We addressed this question by asking CTS patients and healthy controls to grasp, lift, and hold a grip device  for several consecutive trials. We found that CTS patients were able to successfully adapt grip force to object weight. However, multi-digit force coordination in patients was characterized by lower discrimination of force modulation to lighter object weights, higher across-trial digit force variability, the consistent use of excessively large digit forces across consecutive trials, and a lower ability to minimize net moments on the object. Importantly, the mechanical requirement of attaining equilibrium of forces and torques caused CTS patients to exert excessive forces at both CTS-affected digits and digits with intact sensorimotor capabilities. These findings suggest that CTS-induced deficits in tactile sensitivity interfere with the formation of accurate sensorimotor memories of previous manipulations. Consequently, CTS patients use compensatory strategies to maximize grasp stability at the expense of exerting consistently larger multi-digit forces than controls. These behavioral deficits might be particularly detrimental for tasks that require fine regulation of fingertip forces for manipulating light or fragile objects.The delicate tuning of digit forces to object properties can be disrupted by a number of neurological and musculoskeletal diseases. One such condition is Carpal Tunnel Syndrome (CTS), a compression neuropathy of the median nerve that causes sensory and motor deficits in a subset of digits in the hand. Whereas the effects of CTS on median nerve physiology are well understood, the extent to which it affects whole-hand manipulation remains to be addressed. CTS affects only the lateral three and a half digits, which raises the question of how the central nervous system integrates sensory feedback from affected  Skilled manipulatory behaviors require complex spatial and temporal coordination of the digits that can be flexibly adapted to object properties such as size, friction, and weight. In healthy individuals, visual and somatosensory feedback is processed and integrated with motor commands to control multiple digit forces CTS is a compression neuropathy of the median nerve resulting in sensorimotor impairments in the hand that begin with deficits in sensation in the thumb, index, middle, and lateral half of the ring finger  and progresses, in severe cases, to include motor deficits predominantly in the thumb. Symptoms include aching and burning, tingling, numbness, weakness, and clumsiness in the affected hand. The median nerve is a mixed nerve comprised of both sensory and motor axons innervating most extrinsic hand flexor muscles and some intrinsic muscles. It also relays sensory information from the palmar aspect of the thumb, index, middle and the lateral half of the ring finger. Prolonged mechanical compression of the nerve results in ischemic damage and/or changes in the myelination of the nerve leading to slowing of axonal conduction velocity, nerve block, and in severe cases axonal loss CTS affects several classes of sensory receptors required for grasp control, i.e., tactile mechanoreceptors of the glabrous skin as well as muscle, joint, and tendon receptors of intrinsic hand muscles. While the role played by mechanoreceptors of the glabrous skin for object grasping and manipulation has been extensively studied and established , has to be integrated for effective coordination of multiple digit forces. The extent to which CTS patients are able to coordinate multi-digit forces for grasping by integrating feedback from digits with impaired and intact sensorimotor capabilities remains to be addressed. Another gap in our understanding of the effects of CTS on multi-digit grasp control is the extent to which median nerve compression impacts trial-to-trial adaptation of multi-digit forces to object properties for skilled object manipulation.Despite impaired sensory function in digits, CTS patients are able to modulate grip force similar to controls in anticipation of load force to different frictional conditions between the object and digits (1) force modulation to object weight in CTS patients would be less accurate than controls, as indicated by lower discrimination of force modulation to object weight; (2) larger across-trial variability and consistent use of larger digit forces than controls despite repetitive manipulation of the same object with a given weight due to inability to form accurate sensorimotor memories of manipulations from previous trials; and (3) CTS patients would be less skilled than controls in balancing multi-digit forces, thus leading to the production of moments on the object at lift-off that could interfere with the subsequent manipulation.The present study was designed to quantify trial-to-trial adaptation of multi-digit forces to object weight. We hypothesized that CTS patients would maintain the ability to modulate grip forces to object weight while using larger grip forces than controls with both CTS–affected and –unaffected digits. However, based on impaired median nerve function and expected deficits in tactile sensing, and the above evidence on the role of tactile sensing on the formation of sensorimotor memories we also hypothesized that CTS would affect trial-to-trial adaptation of multi-digit forces as follows: All participants gave their written informed consent according to the declaration of Helsinki and the protocols were approved by the Institutional Review Boards at Arizona State University and Mayo Clinic Hospital.Thirteen Carpal Tunnel Syndrome (CTS) patients  and thirteen age- and gender-matched healthy volunteers participated in this study. Subjects' average height and weight were 167.2±4.2 cm and 84.4±7.2 Kg, respectively, for CTS patients and 168.1±3.4 cm and 75.9±5.5 Kg, respectively, for controls. The gender distribution of our CTS patients reflects the higher incidence of women than men For inclusion in our study, CTS patients had to exhibit at minimum a prolonged median nerve distal sensory latency . Even though some CTS patients received sensory (Semmes-Weinstein monofilaments) and provocative tests , the diagnosis of CTS was ultimately based on clinical symptoms and electrodiagnostic tests. Electrodiagnostic tests are considered the best available diagnostic standard for CTS The electrodiagnostic studies confirmed the diagnosis of CTS in all patients by demonstrating prolonged latency of the median nerve localized to the wrist segment where the carpal tunnel is located . AdditioTo summarize, the electrodiagnostic tests used to select our CTS patients indicated abnormalities in two parameters that are considered important for grasp control: slowing of conduction velocity in sensory afferents and axonal loss. With regard to slower conduction velocity in sensory afferents, experimental and modeling work suggest that the timing patterns of action potentials from tactile afferents may play a significant role in fingertip force control, e.g., discrimination of force direction .We studied the effect of CTS on multi-digit force coordination using a blocked weight presentation where a given object weight was presented over consecutive trials. Subjects were instructed to perform the above task with three different mass conditions: 445 g, 545 g, and 745 g. Each of these weight conditions was obtained by adding a mass  at the bottom of the device. Subjects performed 7 consecutive lifts per weight condition, thus resulting in a total of 21 trials. Subjects were unaware of the object weight on the first trial, but were aware that it would remain the same within the block of 7 trials. The three weight conditions were presented in a counterbalanced order across CTS patients, and in the same order between each CTS patient and his/her matched control. Subjects were given a 10-s rest period between trials and experimental conditions. The entire experiment lasted about twenty minutes to prevent pain, fatigue, or worsening of the CTS symptoms. None of our subjects reported any of these adverse reactions.lift onset and object hold were used to define task epochs within which forces were analyzed. Object lift onset was defined as the time at which the vertical position of the grip device crossed and remained above a threshold  for 200 ms . Analyses were performed using MATLAB, Excel, and SPSS software. r 200 ms . The endr 200 ms .Digit forces. Digit tangential force (Ftan) is the vertical force component parallel to the grip surface produced by each digit to lift the object  is the force component perpendicular to the grip surface ((a) sum of Fn and Ftan exerted by all digits ; (b) difference between FG at object lift onset and FG during object hold (ΔFG); (c) peak FG and FT rate computed within the period from contact of the first digit  and end of object lift; (d) time of peak FG and FT rate relative to object lift onset; (e) load phase defined as the time between onset of load force and object lift onset ; (f) FG across-trial variability defined as the standard deviation of mean FG averaged across trials 2 through 7 ; (g) FG within-trial variability defined as the standard deviation of FG over the last 2 s object hold and normalized by average FG, i.e., the coefficient of variation; and (g) Fn exerted by each finger expressed as percentage of thumb Fn .e object . Digit n surface . We procMoment of forces. Analysis of moment of force focused on moments exerted in the frontal plane (yz plane) about the origin ‘O’  the sum of all fingers normal forces match thumb normal force, (b) the sum of all finger tangential forces match thumb tangential force, and (c) the sum of moments produced by Fn and Ftan is zero, i.e., the net moment (Mnet) exerted on the object is zero. Conversely, deviations from zero net moments would denote subjects' inability to coordinate the partitioning of forces exerted by the thumb relative to forces exerted by all fingers.net produced on the grip device consists of two moment components: digit normal moment (Mn) and digit tangential moment (Mtan). Our task requires Fn generated by the thumb (FnT) and by its opposing fingers (FnIMRL) to be equal but opposite to each other, therefore Mn can be calculated as:tan is the moment of tangential forces produced by the thumb (FtanT) and by its opposing fingers (FtanIMRL) about ‘O’:IMRL and LT denote lever arm of Ftan at the fingers and thumb, respectively.MPeak object roll. Peak roll was used to further quantify the behavioral consequences of multi-digit force coordination implemented at object lift onset and throughout the lift All of the above variables are standard metrics to characterize force coordination and/or behavioral consequences (object roll) used by studies of precision grip (a) peak object roll, (b) FG at object lift onset and hold, (c) within-trial (object hold) variability of FG, and (d) Mnet, Mn, and Mtan object lift onset, with Weight  and Trial  as within-subject factors, and Group (two levels: CTS and controls) as between-subject factor. Linear regression analysis was performed on Mn vs. Mtan at object lift onset for all trials pooled across weight conditions for each subject to quantify the coordination between the two moment components. Negative correlations between Mn and Mtan denote error compensation acting to minimize across-trial variability in the sign and magnitude of the net digit moment r) was z-normalized before averaging across subjects within each group and paired t-test was used to assess group differences in the r-value and intercept of the linear fits.To determine differences between CTS and controls in the multi-digit force modulation to object weight we performed analysis of variance (ANOVA) with repeated measures on n) to object weight, we performed separate ANOVAs with repeated measures with Weight, Finger  as within-subject factors, and Group as the between-subject factor on SFn at object lift onset and hold. We also used ANOVA with repeated measures with Weight and Phase  as within-subject factors and Group as the between-subject factor on FG. Fisher's z-transformation was performed on SFn before performing statistical analysis. To determine the consistency with which subjects coordinated multi-digit forces, we performed separate ANOVAs with repeated measures with Weight as the within-subject factor, and Group as the between-subject factor, on the across-trial variability of FG at object lift onset and during object hold. To quantify group differences in the within-trial temporal evolution of multi-digit forces, we performed paired t-tests on the average difference between forces at object lift onset and hold (ΔFG). For repeated measures analysis, Mauchly's test was used to test for sphericity. Greenhouse-Geisser correction was used when the sphericity assumption was violated. When appropriate, we performed post hoc comparisons with Bonferroni adjustments. A significance level of 0.05 was used for all comparisons.For the following analyses, we computed the mean and variability of digit forces from trial 2 through 7. To quantify the group differences in the adaptation of digit force sharing patterns  than the subsequent trials. Even though larger weights tended to cause greater peak object rolls , no group difference was observed .Subjects were asked to lift the object while keeping the object aligned vertically. On the first trial, subjects have not experienced the object weight. Therefore, a larger object roll occurred on the first trial  from seven trials for each weight condition performed by a representative CTS patient and his control. Although both subjects increased FG as a function of object weight during object hold, the CTS patient exerted larger FG than his control for all weight conditions. Both subject groups . However, post hoc tests showed that significant force adaption in CTS patients occurred only when comparing the 745 g condition vs. lighter weight conditions. In contrast, controls showed significant force modulation for all pairwise weight comparisons. CTS patients exerted at least 8 N larger FG during object hold than controls at all weight conditions .t groups  increasen as a function of object weight in CTS patients could have been due to subjects gradually increasing Fn together with Ftan until the object could be lifted, e.g., a ‘probing’ strategy . However, even though load phase duration increased with object weight , no group differences or interactions with object weight were found . Therefore, the timing of force development prior to object lift onset was similar in both subject groups. To further explore the mechanisms of force modulation to object weight, we examined the modulation of the peak rate of the sum of all digit Ftan (FT) to object weight and its timing relative to object lift onset. T rate across object weights for one representative CTS patient and her control. For these two subjects, peak FT rate increased with increasing object weight and occurred shortly before or at object lift onset. Scaling of peak FT rate with object weight is considered evidence for anticipatory force modulation  and was found in both CTS patients and controls . We also found that peak FT rate occurred before object lift onset in the majority of trials across all object weights in CTS patients and controls .The above scaling of FG rate also increased with object weight in both groups . However, the extent of peak FG rate modulation to object weight was higher in controls than CTS patients . Peak FG rate occurred ∼200-300 ms before object lift onset in both groups  and relative time to lift onset tended to increase with object weight . We also found a main effect of Trial , time to peak FG rate being longer on the 1st vs. the rest of the trials.Peak FG during object hold occurred in either subject group . However, FG at object lift onset varied significantly from trial to trial , as subjects in both groups adopted a ‘default’ grip force at object lift onset on the first trial only across all weight conditions .No significant trial-to-trial changes in FG produced at object lift onset on the first trial together with FG produced at object lift onset and hold averaged from trial 2 through 7 for CTS and control subjects. Both groups modulated FG at object lift onset according to object weight  on all but the first trial . After the first trial, FG at object lift onset in control subjects was of similar magnitude to that exerted during object hold, indicating that force scaling had occurred prior to lifting the object and that no further modulation occurred after object lift. Subjects exerted smaller FG during object hold than at object lift onset, indicating that further digit force modulation occurred throughout object lift and the early part of object hold . We also found that CTS patients exerted larger FG than controls across all trials and weight conditions . We noted that even after the object was lifted, CTS patients still exerted larger FG than controls . Therefore, even though CTS patients could anticipate object weight similarly to controls prior to object lift onset  and during object hold. Within-trial variability of FG during object hold were similar across groups . CTS and controls exhibited higher across-trial variability in FG at object lift onset than object hold  as a function of time for two representative subjects (A) and averaged across all subjects (B). The control and CTS patient shown in n shortly after object lift onset , with the middle and ring finger exerting the largest share of thumb Fn. However, these two subjects also exhibited small differences in how they shared finger forces relative to thumb Fn. Across all subjects, the middle and ring fingers combined exerted over 60% of thumb Fn . Importantly, no group differences were found in force sharing patterns either at object lift onset  or during object hold .net) is equal to zero. However, if a non-zero Mnet is generated at object lift onset, subjects can generate corrective force responses during the lift but only at reaction time latencies. We found that both subject groups exerted non-zero Mnet of force at object lift onset, but improved with practice as smaller Mnet were exerted after the first two trials . However, CTS patients were farther away from the optimal zero Mnet strategy as they exerted significantly larger net moments at object lift onset relative to controls . Further analysis revealed that this group difference was caused by CTS patients exerting a significantly larger Mtan  but similar Mn. We found that CTS patients used a significantly different tangential force sharing pattern relative to controls . Post hoc tests revealed that this interaction was caused by CTS patients exerting Ftan in the downward direction with the index finger relative to the rest of the digits, whereas controls exerted Ftan in the upward direction with all digits (P<0.05).To prevent large object roll during object lift subjects have to coordinate digit normal and tangential forces at object lift onset such that the net moment , i.e., symmetrical Ftan and collinear Fn. However, if one of these moment components is non-zero , a zero Mnet can still be generated but only if the other moment component (Mn) covaries negatively. To determine the extent to which the two subject groups differed in their ability to coordinate Mn and Mtan, we performed linear regression between Mn and Mtan for each subject and across all weight conditions. n and Mtan acted to minimize across-trial deviations from the required zero net moment. Note, however, that the plots in n vs. Mtan relations from CTS patients than controls, part of which reflects their larger across-trial variability in FG (described above). Lastly, even though both subject groups implemented a negative covariation between the two moment components, this coordination was more effective in controls as indicated by the intercept of the linear fit being significantly closer to zero than in CTS patients .One way of generating a zero M(a) digit force modulation to object weight by CTS patients did not discriminate lighter weights as accurately as controls; (b) CTS patients exhibited a larger across-trial variability in digit force control; (c) unlike controls, after the first trial CTS patients consistently exerted larger forces at lift onset than during object hold; and (d) CTS patients were less able to balance digit forces than controls, resulting in unnecessary net moments at object lift onset. These results are discussed in the context of behavioral consequences of chronic median nerve compression on dexterous coordination of multi-digit forces for whole-hand grasping.Controls and CTS patients were able to grasp, lift, hold, and replace objects of different weights by modulating digit forces accordingly, even though CTS patients exerted larger digit forces than controls. Besides this observation, a more detailed analysis revealed differences in multi-digit force coordination relative to controls: G among thumb and fingers in a similar way across object weights G used by CTS patients, the ability to proportionally scale individual digit normal forces indicates an intact ability to coordinate multi-digit forces. The fact that the object did not move laterally nor was tilted during the lift indicates that patients were able to attain normal force equilibrium among CTS-affected and non-affected digits. Based on evidence from studies of two-digit grasping in CTS patients During multi-digit prehension tasks, healthy adults share total FG in an anticipatory fashion to object weight , but only between these and the heaviest object weight (745 g). The more similar peak force rates across object weights during the load phase suggest a sustained larger force compared to controls, which ensures higher FG at object lift onset to prevent potential object slips (see below). This between-group difference in the lower discrimination of force modulation to object weight emphasizes the role of somatosensory feedback from hand mechanoreceptors for fine control of hand muscles and digit forces. This further indicates that behavioral deficits in CTS patients might be particularly pronounced in tasks that require fine regulation of fingertip forces for manipulating light or fragile objects.However, we also found evidence indicating that force modulation to object weight was less accurate in CTS than controls. Unlike controls, FG) by CTS patients than controls was found at object lift onset and during object hold regardless of object weight  the inability to acquire accurate feedback from CTS-affected digits throughout grasping and manipulation which, in turn, leads to (b) storage of inaccurate sensorimotor memories linking object weight with the forces necessary to efficiently manipulate the object, and ultimately (c) prevents patients from adjusting multi-digit forces to object weight on subsequent trials to the same level of accuracy as controls.Exertion of significantly larger grip forces . However, an important difference between CTS patients and controls was that the latter group anticipated FG used to hold the object before lifting the object, whereas CTS patients further decreased FG after object lift. This suggests that, unlike controls, CTS patients consistently overshot, from the second trial onwards, FG before lifting the object. We rule out the possibility that this consistent overshoot was due to CTS patients having problems with timing the development of FG before object lift onset because time to peak FG rate was statistically indistinguishable from controls. Therefore, we offer two alternative interpretations of this finding. One interpretation is that the decrease in FG following object lift could have been due to CTS patients sensing, throughout object lift and in the early part of the hold phase, that FG were larger than necessary to prevent object slip and modulated them accordingly during object hold. However, this interpretation raises the question of why, if CTS patients were able to sense the mismatch between actual and required FG, they continued to overshoot FG prior to object lift on each of trials 2 through 7, and not properly plan FG at lift onset. A non-mutually exclusive and more likely interpretation is that the consistently larger FG at object lift onset than during the static phase is a strategy learned during every-day activity to compensate for the deficit in tactile feedback signaling distinct events of the manipulation, e.g., force development prior to object lift, the dynamic force modulation during object lift, and isometric force generation during object hold. Specifically, CTS patients may prefer to use an extra safety margin of grip forces particularly during the dynamic phase for which the necessary digit forces might be more challenging to accurately anticipate than during the static hold phase. Given the above-described CTS patients' decreased ability to discriminate object weights and larger across-trial force variability, we propose that the consistently larger forces at object lift onset vs. object hold represent a compensatory strategy that reflects different requirements and challenges to grasp stability associated with dynamic vs. static phases of manipulation.Analyses of trial-to-trial force adaptation to a given object weight, as well as across object weights, provided further insights into CTS-induced sensorimotor deficits underlying hand control. Specifically, after experiencing object weight on the first trial, both groups used smaller Fous lift  [30], [4G (Mn) with that generated by tangential forces (Mtan) n and Mtan as required by the task, i.e., by implementing a negative covariation between the two moment components. However, CTS patients exhibited a significantly larger non-zero net moment than controls at object lift onset, thus indicating a lower ability to coordinate grip and tangential forces, as well as significantly different tangential force sharing patterns relative to controls. Importantly, our findings suggest that this grasp execution ‘error’ would occur every time that an object is lifted.Our task required subjects to distribute forces among the digits such that no net normal force or moment would be generated during object lift and hold. When using a whole-hand grasp, production of a net zero moment on the object can be attained through an infinite number of solutions to cancel out the moment generated by FThe generation of larger moments on the object at lift onset could result from the interaction of several factors. For example, the inability to accurately integrate somatosensory feedback from each digit would interfere with the spatial and temporal coordination of hand muscle activity to balance the two moments. Furthermore, intrinsic hand muscles, some of which may be affected in CTS, play an important role in fine regulation of moments of force In conclusion, CTS does not affect macroscopic features of grasp control when adapting multi-digit forces to object weight and we propose that such modulation is mediated by proprioceptive inputs spared by CTS. However, relative to controls, multi-digit force coordination in CTS patients was characterized by the consistent use of excessively large digit forces despite repeated exposure to manipulating the same object weight, higher across-trial digit force variability, lower discrimination of force modulation to lighter object weights, and a lower ability to minimize net moments on the object at lift onset. We interpret these behavioral deficits as resulting from impaired nerve function . Such impairment may account for patients' reduced ability to form accurate sensorimotor memories of previous manipulations, or represent learned compensatory strategy to maximize grasp stability. Further studies are needed to distinguish between these two interpretations and quantify the behavioral effects of CTS on fine manipulation.


Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS  system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications. Most bacteria live in a continuously changing environment. In order to survive, they have evolved many signalling pathways to sense various extra- and intra-cellular signals and then respond accordingly by controlling their cognate downstream gene regulatory networks to adapt to the changes. Proteins involved in cell signalling pathways are typically modular, comprising highly conserved domains and residues. Therefore researchers have engineered variations of these proteins to endow them with customized characteristics. For example, both directed evolution and structure-based rational design approaches have been applied to modify scaffold  and reguTCS  transduction is the most prevalent signalling paradigm that bacterial cells use to sense and respond to the outside world . TCS typIn more complex signalling, additional transmitter and receiver modules are found in phosphorelays, which may serve to integrate additional inputs into the signalling pathway . ImportaIn order to propagate signals, modules of a signalling pathway have to physically interact. Hence binding specificity between modules is a key requirement for signalling fidelity. The specificity between modules is determined by the physicochemical complementarity of their interacting surfaces. Indeed, synthetic rewiring of signalling pathways is challenging because of our limited understanding of how to engineer novel complementary protein or domain surfaces with the desired functionalities. Despite a wealth of available structural data of complementary interfaces between proteins and domains, our knowledge remains largely descriptive and only a few generalizable patterns of protein and domain interfaces exist . ComputaTrans-acting modules are predicted to require high physicochemical specificity between them in order to avoid unwanted cross-talk between different signalling pathways comprising conserved domains. In cis-acting modules, where the components are fixed with one another and in stoichiometric ratios, high specificity may not be required due to the very high local concentrations of the linked modules. However, the covalent link between signalling modules may allow for direct and thus stringent output control. Although this hypothesis has yet to be experimentally proved, it is worth noting that cis-acting modules are much more prevalent at the end (receiver-output) of signalling pathways [Sinorhizobium meliloti [Another important consideration when designing synthetic signalling pathways is the identification of which is the relevant step of the pathway to be rewired. pathways , where cmeliloti . Interesmeliloti .Although building protein signalling circuits as a means to rewiring cell signalling is far from routine, several encouraging examples have demonstrated the feasibility and promise of synthetic protein-based signalling circuits to program cellular behaviours. To date, various signalling and regulatory proteins have been modified by domain combination, swapping or evolution to render them responsive to novel input signals or able to act on non-cognate target substrates. In the following sections, we discuss the current advances and challenges in this area where different chimaeric regulatory proteins are engineered alongside the various stages of the cell signalling pathways.Since the HKs in TCS systems have a modular structure comprising two conserved domains (the sensor and transmitter domains), we can hypothesize that the HK sensor domain can be swapped between different signalling proteins so that the downstream network of a signalling pathway can respond to a non-native input signal.Escherichia coli by fusing the photoreceptor domain of the phytochrome Cph1 protein from cyanobacterium Synechocystis to the intracellular signal transduction domain of the E. coli EnvZ kinase [lacZ reporter under the RR OmpR controlled promoter can express β-galactosidase to turn the cells black when exposed to red light. Using this chimaeric light sensor kinase, the authors then followed up this work by subsequently creating a bacteria-based dark/light edge detection programme [In a pioneering proof-of-principle bacterial photographing system, a synthetic light-sensitive chimaeric sensor kinase (Cph1–EnvZ) was made in Z kinase , yieldinrogramme .E. coli EnvZ kinase can be rationally altered to phosphorylate the non-cognate RRs CpxR and RstA instead of the cognate OmpR in vitro. In another study, Xu et al. [Myxococcus xanthus to chemotactically respond to nitrate, which the wild-type strain cannot respond to, independently of cellular metabolism and physiology. Both of these examples indicate the possibility of rewiring the cell native TCS by rationally designing the signalling kinase to act on a non-cognate RR, thus initiating a reprogrammed signalling response.Alternatively, swapping or modifying the transmitter domains, the downstream RRs can be activated by an alternative input signal. As a proof-of-concept of this idea, Skerker et al.  showed tu et al.  created The output domain of a RR is at the end of TCS pathways and is typically a DNA-binding module needed to trigger transcription of downstream genes A. TherefIn principle, any two or more domains can be fused by introducing tethering linkers between them. This is readily done and of great value for instance in co-localization studies, by fusing GFP to a protein of interest. However, in signal transduction pathways, specificity between modules is essential for signalling fidelity and control stringencies. In the aforementioned Cph8 chimaera, signalling fidelity downstream of the EnvZ module was provided by the native EnvZ/OmpR intracellular signalling pathway. The functional rewiring relied on exchanging the extracellular sensor moiety in a way that it could still communicate signal perception to the HK domain, where the transmembrane domain must play a structural coupling role across the membrane. The authors constructed a small chimaeric library after defining domain boundaries based on sequence alignments and tested a number of constructs in which the Chp1 moiety length differed while the EnvZ transmembrane moiety was fixed. This was necessary to obtain a functional chimaera, indicating that precise design of the domain architecture underlies rewired signalling.Box 1•Study protein functioning and evolutionThe major challenge in rewiring signalling pathways is to design novel interactions capable of faithfully communicating between modules. The nature of the functional engineered constructs can reveal design principles, i.e. how module specificity is achieved and how it may have evolved. Rewiring the phosphotransfer reaction between transmitter and receiver modules in TCS systems  has esta•Inform protein engineering approachesAs the number of synthetically rewired signalling pathways increases, so will our understanding of the principles governing signal transduction pathways, facilitating future approaches. Although every signalling connection is different, the component parts are conserved and the underlying biochemistry is similar, enabling rational design that can be based on first principles.•Design proteins with bespoke functionsIn the present article, we have only discussed the strategies that are currently employed to generate interoperability between signalling modules, in particular those culminating in gene transcription. Such systems may be more accessible since the engineering approach still comprises an element of trial and error and transcriptional output can be readily selected for. However, the potential to recombine other domain functionalities in novel combinations may apply more generally, for example combining metabolic pathways that are hitherto not connected.•Perturbation tools that act rapidly, reversibly and are tunableRewiring signalling pathways offers new ways to perturb and study cells. By making new network connections that are not present in the native cell, we can investigate how the cell copes. Such perturbations are conceptually different from conventional gene deletions of transcription factors, where the information flow downstream of the deleted gene is disrupted. Rewired signalling pathways can allow for temporal and tunable  transcri•Allow regulon-wide transcription controlOur approach aims to rewire signalling that would allow the control of transcription of the extensive nitrogen regulon. Most synthetic biology approaches have focused on changing gene regulatory DNA sequences (mainly promoters) for transcription control. To do so for entire regulons would involve the introduction of many such regulatory elements and also require knowledge of all operons of the regulon. Rewired synthetic transcription factors may therefore represent a more accessible and comprehensive mean for regulon-wide transcription regulation.•Applicable for metabolic engineeringOne of the challenges in metabolic engineering is the complexity of feedback loops that operate in homoeostasis, impairing efforts to optimize the production of desired natural products. Rewired signalling can uncouple such feedback loops while retaining transcription control of genes involved in the pathways leading to the desired product, providing considerable scope for their application in biotechnology.bEBPs are modular, typically comprising a C-terminal DNA HTH (helix–turn–helix) binding domain, a central catalytic AAA+ (ATPase associated with various cellular activities) domain and an N-terminal regulatory domain  Figure A. Upon pin vivo  functional modules is an attractive possibility, in accordance with the conceptual approach in synthetic biology to rationally engineer systems through the assembly of predefined components. Such approaches are also attractive because the majority of proteins in Nature have evolved through the recombination of genetic elements that code for functional protein modules . However


Sarcomas represent a clinically and biologically diverse group of malignant connective tissue tumors. Despite aggressive conventional therapy, a large proportion of sarcoma patients experience disease recurrence which will ultimately result in mortality. The presence of a unique population of cells, referred to as cancer stem cells (CSCs), have been proposed to be responsible for refractory responses to current chemotherapies as well underlying the basis for metastasis and relapse of disease – clinical corollaries to what has been termed the CSC hypothesis. The presence of CSCs have been suggested in a variety of hematologic and solid malignancies, and only more recently in sarcomas. Based on our current understanding of normal stem cell biology and evidence obtained from the study of malignant hematopoietic and solid tumors, researchers have identified candidate cell surface markers , biochemical markers , and cytological characteristics  that may identify putative sarcoma CSCs. In this review, we explore the current state of evidence that may suggest the existence of sarcoma CSCs. We present research in osteosarcoma, the Ewing’s sarcoma family of tumors, rhabdomyosarcoma, as well as other sarcoma subtypes to describe commonly used molecular and biochemical markers, as well as techniques, used in the identification, isolation, and characterization of candidate sarcoma CSCs. We will also discuss the current controversies and challenges that face research in sarcoma CSC. Sarcomas are a biologically and clinically heterogeneous group of malignant connective tissue tumors originating from mesenchymal or ectodermal tissue. Sarcomas clinically span the entire age spectrum with an annual incidence ranging from 1 to 6 per 100,000 persons, and comprising 1.5% of all malignant tumors in adults and up to 8% in children. The categorization of different sarcoma types have classically been based upon the histological resemblance of a given sarcoma subtype to the adult tissue which it most closely resembles. There are currently over 50 sarcoma subtypes described underscoring the clinical and biologic diversity of this group of malignant cancers. Despite conventional multi-modality treatment approaches , patients diagnosed with sarcoma experience disproportionally higher rates of morbidity and mortality compared to patients with other cancers. Long term survival rates average ∼50% in adults and up to 70% in children, but a significant proportion suffer recurrence and dissemination of disease despite aggressive therapy with subsequent rates of survival no higher than 20%. Investigations into the biology of cancer resistance to therapy and cancer relapses despite initial remission of disease have resulted in the development of a cell model that may account for observed cancer treatment failures and disease recurrences – the cancer stem cell (CSC) hypothesis.In this review, the CSC hypothesis is discussed in terms of how it may contribute to investigations exploring the origins of sarcomas. We will also discuss methodologies utilized to characterize and isolate so-called sarcoma CSCs and evaluate the evidence to support the existence of sarcoma CSCs in different sarcoma subtypes.Malignant tumors, like the normal tissues from which they arise, are composed of diverse cell populations. Within any given normal tissue reside a subpopulation of cells with the distinct ability to self-renew and are imbued with the ability to give rise to more differentiated progenitor cells – the tissue stem cell. Similarly, a malignant tumor is composed of a heterogeneous population of cells with different degrees of differentiation, proliferation, and tumorigenic potential. A subset of these cells, termed the CSC, possesses the ability to self-renew and generate the diversity of cancer cells that constitute the malignant tumor reminiscent of the tissue stem cell. The hypothesis that CSCs lie at the apex of a cellular hierarchy wherein the CSC gives rise to distinct populations of tumorigenic and non-tumorigenic cells has evolved over the past decade and has uncovered complex models that seek to account for the phenotypic and functional heterogeneity of tumors  self-renew, and (2) possesses the ability to generate the heterogeneous cell lineages which will constitute a tumor  cells  in conjunction with other cell surface markers or functional assays. However, it is important to note that expression of any or all of the stem cell-associated genes is not sufficient for the identification of sarcoma CSCs as these genes are expressed by normal stem and committed progenitor cells. It is believed that re-expression of stem cell-associated genes by cancer cells may simply reflect the malignant transformation of cells and not necessarily unique to the CSC.One of the characteristic features of CSCs is its inherent or acquired resistance to cytotoxic chemotherapy agents. One explanation for the relative chemoresistant properties of CSCs involves the overexpression of ABC (ATP-binding cassette) multidrug efflux transporters. Capitalizing on this feature of CSCs, several investigators have utilized a technique involving the use of fluorescent DNA-binding dyes Hoechst 33342 or Rhodamine 123 to identify putative CSCs through fluorescence-activated cell sorting (FACS)  catalyzes the oxidation of intracellular aldehydes and biogenic amines and has also been described to play an important role in drug detoxification in a variety of normal and malignant cells. Work in leukemia cells lines, as well as in normal hematopoietic and neural stem progenitor cells, have revealed significant expression of ALDH up to 200-fold  commonly utilized in identifying ALDHHigh cell sub-populations functions primarily as a substrate for the ALDH 1A1 and 3A1 isoforms. It remains to be determined if other ALDH isoforms are equally present and expressed in sarcoma CSCs and if alternate ALDH isoforms can be measured utilizing this technique.Since progenitor stem cells can be characterized by elevated intracellular ALDH activity, investigators have examined if high ALDH activity could identify CSC populations /severe combined immunodeficiency (SCID) mice which leads one to question the validity of CD133 expression in identifying a tumorigenic CSC population in osteosarcoma.A subsequent study by Tirino et al.  evaluateOther cell surface markers have been identified which appear to more selectively enrich for cells with tumor-initiating properties. Adhikari and colleagues examined the expression of two mesenchymal stem cell markers, CD117 and Stro-1, and discovered that mouse and human osteosarcoma cell lines positive for both CD117 and Stro-1 (DP or double positive cells) readily formed spherical colonies, more readily formed tumors in a nude mouse model in comparison to cells negative for both CD117 and Stro-1 (DN or double negative cells), had higher metastatic potential in an orthotopic NOD/SCID mouse model, exhibited increased chemoresistance when exposed to the chemotherapy agent doxorubicin, retained multipotent differentiation ability, and able to reconstitute DP and DN populations upon serial transplantation  and which possessed increased tumorigenic capacity compared to ALDHLow cells, increased proliferative and clonogenic abilities, showed increased expression of stem cell markers, and self-renewal capacity. In contrast, ALDHLow cells were incapable of forming tumors in NOD/SCID mice. In a follow-up study, pre-treatment of ALDHBr cells with bone morphogenetic protein-2 (BMP-2), which belong to a class of molecules known to be important in bone and cartilage formation, can effectively inhibit the tumor-initiating properties of ALDHBr cells . However, in a similar study evaluating the ALDHBr cells derived from the osteosarcoma cell line MG63, ALDHBr as well as ALDHLow cells equally showed the ability to reconstitute the original whole cell population putting into question the validity of ALDH activity as a specific sarcoma CSC marker   derived from pediatric bone marrow samples and demonstrated the generation of a CD133+ subpopulation of cells with stem cell properties. However, CD133+ cells derived from hMSCEWS/FLI-1 were unable to form tumors in NOD/SCID mice suggesting that EWS/FLI-1 expression in human MSCs is insufficient for tumorigenesis and despite the change in phenotype reminiscent of stem-like cells.Similar in approach to the identification of CSC subpopulations in osteosarcoma cells, Suva et al.  were theHigh cell fraction. Interestingly, the tumorigenic CD57High fraction did not consistently correlate with the coexpression of CD133 and CD57High cells absent CD133 expression were highly tumorigenic suggesting that other cell surface markers can similarly be utilized to identify cells with CSC properties. It is important to point out that CD57Low cells were also capable of inducing tumor formation and maintain this ability with serial transplantation, albeit at a much lower capacity, precluding the use of cell surface markers such as CD57 or CD133 in identifying the stem cell population in Ewing sarcoma.In contrast to the previously described work, Jiang et al.  found hein vivo was not performed. SP cells have also been observed in other Ewing sarcoma cell lines . Cells from the ALDHHigh sub-population demonstrated CSC properties and were highly tumorigenic. ALDHHigh cells were also more resistant to the cytotoxic effects of chemotherapy drugs. Interestingly, a selective inhibitor of EWS/FLI-1 (YK-4-279) was effective in inhibiting the growth and tumorigenic capacity of both ALDHHigh and ALDHLow cells indicating the dependence of the ALDHHigh population on EWS/FLI-1 oncogene expression  and the less common PAX7/FOXO1 have classically characterized a more clinically aggressive subset of RMS referred to as the alveolar variant. The more common embryonal RMS variant is not associated with any characteristic cytogenetic abnormalities and tends to have a more favorable prognosis. Also similar to both osteosarcomas and Ewing sarcoma, RMS involves multi-modality treatment with generally favorable survival outcomes in the localized disease setting  and poor outcomes for metastatic disease .Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma and the third most common extracranial solid tumor encountered in children while being exceedingly rare in adults. Similar to Ewing sarcoma, ∼20–30% of RMS cases involve the presence characteristic chromosomal translocations resulting in the production of an aberrant fusion oncogene. The OCT-4, NANOG, c-MYC, SOX2, and PAX3) and formed tumors at lower cell densities compared to adherent cells. The investigators further sorted the cells derived from spherical colonies and isolated a CD133+ cell fraction. The CD133+ cells, in contrast to CD133− and unsorted cells, were able to generate tumors in NOD/SCID mice at lower cell densities and were more resistant to treatment with the chemotherapy drugs cisplatin and chlorambucil. Furthermore, analysis of CD133+ expression in patient-derived tissue samples revealed that high CD133+ expression correlated with inferior survival compared to low or intermediate expression levels in patients with the embryonal RMS variant. Hence, CD133 expression in RMS may not only be useful in enriching for candidate sarcoma CSCs, but may also have clinical utility in predicting survival outcomes. It remains to be seen if selection based on ALDH activity, perhaps in conjunction with CD133 expression, would further enrich for cells with stem cell-like properties.There remains little published work regarding the determination of a CSC-like population in RMS. Komuro et al.  showed tHigh activity (ALDHHigh). Cells doubly positive for CD133 and ALDHHigh were more clonogenic and tumorigenic when compared to parental cells or cells singly positive for either CD133 or ALDHHigh. Similarly, chondrosarcoma cells derived from primary tumors and enriched for CD133 also showed potent tumorigenic potential  will be paramount to any progress achieved in the study of stem cell biology. In the future, the unequivocal identification and validation of the existence of CSCs in sarcomas, or any other cancer, coupled with the development of drugs that will specifically target this population, may allow for a paradigm shift in how we view the evolution of cancers, as well as how we develop future strategies for effectively eradicating cancers in children and adults.Work in normal tissue stem cells and CSCs derived from other solid tumors have expanded our current understanding of both normal and malignant stem cell biology and have led to the development of novel techniques to identify and study the CSC population within a heterogeneous tumor. In this review, we have discussed the data with regards to the promise of cell surface markers such as CD133, CD117, or Stro-1 in the identification of candidate sarcoma CSCs. Refinements in flow cytometric techniques have allowed for the identification of SPs of cells which exhibit stem cell-like properties including self-renewal, tumor initiation and serial propagation, and inherent resistance to cytotoxic chemotherapy agents. At the very least, the techniques and assays described have allowed for the isolation of cell populations that contain within it the ever elusive CSC. Further technical refinements and use of novel techniques such as genetic lineage tracking may someday allow for the identification and isolation of a more pure population of CSCs from sarcomas – a goal yet to be achieved. In light of recent work suggesting the ability of a CSC to interconvert between tumorigenic and non-tumorigenic states depending upon tumor microenvironmental cues (Quintana et al., 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 light of recent research, Gordon Mansergh and colleagues discuss barriers to effective implementation of HIV pre-exposure prophylaxis for men who have sex with men. Pre-exposure prophylaxis (PrEP) with anti-retroviral (ARV) medications is partially efficacious for preventing HIV infection among men who have sex with men (MSM) and heterosexuals.As PrEP becomes available and prescribed for use among MSM a better understanding of willingness to use PrEP and avoidance of condom use are needed so that behavioral programs and counseling may be enhanced for maximum benefit.Targeted messaging will be needed about ARV prophylaxis for various at risk populations, but the general message should be that condoms continue to be the most effective way to prevent HIV transmission through sex and that PrEP is an additional biomedical intervention.As new effective biomedical intervention methods, such as PrEP, become available language about “protected” and “unprotected” sex, which used to exclusively mean condom use, will need to adapt.Pre-exposure prophylaxis (PrEP) to prevent HIV infection with anti-retroviral (ARV) medications was found to be partially efficacious among men who have sex with men (MSM) Colleagues PrEP implementation among MSM poses several challenges, including (a) understanding of PrEP use and use preferences; (b) PrEP implementation costs to individuals and the health care system, and the associated epidemiological impact; (c) effective messaging about PrEP to various MSM-related audiences; and (d) implications of PrEP on the dialogue and language for research and PrEP use in practice.Prior to known efficacy, ARV use and sharing for HIV prophylaxis among MSM was minor More surveillance and assessment is needed to monitor and better understand, among other issues, reasons for differential willingness to use PrEP and avoidance of condom use among MSM—so that behavioral programs and counseling may be enhanced for maximum benefit for both ARV prophylaxis and condom use. Ongoing community-level behavioral and clinical research is needed to determine the prevalence and effectiveness of intermittent dosing; behavioral risk substitution for condom use, including use of seroadaptive behaviors ; preferences about PrEP formulation ; provider knowledge, perceptions, and clinical behavior; and PrEP costs and access through different service provision settings.PrEP should be prescribed and clinically monitored by a health care professional to ensure appropriate use and effectiveness  to an era of multiple efficacious interventions


The plasma membrane is a composite material, which forms a semi-permeable barrier and an interface for communication between the intracellular and extracellular environments. While the existence of membrane microdomains with nanoscale organization has been proved by the application of numerous biochemical and physical methods, direct observation of these heterogeneities using optical microscopy has remained challenging for decades, partly due to the optical diffraction limit, which restricts the resolution to ~200 nm. During the past years, new optical methods which circumvent this fundamental limit have emerged. Not only do these techniques allow direct visualization, but also quantitative characterization of nanoscopic structures. We discuss how these emerging optical methods have refined our knowledge of membrane microdomains and how they may shed light on the basic principles of the mesoscopic membrane organization. Cell functions strongly rely on its capacity to interact with neighboring cells and extracellular environment. Vital interactions such as metabolic material exchange and biochemical signaling are mediated by the plasma membrane, a semi-permeable barrier which covers the cell surface and separates a cell from its surrounding environment. While the seminal observations of a cell membrane dates back to the early 17th century with the first optical microscopy images of fly eyes and cork tissues , the com2+ sparks . The transport of vesicles enriched with raft markers such as cholesterol and sphingolipids has been shown to be polarized toward the apical surface . While s+ sparks , which i+ sparks . In the + sparks  and cell+ sparks . TogetheIn situ measurements using fluorescence microscopy methods, such as fluorescent polarization or fluorescent life time imaging microscopy (FLIM) used to study fluorescent lipid analogs have shown the co-existence of different lipid phases , the calibrated spectra obtained from very simple membrane models composed of only a few types of lipids at predefined ratio are often too simplistic to interpret the spectra obtained on cell membranes, which are far more complex in terms of lipid and protein compositions. Second, biochemical methods, such as nonionic detergent-soluble assays, can induce artificial clustering . Third,  objects . MoleculFigures 1A,D) and then allow the reconstruction of images at higher resolution  can decode the conventional inaccessible high-resolution structural information into Moiré images obtained by individual excitation pattern , stimulated emission depletion (STED) microscopy can improve the lateral resolution down to a few ten nanometers  can be very toxic for live samples and can cause photo-bleaching during imaging. Finally, the efficiency of STED effect requires a perfect alignment of the excitation and the depletion lasers, which might be complex to achieve.Resolution can also be increased in another elegant way. In fluorescence microscopy, the size of the focal spot at the sample plane usually defines an unresolvable region. The resolution can, however, be improved by reducing the size of the region from which the excited molecules fluoresce. By superimposing a hollow-patterned laser  on the conventional excitation laser to specifically quench excited molecules at the rim of the focal spot through stimulated emission . To date, there are two types of microscopies, which implement these principles using photoswitchable molecules. The first type called photoactivated-localization microscopy (PALM), is based on photoactivable fluorescent proteins  has provided the best spatial resolution among other super-resolution optical microscopy techniques, its time resolution is still limited (>0.05 Hz) due to the need to collect a large amount of single molecule images. Furthermore, PALM/STORM data require cautious interpretation. There are indeed concerns about clustering artifacts, which might arise from the oligomeric nature of fluorescent tags . MoreoveThe development of localization microscopy (PALM and STORM) is currently a very active field with rapid development in different areas. The possibility to photoswitch a large range of commercially available fluorescent tags using reducing buffer without the need of oxygen scavengers  makes itIn the near future, the combination of PALM/STORM with EM . These microdomains/clusters have been observed not only in fixed but also in live cells  have been shown to form either elongated structures with size up to few micrometers or point-like puncta of 100–300 nm in size. While elongated nascent adhesion complexes exhibit growth, fusion and dissolution at few minutes time scale, the punctae can be stable during a few 10s of minutes. The dynamics of theses structures differs from one cell type to another  with different protrusive motions . Some otTable 1). Measuring the relative distance between protein components in complexes is key to determining the existence of molecular interactions. Some proteins, which colocalize at the diffraction-limited resolution turn out to have little overlap or even to create interwoven arrangements as revealed by high-resolution image . Also, proteins that were suggested to be in separate microdomains as observed by immunoelectron imaging, a method which is prone to clustering artifact . Superresolution optical methods form currently an active field with fast and continuous improvements. In a near future, it is likely that we will gain access to the dynamics of objects with molecular scale precision (2–10 nm) at subsecond time scale. Moreover, combining super-resolution fluorescence microscopy for specificity and localization precision with EM or AFM for resolution will provide molecular details on the organization of supramolecular structures (Emerging optical methods have provided a powerful palette to study different aspects of membrane microdomains (ructures . CombinaThe 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 hematopoietic stem cell transplantation, donor selection is based primarily on matching donor and patient HLA genes. These genes are highly polymorphic and their typing can result in exact allele assignment at each gene (the resolution at which patients and donors are matched), but it can also result in a set of ambiguous assignments, depending on the typing methodology used. To facilitate rapid identification of matched donors, registries employ statistical algorithms to infer HLA alleles from ambiguous genotypes. Linkage disequilibrium information encapsulated in haplotype frequencies is used to facilitate prediction of the most likely haplotype assignment. An HLA typing with less ambiguity produces fewer high-probability haplotypes and a more reliable prediction. We estimated ambiguity for several HLA typing methods across four continental populations using an information theory-based measure, Shannon's entropy. We used allele and haplotype frequencies to calculate entropy for different sets of 1,000 subjects with simulated HLA typing. Using allele frequencies we calculated an average entropy in Caucasians of 1.65 for serology, 1.06 for allele family level, 0.49 for a 2002-era SSO kit, and 0.076 for single-pass SBT. When using haplotype frequencies in entropy calculations, we found average entropies of 0.72 for serology, 0.73 for allele family level, 0.05 for SSO, and 0.002 for single-pass SBT. Application of haplotype frequencies further reduces HLA typing ambiguity. We also estimated expected confirmatory typing mismatch rates for simulated subjects. In a hypothetical registry with all donors typed using the same method, the entropy values based on haplotype frequencies correspond to confirmatory typing mismatch rates of 1.31% for SSO versus only 0.08% for SBT. Intermediate-resolution single-pass SBT contains the least ambiguity of the methods we evaluated and therefore the most certainty in allele prediction. The presented measure objectively evaluates HLA typing methods and can help define acceptable HLA typing for donor recruitment. The typing has historically been performed using serological antibody tests, which are able to identify HLA protein variants on the surface of the cell using antigen-specific antibodies DNA-based methods identify HLA alleles by interrogating the nuclear DNA sequence and can result in different levels of ambiguity depending on typing methodology or test kits used. HLA typing methods, their corresponding formats and abbreviations used in this paper are given in In HSCT the selection of donors for a patient in need of a transplant is based primarily on HLA matching, and the lower the ambiguity of typing the easier it is to determine the probability of allele level match during the donor search Some previous work has been done in measuring typing ambiguity – first a measure developed by Helmberg et al. Both previous typing ambiguity studies used allele frequencies in their computations and, as we will later show, fail to demonstrate the advantage of linkage disequilibrium information contained in haplotype frequencies when it comes to reducing ambiguity and improving predictions of patient-donor matching. We use haplotype frequencies and show that the ambiguity is reduced considerably compared to using allele frequencies, proving that this advance in strategy for identifying matched donors has had a significant positive impact. In addition, we take this methodology further and use it to evaluate several different typing methods, and directly compare them with respect to the inherent ambiguity measured by entropy. To measure the impact of the typing method ambiguity in more relatable terms, we also developed a measure we call Confirmatory Typing (CT) Mismatch Rate, which gives the average probability across a set of patients that a mismatch would occur between the patient and donor when a high resolution confirmatory typing is performed on the ambiguously-typed donors in a uniformly-typed registry.We show that entropy can be used to objectively compare methods of HLA typing to each other in terms of the information they provide, in the context of each individual population. Our results show that intermediate-resolution single-pass sequence-based typing (SBT) reported in genotype list format contains the least ambiguity and, therefore, the most certainty in allele prediction across all populations. We examine the benefit of using haplotype frequencies in entropy calculations versus allele frequencies. Neighboring HLA and non-HLA genes are highly correlated and major efforts have been directed at describing linkage disequilibrium (LD) across the region The naming of HLA alleles is standardized and regulated by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System Before DNA-based HLA typing methods were developed, serological testing identified sets of alleles with similar reactivity. Two-digit level resolution is the lowest HLA typing resolution reported by typing laboratories today. For these lower-resolution formats, alleles in the same family as A*01:01 are reported as A1 using serological methods (abbreviated SERO), or as a truncated result of intermediate-level DNA-based typing (referred to as DNA2 in this text) as A*01 or A*01:XX.allele codesA commonly used intermediate-resolution format is the one using NMDP Sequence-specific oligonucleotides (SSO) typing results are reported in this format for this study, where each allele code represents two or more alleles. For example, an allele reported as A*01:AB can be either A*01:01 or A*01:02, and an allele reported as A*26:JGSJ can be any of the following three: A*25:13, A*26:01, A*26:52. Therefore, the number of combination for an ambiguous allele pair reported in this format increases multiplicatively, that is, the allele pair  will have six possible pairwise combinations . Ambiguous sequence based typing (SBT) is reported in the format of genotype lists for this study, that is, in the form of several possibilities for pairs of alleles an individual carries . Because in single-pass SBT results, some ambiguous genotype lists cross several allele families, allele codes could be used to represent all typing results. However, genotype list representation has the advantage of showing that some genotype possibilities, added implicitly when compressing to allele code format, are not possible.We used high-resolution haplotype frequencies generated from unrelated donors from the National Marrow Donor Program (NMDP) database for four principal population categories defined by the United States census: African American (AFA), Caucasian (CAU), Hispanic (HIS) and Asian/Pacific Islander (API) To generate simulated typings for different HLA typing methods, we first sampled 2 haplotypes from high resolution population haplotype frequency data set http://www.ebi.ac.uk/imgt/hla/ambig.html) and reported the typing in the genotype list format.While we use HLA nomenclature Version 3 style formatting to describe HLA alleles in this paper, we simulated the four typing methods for Version 2.28 of the IMGT-HLA database, to more closely match the time in which the typing results used to generate the haplotype frequencies were reported. To generate serologic typing (SERO), we used the HLA dictionary, which allows each HLA allele to be mapped to a serologic equivalent (e.g. B*15:02 maps to B75) X, Shannon's entropy (H) is defined as:Shannon's entropy quantifies the amount of uncertainty or disorder associated with a particular system, and is widely used in a variety of applications, such as genetics Entropy can be thought of as the ambiguity or impurity present in a system of interest. If, in a set of typing results for one individual, all of them are equally likely (frequent) then the entropy is the highest as we have the least information to choose the most likely real genotype.nH is the entropy of n typings, then it is maximal for outcomes of equal frequencies, that is, To illustrate the usefulness of entropy in measuring typing ambiguity we show an example of two typing results with the same number of ambiguities . Both ofA, B, and D are typed loci, N is the total number of haplotypes and allele entropy for each locus.The HLA haplotype frequencies we used in this study were estimated by the expectation-maximization algorithm (EM) described in haplotype entropy, we do the following. For each ambiguously typed three-locus genotype, g = (AaBbDd), we generated all possible haplotype pairs and their frequencies using imputation as described in haplotype entropy in the same manner as described for the case of allele entropy.To compute locus entropy using haplotype frequencies, or As a side note, an ambiguous typing with many possible alleles at each locus can result in a large combinatorial number of possible haplotype pairs. Given a fully heterozygous case of three-locus un-phased genotype with Besides objective evaluation of typing methodologies employed in typing the HLA region, using the entropy approach to measure ambiguity has another application from a clinical perspective, namely its direct relationship to confirmatory typing (CT) mismatch rates. For a given patient, high resolution CT is done to confirm the patient-donor match from a selected set of donors. A case where the high resolution typings mismatch is called a CT mismatch. We compute CT mismatch rates on the same simulated donor sample by comparing the ambiguous typing and the exact haplotype pair that was used to generate that ambiguous typing. As described in a previous section, each ambiguous genotype can generate multiple HLA haplotype pairs, the true one being the haplotype pair we used to simulate the ambiguous typing. The CT mismatch rate for each locus is computed as the summation of frequencies of all allele pairs . SBT had the lowest entropy and therefore the least inherent ambiguity when it comes to resolving HLA alleles at the locus level, across all populations. As expected, serology produced the lowest resolution typing and had the highest entropy. SSO typing was far more ambiguous than SBT across all evaluated datasets, reflecting both SBT's more complete coverage of polymorphic positions in the exons and the benefits of using genotype list format for SBT typings rather than the NMDP allele code format we used for SSO. Locus entropies obtained for SBT, SSO, allele family level DNA2 and SERO typing methods are shown in haplotype entropy). However, a dramatic decrease in entropy occurred across all typing methods when we used haplotype instead of allele frequencies. For example, the allele entropy of SSO typing in Caucasian group is 0.49 while the haplotype entropy is an order of magnitude lower at 0.0477. This decrease is due to some ambiguity being resolved by LD information provided in haplotype frequencies, and is successfully captured by Shannon's entropy. When we used haplotype instead of allele frequencies, we got the same ambiguity ranking  for averTo demonstrate the impact of typing method ambiguity in a clinical setting we computed CT mismatch rates, which give the average probability that a mismatch would occur between a patient and donor when high resolution confirmatory typing is performed on the ambiguously typed donors in a uniformly typed registry. CT mismatch rates computed on the same sample of 1000 simulated donors are shown in We have shown that entropy can be used to objectively compare methods of HLA typing in terms of the information they provide. The calculation of per-locus entropy using haplotype frequencies has a direct application in measuring the impact of using haplotype frequencies to predict the likelihood of allele match for stem cell registry matching algorithms. The LD information contained in haplotype frequencies reduces the entropy considerably compared to using allele frequencies, showing that this strategy for identifying matched donors has a significant positive impact.No objective quantitative comparisons between SBT and SSO methods have been available to date. Typing laboratories may choose SSO methods over SBT methods primarily based on cost savings achieved due to easier set-up, staff training, pre-packaged kits, and automation. However, these apparent cost savings may have a price of higher typing ambiguity. We have shown that single-pass SBT typing performs far better at distinguishing alleles compared to mid-1990's-era SSO typing. However, currently available SSO typing kits used for recruitment typing have more oligonucleotide probes and thus are able to distinguish more alleles, which may result in entropy as low as that of single-pass SBT. Given equal cost, registries should utilize laboratories that employ HLA typing methods that achieve lower entropy for their population.Our objective measure of typing ambiguity can be advantageously applied to the continual improvement of all methods of HLA typing. Design of SSO kits could be done in silico using population haplotype frequencies and sequence information from the IMGT-HLA database As new alleles are discovered, SSO kits are often altered to add more probes so that typing results do not cross allele families and thus meet current guidelines for acceptable recruitment typing. These new probes will not decrease entropy appreciably as the frequency of a newly discovered allele tends to remain very low.p contributes p. More formally, Because of sample size limitations, many of the rare alleles described in IMGT-HLA were not observed in our samples. However, rare alleles do not have a significant impact on entropy calculations. Owing to the logarithmic nature of Shannon's entropy, an allele with a very small frequency Our methods of HLA typing method evaluation can also be applied to next-generation sequencing technologies. Recently the Roche 454 sequencing platform has been employed for HLA typing in research rather than recruitment A consideration specifically related to the HLA typing method is the representation of the ambiguous allele data derived from the HLA typing, which was also measured using entropy. The 2-digit DNA typing resolution is in practice a result of incomplete reporting of SSO, SSP, or SBT typing data. The higher entropy of this type of data shows the value in reporting the complete information available from the HLA typing platform rather than rounding to the allele family level. The genotype list representation yields a slightly lower entropy than the NMDP allele code representation. Genotype list representation allows for the exclusion of some genotypes that have been ruled out by the HLA typing method, but would still be included in the Cartesian product of the alleles listed in the NMDP allele codes.Note that, in some populations, the HLA-B locus presents higher values of entropy when typed using 2-digit DNA methods than when typed at the serological level . This isThis analysis provides a path for defining acceptable HLA typing for recruitment as minimum requirements for entropy scores as a measure of typing ambiguity and for HLA data representation guidelines as a way to ensure that genotype lists are reported. Single-pass or highly automated SBT can result in HLA typings that cross allele families, which does not meet current minimum standards for recruitment typing at NMDP, yet we show that it provides a high-quality low-entropy HLA typing. In fact, we had to use the genotype list representation for the simulation of single-pass SBT typings because some allele combinations result in HLA typings for which no NMDP allele codes have been created due to required minimum standards that HLA allele codes do not generally cross allele families We observe variation in entropy for the same HLA typing method between populations and loci. For example, In addition to absolute differences in entropy between populations, we also observed differences between population groups in the effectiveness of using haplotype frequencies in decreasing haplotype entropy. Having higher levels of LD can improve the predictive capability of haplotype frequencies, and so African population samples with lower LD could have higher entropy than European populations, with higher LD, for this reason. In the opposite direction, higher HLA diversity would lead to higher entropy in African population samples than in European samples. The API sample may have relatively higher entropy than other population samples because the API frequency distribution constitutes an average of the frequency distributions of multiple distinct populations, and thus may be skewed more towards rare types than other populations in this study. If API entropy were evaluated using more detailed race subcategories , we would expect lower entropy values because the HLA diversity of each respective sub-region would be lower. The size of the population sample used to generate haplotype frequencies also plays a role in the entropy calculations in that a relatively larger sample, as we had for CAU compared to the other races, would give higher entropy. Because of these multiple confounding factors affecting entropy, we urge caution in using entropy as a measure to compare the HLA characteristics between samples of different ancestry. There are some caveats in that the simulation framework implicitly has no sampling error or estimation error in the haplotype frequencies. In practice, uncertainty in the frequency estimates will lead to higher entropy, so our results should be treated as a practical lower bound.For interpretation of between-locus entropy differences, we turn to the history of HLA nomenclature in that the allele families and serologic types were defined primarily using European samples. The naming of allele families was based loosely on serologic categories, and at some point in history newly discovered serologic patterns were no longer used to split up allele families. The discovery of new alleles also has an impact on entropy in that some populations have not been well-characterized for HLA and some individuals may have as yet un-described alleles that can result in some hidden entropy. In evaluating entropy at the locus level, we see that at the allele family level, the HLA-DRB1 locus has a higher entropy than HLA-A and HLA-B loci. The number of allele families defined for HLA-A and HLA-B is higher than that of HLA-DRB1, giving a lower entropy for typing resolution at the 2-digit or serologic levels, all else being equal.Stem cell registries have been accruing HLA typing results for over 25 years, with continual advancement in typing methods during this period. The proportion of donors typed by each method changes over time in a searchable registry due to new donor recruitment, roll-off of donors exceeding the maximum age, reporting of primary HLA data, prospective typing, and high resolution typing on behalf of patients. With analysis of changes in HLA typing data for each donor over their time on the registry, it becomes possible to chart decreasing entropy in HLA typing over time and determine which typing methods were primarily responsible for this decrease. Entropy could also be applied as a selection factor for prospective typing projects in which some donors are upgraded to lower ambiguity typings.In summary, the application of Shannon's entropy as a measure of HLA typing ambiguity has benefits throughout the lifecycle of HLA typing: in reagent design, lab reporting standards, donor recruitment typing guidelines, and registry matching algorithm performance evaluation.Text S1Frequency Inferred Typing (FIT) Index. FIT index is proposed as the first attempt to characterization of HLA typing kits. It describes the probability of correct allele pair assignment for an ambiguous typing result, and is calculated as the negative log of the probability of a wrong allele pair prediction.(DOC)Click here for additional data file.