Previous studies demonstrated that while the mandible assumes its resting position in space, antagonistic muscles should assume minimal muscle activity within a spatial range. This zone of mandibular rest has been mapped using physiologic parameters of muscle activity and incisal spatial kinematics. This case study expands on previous research by monitoring incisal and posterior jaw position and includes lateral pterygoid muscle activity, thus allowing for determining the spatial range including additional relevant coordinates and muscle activity. Four positions were evaluated: a maximum physiologic open position, a maximum physiologic closed position, physiologic rest position, and maximum physiologic protrusion position. Within the physiologic zone of rest formed by these 4 positions, the vertical and anterior borders of the envelope of function may be documented for the incisal and posterior mandible in true 3-dimensional fashion to assist the clinician in determining a physiologic interocclusal freeway space and vertical dimension of occlusion. Advantages and limitations are discussed. Clinical application of a maxillomandibular orthopedic relationship including nerves, muscles, ligaments, dental occlusion, and both temporomandibular joints for optimal function can be challenging. However, failure to consider these basic dynamic elements of dental occlusion in providing temporomandibular dysfunction (TMD) treatment may result in a functional compromise. Functional compromise may either predispose or ultimately lead to muscle pathology, destruction of the dentition, supporting structures and/or the temporomandibular joints. Even mild forms of compromise result in dysfunction of the neuromuscular apparatus and associated structures .In an age that demands evidence-based care, combining objective measurements of physiologic and anatomic components is a natural evolution from treatment based on subjective and mechanical concepts, to computer-assisted technologies when seeking objective answers to complex physiologic functions .Posture may be defined as the relative position of the various parts of the body with respect to the egocentric coordinate system, the exocentric coordinate system, and the geocentric coordinate system . PosturaThe orientation of any body part, such as the mandible, may be described in terms of these frameworks and it reflects a dynamic process.The habitual postural position of the mandible when at rest in the upright position and the condyles are in a neutral unstrained position in the mandibular fossae is also called “postural position” . AnatomiThrough the use of surface electromyography (SEMG), muscle activity of the masticatory system can be objectively measured. The repeatable postural range at minimum baseline SEMG levels has been shown to be consistent with physiologic rest position and jaw posture . PhysiolTo achieve physiologic rest, the masticatory and cervical muscles must be balanced and coordinated with respect to left and right sides and the muscles of the posterior neck must be in balance with the anterior cervical muscles. This represents a stable mandibular position concurrent with a stable head position , 8 as onAdditional research supporting SEMG clinical techniques in dentistry are numerous and conveniently provide guidelines where SEMG may be employed within treatment approaches including appliance therapy –19, orthThrough the use of incisal based, magnetic kinematic technology and SEMG, Mazzocco et al.  proposedA postural envelope defines the limits of stability in space in all directions. As long as the sway envelope stays within the limits of stability, balance is maintained. If sway exceeds these limits, strategies involving neighboring regions must be employed to regain balance. If an individual's center of gravity is more anterior, posterior, or lateral than center, a smaller sway envelope is tolerated before losing balance . The demA stable, physiologic occlusion would consider the spatial position from which the postural resting position of the mandible is maintained within a spatial zone that offers relatively minimal, balanced muscle activity in order to be able to provide symmetrical and prompt activation of the jaw musculature as the teeth are brought into occlusion, where maximal muscle recruitment is available and where the mandible may return to the physiologic resting range promptly and symmetrically.Previous clinical works have been limited to incisal vertical dimension and lack documentation of posterior jaw position and an anterior boundary. Knowledge of the anterior border would enable clinicians to establish a jaw position within a boundary which, if exceeded, would require the neighboring systems to accommodate to maintain stability. Furthermore, posterior mandibular position is also of great importance as loss of posterior occlusal support leads to noticeable cranial condyle movement  as well The concept that disturbance to the activity of the lateral pterygoid muscles plays an important role in the etiology of TMD and is widely accepted; however, there is a dearth of scientific evidence to support this clinical notion and the role of the lateral pterygoid muscle in normal function remains controversial. Motor unit studies at computer tomography confirmed that sites demonstrate and confirm that the inferior head of the lateral pterygoid (IHLP) and the superior head of the lateral pterygoid (SHLP) are active during opening, protrusion, and contralateral jaw movements, while a minority of SHLP were active during closing movements. These percutaneous results also provide clinically valuable information and have demonstrated that the SHLP and IHLP were minimally active when the jaw was in the clinically determined postural jaw position –33.The presence of elevated activity in the lateral pterygoid muscle at resting jaw posture may be of clinical significance in that variations in the level of lateral pterygoid muscle activity could influence and/or assist in determining the anterior positioning of the condyle in relation to the disc and eminence, as well as the positioning of the jaw at postural position.Through the works of Hiyama et al. , 35 and In this case study, the physiologic window of postural rest position inclusive of anterior and posterior mandibular position relative to the maxilla and muscle activity of the intraoral lateral pterygoid placement was explored.Case study design including a 43 year old male with normal occlusion, with temporomandibular dysfunction. The subject was a unilaterally involved combined myogenous and arthrogenous patient (groups I and II according to RDC/TMD)  and repoAdditional objective data included right joint sounds analysis that revealed possible crepitus, adhesions, scarring, and degenerative issues. Imaging results revealed slight osteogenic degeneration, sclerosis, and posterior and superior displacement of the right condyle. SEMG data was collected from the bilateral temporalis anterior (TA), masseter (MAS), and suprahyoid (DIG) sites using pregelled bipolar Ag/AgCl surface electrodes (Noraxon USA) with a diameter of 1 cm and an interelectrode distance of 2 cm center to center, parallel to muscle fiber direction over the muscle belly as recommended . The ref2 and a decentralized snap. The electrodes were trimmed and adhered to reach an interelectrode distance of approximately 1 cm and secured on the mucosa in the buccal vestibule distal to the maxillary tuberosity with an adhesive. Prior to electrode placement, the mucosal tissue was dried using a standard paper towel and then cleaned with a sterile alcohol prep pad.The activity of the bilateral lateral pterygoid (LP) sites was recorded intraorally by way of surface electrodes per Hiyama et al. , 35. TheAll SEMG data was collected with a telemetry device, preamplified and band-pass filtered in hardware between 20 and 500 Hz (Noraxon USA) with input impedance greater than 100 Mohms and sampled at 1000 Hz with an overall gain of 1000. Data was rectified and smoothed at 100 ms (movag) in WinNMBite software (Zebris GmbH). Spatial analysis of the lower jaw was determined in conjunction with SEMG using the Jaw Motion Analysis system (JMA) from Zebris Medical GmbH . The JMAThe JMA utilizes ultrasonic sensors attached to a light head frame (approximately 3.9 ounces) and emitters (weight—approximately 10 grams) that were attached to the mandibular dentition via Stomahesive (Convatec Inc). The resolution of the ultrasonic path is approximately 0.085 mm with a maximum sampling rate of 200 Hz (sampling rate was 75 Hz for the case study).Documentation of the subject's maxillary right and left distobuccal molars and the incisal points were determined using the pointer system. The lower jaw is then referenced to these three points in space. These coordinates used are relative to the maxillary plane and provide incisal and posterior mandibular coordinates relative to the maxilla.The subject was seated upright comfortably in a straight-back chair without head support, with the Frankfurt occlusal plane parallel to the floor. After SEMG electrode placement was completed, baseline/postural resting values of each site were recorded .Following the baseline recording, the subject was instructed on how to perform a maximal voluntary contraction (MVC) for the masseter, temporalis anterior, and pterygoids. SEMG baseline/postural data of the temporalis anterior and masseter sites were normalized to their corresponding mean SEMG values recorded during a two second maximal clench. With the teeth apart where maxillary and mandibular incisal position was even, a recording during a 2 second maximally resisted protrusive effort was performed . SEMG baUltralow frequency transcutaneous electrical stimulation  was chosen as the relaxation method due to research demonstrating the efficiency in relaxing masticatory muscles –44. FollPhysiologic rest position of the mandible may be described as a position assumed by the mandible within the physiologic window . This poAs the masticatory muscles are stimulated to contract, under the influence of ULF-TENS, the mandible is propelled through interocclusal rest (freeway space) forming an extended computed trajectory . The firThe second position, “maximum physiologic closure” was identified based upon resting SEMG parameters and has been described by Mazzocco et al. . MaximumA third position, “maximum physiologic protrusion”  was idenSpatial position for incisal, right and left posterior jaw positions relative to their corresponding physiologic positions are presented in Physiologic rest was spatially determined when all elevator muscle levels were at relatively minimal levels and balanced. Incisal vertical demonstrated the greatest discrepancy. When establishing a spatial position of “maximal physiologic opening”, the suprahyoids demonstrated an initial elevation in activity in 2 of the 3 efforts. The 2nd effort demonstrated a simultaneous response of the right lateral pterygoid and suprahyoids. When establishing a spatial position of “maximal physiologic closing”, the lateral pterygoids consistently demonstrated a bilateral elevation in activity prior to the temporalis anterior and masseter recording sites in all 3 trials. When establishing a spatial position of maximal physiologic protrusion, the right lateral pterygoid site was consistently the first to demonstrate an elevation in activity over all muscles, in all 3 trials.As previously indicated, once adaptive capacities have been surpassed, pathology develops. This is obvious within the masticatory and associated structures. Shortened muscles can initiate pain by affecting intramuscular nociceptors  or throuIn the case study presented, each spatial position was documented when there was evidence of muscle activity above clinically acceptable baseline levels (hypertonicity). Therefore, the resting physiologic envelope of postural jaw function in this subject would be slightly superior to maximal physiologic opening, slightly inferior to maximal physiologic closing, and posterior to maximal physiologic protrusion, and it approximates the computed trajectory to be within a physiologic muscular position .The data would suggest that in this subject, an increase in vertical dimension may be considered to be compatible within the physiologic range. The vertical dimension compared to anterior/posterior positioning may require the largest change to reach a homeostatic state with mild differences between right and left posterior mandibular positions. If vertical dimension changes were to be considered on this subject, the vertical dimension would allow relatively moderate changes  and suggest mild changes in 3-dimensions to the subject's right in 3 of the 4 positions documented. Also, any anterior change may be limited in this subject as all positions were less than a millimeter anterior to CO to be within the physiologic range. These data suggest that the vertical dimension of both the anterior and posterior jaw may endure the greatest change, where the anterior/posterior range is much more sensitive to change.Furthermore, it was found that in establishing the resting physiologic envelope, during maximal physiologic closing, the lateral pterygoid sites, not investigated in previous studies, were consistently found to be responsive prior to prime mover sites . During maximal physiologic protrusion, the lateral pterygoid consistently demonstrated earlier activation than any other site, and during maximal physiologic opening, it was found to be elevated approximately 30% of the time concurrent with the suprahyoid sites.This physiologic resting range of the anterior and posterior mandibular positions may be documented utilizing jaw motion analysis  when combined with SEMG. The vertical physiologic envelope may be smaller than previously suggested in TMD subjects. The function and activity of the lateral pterygoid suggest that this may be the limiting factor. The anterior border (maximum physiologic protrusion) may be documented if concurrent lateral pterygoid activity is recorded. Caution should be exercised if jaw positioning is considered anterior to a trajectory created by TENS if the lateral pterygoid activity is not measured.Intraoral lateral pterygoid recordings may lend important information in the construction of “pull-forward” appliances for sleep apnea patients to ensure the chosen appliance and/or position is within the physiologic range.Establishing the physiologic borders through the combined use of SEMG and posterior jaw position during a bite registration procedure may allow the clinician to objectively document the range of physiologic rest of the anterior and posterior lower jaw in the vertical and anterior dimensions. Theoretically, this would allow the clinician to ensure the chosen bite registration position is within physiologically tolerable limits, thereby lessening the amount of adaptation that would be required. Within the physiologic zone of rest formed by these 4 positions, the vertical and anterior borders of the envelope of function may be documented for the incisal and posterior mandible in true 3-dimensional fashion to assist the clinician in determining a physiologic interocclusal freeway space and vertical dimension of occlusion.This information presents a number of appealing implications as follows.This and previous works demonstrated tremendous promise for clinical applications for intraoral SEMG of the lateral pterygoid sites if commercially available customized miniature intraoral surface electrodes became available and an established electrode placement protocol based on verified CT sites is tested more thoroughly for clinical applications. Should these parameters be met, use of surface electromyography of the lateral pterygoids would provide noninvasive, functionally diagnostic treatment information not attainable by other means.In the computed trajectory, one limitation is that functional anatomy dictates that the mandible does not move in a straight line. Therefore, it is seemingly intuitive that any sort of computer generated trajectory is an approximation and the addition of SEMG may be necessary to ensure that any chosen treatment position is within physiologic parameters.Furthermore, there are obvious biomechanical implications related to the cranial-skeletal system that should be considered. Cranial asymmetries are not uncommon, often associated with craniofacial asymmetries, as well as cerebral asymmetries –51. In vThe author acknowledges some clear limitations. In addition to the aforementioned parameters, the dimensional changes by way of orthodontic or reconstructive or other methods that were considered when referring to the data are speculative only and were not explored and thus, the rigorous research is required to establish validity of the concept. The analyzed individual represents only a convenient sample, and the extrapolation of the results or to different TMD diagnostic groups, should be done with caution. However, the concept does follow basic physiologic principles and appears to be promising.


It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity. The mature brain undergoes continuous morphological changes in response to external and internal stimuli through the turnover and reorganization of neuronal networks and synapses -3. Thesevia heterotrimeric G-proteins in the so-call Wnt/Ca2+ pathway . DKK1 is hardly detectable in the healthy brain, but it is strongly induced in brain tissue from AD patients or from patients with temporal lobe epilepsy and hippocampal sclerosis . One of the neurodegenerative diseases in which Wnt signaling has been extensively documented is AD. More than a decade ago, it was suggested that sustained loss of function of Wnt signaling would determine the onset and development of AD . PreseniMany other components of the Wnt signaling pathway have been implicated in the molecular pathology of AD. In Drosophila models of neurodegeneration, Wnt dysregulation can lead to neurofibrillary pathology  and the Recent studies have found that the Wnt5a ligand and its receptor Fz5 were up-regulated in the brain of a mouse model of AD and in cultured cortical neurons by Aβ exposure . Intereset al., [Microglia plays an important role in inflammation and the hippocampus is densely populated with this type of cell. During aging and pathological conditions, the activated form of microglia has been found to be increased in the hippocampus ,134 and et al.,  reportedRecently, an elegant functional genomic analysis has shown a major role of Wnt dysregulation in brain samples from FTD associated with mutations in the progranulin gene and an important increase of the Fzd2 in a knockout progranulin gene mouse model . Together, the above revised studies suggest that excessive and reduced Wnt function acting through the three signaling pathways may be involved at different steps of the neurodegenerative process in the adult brain. The specific disease outcome of such Wnt uncontrolled function may depend on the neuronal metabolic context and the cellular type  where Wnt signaling is exacerbated or inhibited. in vitro inducing them to become neurons at particular concentrations. Moreover, experiments in which lithium is administered to a mouse strain encoding a double-mutant form of APP, have shown to stimulate adult hippocampal progenitor cells proliferation and neuronal differentiation along with the improvement in cognitive functions through the inhibition of GSK3β and subsequent activation of Wnt/β-catenin signaling [et al. [An ideal compound with neuroprotective potential against hippocampal dysfunction should decrease or delay neuronal death, enhance neurogenesis, improve cognitive function, and control neuroinflammation. In view of the role played by Wnt pathways in maintaining neuronal homeostasis in the healthy hippocampus and their potential participation in brain disease it is feasible to suggest the targeting of Wnt signaling pathways to modify the progression of synaptic impairment, inflammation and ultimately neuronal death. In addition, the fact that Wnt signaling is involved in different steps of neural stem cell renewal suggests that purified Wnt proteins can be used as tools for cell replacement therapies in the treatment of neurodegenerative diseases and brain injury . Adult nignaling . In a re [et al.  show tha [et al. . Wnt signaling also mediates a positive neuron-astrocyte crosstalk for neuroprotection as has been found in mesencephalic neurons where Wnt1 effects depends on the astroglial response to oxidative stress and inflammation after neuronal injury, and requires Fz1 receptor and β-catenin stabilization to transmit pro-survival signals into the neuronal nucleus .However to deep into the design of a molecule with Wnt positive effect, it should be considered that a fine balance between different Wnt ligands exists in the brain. For example, experimental models of behavior suggest that the inability of rats to tolerate stressful environments and the resulting anxiety may be associated with the enhanced expression of the Wnt2 gene and the resistance to the increase of Wnt7b levels in the ventral tegmental area  thus strPrevious work has proposed that different canonical and non-canonical Wnt agonists, acting at different levels can be protective in AD . ParticuGiven that Fz2 was found selectively elevated in the brain of patients with FTD and Fz2 activates a non-canonical Wnt pathway it is suggested that modulation of this pathway could be therapeutically beneficial for this demential illness.However pharmacotherapy aimed at modulating Wnt pathways should consider the timing for application as well as the targeted cell type and brain region in order to provide specificity to the molecular processes underlying a specific neurodegenerative disease. For example, in terms of brain inflammation enhanced β-catenin signaling in microglia could be either beneficial or detrimental for the disease outcome. Also, Wnt activates proliferative pathways that may lead to uncontrolled cellular proliferation and tumor growth but as previously discussed may also represent a powerful therapeutic strategy for neuronal protection.Wnt signaling regulates many aspects of hippocampal development and continues critically involved in the adult regulating plasticity mechanisms. However the complexity of events that are under the control of the Wnt pathways poses the need to perform further studies that unravel the biological roles of the highly dynamic Wnt signaling through different stages of brain maturation as well as in disease. The expression of different genes and proteins of the Wnt signaling pathway in regions of the central nervous system that control learning and memory opens the possibility to understand how they can influence the processes of neuronal plasticity that are altered in dementias and neuropsychiatric disorders. Thus, new pharmacological developments can be specifically targeted at a particular disease entity to enhance treatment efficacy.


The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex.Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14.The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant  and the Vif PPL → AAA mutant  could reduce CBF-β binding. (3) EloB-EloC but not CBF-β could greatly enhance the folding of full-length Vif in Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization. Vif, as an accessory gene of HIV-1, plays pivotal roles in both the early and late steps of the HIV-1 life cycle [The human immunodeficiency virus type 1/acquired immunodeficiency syndrome (HIV-1/AIDS) epidemic is a global health concern. fe cycle . Vif is fe cycle , and APOfe cycle  are sufffe cycle -11. Theyfe cycle . Vif coufe cycle , the trafe cycle ,13-17.As the formation of the E3 ubiquitin ligase complex is a prerequisite for Vif to neutralize APOBEC3 proteins, the functional domains that Vif utilizes to recruit the E3 complex have been well studied ,19. The EloB and EloC are two ubiquitous proteins that form a heterodimer complex. They were originally found in the Elongin (SIII) complex, which was proposed to contain three subunits: EloA, EloB and EloC ,38. EloADrosophila melanogaster and Caenorhabditis elegans[EloB is a highly conserved cellular factor in humans, rats, s elegans. As a mes elegans,48. The s elegans,48, whers elegans,50. In ts elegans.Escherichia coli, and in cells, both the introduction of the VifΔSLQ mutant, which dramatically impairs Vif-EloB-EloC binding, and that of the VifΔPPL mutant, which is considered to reduce Vif-EloB binding, were found to impair Vif-CBF-β binding [In this study, we demonstrated that the interactions between Vif and EloB-EloC are important for CBF-β recruitment to Vif. Both knockdown of endogenous EloB (together with EloC) and over-expression of EloBΔC34, an EloB mutant with a 34-residue deletion in the COOH-terminal tail, impaired the Vif-CBF-β interaction and stabilized the A3G level. The interaction with EloB-EloC, but not CBF-β, was shown to greatly increase the solubility of full-length Vif in  binding ,36. Moreet al. [EloB mRNA  or empty plasmid VR1012 in the presence of siEB1 or siNC. In the siNC-transfected 293 T cells, the over-expressed Vif could efficiently reduce the intracellular level of A3G  is important for Vif-CBF-β binding. To verify this hypothesis, we transfected vectors expressing Vif or Vif-SLQ into 293 T cells. Both of these expressed proteins were tagged with a C-terminal Myc epitope. Vif-SLQ contains mutations of critical residues in the Vif SOCS box (SLQ to AAA), which can significantly disrupt the interaction of mutant Vif with EloB-EloC. The results showed that Vif and Vif-SLQ were expressed at comparable levels . However, we observed that the protein level of E4orf6 could be slightly down-regulated by over-expression of EloBΔC34 (data not shown).We have presumed that EloB prevented degradation of Vif through the same sites by which it interacted with EloC. To verify this hypothesis, we constructed three mutants, VR-EBΔ9-17-HA, VR-EBΔI34-HA and VR-EBΔ69-72-HA, each of which contained a deletion in a region in the EloB UbL domain involved in the contact with EloC  are highly important for the recruitment of CBF-β to Vif. Based on our results, we conclude that EloB can directly affect the assembly of the Vif-CBF-β-Cul5 E3 complex as EloC does by interacting with the PPLP motif of Vif. We identified that the EloB C-terminus, which is not necessary for EloC binding and for E4orf6-mediated depletion of P53, may play a role in improving Vif function. Therefore, the EloB C-terminal tail may be investigated as a promising drug target. Moreover, we found that over-expressed EloB could remarkably stabilize Vif mainly though the same region (residues 9-14) it used to bind EloC. And this important point further expands the diversity of functions of UbL proteins. Thus, our observations extend the current understanding of the function of the Vif-CBF-β-Cul5 E3 ligase.E. coli. In these plasmids, sequences encoding the following proteins were cloned between the indicated restriction sites: Vif, XbaI and BamHI; CBF-β, EcoRI and HindIII; EloC, SacI and KpnI; and EloB, BspEI and MluI [The polycistronic expression system pST39/pET3aTr  was used to produce the plasmids pST39-Vif, pST39-EloB, pST39-EloC, pST39- CBF-β, pST39-Vif-CBF-β, pST39-Vif-EloB, pST39-Vif-EloC, pST39-Vif-EloB-EloC, pST39-Vif-EloB-CBF-β, pST39-Vif-EloC-CBF-β and pST39-Vif-EloB-EloC-CBF-β for expression of proteins or protein complexes in and MluI . The CBFPstI and BamHI restriction sites. VR-Vif-HA, VR-Vif, VR-VifΔSLQ, VR-Vif-Myc, VR-VifΔSLQ-Myc, VR-EloB-Myc, VR-EloB-HA, VR-EloC-HA and pcDNA3.1-A3G-HA have been previously described [VR-EloB/VR-Flag-EloB and VR-EloBΔC34/VR-Flag-EloBΔC34 were generated by subcloning EloB/Flag-EloB and EloBΔC34/Flag-EloBΔC34 into the VR1012 vector at the escribed ,27,77. Vescribed .The infectious molecular clone pNL4-3ΔVif was obtained from the National Institutes of Health AIDS Research and Reference Reagents Program (NIH-ARRRP), Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID) in Germantown, MD.2. The following antibodies were used in this study: anti-HA mouse monoclonal antibody (mAb) , anti-Myc mouse mAb , anti-Flag mouse mAb , anti-P53 mAb , anti-human ribosomal P antigen , anti-tubulin mouse mAb (Covance), anti-Cul5 rabbit polyclonal antibody (pAb) (Santa Cruz Biotechnology), anti-ElonginB rabbit pAb , anti-ElonginC , anti-CBF-β (Santa Cruz Biotechnology), anti-Vif antibody  and a monoclonal anti-HIV-1 capsid antibody generated from an HIV-1 p24 hybridoma (NIH-ARRRP) to detect Pr55Gag and CAp24. Plasmid transfections into 293 T cells were performed with Lipofectamine 2000 according to the standard protocol provided by the manufacturer .HEK293T  cells were purchased from the American Type Culture Collection . MAGI-CCR5 cells  were obtained through the NIH-ARRRP. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% COEloB and β-actin was performed using the 2× QuantiTect™ SYBR Green PCR Master Mix (Qiagen), C1000™ Thermal Cycler  and PCR primers for EloB  and β-actin .The following double-stranded siRNAs specific to human EloB were purchased from Shanghai GenePharma Co., Ltd : EloB-siRNA1 (siEB1), GGGAAGCAGUGCCAAUGAATT; EloB-siRNA2 (siEB2), UGACCAACUCUUGGAUGAUTT; EloB-siRNA3 (siEB3), GACGAUGGCCAAGAGCAGATT. The AllStars Negative Control siRNA was obtained from Qiagen . Hiperfect transfection reagent (Qiagen) was used for transfection of 293 T cells with EloB siRNAs at a final concentration of 20 nM according to the manufacturer’s recommendations (Qiagen). Protein expression was monitored by immunoblotting 3 days after transfection. Efficiency of siRNA silencing of EloB was evaluated both by evaluating relative gene expression using qRT-PCR and protein expression by Western blot. Total cellular RNA was isolated from the siRNA-transfected 293 T cells using TRIzol (Invitrogen) and used as the template in cDNA synthesis with the Promega Reverse Transcription System . qRT-PCR analysis for NdeI and BamHI restriction sites (in bold) were included in the primers to facilitate cloning into pET3aTr.The cDNA acquired above was also used as the template for CBF-β amplification using the following primers based on primers described before : forwardg and then incubated with anti-HA Ab-conjugated agarose beads (Roche) at 4°C for 3 h. Alternatively, the pre-clarified lysates were incubated with mouse anti-Myc (Millipore) for 3 h, and Protein G-agarose was then added for a 3-h incubation at 4°C. Subsequently, the samples were washed three times with washing buffer , boiled in SDS sample buffer and then analyzed by SDS-PAGE and immunoblotting.At 48 h post-transfection, 293 T cells were harvested, washed with cold PBS and solubilized in lysis buffer  plus Complete protease inhibitor cocktail tablets  at 4°C for 1 h. The cell lysates were clarified by 30 min of centrifugation at 10,000 × Virus particles were harvested 48 h after transfection, and culture supernatants obtained were clarified by centrifugation at 3000 rpm for 10 min and then filtered through a 0.22-μm pore size membrane. Virions were pelleted through a 20% sucrose cushion by ultracentrifugation at 26,000 rpm for 2 h at 4°C in a Beckman SW40 rotor. For immunoblotting, virions were disrupted in radioimmunoprecipitation assay (RIPA) buffer. Virus infectivity was examined by the MAGI assay as described previously ,78. Two 293 T cells were grown to confluence in 6-well culture dishes. At 36 h post-transfection, cells were treated with CHX (Sigma–Aldrich) at the final concentration 100 μg/ml for various time periods, lysed and analyzed by Western blotting.E. coli BL21 (DE3) cells containing appropriate pST39 constructs. Cells were grown in Luria-Bertani (LB) medium with 50 mg/L ampicillin at 37°C until an optical density (OD) of about 0.8 was reached. The proteins were over-expressed at 37°C for 7 h by induction with isopropyl-D-thiogalactopyranoside . Harvested cells were resuspended in PBS (pH 7.4), homogenized by sonication and then clarified by centrifugation at 16,000 × g for 1.5 h. For solubility analysis, the supernatant was carefully removed, and the pellet was washed with PBS twice and then resuspended in a volume equal to the original volume of the supernatant.Individual proteins and protein complexes were expressed in Cells and viruses were harvested at 48 h post-transfection and solubilized with RIPA buffer. Samples were boiled in SDS sample buffer for 20 min, subjected to Tricine-SDS–PAGE and then transferred onto nitrocellulose membranes . Secondary antibodies used in this study were alkaline phosphatase-conjugated affinipure goat anti-rabbit IgG, goat anti-mouse IgG and goat anti-human IgG . Immunoreactions were detected with 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium chloride (NBT) solutions.CBF-β/CBF: Beta core binding factor beta; Cul5: Cullin5; EB/EloB: ElonginB; EBΔC34: ElonginB mutant with a deletion of the 34 amino acids in the C-terminal tail; EC/EloC: ElonginC; HIV-1: Human immunodeficiency virus type 1; mAb: Monoclonal antibody; pAb: Polyclonal antibody; qRT-PCR: Quantitative real-time reverse transcription polymerase chain reaction; RNAi: RNA interference; siEB: EloB-specific siRNAs; siRNA: Small interfering RNA; siNC: Negative Control siRNA; UbL: Ubiquitin-like; Vif: Virus Infectivity Factor; VifΔSLQ: The Vif SLQ → AAA mutant; VifΔPPL: Vif PPL → AAA mutant.The authors declare that they have no competing interests.XDW, XHY and WK conceived and designed the experiments and wrote the paper. XDW, XYW, TZ and HW performed the experiments. XDW, HHZ and MYL analyzed the data. JWW, DLL, JYZ, XL, JXW and BY helped design the study and provided reagents. All authors read and approved the final manuscript.


Legionella pneumophila and Vibrio cholerae, which cause Legionnaires’ disease and cholera, respectively. The “Legionella quorum sensing” (lqs) or “cholera quorum sensing” (cqs) genes encode enzymes that produce and sense the AHK molecules “Legionella autoinducer-1”  or cholera autoinducer-1 . AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic “fitness island” and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens  QS-depeN-acyl-L-homoserine lactones (AHLs) ..N-acyl-LLegionella pneumophila and Vibrio cholerae colonize several environmental niches and are important opportunistic human pathogens that cause the life-threatening diseases “Legionnaires’ pneumonia” or “cholera”, respectively . Th. ThV. chin vitro the enzyme catalyses the formation of a carbon-carbon bond between (S)-2-aminobutyrate (SAB) and decanoyl-Coenzyme A (CoA) to produce (S)-3-aminotridecan-4-one (Am-CAI-1)  . Synthetbacteria .S)-adenosylmethionine (SAM) was identified as a much more efficient biosynthetic co-substrate than SAB for CqsA to produce the novel compound 3-aminotridec-2-en-4-one (Ea-CAI-1)  followed by an NADPH-dependent reduction catalysed by a dehydrogenase .,87.S)-ad10 acyl tail , V. cholerae produces and responds to AHKs with other acyl tail lengths [V. cholerae CqsA shows promiscuous substrate specificity and also forms Ea-C8-CAI-1 from SAM and octanoyl-CoA. Still, V. cholerae CqsA prefers the acyl CoA substrate C10-CoA over C8-CoA, and this substrate preference is also reflected by the in vivo production profiles of CAI-1 molecules detected in native cultures supernatants (concentration ratios of C8-CAI-1:Ea-CAI-1:CAI-1 = 1:7.4:11.8).In addition to CAI-1 derivatives harbouring a C lengths ,70,84. VL. pneumophila LqsA, which produces either LAI-1 (C12 acyl tail), or to a smaller extent CAI-1 (C10 acyl tail) and derivatives with C11 or C13 acyl tails [E. coli. The physiological specificity of LqsA might be more stringent, and presumably, dodecanoyl-CoA is used preferentially by L. pneumophila as a co-substrate to synthesize LAI-1 favored over the hydroxyl modification , which only allows binding of CAI-1 derivatives with a C8 but not with longer acyl tails [V. harveyi CqsS preferentially binds Ea-C8-CAI-1, and the presence of the enamine group at the C3 moiety is critical for downstream signaling.The position corresponding to cyl tail . Thus, is C8-CoA , V. cholyl tails . FurtherL. pneumophila sensor kinase LqsS by LAI-1 has not been shown experimentally. However, in analogy to V. cholerae CqsS, the motifs located in the first three trans-membrane domains that are essential for the interaction with CAI-1, are conserved at the corresponding sites in LqsS . In contrast, the tryptophan at position 104 in CqsS is replaced by a serine in LqsS (S111), which possibly indicates a preference for LAI-1 molecules with hydroxyl modifications. Furthermore, at the position that discriminates acyl tail length in Vibrio spp. , LqsS harbors small amino acids (T175/L176), suggesting that the LqsA/LqsS system might show relaxed specificity similar to V. cholerae CqsA/CqsS. Finally, LqsS and CqsS also differ in the gatekeeper motif, which defines binding of the polar head group (F160/F162/F166 vs. I166/F168/A172).Direct binding and stimulation of the  in LqsS . MoreoveV. cholerae CqsA/CqsS system produces and detects three different AHK molecules: (1) CAI-1, (2) amino-modified CAI-1 analogues , and (3) Ea-C8-CAI-1 (but not C8-CAI-1). V. cholerae CqsA prefers the acyl CoA substrate C10-CoA over C8-CoA, and this substrate preference also correlates with the ligand selectivity of CqsS, which binds CAI-1 with C10 acyl tails more efficiently than molecules with C8 acyl tails. The chain length discrimination is defined by the amino acid residue at position 170 located in the sixth trans-membrane helix of the CqsS receptor domain. V. cholerae C170 allows the detection of ligands with C10 or C8 but not shorter  or longer (C12) acyl tails. The motifs F160/F162/F166 (polar head group) and W104/S107  ensure the exclusive binding of CAI-1 analogues with a relaxed specificity for amino and hydroxyl modification.In summary, the 5.V. cholerae CqsA/CqsS system shows a rather relaxed signal discrimination, and multiple CAI analogues can interact with the sensor kinase CqsS, including molecules produced by other Vibrio spp. Since V. cholerae and V. harveyi produce and detect Ea-C8-CAI-1, this CAI-1-derivative might be used for communication among different Vibrio spp. Potential ecological interactions among Vibrio spp. might indeed take place in river deltas and other aquatic ecosystems [Vibrio spp. are most sensitive for AHK molecules produced by their cognate CqsA AI synthases. The coordination and fine-tuning of cognate AI synthases and AI receptors ensures efficient ligand-receptor interactions, avoids inappropriate communication and maintains high fidelity QS signaling.The osystems . Still, V. harveyi produces higher concentrations of the hydroxyl-modified C8-CAI-1 compared to Ea-C8-CAI-1, even though the hydroxyl molecule is only a weak agonist. Similar CAI-1 production profiles were found in supernatants of several other marine Vibrio spp., e.g., V. parahaemolyticus, V. alginolyticus or V. anguillarum [Vibrio spp. employ three parallel QS systems . As LuxS/LuxPQ-based AI-2 signaling is broadly used, the stringency imposed by the AHK (and AHL) systems might be essential for an accurate integration of multiple and similar AI signals.uillarum ,87. FurtVibrio spp., which employ several QS systems, L. pneumophila apparently uses only the Lqs system. Perhaps, this reflects the distinct lifestyles of the bacteria. While Legionella spp. can colonize extracellular niches, such as multi-species biofilms, their preferential habitat is likely the resident amoebal fauna in biofilms [L. pneumophila might avoid competitors, which are normally encountered in nutrition-rich extracellular ecological niches, and consequently, the bacteria might have lost (or never acquired) QS systems that support broad inter-species communication. In agreement with the notion that (inter-species) communication systems are dispensable for intracellular bacteria, neither L. longbeachae [Legionella spp. other than L. pneumophila apparently possess the lqs system [In contrast to biofilms ,90. Thusgbeachae , nor Legs system ,91.lqs genes are located in a genomic region of L. pneumophila that seems to represent a hot spot for recombination. The corresponding site in L. longbeachae harbours a homologue of the putative effector gene legG2 , which is flanked by transposase elements and localizes upstream of a homologue of E. coli hdeD. The function of L. pneumophila hdeD is unknown [lqsS and lqsR genes  yields insights into a QS strategy likely used by a number of environmental bacteria, (ii) provides tools to analyze the mechanism of signal transduction by AHK ligands and TC system sensor kinases, and (iii) might lead to the development of compounds useful in clinical or environmental settings to interfere with virulence and persistence of important human pathogens.The water-borne opportunistic pathogens


HTLV-1 proviral DNA, HTLV-1 protein, and viral particles were detected from infiltrating cells in eyes with HU. HTLV-1-infected CD4+ T cell clones established from infiltrating cells in eyes with HU produced large amounts of various inflammatory cytokines, such as IL-1, IL-6, IL-8, TNF-α, and interferon-γ. Taken together, HU is considered to be caused by inflammatory cytokines produced by HTLV-1-infected CD4+ T cells that significantly accumulate in eyes; therefore, topical and/or oral corticosteroid treatment is effective to treat intraocular inflammation in patients with HU. Further investigation is needed to establish a specific treatment for HU.Human T cell lymphotropic virus type 1 (HTLV-1) is the first retrovirus described as a causative agent of human disease. Following adult T cell leukemia/lymphoma and HLTV-1-associated myelopathy/tropical spastic paraparesis, HTLV-1 uveitis (HU) has been established as a distinct clinical entity caused by HTLV-1 based on seroepidemiological, clinical, and virological studies. HU is one of the most common causes of uveitis in endemic areas of Japan and can be a problematic clinical entity all over the world. HU occurs with a sudden onset of floaters and foggy vision, and is classified as an intermediate uveitis. Analysis of infiltrating cells in eyes with HU revealed that the majority of infiltrating cells were CD3 Retrovirus was first described in the 1970s , but itsHTLV-1 uveitis, the third clinical entity of HTLV-1 infection, was established by a series of studies in the highly endemic area of southern Kyushu, Japan. Clinical case reports from this area suggested possible associations of HTLV-1 carriers with various ocular manifestations . In the Figure 1). This was the first clue suggesting that HTLV-1 infection is significantly related to uveitis. Uveitis is now recognized as a distinct clinical entity related to HTLV-1 and is designated as HU. The seroprevalence of HTLV-1 in the general Japanese population is known to have decreased after serological screening tests of HTLV-1 in blood donors started in 1987, as blood transfusion and breastfeeding from mother to child are major routes of viral transmission  and exclusion of other uveitis entities with defined causes. Therefore, all clinical entities of uveitis with defined causes should be excluded by careful ophthalmic and systemic examinations. Patients with HU should not have ophthalmic and systemic symptoms that are compatible with other types of uveitis such as Behçet’s disease, Vogt–Koyanagi–Harada syndrome, and sarcoidosis.+ T cells . In addition to this molecular biological/immunological evidence, virological research supported the pathogenicity of HTLV-1 in the eye by the following three pieces of evidence: (1) the HTLV-1 provirus load in patients with HU is significantly higher than that in asymptomatic carriers without uveitis  the uveitis ; and (3) uveitis . Serolog uveitis . Antibod uveitis . Taken t+ T cells that significantly accumulate in the eyes of the patients. Furthermore, the addition of corticosteroids in the culture medium suppressed the cytokine production . We may be able to find more effective treatments if we can understand the mechanism of HU in more detail. Recent studies have shown new insights into HTLV-1 infection and pathogenesis by pursuing the molecular functions of HTLV-1 basic leucine zipper factor and Tax (We reviewed the seroepidemiological, clinical, molecular biological, and virological studies that established the HU entity and clarified the immunopathogenesis and the clinical management of HU. Corticosteroid is the only effective treatment for HU to suppress the cytokines produced by infiltrating HTLV-1-infected cells; however, it is unknown whether long-term corticosteroid treatment adversely affects patients with HU. Many mechanisms in HU remain unclear, such as how HTLV-1-infected CD4 and Tax . HoweverThe 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.


Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK  transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration. Computational modeling of the dynamics of intracellular signaling pathways is an area of active research. Chemical equations such as the law of mass action or other higher-order reactions have been used to simulate the various intermolecular interactions involved in signaling pathways Such transport of proteins and organelles is especially important in neuronal cells. Most axonal proteins are synthesized within the neuronal cell body and mechanisms need to be in place to direct these proteins to the growing axon tips Often, proteins that are transported by motor proteins are also bound to scaffold proteins. Scaffold proteins have been known to interact and/or bind with various players of a signaling pathway and to tether them into complexes. In doing so, they regulate signal transduction and aid in localization of signaling cascades to specific parts of the cell. Signal activation by irrelevant stimuli can also be prevented, thus providing the cell with spatial and temporal control of signaling Jip1 gene in mice prevented JNK activation One specific example of a signaling cascade that makes use of both scaffold proteins and motor proteins is the JNK  signaling pathway. The JNK group of mitogen-activated protein (MAP) kinases modulate a number of cellular processes in mammalian cells such as early embryonic development, apoptosis, oncogenic transformation and the immune response in vivoJIP localization to the cell periphery could be modulated via its association to kinesin. In fact, JIP1, JIP2 and JIP3 have been identified as binding partners to kinesin using yeast two-hybrid procedure with kinesin light chain as bait In this article, using the JNK signaling pathway as a model system, we study how the activity of JNK is being modified by being scaffolded via JIP1 and, in addition, transported to a distant part of the cell along the cytoskeleton via KIF5. Therefore, we seek to understand how the combined effects of both scaffolding and cytoskeletal transport modify signaling activity compared to the case if JNK is to diffuse to the distant part of the cell without scaffolding or cytoskeletal transport. We model the activation of JNK that is being scaffolded by JIP1 and then transported along the cytoskeleton via KIF5 by a set of reaction-diffusion-advection equations, and investigate how signaling rate and signal amplification are modified by the presence of scaffold and motor proteins. In Section II, we describe our model as well as the algorithm used to simulate the scheme effectively. In our model, the signaling protein JNK exists in either an inactive (unphosphorylated) or active (phosphorylated) state, denoted by JNK and JNK*, respectively. The activation of JNK is catalyzed by an upstream activated kinase MKK7 and the inactivation from JNK* to JNK is catalyzed by a phosphatase M3/6. Both the activation enzyme MKK7 and the inactive signaling protein JNK can bind to the scaffold protein JIP1. The scaffold protein JIP1 is assumed to possess catalytic properties such that the rate of activation of JNK by MKK7 is higher within the scaffold than that outside of the scaffold. Enhancement of catalysis within the scaffold has been observed experimentally where the prototypical scaffold Ste5 unlocks the Fus3 MAP kinase for activation by Ste7 MAPKK, thereby increasing the phosphorylation rate In the JNK signaling cascade, three kinases are successively activated under stimulus. The cascade starts with activation of MAPKKKs such as MLK3 which go to on to phosphorylate and activate the MAPKKs MKK4 and MKK7 which finally phosphorylate and activate JNK In the model, the reactions in Species that are not bound to KIF5 move by diffusion only. They follow the Neumann boundary condition The initial distribution of all the species with the exception of M3/6 is assumed to follow a Gaussian distribution centered at We solve the set of reaction-diffusion-advection equations listed in Next, we want to understand the relative importance between protein diffusion and cytoskeletal transport in the arrival and accumulation of JNK* at the cell periphery. Specifically, we are interested in how two parameters, the concentration of scaffold protein JIP1 and the speed We first solve for the purely diffusive scenario where We shall now proceed to quantify the transport activities more carefully. In particular, we define and make use of two metrics, namely, signaling rate and signal amplification. Signaling rate, If we fix Similarly, if we now fix Intuitively, one would expect that an increase in the motor speed  from low signaling rates (Region 4) depends on both Next, we look at the signal amplification, Similarly, if we now fix Similar profiles are observed for other values of We seek to understand how the values of scaffold concentration Increasing concentration of M3/6 increases the value of A higher concentration of KIF5 motor protein increases the value of Next, we look at how JNK concentration modifies the values of C. elegans also suggest that axonal transport depending on KIF5 is upregulated by the JNK pathway Thus, one can foresee a scenario where the cell upregulates JIP1 scaffolds and KIF5 motors when JNK concentration is increased at the cell body. Increasing the amount of JIP1 scaffolds serves to increase amplification of JNK* at the cell periphery while increasing KIF5 serves to increase the optimal speed of transport of associated kinases for faster delivery. JIP1 is observed to be upregulated with an increase in phosphorylation of JNK when GLUT1 (glucose transporter1) is overexpressed The combination of scaffolding by JIP1 and transport by motor protein KIF5 can be summarized as follows. At low JIP1 scaffold concentration, few JNK are recruited to JIP1 for subsequent phosphorylation and transport by KIF5. Thus, majority of the JNK* reaches the axon terminals by free diffusion, resulting in low signaling rate and signal amplification. However, at high JIP1 concentrations, JNK and MKK7 are spread out too widely amongst the scaffold proteins, leading to ineffective phosphorylation and a corresponding suppression of phosphorylation activity in the entire system. Active transport of JNK* still occurs, although scaffold-assisted phosphorylation is now suppressed.On the other hand, at low KIF5 speed, both forms of JNK (activated or unactivated) and MKK7 diffuse freely along the axons, such that they are far beyond encounter distance from KIF5 which are concentrated near the cell body. Under such circumstances, signaling proceeds via free diffusion coupled with limited active transport, resulting in low signaling rate and signal amplification. At high KIF5 speed, however, motor proteins translocate along the cytoskeleton before the kinases can bind onto the motor. Here, we witness the other extreme case whereby free diffusion coupled with limited active transport prevails.Finally, an ideal scenario should comprise an optimum JIP1 concentration to concentrate both JNK and MKK7 effectively and an optimum KIF5 cytoskeletal transport speed, such that most of the corresponding scaffold complexes are recruited by the motor proteins and actively transported along the axons. Such a scenario is observed at the maxima region in the phase diagrams of signal amplification and lies within the high signaling rate region of the signaling rate plot. This is depicted in Region 3 of Even though our model is constructed specifically for the analysis of the JNK signaling cascade, we believe that our model is generic enough to be applied to other signaling pathways that also make use of scaffold proteins and cytoskeletal transport. Features extracted in our model such as the biphasic behavior in scaffold concentration and cytoskeletal transport speed should be universal features in other motor proteins-assisted scaffolded signaling complexes. In recent years, an increasing number of scaffold proteins that associate with motor proteins have been uncovered. A yeast-two-hybrid screen to identify proteins that interact with the KIF1C C-terminal domain identified proteins of the 14-3-3 family as binding partners The role of JIP1 in modulating the JNK pathway has been well studied. JIP1 was originally assumed to be an inhibitor of JNK. JIP1 has been shown to suppress signal transduction of the JNK pathway by competing with substrates that interact with JNK. JIP1 overexpression has also been proposed to be a cytoplasmic anchor for JNK as overexpression of JIP1 caused retention of JNK in the cytoplasm Understanding cytoskeletal-assisted protein transport is important, because axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative disease. Examples include polyQ aggregates in Huntington disease, synuclein in Lewy bodies found in Parkinson's disease, amyloid beta and tau protein deposits in Alzheimers disease. These observations suggest that defects in axonal transport may contribute to neuronal inclusions and plaques Finally, we discuss how the two parameters We have studied computationally the various strategies that JNK may be transported from the cell body to the cell periphery. We have shown that binding to a scaffold JIP1 and then having the whole protein-scaffold cargo being transported by motor proteins KIF5 along the cytoskeleton is superior to relying on transport by protein diffusion, but only in a limited range of JIP1 concentration and KIF5 motor speed. We defined two metrics to quantify transport, namely signaling rate and signal amplification. It is only possible to achieve maximum amplification at a specific range of JIP1 concentration and KIF5 motor speed. These findings are summarized in


Epidemiological studies show that human T-lymphotropic virus type 1 (HTLV-1) is closely associated with polymyositis (PM). However, the pathogenic roles of HTLV-1 in PM remain unknown. This study aims to assess skeletal muscle morphology in the presence of HTLV-1 infection to compare the histopathological findings of HTLV-1-positive and HTLV-1-negative PM.Among the 68 patients with inflammatory myopathy diagnosed through muscle biopsy over the previous 10 years at Kagoshima University Hospital, we retrospectively selected 21 patients with PM not associated with any other disease; we evaluated HTLV-1 positivity through serology, confirmed it by nested PCR using DNA extracted from muscles, and then assessed the tissue viral load. Meticulous histopathological examination was performed using routine histochemical and immunohistochemical staining, and specimens from selected cases were examined by electron microscopy.The clinical and histopathological findings of muscle biopsy specimens of HTLV-1-positive (n = 11) and HTLV-1-negative PM cases (n = 10) were compared. Compared with HTLV-1-negative patients, HTLV-1-positive patients exhibited protracted clinical courses, prominent endomysial infiltrates, infrequent necrotic fibers, and prominent regenerative activities. Moreover, they showed frequent cytochrome c oxidase deficiency and ultrastructural abnormalities in mitochondria.These differences are significant but not specific to HTLV-1-positive PM. Therefore, HTLV-1 may induce the clinical and histopathological modifications of PM observed in this study.


P ≥ 0.17). It was found that using a multi-wavelength and processed signal approach leads to more accurate results, and therefore these approaches should be used in future.The aim of this study was to estimate the concentration of uric acid (UA) optically by using the original and processed ultraviolet (UV) absorbance spectra of spent dialysate. Also, the effect of using several wavelengths  for estimation was examined. This paper gives an overview of seven studies carried out in Linköping, Sweden, and Tallinn, Estonia. A total of 60 patients were monitored over their 188 dialysis treatment procedures. Dialysate samples were taken and analysed by means of UA concentration in a chemical laboratory and with a double-beam spectrophotometer. The measured UV absorbance spectra were processed. Three models for the original and three for the first derivate of UV absorbance were created; concentrations of UA from the different methods were finally compared in terms of mean values and SD. The mean concentration (micromol/L) of UA was 49.7 ± 23.0 measured in the chemical laboratory, and 48.9 ± 22.4 calculated with the best estimate among all models. The concentrations were not significantly different ( Uric acid (UA), a final product of the metabolism of purine, is a very important biological molecule present in body fluids. It is mostly excreted from the human body through the kidneys in the form of urine. The concentration of UA in blood increases when the source of UA increases or the kidneys malfunction. Hyperuricemia is a symptom when the UA concentration is above 7 mg/dL. UA is hard to dissolve in blood and will crystallise when supersaturated. The UA crystallites are deposited on the surface of the skin, in joints, and particularly in the toes, resulting in gout. Analysis of the UA concentration in blood helps to diagnose gout. In addition to gout, hyperuricemia is connected with lymph disorders, chronic haemolytic anaemia, an increase in nucleic acid metabolism, and kidney malfunction. Elevated serum UA contributes to endothelial dysfunction and increased oxidative stress within the glomerulus and tubulointerstitium, with associated increased remodelling fibrosis of the kidney . A high It would be advantageous to measure the concentration of UA during dialysis online. For creating this opportunity it is necessary to create accurate and reliable models. UA may be the novel marker molecule for estimating the quality of dialysis procedure, since the UA is uremic toxin itself, removal pattern and amount of this compound during the dialysis are informative for patients and medical personnel.Ways of monitoring UA, dialysate, and other biological fluids with optical tools have been shown previously by our and other groups –15. If yThe aim of this study was to estimate the concentration of uric acid (UA) optically by using the original and processed ultraviolet (UV) absorbance spectra of spent dialysate. Data from different dialysis centres and over a long period was used to build models to increase general validity and reliability.All of the studies were performed after approval of the protocol by the Regional Ethical Review Board, Linköping, Sweden, and by the Tallinn Medical Research Ethics Committee at the National Institute for Health Development, Estonia. Informed consent was obtained from all participating patients.During the period 1999–2009 seven studies were carried out in the Department of Dialysis and Nephrology at the Linköping University Hospital in Sweden and at the North Estonian Medical Centre in Estonia. Clinical setup of the experiments is presented in The dialysers used in the studies, the effective membrane areas of the dialysers, the number of sessions when the respective dialyser was used, the type of dialysis machine used, and blood flow for the studies are presented in For all of the studies, samples of spent dialysate were taken at discrete times for analysis . The numThe concentration of UA was determined in the Clinical Chemistry Laboratories at the North Estonian Medical Centre and at Linköping University Hospital using standardised methods. The accuracy of the methods for the determination of UA in dialysate was ±5%.Double-beam spectrophotometers  were used for the determination of UV absorbance. Spectrophotometric analysis over a wavelength range of 190–380 nm was performed by an optical cell with an optical path length of 1 cm. A lower UV absorbance value is obtained at all wavelengths versus time due to a decreased concentration of UV-absorbing compounds in the blood when transported through the dialyser into the dialysate and removed from the blood during the dialysis treatment. The treatments were also monitored with a single wavelength online, and thereby all interruptions, self-tests, alarms, and so forth could be identified directly on a screen. Some of the measured values (absorbance or concentration) were excluded from data before analysis. The exclusion criteria were incorrect or illogical values of measured concentration or absorption, for example, sampling coexisting with self-tests of the dialysis machine.The obtained UV spectra were processed with a signal-processing tool using a Savitzky-Golay algorithm for smoothing, and the first derivative calculation wherein a smoothing window with nine points was used . PanoramOn the basis of the UA concentrations measured in the laboratory, measured UV absorbance spectra and processed UV absorbance spectra, multiple regression analysis was carried out on the calibration set of material . UA was set as a dependent variable, and UV absorbance values between 190–380 nm were set as independent variables. Multiple linear regression (MLR) analysis using the forward stepwise regression method was employed to determine the best wavelengths for the models –25. UsinY) are in the formModels for the calculation of the concentration of UA .The obtained models were used on the data from the remaining 113 dialysis procedures  to calculate the concentration of UA and compare these values with the laboratory results and validate different models.Systematic error was calculated for the models as follows :ei is the residual and N is the number of observations.Standard error was calculated for the models as follows:t-test for residuals, which were significantly different (at P level 0.05) between models that used an absorbance or first derivate of absorbance value from one, two, or three wavelengths and which were not different in the case of models which used four wavelengths.During regression analysis, three steps were considered sufficient after estimation of the behaviour of the root mean squared error (RMSE). From As a result of regression analysis, three models for UV absorbance and three models for derivate of UV absorbance were found wherein each used an absorption or derivate of absorption value from one, two, or three wavelengths, respectively . The modFigures R2, BIAS, SE, and RMSE. The results are presented in The models presented in Figures P = 0.17–0.48) from the observed concentrations in the laboratory for any model.The concentrations achieved by the models were not significantly different (P level 0.05) in the following cases :The systematic and root mean squared errors were significantly different  are presented in The root mean squared error decreased as wavelengths were added to the models in the case of both the UVa and UVd models, and the decrease was slightly greater in the case of UVd models.These results demonstrate that using UV absorbance from several wavelengths provides more accurate results in the estimation of the concentration of UA. Also, using information from the first derivate of spectra instead of original UV absorbance spectra produces a notable effect.The results in R2, between the laboratory and calculated values of UA are higher or equal in the case of the UVd (single/two/three) compared to the UVa (single/two/three) (0.86/0.88/0.92 versus 0.91/0.93/0.93)  was created  analysis using the forward stepwise regression method was used to determine the best wavelengths for models. Using the stepwise regression method helps us to avoid mistakes in the models due to the possible collinearity of independent variables. It seems that models developed with MLR are relevant and work well in a validation set of material, although using other approaches like partial least squares regression (PLS-R) or principal component regression (PCR) to create models should be considered in the future .The clinical aim in the future is to develop an online monitoring system that offers an estimation of the removal of clinically important solute and marker UA during haemodialysis.Also, regarding the optical properties of UA, it is possible to develop an optical system to measure the UA concentration in blood and/or urine. This makes it possible to rapidly detect hyperuricemia widely and at an early stage. This is very important in preventing serious clinical issues caused by hyperuricemia , 12, 31.An accurate optical method makes it possible to measure UA rapidly online without the need for blood samples and disposables or chemicals. Using a simple signal-processing tool and UV absorbance values from several wavelengths could be very helpful in achieving more accurate and reliable results.This study investigated the effect of using several wavelengths and a simple signal processing to estimate the concentration of UA in dialysate using an optical method. The data analysed were collected over 10 years: 60 patients participated and 188 dialysis sessions were monitored in various centres in different countries. It was found that using a multi-wavelength and processed signal approach leads to more accurate results. This approach enables us to develop an advantageous, reliable, and cost-effective method of measuring the concentration of UA, an independent risk marker of cardiovascular and renal diseases and also a novel risk factor for type 2 diabetes mellitus. Developed algorithms could be used in optical dialysis quality monitors; these monitors should be integrated to dialysis machines and with these several parameters; UA among them is possible to monitor during the dialysis. No blood will be monitored; removal on substances is possible to estimate only by monitoring the spent dialysate. A future method evaluates the treatment dose and makes it possible to control treatments against set target values.


In an earlier study, we detected an association between human T-cell lymphotropic virus (HTLV) infection and cervical human papillomavirus (HPV) in indigenous Amazonian Peruvian women of the Shipibo-Konibo ethnic group. As both HTLV and HPV can be transmitted sexually, we now report a population-based study examining the prevalence and risk factors for HTLV-1 and HTLV-2 infection in this population.Between July and December 2010, we conducted a comprehensive screening for HTLV among Shipibo-Konibo women 15 to 39 years of age living in two communities located in Lima and in 17 communities located within four hours by car or boat from the Amazonian city of Pucallpa in Peru.We screened 1,253 Shipibo-Konibo women for HTLV infection 74 (5.9%) tested positive for HTLV-1, 47 (3.8%) for HTLV-2 infection, and 4 (0.3%) had indeterminate results. In the multivariate analysis, factors associated with HTLV-1 infection included: older age : 1.04, 95% CI 1.00–1.08), primary education or less , younger or same age most recent sex partner , and having a most recent sex partner who worked at a logging camp . The only factor associated with HTLV-2 infection was older age .HTLV infection is endemic among Shipibo-Konibo women. Two characteristics of the sexual partner (younger age and labor history) were associated with infection in women. These results suggest the need for implementation of both HTLV screening during the antenatal healthcare visits of Shipibo-Konibo women, and counseling about the risk of HTLV transmission through prolonged breastfeeding in infected women. We also recommend the implementation of prevention programs to reduce sexual transmission of these viruses. Human T-cell lymphotropic virus type 1 (HTLV-1) was the first oncogenic human retrovirus to be discovered HTLV-1 and -2 infections are transmitted via breastfeeding, sexual contact, and parenteral transmission via blood transfusion and intravenous drug use In Peru, most HTLV infections have been reported in indigenous people from the Andean Region (Quechua and Aymara ethnicities), and among African-Peruvian and mestizo populations (a mix of Spanish and Andean indigenous blood) As part of a previous study conducted among ten indigenous groups in the Peruvian Amazon, we found 18 HTLV infections restricted to the Shipibo-Konibo people, one of the largest Peruvian Amazonian ethnic groups residing along the Ucayali river Our study was approved by the Institutional Review Boards (IRB) of the University of Washington in Seattle, the Universidad Peruana Cayetano Heredia, and U.S. Naval Medical Research Unit No.6 (NAMRU-6) in Callao, Peru. All enrollees provided written informed consent prior to their participation in our study. If participants were at least 18 years old, or between 15 and 17 years of age and married/cohabitating for two or more years, and they agreed to participate, they were invited to participate and asked to sign the informed consent. Other consenting eligible women signed an assent and their parent or legal guardian signed an informed consent. The IRB considered participants between 15 and 17 years of age and married/cohabitating for two or more years as emancipated, not requiring parental permission for enrollment. In the context of Amazonian ethnic groups, it is common for women to become emancipated before 18 years of age, usually through marriage or cohabitation. At this age they leave their parent’s home, raise their children and provide economic support for their families.As part of a study to determine the association between HTLV and cervical HPV infection, between July and December 2010 we conducted a comprehensive screening for HTLV infection among Shipibo-Konibo women 15 to 39 years old who agreed to participate and lived in any of the Shipibo-Konibo communities located in Lima or within four hours (most often within one hour) by car or boat from the city of Pucallpa. The screening was conducted through household visits. We defined women as belonging to the Shipibo-Konibo ethnic group if they had at least one of the following characteristics: 1) they self-identified as Shipibo-Konibo, 2) they spoke Shipibo-Konibo language, or 3) at least one of their parents belonged to the Shipibo-Konibo ethnic group.Details of the recruitment process and laboratory techniques are described elsewhere Venous blood samples were collected using 10 ml vacuum blood collection tubes, transported in coolers and delivered within 6 hours of collection to the laboratory in Lima or Pucallpa. Serum was promptly separated, divided into aliquots in vials and frozen at −20°C. Frozen serum aliquots were sent weekly for HTLV testing from Pucallpa to the NAMRU-6 laboratory in Callao via air courier in Styrofoam coolers wrapped in metal. Serum samples obtained in Lima were sent directly to NAMRU-6 for processing and analysis.HTLV testing was performed at NAMRU-6 in Callao, Peru. A woman was considered to be HTLV infected if she had a positive test for HTLV ELISA , and the confirmatory Western blot assay  revealed bands representing Gag  and Env proteins . An individual was considered HTLV-2 seropositive if the ELISA assay was positive and confirmatory Western blot assay revealed bands representing p24, GD21 and rgp46-II. If Gag and Env proteins were absent, but other HTLV specific bands, such as p53 or p19 were present, the individual was considered indeterminate. Subjects with indeterminate results were excluded from the analysis. PCR testing was not performed since the specimens needed for this testing were not collected in the study.Approximately two months after the first visit, we carried out a second visit to the communities to deliver HTLV test results Analyses were performed using Stata . To identify factors associated with HTLV-1 and HTLV-2 infections, crude and adjusted prevalence ratios (PR) and their corresponding 95% confidence intervals were estimated using generalized linear models adjusting by community of residence. Analysis of HTLV-1 and 2 infections were conducted separately because of their potential differences in risk factors. Variables independently associated with either HTLV infection at a level of significance of 0.1 were included in a multivariate model that was refined using backward stepwise selection process. Predictors significant at the 0.05 level were retained in the final model.Between July and December 2010, we approached leaders of 21 Shipibo-Konibo communities and 19 consented participation of their communities. Two communities were located in Lima, and 17 were Amazon jungle communities located near the city of Pucallpa. In these communities we approached 1,460 women, of whom 58 refused to participate. Of the 1,402 consenting women 149 were excluded because they did not belong to the Shipibo-Konibo ethnic group. All the remaining 1,253 eligible women could speak Spanish. Of these women, 74 (5.9%) tested positive for HTLV-1, 47 (3.8%) for HTLV-2 infection, and four (0.3%) had indeterminate results. Participants with indeterminate results were excluded from further analysis. The combined HTLV-1/2 prevalence was 9.7%. There were no dual infections. HTLV infected participants were born in seven out of 10 districts included in the Ucayali region and in 4 out of 11 districts included in the Loreto region. The distribution of HTLV within communities did not follow a clear geographical pattern. HTLV-1 infected women were more likely than HTLV-negative participants to be older , less educated, married or cohabiting, to have a last sexual partner either younger or of the same age than HTLV negative women, to live with current sex partner, to not use a condom in their last sexual intercourse, to have a last sex partner who worked at a logging camp and to self-report a history of tuberculosis .HTLV-2 infected women were more likely to be older and not living in the community where they were born. They were also more likely to be sexually active, to have a last sexual partner younger or of the same age than HTLV negative women, to report a history of bleeding during a dental procedure and a history of crusted scabies .We did not find an association between having had a partner who used intravenous drug use, having tattoos, piercing or surgery and HTLV-1 or -2 infection (data not shown). We were not able to evaluate duration of breastfeeding as a risk factor for HTLV-1 or -2 infection, since 850 (68.1%) of participants did not recall this information. When analyzing women who knew the duration of breastfeeding, the mean duration of breastfeeding was similar for both HTLV seropositive and seronegative participants .Overall, 15.0% (17) participants who reported they were pregnant at the time of the interview were infected with HTLV, 10 with HTLV-1 and 7 with HTLV-2. The mean number of sexual partners during the last 12 months among HTLV negative, HTLV-1 and HTLV-2 positive participants was similar (1.0 vs. 1.0 vs. 1.2 partners). Among sexually active participants, the combined HTLV-1 and -2 prevalence was 10.2%, whereas among participants reporting not having initiated sexual activity the prevalence was 4.2%, suggesting vertical transmission is a common cause of HTLV-1 and -2 infection.In the multivariate analysis, factors associated with HTLV-1 infection were age, having primary education or less, having the most recent sex partner either younger or of the same age, and having the most recent sex partner who worked at a logging camp. The only factor independently associated with HTLV-2 infection was age .To our knowledge, this is the largest population-based study to assess the prevalence of HTLV-1 and -2 infections in indigenous populations in Latin America. We found a combined HTLV-1 and -2 prevalence of 9.7% among Shipibo-Konibo women, the highest prevalence described in an indigenous group in Peru.In the multivariate analysis, older age was associated with both HTLV-1 and -2 infections. Other studies that have assessed risk factors for HTLV infection have reported that seroprevalence increases with age, suggesting that sexual intercourse plays an important role in the transmission of this virus We also found that lower education level was associated with HTLV-1 infection. In a study in Salvador, Brazil, formal education of less than 7 years was associated with HTLV-1 infection in females In our study, having a most recent sex partner who was younger or of the same age, and having a most recent sex partner who worked at a logging camp were both associated with HTLV-1 infection. While the first finding is intriguing, a previous study that assessed risk factors for STI among men from 10 indigenous groups in the Peruvian Amazon found that illegal logging camps were venues where heterosexual males were often exposed to unprotected sex with FSW and MSM, and therefore at higher risk for acquiring HTLV and other STI Sarcoptes scabiei var. hominisHTLV-2 infection was associated with a self-reported history of crusted scabies and HTLV-1 infection was associated with a history of tuberculosis. Crusted scabies is a rare but severe infection caused by massive infestation with Although we included non-sexually active women of reproductive age, one limitation of our study is the exclusion of participants under 15 years of age that would have provided valuable data regarding vertical transmission.In conclusion, HTLV infection is endemic among Shipibo-Konibo women, particularly among older and less educated women. Remarkably, two characteristics of the sexual partner (younger age and labor history) were associated with infection in women. These results suggest the need for implementation of both HTLV screening during the antenatal healthcare visits of Shipibo-Konibo women, as well as counseling about the risk of HTLV transmission through prolonged breastfeeding in women infected with the virus. We also suggest the implementation of programs oriented towards preventing sexual transmission of this virus through educational interventions that cover safe-sex behavior and condom use. Additionally, we suggest clinical follow-up of HTLV infected people in order to provide early diagnosis and treatment of HTLV-associated diseases. For an intervention to be successful and tailored to this indigenous community, researchers should integrate input from the Shipibo-Konibo community into the development of evidence-based interventions.