rate and the precision rate for all types of tamper attack. The daily false alarm rate is sufficiently low for the proposed algorithm to be applied to real video surveillance systems, in which the DFARs in conventional studies are too high to be acceptable.


The impact of video game playing on cognitive abilities has been the focus of numerous studies over the last 10 years. Some cross-sectional comparisons indicate the cognitive advantages of video game players (VGPs) over non-players (NVGPs) and the benefits of video game trainings, while others fail to replicate these findings. Though there is an ongoing discussion over methodological practices and their impact on observable effects, some elementary issues, such as the representativeness of recruited VGP groups and lack of genre differentiation have not yet been widely addressed. In this article we present objective and declarative gameplay time data gathered from large samples in order to illustrate how playtime is distributed over VGP populations. The implications of this data are then discussed in the context of previous studies in the field. We also argue in favor of differentiating video games based on their genre when recruiting study samples, as this form of classification reflects the core mechanics that they utilize and therefore provides a measure of insight into what cognitive functions are likely to be engaged most. Additionally, we present the Covert Video Game Experience Questionnaire as an example of how this sort of classification can be applied during the recruitment process. Video games are one of the most popular free-time activities, with 42% of Americans playing at least 3 h per week  game experience” Boot, . UnswortAddressing this issue, we analyzed a random sample of non-declarative data concerning gameplay times that was provided to us by the Valve Corporation. Their online platform Steam is a digital game distribution service for PC and Macintosh with over 125 million  and removed those who reported playing more than 112 h per week (nexcluded = 4), assuming that this volume of play (16 h per day on average) is unlikely and unsustainable. The final sample included 1254 participants ranging in age from 18 to 64 . Two hundred and sixteen of these participants reported playing 5 h or less, and 18 reported playing more than 80 h per week  with a SD of 18.78. This suggests that video games are played more frequently on average than the generally adopted minimum recruitment criteria of 5–7 h weekly, though the playtime does vary substantially as suggested by Unsworth et al. , with men playing on average 23.91 h per week (SD = 19.35) and women 13.42 h per week (SD = 14.66). Second, we analyzed players of each genre. Gender differences in gameplay times were not dependent on genre . However, the results for Turn-Based Strategies  indicated anecdotal evidence for differences between groups . Finally, both men and women in our sample were of similar age , and we found that overall gameplay time does not decrease with age .There was also evidence for a number of additional interesting effects. First, women and men were found to decisively vary in their gameplay time  are characterized by navigation in a three dimensional environment from the first person (egocentric) perspective, aiming and shooting coordination, and focus on the avatar and its surroundings, with physics often being similar to the real-world. Another popular genre, Real-Time Strategy , requires players to view the environment from a top-down  perspective and use strategic planning skills in order to manage multiple units within a visible portion of the environment, all while under time pressure. It is important to note that both of these genres qualify for the “action video game” category proposed by Green and Bavelier  consists of one block of questions about demographics and four blocks of questions about free-time activities . Each category begins with an initial filtering question about the frequency of a particular activity and is followed by more questions if the participant declares it to be more than once a week (with the exception of cinema being more than once a month). Participants are asked similar questions within each category, and the video game category is presented in the middle.Information gathered from the questionnaire allow recruiters to determine mean weekly gameplay time in the preceding 6 months and gameplay time in individual genres (chosen by the participant), which allows for players from specific genres to be filtered and recruited. Additionally, the questionnaire collects data regarding years of video game experience, a topic that was recently the focus of research (Latham et al., In our opinion, current research practices have benefitted greatly from the methodological discussions of recent years. However, the remaining issues are substantial. The way in which VGPs are defined and recruited likely does not represent the typical player that researchers aim to include in cross-sectional research. It seems that many VGPs spend more time on video games than initially thought, and that this tendency should be kept in mind when interpreting research results coming from player samples that were not well controlled. Additionally, in line with the common demands hypothesis, researchers should pay closer attention to the games that are most often played by their participants and classify them based on the similarity of their core mechanics. Following these recommendations can allow researchers to reduce the error coming from within-group variability in their samples and make finer predictions about the sources of cognitive enhancement resulting from video game training.BS developed the concept of the manuscript, contributed to the data acquisition, performed all data analyses and interpretation, and drafted the manuscript. PD, MS, JM, AB substantially contributed to the concept of the manuscript and provided critical revisions and changes to the manuscript. Additionally, PD contributed to drafting of the work and preparation of the Steam database and together with MS contributed to the data acquisition. All authors approved the final version of this manuscript for submission and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Four subtypes of hepatocellular adenomas (HCA) are recognized: hepatocyte-nuclear-factor-1α mutated (H-HCA), β-catenin-mutated type with upregulation of glutamine synthetase (b-HCA), inflammatory type (IHCA) with serum-amyloid-A overexpression, and unclassified type. Subtyping may be useful since b-HCA appear to have higher risk of malignant transformation. We sought to apply subtype analysis and assess histological atypia, correlating these with next-generation sequencing analysis.Twenty-six HCA were stained with serum amyloid A (SAA), liver fatty acid-binding protein (LFABP), glutamine synthetase (GS), and β-catenin IHC, followed by analysis with a targeted multiplex sequencing panel.HNF1A mutations in all H-HCA, confirming reliability of LFABP IHC in identifying these lesions. CTNNB1 mutations were detected in 1 of 4 (25 %) of GS/β-catenin-positive cases, suggesting that positive GS stain does not always correlate with CTNNB1 mutations.By IHC, 4 HCA (15.4 %) were classified as b-HCA, 11 (42.3 %) as IHCA, 9 (34.6 %) as H-HCA, and two (7.7 %) unclassifiable. Eight HCA (30.8 %) showed atypia . Targeted sequencing confirmed Immunohistochemistry does not consistently identify b-HCA. Mutational analysis improves the diagnostic accuracy of β-catenin-mutated HCA and is an important tool to assess risk of malignancy in HCA. Hepatocellular adenomas (HCA) are rare benign liver tumors with the capacity to undergo malignant transformation. Epidemiologic studies report the prevalence of HCA is approximately 3–4 cases per 100,000 people in Europe and North America, . The higHNF1A (TCF1) gene causing loss of hepatocyte nuclear factor 1α (HNF-1α) expression define H-HCA, which are characterized histologically by marked steatosis and bland hepatocyte cytology [IL6ST, FRK, STAT3, JAK1, and GNAS [. Whole exome sequencing has also demonstrated that there is overlap between b-HCA and IHCA in some adenomas that harbor mutations in both the β-catenin and IL6ST genes [CTNNB1; however no increased risk of malignant transformation was noted in these patients [In the last decade, four subtypes of HCA have been identified based on genotype-phenotype analyses . Inactivcytology , 13. A scytology , 14–16. and GNAS , 18. WhoHNF-1A mutation in H-HCA, and increased SAA immunoreactivity serves as a marker for IHCA. Upregulation of a downstream CTNNB1 target gene, glutamine synthase (GS), is seen in b-HCA, as well as nuclear β-catenin staining. It has been postulated that the immunophenotypic subtypes closely parallel specific histologic features and molecular alterations, but limitations have been observed by numerous studies and detailed studies correlating morphology, immunohistochemical profile and mutation analysis are lacking [Based on this classification, subtyping of HCA by immunohistochemistry has been used with some success . Briefly lacking , 20, 21.HCA cases diagnosed between January 1994 and December 2012 were retrieved from our pathology department archives. For resection specimens, representative sections of tumor and non-tumorous liver were reviewed for histological features. The presence of multiple adenomas (2 or more tumors) had been previously assessed by gross organ review and representative sections of each adenoma were examined by microscopy. For biopsies, only tumor tissue was available for review and radiology reports were reviewed to determine the presence of multiple adenomas. Retrospective chart reviews were performed to collect additional demographic data including age, gender, related medical history and clinical follow-up. The study was approved by the Institutional Review Board at Columbia University Medical Center.Hematoxylin and eosin (H&E) stained slides, reticulin and Masson’s trichrome stains as well as immunohistochemical studies (IHC) were used to evaluate general morphologic and immunophenotypic features. All cases were reviewed by 3 pathologists . Tumor characteristics evaluated on routine H&E stained slides included: steatosis , inflammation, sinusoidal dilatation (telangiectasia), ductular proliferation, nuclear atypia  and architectural atypia (gland-like or acinar growth). Atypia was defined as the presence of any of the following: (1) nuclear atypia, (2) any degree of architectural atypia, and/or (3) focal loss of reticulin staining.Immunohistochemistry for LFABP , SAA , β-catenin  and GS  was performed in all cases using standard laboratory techniques in the Ventana Benchmark Ultra platform . GS IHC was scored as 0 , 1+ (perivascular staining or pseudo-maplike pattern of >10 % of the tumor), and 2+ (diffuse strong staining), as previously described . Pseudo-HNF1A gene, and one amplicon representing exon 3 of CTNNB1 gene.Multiplex targeted DNA next generation sequencing was performed in 18 of 26 cases. DNA was extracted from frozen and/or formalin-fixed paraffin-embedded tumor tissue using QIAamp DNA Mini Kit  according to manufacturer’s specifications. DNA was quantitated by fluorometry with the Invitrogen Qubit fluorometer and Quant-iT dsDNA BR Assay Kit , as recommended by the manufacturer. The samples were sequenced using the TruSeq Amplicon Cancer Panel , which covers 225 amplicons within 48 cancer-related genes, including 2 amplicons corresponding to exons 3 and 4 of HNF1A, CTNNB1 , IL6ST, STAT3, GNAS, and JAK1, was performed in a subset of cases. This test requires higher amount of DNA and could only be performed in 11 cases with sufficient DNA . Target capture and enrichment were performed with the SureSelect Hybrid Capture system  using custom probes. cDNA Libraries were then quantified using qPCR, diluted to 2nM and pooled for analysis on Illumina HiSeq 2500 using Illumina TruSeq v3 chemistry .Whole exome sequencing on an expanded panel of 467 tumor-specific genes, including Data deconvolution was performed using CASAVA Software . Files meeting QC metrics were used for mapping and variant calling using NextGENe Software . Reads were aligned to the hg19 reference genome. Variant calls with allele prevalence >1 % in the 1000 Genome Project, <3 variant reads, ambiguous alignments, quality score <10, and allele frequency <10 % were excluded. Variants were cross referenced with COSMIC, PROVEAN, and SIFT prediction tools .t-test or one-way ANOVA, as appropriate. P <0.05 was regarded as statistically significant.For categorical variables, Fisher’s exact test was used. One-way Kruskal-Wallis test was used for nonparametric data, including immunohistochemical scoring. Continuous variables were compared using a two-tailed student A total of 26 HCA cases (22 resections and 4 biopsies) were included in this study . Ten patients had multiple adenomas (38.5 %) with an average number of 6 lesions per patient (range: 2–14). The average size of HCA was 8.2 ± 4.5 cm (range: 1-18 cm). Detailed clinical history and follow-up data was available in 22 cases. One or more risk factors for the development of HCA at the time of resection  was identified in the majority of cases . Among the female patients, use of oral contraceptives (OCP) was identified in 12 of 21 (57.1 %) cases. One of 5 male patients (20 %) reported anabolic steroid use. We did not identify any case of glycogen storage disease, vascular diseases or mature-onset diabetes of the young type 3 (MODY3). None of the 22 patients with follow-up data developed hepatocellular carcinoma . One patient died of colon cancer. The patient’s demographics and follow-up data are summarized in Table First, we attempted to classify each adenoma based on IHC pattern, as previously described . BrieflyBy applying the above criteria, we identified 4 b-HCA (15.4 %), 11 IHCA 42.3 %), and 9 H-HCA (34.6 %). Two cases (7.7 %) were negative or weakly positive for GS, negative for SAA, and positive for LFABP, and were therefore unclassifiable  was present in 2 out of 4 b-HCA (cases 1 and 2). Conversely, 5 IHCA with 1+ or 2+ SAA staining showed weak, heterogeneous GS expression (score 1+) . We chose not to classify these as mixed β-catenin/inflammatory HCA based solely on immunohistochemistry because none of them demonstrated strong positivity for both GS and SAA. Cases with loss of LFABP did not show any staining for GS, except for one H-HCA case with strong (2+) GS staining Table .During the interpretation phase of this study, we encountered two technical difficulties. First, we found that LFABP IHC can sometimes be faint in the non-neoplastic liver resulting in an inconspicuous gradient between native liver and tumor. In such cases, LFABP staining was repeated in multiple tumor sections in order to obtain acceptable results. Second, the few cases with nuclear β-catenin positivity were difficult to evaluate since nuclear positivity could only be appreciated in less than 5 % of the tumor cells.HNF1A mutations were identified in all analyzed H-HCA  and in no other HCA subtype . The codon alterations were Gly207Asp, Asn237Ser, Asn237Lys, Leu281Gln and Tyr286Cys, 3 of them previously reported in H-HCA [Results of targeted DNA sequencing analysis were correlated with the classification obtained by immunohistochemical analysis Table . HNF1A min H-HCA .CTNNB1 gene alterations were identified in 2 cases: one b-HCA harboring both a CTNNB1 gene deletion  and a TP53 gene mutation (Ile251Phe) (case 1); and a second case originally classified as IHCA based on IHC results , found to have a missense CTNNB1 mutation (Ser45Pro). This case also carried an IL6ST mutation  and was thus reclassified as a mixed β-catenin/inflammatory HCA (bIHCA)  and were reclassified as IHCA. The third case had no alterations in CTNNB1, HNF1A, IL6ST, STA3, GNAS or JAK1 genes and final classification could not be confirmed by molecular studies , were reclassified because molecular analysis demonstrated no abnormalities in the ies Fig. .n = 9) showed features of telangiectasia , focal inflammation  and ductular reaction . Mild to moderate steatosis was occasionally seen IHCA . Telangiectasia, a feature commonly associated with IHCA, was seen in the bI-HCA (case 7). No cases classified as b-HCA, IHCA or UHCA showed marked steatosis.Following classification by molecular testing Table , 2 and 3In rare occasions, limited DNA concentration or DNA degradation may limit detection of mutations and deletions in tumor samples. To circumvent this issue, DNA was obtained from both frozen and formalin-fixed tumor samples and sequenced using two different platforms. This approach was feasible in 12 of our 18 analyzed samples and yielded reproducible sequencing results.p = 0.001 and 0.036, respectively). Not surprisingly, telangiectasia was preferentially present in IHCA (p = 0.0002) . Similarly, patient age was not significantly different in patients with atypical HCA .Eight cases (30.8 %) displayed one of more atypical features Table . By moleCTNNB1 mutations in HCA may offer crucial information regarding management of some of these patients.Hepatocellular adenomas have a small risk of malignant transformation with estimated rates of less than 5 % , 25–27. CTNNB1 and HNF1A gene sequencing [CTNNB1 gene in the b-HCA subtype, and defects in the IL-6/JAK/STAT3 pathway in IHCA. Our study is one of the first to use a comprehensive targeted next-generation sequencing panel to evaluate the mutational status of HCA and correlate with immunohistochemistry results.The current HCA classification scheme is based on the tumor’s morphologic and immunohistochemical characteristics and was originally validated with quencing . More requencing , 18 inclHNF1A gene mutations in all analyzed H-HCA cases, confirming high accuracy of this marker in the diagnosis of H-HCA. However, our results suggest limited reliability of GS and β-catenin immunohistochemistry in predicting β-catenin mutations. The utility of both markers has been challenged by other researchers [et al. showed strong GS positivity in over 50 % of HCA and Joseph et al. described patchy to diffuse GS staining in 23 % of IHCA, but no correlation with molecular studies were available in these studies [CTNNB1 alterations in 4 cases, one of which was a biopsy specimen (case 13). These somewhat controversial staining patterns and their correlation with β-catenin activation have not been fully characterized and may be a result of localized metabolic alterations within the tumor [As expected, our study demonstrated earchers , 29, 30. studies . In the he tumor –22, 31.et al. described strong GS staining in a peliotic HCA and suggested that vascular flow alterations and hepatic parenchymal remodeling may explain increased GS expression [In addition, we identified strong GS positivity in four β-catenin wild-type tumors, including one H-HCA. We speculate whether these tumors may have activation of an alternative pathway resulting in GS expression. Berry pression , 32.CTNNB1-mutated HCA. Low GS expression in b-HCA has been previously reported in HCA carrying the Ser45Pro mutation [CTNNB1 alterations may not disrupt GS and β-catenin expression patterns [CTNNB1 deletions or mutations in exons 7 or 8, the latter described in a minority of bHCA and previously shown to activate β-catenin in HCC cell lines [In our study, GS staining failed to identify 1 of 2 confirmed mutation . Interespatterns . None ofll lines . Cautionll lines .IL6ST alteration. Three cases initially categorized as b-HCA lacked CTNNB1 gene alterations. Two of them were re-subtyped as IHCA following identification of IL6ST gene alterations, all of which previously described in IHCA [HNF1A or any of the IHCA-related genes. In this challenging case, the diagnosis of a β-catenin-activated HCA is favored, but the lack of molecular evidence for CTNNB1 alterations suggests alternative mechanisms of β-catenin activation and GS overexpression in the tumor [Following molecular analysis, four tumors were reclassified. One case phenotypically classified as IHCA was reclassified as bIHCA because it carried both a β-catenin mutation and an  in IHCA , 35. Thehe tumor . This adhe tumor , 27.CTNNB1 activating mutations [A large percentage of HCA showed atypical features (30.8 %) but no correlation with a specific HCA type was identified. Other histological features, such as mild steatosis and telangiectasia, were not reliable predictors of HCA subtype as defined by IHC and molecular analysis. In addition, we identified significant immunohistochemical overlap between the b-HCA and IHCA subtypes. Some of these “overlap” lesions, with immunohistochemical features of both IHCA and b-HCA, have been previously described as carrying utations . Both β-CTNNB1 exon 3 deletion (Gly38_Thr40del) and a TP53 mutation (Ile251Phe), the latter located in a known ‘hotspot’ for cancer-related alterations and previously described in HCC [et al. described HCC-related genetic alterations in β-catenin-activated adenomas suggesting that some of these lesions more likely represent low-grade hepatocellular carcinomas [There is a strong association between b-HCA and hepatocellular carcinoma at the time of diagnosis or during follow-up . In our d in HCC . For theetion Gly_Thr40delImmunohistochemistry remains the mainstay for HCA classification due to its relative reliability and rapid turnaround time, especially when differentiating HCA from other lesions, such as focal nodular hyperplasia and hepatocellular carcinoma , 39. HowIn summary, while LFABP immunohistochemistry is reliable in identifying H-HCA, GS and β-catenin stains are weak predictors of β-catenin mutations. Our molecular analysis demonstrates that the currently proposed IHC criteria may be somewhat oversimplified. Until other reliable and specific immunohistochemical markers of malignant transformation in HCA are identified, molecular testing of LFAPB-positive HCA may be the best strategy to identify some of the lesions requiring further clinical management. In this context, next-generation sequencing offers a straightforward, highly sensitive, and accurate option for biopsies with equivocal immunohistochemical results.


Introduction: Malnutrition, a state of under or over nutrition caused by improper food intake, causes significant morbidity and mortality in developing countries. It leads to a number of diseases which can be further divided into those caused by protein-caloric malnutrition and those caused by vitamin deficiencies, micronutrient, and mineral deficiencies. The purpose of this study was to identify the factors which contribute to malnutrition and to assess the dietary pattern in the pediatric population from birth up to five months belonging to poor socioeconomic areas. The children in this sample presented to a tertiary care hospital in the district of Sargodha, Pakistan. The findings in this cohort will support the development of an effective plan to tackle these issues.Methods: This cross-sectional study was undertaken between June 2014 and December 2014 at the inpatient pediatric department of District Headquarter Hospital Sargodha. Data were collected and recorded on a predesigned form which consisted of four sections to record 1) demographics: parents' level of education, monthly income, number of dependent household members, and birth interval; 2) anthropometric and personal history, birth history, and degree of malnutrition; 3) any secondary causes of malnutrition; and 4) feeding history. The data were analyzed in SPSS v. 20. Chi-squared, phi statistics, and logistic regression analysis were run to analyze the data.Results: A total of 294 participants were included in the study. Logistic regression analysis showed that the degree of malnutrition was associated negatively with increasing age and positively with family size. A majority of children  were being breastfed for less than 5 minutes followed by 38 (13%) > 5 minutes to 15 minutes, and 2 (0.7%) > 15 minutes while 110 (37 %) infants were not breastfed. Children who were breastfed were less likely to have severe malnutrition than those who were given formula, fresh cow's or goat's milk, or more than one type of food.Conclusion: Children who were breastfed were less likely to have severe malnutrition. The degree of malnutrition was negatively associated with age and positively associated with family size. South Asian countries have the highest burden of malnourished children under five years of age -3. Half Pakistan has the highest child and infant mortality rate in South Asia . The morThe present study was designed to determine the feeding patterns and predictors of malnutrition in children from birth up to five months of age in Pakistan.This cross-sectional study was undertaken between June 2014 and December 2014 in the District Headquarter Hospital (DHQ) Sargodha, Pakistan. Ethical approval was obtained from the Ethical Review Committee of Yusra Medical and Dental College, Islamabad. Two hundred and ninety-four children under five months of age followed at the inpatient department of DHQ Sargodha were included in the study. The minimum sample size required for the survey was calculated to be 384 based on a 95% confidence level, 5% margin of error, and a population of 11.2 million children suffering from malnutrition in Pakistan [Written consent was obtained from all participants following a comprehensive explanation of the purposes of the study. Children with serious illness or comorbidities were excluded from the study. Detailed history and anthropometric measurements were taken by a physician.Data were collected and recorded on a pretested, predesigned form which consisted of four sections. The first section recorded demographics, parents’ level of education, monthly income, the number of dependent household members, and birth interval. The second section recorded anthropometric data and personal history, birth history, and degree of malnutrition. The degree of malnutrition were categorized as first degree (75-90% of expected weight), second degree (60-75% of expected weight), and third degree (<60% of expected weight). The third section recorded any secondary cause(s) of malnutrition. The fourth section recorded feeding history, including details about breastfeeding, formula feeding, and fresh cow’s or goat’s milk feeding. The data were analyzed in SPSS v. 21 . Frequencies, percentages, and the chi-squared goodness of fit test were run for participants’ characteristics and feeding patterns. Logistic regression analysis was used to elucidate the predictors of severity of malnutrition in the infants. Covariates entered into the logistic regression model were the age of the infant, gender, mother’s education, income, family size, background, gestation, mode of delivery, and type of feeding. The degree of malnutrition was dichotomized as “first degree” and “severe”  and entered as a dependent variable.The mean age of the infants was 3.51 months and the mean number of family members was 7.14. Most of the infants included in the survey were males, had illiterate mothers, had family income < Rs. 20,000/month (200 USD) and belonged to rural areas. Most of the respondents had SVD and partial immunization status. Almost 45% of the infants were being breastfed. Detailed results are given in Table Table Less than 40% of the infants were receiving fresh food. Most of them were receiving cow’s milk, < 5 times/day and 2 times/night. Most of the infants (104/109) received diluted fresh food and the most cited reason for over-dilution was family pressures/financial constraints. Table Many of the infants  were receiving formula food, < 5 times/day and 2 times/night. Nine (9/56) infants were receiving over-diluted food and the most commonly cited reason for over-dilution was no awareness and family pressures/financial constraints. Table According to logistic regression analysis, the degree of malnutrition was negatively associated with increasing age and positively with family size. Children who were taking mother’s food were less likely to have severe malnutrition than those who were having formula food, fresh food, or more than one type of food.Malnutrition in children is a serious health hazard in a developing country like Pakistan. We searched for factors that contribute to malnutrition in children under five months of age from families with poor socioeconomic status. Our analysis showed that malnutrition was negatively associated with increasing age and positively associated with family size. We also found that children who were breastfed were less likely to have severe malnutrition.Among the factors associated with malnutrition are the parents’ education, income, family size, the birth interval with the older sibling, and feeding pattern. Many studies have shown a significant association between malnutrition and the parents’ education, especially the mother’s. Ali, et al. reported a significant difference in malnutrition (as shown by low weight) between children whose mothers were illiterate compared to educated mothers in Karachi (P < 0.016) . Islam, Family income is also important for child nutrition. Less income is associated with inadequate nutrition, poor sanitation, and an increased risk of infection, which leads to stunting in children -18. KavoFamily size is also an important factor in malnutrition. A study done in India showed that the overall prevalence of protein-energy malnutrition was significantly higher (P < 0.001) in infants from joint families than those from nuclear families. In large families, 74.6% of children had malnutrition vs. only 21.96% in small families . Our stuRegarding birth interval, a study of 1,884 children showed that children who had less than a 24-month birth interval with the older sibling had increased the risk of malnutrition – a result that points toward the potential benefits of longer birth intervals .Breastfeeding is a cost-effective mechanism to reduce malnutrition in developing countries. The World Health Organization recommends that exclusive breastfeeding should be started soon after birth and continued for six months, followed by the addition of complementary food up to two years of age . A previPakistan is an underdeveloped country where most of the population lives in rural areas. Mothers in these areas often have a low level of education and live in poverty, and these factors have an especially marked influence on infant and child nutrition. In addition, people in rural areas have less access to basic health care facilities. There are also misconceptions about the foods given to children, according to traditional practices without an actual scientific basis. Because of the multifactorial origin of malnutrition, appropriate intervention to reduce its incidence and prevalence in Pakistan should include: 1) education of mothers about healthy feeding practices by the Lady Health Visitors (LHV) in rural areas, 2) public awareness through mass media and community participation, and 3) the development of programs by the government and non-government organizations to provide care to malnourished children. Although great efforts are being made by WHO and the government, continued work is still needed.Malnutrition in children is associated with poor breastfeeding practices. Children who were breastfed were less likely to suffer from malnutrition. More than one-third of the infants were not breastfed, mainly because of the personal preference of the mother and less amount of breast milk. These poor feeding practices can be improved by education of the mothers. The degree of malnutrition was negatively associated with age and positively associated with family size.


Osteoarthritis (OA) is a multifactorial disease, and recent studies have suggested that cell cycle–related proteins play a role in OA pathology. p21 was initially identified as a potent inhibitor of cell cycle progression. However, it has been proposed that p21 is a regulator of transcription factor activity. In this study, we evaluated the role of p21 in response to biomechanical stress.Human chondrocytes were treated with p21-specific small interfering RNA (siRNA), and cyclic tensile strain was introduced in the presence or absence of a signal transducer and activator of transcription 3 (STAT3)-specific inhibitor. Further, we developed an in vivo OA model in a p21-knockout background for in vivo experiments.MMP13) mRNA increased in response to cyclic tensile strain following transfection with p21 siRNA, whereas the expression of aggrecan was decreased. Phospho-STAT3 and MMP-13 protein levels increased following downregulation of p21, and this was reversed by treatment with a STAT3 inhibitor. p21-deficient mice were susceptible to OA, and this was associated with increased STAT3 phosphorylation, elevated MMP-13 expression, and elevation of synovial inflammation. The expression of p21 mRNA was decreased and phosphorylation of STAT3 was elevated in human OA chondrocytes.The expression of matrix metalloproteinase  contains supplementary material, which is available to authorized users. The development of osteoarthritis (OA) is related to genetic factors, biomechanical stress, and biological factors within the joints –3. Previp21 induced a regenerative response in an appendage of an otherwise nonregenerating mouse strain [p21 transcription is activated by p53, and p21 is part of a negative feedback mechanism that controls p53 activity during apoptosis [1-to-S phase shift in chondrocytes, accompanied by a reduction of p21, and reduction of p21 caused delayed cell differentiation, manifested by decreases in proteoglycan synthesis, mineralization, alkaline phosphatase activity, and matrix proteins [The cyclin-dependent kinase inhibitor p21 was initially identified as a potent inhibitor of cell cycle progression –13. Knoce strain . Subseque strain . p21 trapoptosis . It is apoptosis . Recentlpoptosis –21. p21 poptosis . Furtherpoptosis . A previpoptosis . Given tpoptosis . Anotherproteins .We initiated the present study to investigate the function of p21 in cartilage homeostasis. Specifically, we focused on the relationship between p21 and STAT3 in chondrocyte and joint biology in response to mechanical stress both in vitro and in vivo.Cartilage tissues were obtained during total hip joint replacement surgery from 11 patients with OA. Diagnosis of OA was based on clinical, laboratory, and radiographic evaluations. Normal cartilage tissues were obtained during surgery for femoral neck fractures from eight patients with no history of joint disease and with macroscopically normal cartilage. All the samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. The study protocol was approved by Kobe University Graduate school of Medicine Ethics Committee, and all patients gave their informed consent.5 cells/well). All experiments were conducted using first-passage cells. To characterize the chondrocytes, we confirmed that the type II collagen showed higher expression in the normal human hip chondrocytes than in the OA knee chondrocytes and that type X collagen showed higher expression in the OA chondrocytes than in the normal chondrocytes.Primary chondrocytes were isolated and cultured from the cartilage tissues. Tissues were minced and incubated with trypsin  for 15 minutes at 37 °C, after which the cartilage was treated with Dulbecco’s modified Eagle’s medium  containing 0.2 % collagenase (Sigma-Aldrich) at 37 °C for 15 h. Dissociated cells were cultured in DMEM supplemented with 10 % fetal bovine serum  and 100 U/ml penicillin-streptomycin. After overnight culture, nonadherent cells were removed and adherent cells were further incubated on a 6-well plate in fresh medium (3 × 102 and 95 % air at 37 °C in a BulletKit (Cambrex). NHAC-kn were derived from a single-donor knee articular cartilage and used as an established normal chondrocyte cell line [Normal human knee chondrocytes  were cultured in a humidified atmosphere of 5 % COell line , 28. To 5 cells/well) in a 6-well plate and were then plated onto a silicon chamber coated with fibronectin (Sigma-Aldrich) at a density of 3 × 105 cells/well in DMEM/F-12 supplemented with 10 % FBS and 100 U/ml penicillin-streptomycin. Cyclic tensile strain experiments were performed using an ST-140 cyclic tensile strain system . Cyclic tensile strain was enforced at 3 %, 5 %, 8 %, and 10 % elongation for 3 h (0.5 Hz) according to previous studies [Before we performed the experiments, cells were grown to a subconfluent state  and nonspecific siRNA control into normal human knee chondrocyte monolayers according to the recommendations of the manufacturer . Briefly, 1 day before transfection, cells were plated in a 6-well plate in growth medium without antibiotics to attain 30–50 % confluence at the time of transfection. Subsequently, 100 pmol of siRNA and Lipofectamine 2000 complexes were prepared and added to each well. After 12 h of transfection, the complexes were removed, and fresh medium containing 10 % FBS was added. After an additional 12 h, the cells were replaced onto a silicone chamber coated with fibronectin at a density of 3 × 102, 0.5 mM deoxyribonucleotide triphosphate mix, 0.5 U of RNase inhibitor, and 1.25 U of murine leukemia virus reverse transcriptase  at 42 °C for 60 minutes.Chondrocytes were cultured in 6-well plates with various stimulations, and RNA was extracted using a QIAshredder and RNeasy Mini Kit  according to the manufacturer’s protocol. Briefly, 1 μg of total RNA was reverse-transcribed to first-strand cDNA with 1.25 μM oligo(dT) primer in 40 μl of polymerase chain reaction (PCR) buffer II containing 2.5 mM MgC1p21, collagen, type II, alpha 1 (COL2A1), ACAN, MMP3, MMP13, ADAMTS4, and ADAMTS5 were analyzed by SYBR Green real-time PCR using an ABI Prism 7700 sequence detection system (Applied Biosystems). The relative expression of the genes of interest was normalized against the GAPDH housekeeping gene by using the comparative cycle threshold (Ct) method. The difference between the mean Ct values of the gene of interest and the housekeeping gene is denoted as ΔCt, and the difference between ΔCt and the Ct value of the calibrator sample is denoted as ΔΔCt. The log2 (ΔΔCt) value gives the relative level of gene expression. The primer sequence for detection of human p21, COL2A1, ACAN, MMP3, MMP13, ADAMTS4, and ADAMTS5 are described in the Additional file The relative expression levels of mRNA encoding human Chondrocytes were washed three times with phosphate-buffered saline and lysed in a hypotonic lysis buffer  supplemented with a protease and phosphatase inhibitor mix  on ice for 20 minutes . The lysHorseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) Ab  or HRP-conjugated rabbit anti-mouse IgG Ab  was used as a secondary antibody, and the signals were visualized using Amersham ECL Plus reagent  with the LAS-3000mini chemiluminescent image analyzer . Actin was used as a control to estimate protein loading on the gel.Cdkn1atm1Led/J mice were obtained from The Jackson Laboratory . We backcrossed the mice against a C57BL/6 background and studied male mice at 10 weeks of age. All the mice used in this study were backcrossed over 20 generations. p21+/+ littermates were used as wild-type controls. Genotyping was performed by PCR amplification of mouse-tail DNA by using allele-specific probes. Each experimental group contained at least six mice.Homozygous B6.129S6(Cg)-−/− and p21+/+ mice were used in these experiments. Mice were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg). Destabilization of the medial meniscus (DMM) was induced in the right knee joint by transecting the anterior attachment of the medial meniscotibial ligament as described previously [sham. All mice were subjected to weight-bearing following recovery from anesthesia. The mice were sacrificed 8 weeks after DMM or sham surgery, and their tissue was subjected to histological evaluation. Six mice were analyzed for each group: p21+/+ sham, p21+/+ DMM, p21−/− sham, and p21−/− DMM .This study was carried out in strict accordance with the recommendations contained in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the Animal Studies Committee of Kobe University, Japan (permit number P131104). Ten-week-old male p21eviously . For theMouse knee joints were fixed with 4 % paraformaldehyde for 24 h, decalcified with 14 % ethylenediaminetetraacetic acid for 7 days, and embedded in paraffin. Coronal histological sections were taken through the joint at 80-μm intervals and stained with Safranin O and Fast Green. OA histopathology was evaluated by using the Osteoarthritis Research Society International (OARSI) cartilage OA histopathology scoring system . HistoloDeparaffinized sections were digested with proteinase  for 10 minutes and treated with 3 % hydrogen peroxide  to block endogenous peroxidase activity. The sections were treated with a 1:100 dilution of antiphosphorylated STAT3 , anti-MMP-13 , and anti-F4/80  antibodies at 4 °C overnight and were subsequently treated with peroxidase-labeled antirabbit Ig  at room temperature for 30 minutes. The signal was developed as a brown reaction product by using the peroxidase substrate 3,3′-diaminobenzidine , and the sections were examined microscopically. Methyl green stain was used as a counterstain. One sagittal section from the center of the most severe OA lesion in each tibial plateau was scored. The number of stained cells was counted in three areas of high-magnification fields at both superficial and deep zones of the cartilage tissue individually by three blinded observers. The average percentage of p-STAT3– and MMP-13–positive cells/total cells were calculated. One sagittal section each from six mice was evaluated in each group. These positive cells were included superior to the tidemark and were included for both femur and tibia.U test was used for comparisons between the two groups  and were considered statistically significant at P < 0.05.Statistical analysis was performed using one-way Fig.  or two-wups Fig. . P valueCol2a1 in untreated control mice did not change in response to tensile strain. ACAN in untreated control mice increased after 3 %, 5 %, and 8 % strain; however, the expression levels were decreased after 10 % strain. Moreover, MMP3 in untreated control mice was increased after 8 % and 10 % strain, and MMP13 was increased after 10 % strain. ADAMTS4 in untreated control mice was increased after 5 %, 8 %, and 10 % strain, and ADAMTS5 was increased after 8 % and 10 % strain -STAT signaling pathway. Recently, it was reported that the JAK-STAT signaling pathway plays a critical role in MMP13 expression in chondrocytes [MMP13 expression in chondrocytes [ACAN expression. Therefore, p21-regulated ACAN expression does not appear to be controlled by STAT3 signaling. Dai et al. demonstrated that the catabolic stress induced by IL-1β or hydrogen peroxide increased caveolin 1 and p21 expression in chondrocytes and that overexpression of caveolin 1 increased p21 expression and p38 mitogen-activated protein kinase activation, as well as impaired the ability of chondrocytes to induce type II collagen and ACAN expression [To determine the mechanism underlying the changes in drocytes . Legendrdrocytes . These rpression , we demoWe established a mouse OA model and examined the role of p21-knockout in this setting. We showed that p21-deficient mice were susceptible to OA and this was associated with increased STAT3 phosphorylation and MMP-13 expression. These results supported our in vitro experiments.MMP13 expression in chondrocytes.Lee et al. reported that IL-17 increased the expression of Toll-like receptor 3 via the STAT3 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes and that STAT3 phosphorylation was increased in the synovial samples from patients with RA compared with patients with OA . HoweverMacrophages play a critical role in inflammation with three major functions—antigen presentation, phagocytosis, and immunomodulation—through the production of various cytokines and growth factors . RecentlThe lack of p21 has catabolic effects by regulation of ACAN and MMP-13 expression through STAT3 phosphorylation in the cartilage tissue. p21 may function as a regulator of transcription factors in addition to being an inhibitor of cell cycle progression in the cartilage tissue. Thus, the regulation of p21 may be a therapeutic strategy for the treatment of OA. However, p21 also plays a role as a cell cycle regulator and as an oncogene. Therefore, further work is required to verify that the stabilization of p21 would be a viable and safe strategy for OA treatment.