Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS) and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of Ranvier, and how abnormalities in these processes may contribute to disease. Neurons are polarized cells made up of structurally and functionally distinct processes, dendrites and axons, which direct the flow of information throughout the nervous system. The dendrites are often composed of multiple branches and dendritic spines that receive signals from upstream synaptic inputs and transmit this information to the axon. The axon propagates electrical signals, known as action potentials, to downstream neurons by the opening of voltage gated-sodium channels at specialized excitable microdomains referred to as the axon initial segment (AIS) and nodes of Ranvier Figure . Action Despite similar structural composition between the AIS and the nodes, the mechanisms by which these microdomains form are quite different. The formation of the nodes of Ranvier is strongly influenced by both intrinsic and extrinsic factors, whereas localization of these same proteins to the AIS relies mainly on factors intrinsic to the neuron. The intricate formation and function of excitable axonal microdomains of the vertebrate nervous system plays a critical role in fast neuronal signaling and higher order cognitive processing. Several excellent detailed reviews about the structural organization and physiology of the AIS and nodes of Ranvier have been published  indicated by the clustering of ankyrin-G, the first detectable marker of the AIS . Alternative splicing is a key mechanism underlying the functional diversity and cellular distribution of ankyrins. In addition to the canonical 190 kDa ankyrin-G, alternative splicing of the giant 7.8-kb exon produces a 270 kDa isoform, which only utilizes the first ~2700 nucleotides of the giant exon due to in-frame splicing, and a giant 480 kDa isoform which utilizes the entire giant exon. Similar alternative splicing of ANK2 gives rise to a 220 kDa isoform and a 440 kDA isoform of ankyrin-B. The giant ankyrin-G differs from the large isoform of ankyrin-B due to the presence of a 40 kDa serine and threonine rich domain located on the N-terminal side that is modified by O-linked N-acetylglucosamine residues with unknown function , there is no significant sequence homology outside of a composition bias of increased usage of serine and glutamic acid and there is a huge variation in exon size . Although the Drosophila giant splice variants also show some ability to restrict ion channel mobility within the axon  and phosphorylation of Nav channels by CK2 increase binding affinity to ankyrin-G. Bréchet et al.  showed tin vivo will be important for the understanding of human diseases involving dysfunction of ankyrins and spectrins. In addition, it remains unclear how 480 kDa ankyrin-G, found both at the AIS and at the distal nodes of Ranvier, is able to avoid the restriction at the proximal axon by the ankyrin-B/spectrin cytoskeletal boundary. It is attractive to speculate that nascent ankyrin-G protein is locally translated at the node of Ranvier or perhaps locally palmitoylated; however, the exact mechanisms controlling AIS ankyrin-G vs. that found at the node of Ranvier remains poorly understood.Lastly, the distal axon cytoskeleton, which is composed of ankyrin-B, αII-spectrin and βII-spectrin complexes, has been proposed to create a boundary that restricts ankyrin-G to the proximal axon Figure . ManipulA primary function of the AIS is to maintain polarity of the proximal axon. One potential mechanism is the role of the AIS as a diffusion barrier to inhibit the mobility of membrane-associated proteins from dispersing from one neuronal domain to another. To support this theory, Kobayashi et al.  first suin vivo results in disassembly of the AIS and causes the proximal portion of the axon to acquire dendritic characteristics including dendritic spines and the presence of MAP2  lysosome, were indistinguishable between the AIS and distal axon in hippocampal neurons . They revealed the actin filaments form “actin rings” that distribute consecutively throughout the entire length of the AIS and are spaced roughly 190 nm apart. In addition, they also determined that ankyrin-B was also found to be periodically localized between the adjacent actin rings in the distal axon  in olfactory bulb dopaminergic interneurons. In addition, heightened cdk5 activity extended AIS length by as much as 100% in mushroom body neurons of Drosophila, while depleting cdk5 causes the AIS to significantly shrink or disappear altogether  and oligodendrocytes in the central nervous system (CNS). The clustering of Nav channels to the node is critically important for the rapid, saltatory propagation of action potentials. Myelination divides the axonal membrane into distinct domains including nodes of Ranvier, paranodes, juxtaparanodes and internodes. The nodes of Ranvier are the sites of action potential repolarization and depolarization due to the clustering of high concentrations of ion channels, including Nav and Kv channels. The complement of Nav and Kv channels at the node are diverse and can include Nav1.1, Nav1.2, Nav1.6, Nav1.7, Nav 1.8 and Nav1.9 which interact with β-subunits Navβ1, Navβ2 and Navβ4  GABAergic interneurons. Interestingly, mice lacking the exon 1b isoform loose Nav channel clustering at the AIS of PV interneurons and demonstrate behavioral characteristics of bipolar disorder, epilepsy and sudden death  and sleeping problems  are associated with Dravet syndrome, a severe myoclonic epilepsy of infancy, as well as West syndrome, genetic epilepsy with febrile seizures plus (GEFS+) and others  and SCN8A (Nav1.6) mutations are found in patients with early infantile epileptic encephalopathy  pathology; however, the mechanisms that contribute to axonal loss remain elusive (Dutta and Trapp, In a rat model of mild traumatic brain injury, Baalman et al.  showed tOverall, changes in excitable domains of the axon or their constituent proteins have profound impact on neurological function. Although many of the proteins of the AIS and nodes of Ranvier have important functions in other cellular domains, the overlapping phenotypes seen with loss of function of different AIS and nodal components suggest that dysfunction of these axonal membrane domains is a major factor in the development of disease. As we increasingly understand the genetic basis of neurological disorders, we will likely uncover more genes involved in the formation and function of axonal domains that can give us more insight into the etiology of human disease.The structural assembly and maintenance of the axon relies on the precise organization between ankyrins, spectrins, membrane-associated proteins and actin and microtubule cytoskeletal proteins. The mechanisms underlying the interaction between these components at the AISs and nodes of Ranvier are now becoming more apparent. A better understanding of the organization and maintenance of axonal excitable domains as well as how abnormalities in their signaling may lead to altered axonal function will provide insight to novel therapeutic targets for the treatment of human diseases of the nervous system.ADN and PMJ wrote the manuscript.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.


This study also revealed that the activity of the two-component system (TCS) responsible for its induction is retro-inhibited by the level of pyruvate influx. Following up on the open question which is whether this retro-inhibition is a generic mechanism for TCSs, we will discuss the implications in metabolic engineering.Synthetic Biology (SB) aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (over)producing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM) to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5): e00976-17), which identified and characterized a pyruvate transport system in the Gram-positive (G Subsequent mechanistic studies in bacteria showed that preferred carbon source(s) (most often glucose) are able to restrain transport and catabolism of alternative carbon sources. This regulation, called carbon catabolic repression (CCR), imposes a strict hierarchy in the use of carbon sources and allows for optimal adaptation (i.e. fitness and growth rate) to environmental conditions. Usually, CCR involves transcriptional regulation to prevent transcription of catabolic genes and operons (catabolite represssion sensu stricto) and post-transcriptional regulation to prevent uptake or formation of the specific inducers of catabolic operons (inducer exclusion). In most G+ve bacteria and in particular in B. subtilis, the glucose-mediated CCR operates at the transcriptional level via the master regulator of carbon metabolism CcpA. Repression of several targets, among which are the genes encoding the transporters of alternative carbon sources, then occurs upon binding of CcpA to regions in promoters called catabolite responsive elements (cre sites). Hence, CcpA can regulate up to 10% of the bacterial genome. While organic acids are considered to be low in the hierarchy of the CCR, malate is an exception in B. subtilis. Malate is rapidly metabolized by the cell, can be co-metabolized with glucose, and represses the use of alternative substrates by hijacking the usual glucose-mediated CcpA-dependent CCR. In addition to regulation by the CCR, uptake systems are usually induced in the presence of the metabolite they transport. This creates an additional level of regulation that avoids wasting resources by producing unnecessary carriers. It is a fundamental principle of design that can be understood in biology as the result of the optimization of fitness. In bacteria, signal transduction mechanisms, known as two-component regulatory systems (TCS), represent the major signal response cascade to environmental signals. TCSs are found in a wide variety of processes such as virulence, mobility, growth and metabolism. The canonical form of a TCS consists of a sensor kinase (SK) and a response regulator (RR). The SK detects an environmental signal and is capable of autophosphorylating from an ATP molecule to a histidine residue in its catalytic domain. The phosphate is then transferred to the aspartate of the RR, which often functions as a phosphorylation-dependent DNA-binding transcription factor (i.e. activator or repressor). In B. subtilis, there are at least thirty-four TCSs involved in regulating the central carbon metabolism and more particularly the entry of gluconeogenic substrates. Pyruvate is a small-molecule metabolite at the heart of the CCM and a gluconeogenic substrate assimilated by several bacterial species. Our initial results from a study to address the identification and characterization of pyruvate uptake and regulation in B. subtilis were recently reported in mBio (MBio 8(5): e00976-17) and are summarized and expanded here.The central carbon metabolism provides energy and biomass precursors to the entire cellular network. Its fine regulation is therefore essential to ensure optimal use of carbon sources and proper allocation of resources to the metabolic pathways. Most living organisms including bacteria can use various compounds as carbon sources, where these can be either co-metabolized or selectively used. The later phenomenon was first described by Jacques Monod, who observed a glucose-over-lactose preference for the Gram negative , whose products are predicted to be membrane proteins, are specifically induced in the presence of pyruvate. This prompted us to question whether PftA and PftB were involved in extracellular pyruvate utilization. As expected, deletion of either pftA or pftB drastically reduces growth on pyruvate as sole carbon source. We also learned from various purification and co-purification experiments that PftA and PftB form a hetero-oligomeric membrane protein complex. The next step was to show that the PftAB membrane complex effectively codes for a pyruvate transport system. During growth on pyruvate of the G+ve bacterium, Corynebacterium glutamicum, pyruvate is taken up by the monocarboxylate transporter MctC, and as a result, a mctC mutant is unable to grow on pyruvate as the sole carbon source. When expressed in C. glutamicum, the pftAB operon functionally replaces mctC and enables pyruvate uptake and cell growth. As the gradient of pyruvate drove the PftAB-mediated transport of pyruvate across the cell membrane, we concluded that PftAB operates as a pyruvate-specific facilitated transporter . Hence, PftAB is able to either import or export pyruvate in response to environmental changes in pyruvate . What happens if PftAB is overproduced in a bacterial species where the intracellular pyruvate concentration is much higher than the Km of PftAB as it may lead to significant pyruvate outflow? Several lines of evidence suggest that the intracellular level of pyruvate in E. coli is at least 10 times higher than in B. subtilis (i.e. between 5 and 10 µM). Expression of pftAB under the control of inducible promoters in E. coli grown in rich medium revealed that the higher the level of expression of pftAB, the more toxic . Although not yet demonstrated, it is reasonable to assume that PftAB overproduction in E. coli disrupts pyruvate homeostasis, and as a consequence leads to cell growth inhibition or cell death.We knew from previous genome-wide studies in lytST operon, which codes for a putative TCS, is involved in the induction of pftAB. Combining various in vitro and in vivo experimental approaches, we learned that extracellular pyruvate acts as the signal molecule for the LytS SK, which in turn activates the LytT RR. Induction then occurs by binding of LytT onto two boxes upstream of pftAB. The promoter of pftAB also contains a putative cre site overlapping the -35 region. Consistently, both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream pftAB which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate but not glucose. As an active malic enzyme replenishing the pyruvate pool is required for this repression, we hypothesized that the end product of the reaction (i.e. intracellular pyruvate) is responsible for this repression. Interestingly, a general principle of design of metabolic systems resides in feedback regulations that avoid metabolic systems to run out of control. The reader will find in the literature excellent reviews that cover the area of metabolic feedback regulations, especially where the end products are the signal molecules. Lesser known but not less important are feedback regulations where the inducer metabolite also mediates the feedback control. For instance, sugars such as fructose or sucrose induce expression of their own transporter but also activate the CcpA-dependent CCR to repress the use of alternative carbon sources. By doing so they partially repress their own assimilation. An attractive hypothesis was therefore that pyruvate is capable of feedback regulationWe knew from early studies that the pftAB, this induction is strongly inhibited by the activity of PftAB . Hence, the higher is the influx of pyruvate, the stronger is the retro-inhibition of pftAB induction via LytST.on LytST to avoid the metabolism to run out of control in the presence of high amount of pyruvate. We tested this hypothesis and although extracellular pyruvate triggers the induction of B. subtilis suggest that the TCS retro-inhibition is not restricted to LytST. We are currently investigating the underlying molecular mechanism of these retro-inhibitions and whether these are widespread in the bacterial kingdom. This is an important issue in metabolic engineering as the induction of synthetic circuit expression by heterologous TCSs may be retro-inhibited by the cognate inducer metabolite if naturally present at high concentration in the host cell . In addition, unless the inducer metabolite is not taken up by the cell , the activator activity of a TCS may be reduced by the inducer metabolite influx. This regulation also complicates the development of biosensors for health or for environmental bioremediation. Such biosensors are often inspired from sensor proteins such as TCSs, and the distortion between the expected and effective induction levels challenges the rational design of novel nature-inspired sensors .Our unpublished data in


Gap junctions are intercellular channels that allow passage of ions and small molecules between adjacent cells. Gap junctions in vertebrates are composed of connexons, which are an assembly of six proteins, connexins. Docking of two connexons on the opposite cell surfaces forms a gap junction between the cytoplasm of two neighboring cells. Connexins compose a family of structurally related four-pass transmembrane proteins. In mammals, there are ~20 connexins, each of which contributes to unique permeability of gap junctions, and mutations of some connexin-encoding genes are associated with human diseases. Zebrafish has been predicted to contain 39 connexin-encoding genes; the high number can be attributed to gene duplication during fish evolution, which resulted in diversified functions of gap junctions in teleosts. The determination of body shapes and skin patterns in animal species is an intriguing question. Mathematical models suggest principle mechanisms explaining the diversification of animal morphology. Recent studies have revealed the involvement of gap junctions in fish morphological diversity, including skin pattern formation and body shape determination. This review focuses on connexins in teleosts, which are integrated in the mathematical models explaining morphological diversity of animal skin patterns and body shapes. Gap junctions are intercellular channels that mediate the transfer of small molecules between adjacent cells  and jaguar (kir7.1) mutants  channels through binding to the channel pore  mutant who has a short fin segment  axis was decreased , manifested by small eyes, underdeveloped teeth, and malformation of fingers (Paznekas et al., The mutation in the human It is very interesting that two independent projects which aimed to disclose the molecular mechanism hidden in mathematical models, found the involvement of gap junction proteins: Cx41.8 in the R-D model and Cx43 in the theory of transformation.leopard mutant (Figure In the R-D model, positive interactions between two factors are important. As mentioned above, Cx41.8-gap junctions might be formed between xanthophores and melanophores, and control the directional flow of small molecules from a xanthophore to a melanophore depending on spermidine concentration. In addition, the R-D model predicts that Cx41.8 might be involved in melanophore differentiation. The reduction of basal level synthesis of a factor also causes pattern changes in the R-D model (Asai et al., t Figure .stp mutant might be the simplest example. It is possible that the reduction of osteogenic activity causes the formation of shorter vertebra in the stp mutant, although the difference in the mechanisms underlying the formation of shorter vertebra in the stp fish and shorter fins in the sof fish is unclear. Recent gap junction studies have revealed that hemichannels formed by connexins and/or pannexins function as sensors of mechanical stress (Jiang et al., Regarding the theory of transformation, the Many questions still remain, including functional differences among connexin proteins, the type of transferred molecules, and the evolutionary events that have led to the acquisition of this intercellular communication system in the animal kingdom. Future mechanistic and genetic studies would address these questions.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A sinus of Valsalva aneurysm is a rare cardiac anomaly which may be acquired or congenital. The main associated symptoms are conduction disturbances, myocardial ischemia, and syncopes.In this report we describe a 52-year-old Albanian woman from Kosovo with an unruptured aneurysm of 74×60 mm of the noncoronary sinus of Valsalva presenting dyspnea, jugular distension, and tachycardia due to cardiac compression. She underwent successful closure of the orifice and sinus remodeling with a Dacron patch.To the best of our knowledge this is the largest reported isolated unruptured aneurysm of the coronary sinus causing severe compression of the cardiac chambers undergoing successful surgical correction. A sinus of Valsalva aneurysm is a rare cardiac anomaly which may be acquired or congenital, intracardiac or extracardiac . In 65 tA 52-year-old Albanian woman from Kosovo presented to our hospital with dyspnea, tachycardia, and jugular distension. On admission she was in New York Heart Association class III, with normal blood pressure and absence of aortic murmur. She was in sinus rhythm and a chest X-ray demonstrated cardiomegaly with right atrium enlargement. A transthoracic echocardiography demonstrated increased pericardial effusion of almost 35 mm and a giant mass above her left atrium communicating with the ascending aorta. A transesophageal echocardiogram showed a large unruptured aneurysm/pseudoaneurysm 61×77 mm of the noncoronary sinus of Valsalva, without thrombosis or shunts, above her left atrium Fig. . Her aorShe underwent femoral artery and vein cannulation and on cardiopulmonary bypass a sternotomy was performed. Her pericardium was then opened. An unruptured aneurysm of the noncoronary sinus of Valsalva was identified Fig. . On intrTo the best of our knowledge there are no previous published data of an acquired unruptured aneurysm of the noncoronary sinus of Valsalva of such dimensions, which first presented as a cardiac tamponade with the additional symptoms and signs that are typical in these settings, such as dyspnea, tachyarrhythmias, and jugular distension. The most frequently found symptoms of an unruptured aneurysm of the sinus of Valsalva are exertional dyspnea , palpitaSinus of Valsalva aneurysms can be acquired secondary to infections, degenerative conditions, or trauma. Most cases are congenital due to a defect in the aortic wall, more precisely of the continuity between the aortic media and aortic annulus. The essential lesion of a sinus of Valsalva aneurysm is separation of the aortic media of the sinus from the media adjacent to the hinge line of the aortic valve cusp. The differential diagnosis of sinus of Valsalva aneurysm includes isolated pseudoaneurysm of the sinus of Valsalva. In the case of a pseudoaneurysm the orifice is apart from the aortic annulus, which is not the case in a sinus of Valsalva aneurysm where the orifice of the aneurysm is adjacent to the aortic annulus. In a pseudoaneurysm of the sinus of Valsalva there are no specific histologic changes in the aneurysmal wall. An association with Marfan and Ehlers–Danlos syndromes, bacterial endocarditis, syphilis, and mycotic infection has been reported . In our Two-dimensional Doppler echocardiography can adequately predict sinus of Valsalva aneurysms. Cardiac catheterization confirms the diagnosis and the hemodynamic significance of the lesion, the associated cardiac anomalies, and the coronary anatomy can be precisely evaluated. A contrast-enhanced angio-CT scan or MRI, when feasible, can accurately assess aneurysm size, sinus of origin, aortic valve involvement, and the presence of associated cardiac abnormalities.Rupture is the most frequent complication of a sinus of Valsalva aneurysm. An unruptured aneurysm of the sinus of Valsalva may cause right ventricular outflow tract obstruction, infective endocarditis, malignant arrhythmias, myocardial ischemia/infarction due to severe distortion of coronary ostia or compression of the coronary trunks, and dilatation of aortic annulus due to anatomical change. Our case presented a syndrome similar to a precardiac tamponade, due to a significant compression of her right atrium and due to the pericardial effusion. In such a case it was difficult to differentiate an unruptured aneurysm from a ruptured aneurysm or pseudoaneurysm of the sinus of Valsalva. Intrapericardial rupture or pseudoaneurysm carries a high rate of mortality, therefore, urgent surgical intervention is recommended in this subset of cases as in our case.We decided to repair the affected noncoronary sinus, avoiding the possible drawbacks related to reduced aortic root pulsatile expansion . The indIn other cases in which the aortic root is dilated, the root replacement procedures should be applied according to established guidelines. In most of the cases the aortic annular ectasia is present and valve-sparing procedures might be applicable ; in otheIn conclusion we report the largest unruptured aneurysm of the noncoronary sinus of Valsalva, causing severe compression of the cardiac chambers, undergoing successful surgical correction.


Therefore, we propose to use PI as an additional and essential diagnostic quantity for improved predictability of the reciprocating WSS.Arterial wall shear stress (WSS) parameters are widely used for prediction of the initiation and development of atherosclerosis and arterial pathologies. Traditional clinical evaluation of arterial condition relies on correlations of WSS parameters with average flow rate (Q) and heart rate (HR) measurements. We show that for pulsating flow waveforms in a straight tube with flow reversals that lead to significant reciprocating WSS, the measurements of HR and Q are not sufficient for prediction of WSS parameters. Therefore, we suggest adding a third quantity—known as the pulsatility index (PI)—which is defined as the peak-to-peak flow rate amplitude normalized by Q. We examine several pulsating flow waveforms with and without flow reversals using a simulation of a Womersley model in a straight rigid tube and validate the simulations through experimental study using particle image velocimetry (PIV). The results indicate that clinically relevant WSS parameters such as the percentage of negative WSS (P[%]), oscillating shear index (OSI) and the ratio of minimum to maximum shear stress rates ( For example, Finol et al. ), and c) the measure of overall oscillation . The first two parameters, min/max and P[%] were defined by Gharib and Beizaie  and most importantly, the OSI parameters are very well related to the 2nd order polynomial multi-variable model that involves the three hemodynamic parameters. Large OSI and P[%] values for large PI values imply that the global flow reversal is a major contributing mechanism to WSS variability during the cycle as well as to the duration of the negative WSS. The results undoubtedly reflect the need for an additional, third, parameter that influences WSS parameters in pulsating flows independently of the HR or Q.The The application seems to be straightforward: by means of sampling flow waveform (e.g. using ultrasound Doppler) at several phases during the cardiac cycle, one can readily obtain PI in addition to the traditional HR and flow rate measurements. Joint analysis of these three values will then provide the necessary prediction of negative WSS and severity of associated WSS-related parameters.Q, extracted from Doppler measurements with vascular dysfunctions.Addition of PI to the measurement list may improve significantly prognosis and prediction of vascular disease development due to vascular remodeling and variations in flow pulsatility. Future research could implement clinically the above suggestion and investigate correlations of local PI, HR and The inverse Womersley method in the present study considers a simplified geometry of a straight long circular tube. We do not take into account complicated anatomical geometries, arterial stenoses, arterial curvatures, bifurcations and nonuniform cross sections—which may contribute vortical or helical 3D flow, skewed flow profiles, separation regions, etc. In addition, wall motion may affect WSS parameters in up to 15% . TherefoAnother limitation relates to the working fluid. The working fluid used as a model for blood is not perfectly matched with the Tygon tube. The small refractive mismatch resulted with some light reflection from the pipe walls. Static and dynamic calibrations were implemented to ensure accurate measurement. However, measurement resolution at the near wall region was limited.spatial resolution of the PIV method and the inverse Womersley method, we overcame this limitation and succeeded identifying an important improvement in correlation between the WSS and WSS-related parameters, and nondimensional numbers, using the addition of the pulsatility index.Due to limited time resolution of the PIV system, the velocity profiles were measured only at seven time instants during the cycle. It could be beneficial to achieve a better accuracy with the time-resolved PIV systems . NeverthThe inverse Womersley method is based on Womersley's simplified model of a straight, rigid and infinitely long tube. It was shown in previous studies that the goodness of fit of the models may be affected by the geometry and boundary conditions , SahtoutRecent studies show the feasibility of using the inverse Womersley method in conjunction with the Doppler ultrasound measurements to assess the flow rates and the velocity profiles in rigid tubes . Our conS1 FigThe aberrations are negligible due to very small differences in index of refraction (less than 0.001).(TIF)Click here for additional data file.


This paper provides an overview of the current research in the field of optical techniques for cervical neoplasia detection and covers a wide range of the existing and emerging technologies. Using colposcopy, a visual inspection of the uterine cervix with a colposcope (a binocular microscope with 3- to 15-fold magnification), has proven to be an efficient approach for the detection of invasive cancer. Nevertheless, the development of a reliable and cost-effective technique for the identification of precancerous lesions, confined to the epithelium  still remains a challenging problem. It is known that even at early stages the neoplastic transformations of cervical tissue induce complex changes and modify both structural and biochemical properties of tissues. The different methods, including spectroscopic  and imaging techniques , probe different tissue properties that may serve as optical biomarkers for diagnosis. Both the advantages and drawbacks of these techniques for the diagnosis of cervical precancerous lesions are discussed and compared. Cervical cancer remains one of the major health issues, causing 266000 deaths of women worldwide in 2012  Figure .human papillomavirus (HPV) [There is conclusive evidence that the majority of cervical cancer cases 95–98%) is caused by the infection with cancerogenic strains of us (HPV) –6. Most us (HPV) –9. The l5–98% is The transformation zone, defined as an area limited by the positions of original and the active squamocolumnar junctions is most susceptible to HPV infection. When HPV gets a foothold and reproduces itself, it can invade the cells of the basal layer, which separates epithelium from underlying connective tissue and, eventually, rise to the epithelium surface with the mature squamous cells. The virus infection gradually induces severe damage. HPV-infected cells may become malignant if the virus inserts its cancer-causing genes into the DNA of the host cell.The staging of the disease is based on morphological criteria and tissue architecture, namely, on the thickness of the involved epithelium layer . When onLeft untreated, severe dysplasia will grow and break a basal membrane and eventually evolve into an invasive cancer. This process is very slow and may take over ten years after the infection. It makes cervical cancer perfectly suited for the effective management by screening according to criteria defined by the World Health Organization –12.Recent discovery and subsequent mass use of the vaccines against HPV hold promise for the prevention of cervical cancer and will significantly improve the situation at large . Those vIn high-resource settings a regular screening by the cytopathological Pap test is performed for an early detection and prevention of cervical cancer. Cells collected from the external os of the cervix are studied under a microscope. If abnormal cells are detected, further diagnostic testing in the form of colposcopy is often recommended for the localization and marking out of metaplasia.The visual examination of the cervix for metaplastic lesions with a colposcope is done after the application of acetic acid and then repeated after the application of iodine Lugol’s solution (both work as contrast enhancing agents). Normally, the biopsies  are taken from the areas whitened by acetic acid and those which are not colored by iodine. If the analysis of histological cuts by pathologists ultimately confirms the presence of a high grade malignant lesion (CIN 2–3), the abnormal zone is surgically removed by cervical conization. This is a minimally invasive curative treatment which can completely eliminate the disease provided it was diagnosed at an early stage before the transformation into an invasive cancer. This treatment has minimal adverse effect on fertility and reproductive functions of women.It is worth to mention that the results of colposcopy may also be affected by the presence of non-neoplastic cervical diseases and demographic factors such as age and parity. Thus, the accuracy of colposcopy strongly depends on the level of training and experience of clinicians performing the test. As stand-alone diagnostic method colposcopy has a quite high sensitivity  of over 90% in detecting HSIL and cancer (CIN 2+). But the specificity  of colposcopy for the detection of CIN 2–3 is reported to be relatively low (23–87%) –21. EvenCurrent management of cervical cancer  has significantly decreased the mortality rate in highly developed countries during last decades. At the same time the incidence and mortality rates in the middle and low-income countries did not improve and remain significantly high due to insufficient awareness about cervical cancer among women and health providers, lack of access to HPV vaccination, absence of screening and treatment programs. This puts women at the increased risk of developing invasive cervical cancer .In current programs for screening and diagnosis of cervical cancer the critical issue is an increase of efficiency and accuracy of screening and diagnostics techniques. Typically it requires up to three visits to a medical professional and several weeks in total in order to obtain the diagnosis and treatment, if necessary . The impCurrently several optical methods such as diffuse reflectance spectroscopy, fluorescence spectroscopy, Raman spectroscopy, in vivo confocal microscopy, optical coherence tomography and multi-wavelength imaging Mueller polarimetry, as well as the combination of different techniques have been explored to improve the detection of cervical neoplasia. The results of these studies as well as current trends to miniaturization of diagnostic instruments will be discussed further.In vivo diffuse reflectance optical spectroscopy (DRS) exploits the fact that abnormal zones of the cervical epithelium illuminated with a low-power broadband light source produce different backscattering spectra compared to normal cervical tissue in the visible wavelength range. Such difference in spectra detected by an optical sensor can be used in order to identify neoplastic lesions of the cervical epithelium. DRS is an indirect optical technique and may require either fitting of measured spectra with multi-parametric models describing the realistic optical properties of tissue  or usingIt has been demonstrated that the total concentration of Hb, which is responsible for absorption in tissue in the visible wavelength range, was statistically higher in CIN 2–3 compared to normal cervical tissue ,27. ThisDespite the observed common trends for DRS optical markers with the evolution of CIN lesions there is a significant variability of parameter values in different patients depending on their age as well as presence of non-neoplastic lesions . The shoPoint-probe optical spectroscopic instruments may also be used for scanning the suspicious sites of the cervix. However, this approach is laborious and time-consuming and the possibility to miss the potential lesions is not negligible. Hence, these techniques are not suitable for CIN screening in real settings. The instruments that perform a multi-spectral wide-field imaging of the whole cervix are required to address these issues. Park et al.  developeThe advanced version of the automated domain-specific image analysis algorithm for the detection of cervical precancerous lesions identified first the regions of squamous and columnar epithelium . TransfoWhile the scattering of light by biological tissue plays the main role in DRS, the absorption and emission of light by matter are the key steps in fluorescence spectroscopy. The use of fluorescence spectroscopy for the screening and diagnosis of cancer is related to the ability of this technique to probe the molecular composition of tissue and observe the distribution of specific molecules. When light of a chosen excitation wavelength illuminates the sample, the tissue molecules are exposed to light having an energy that may match a possible electronic transition within the molecule. Consequently, part of incident radiation will be absorbed as the electron is lifted to a higher energy orbital. During de-excitation (return of electron to the ground state) those molecules release energy in the form of light of a specific emission wavelength , which can be measured by a detector. The fluorescence signal is a superposition of various emission signals of different wavelengths and intensities. It depends on the excitation wavelength and on the presence and concentration of fluorophore molecules in the tissue.Depending on the type of investigated fluorophores  light-induced fluorescence spectroscopy can be classified either as autofluorescence spectroscopy or as induced fluorescence spectroscopy, respectively. The information on fluorescence lifetimes may be obtained using instruments with pulsed mode illumination and time-resolved detection . The dirThere is experimental evidence that the intensity of autofluorescence of normal cervical tissue is altered by the precancerous modifications of cervical epithelium . The difChidananda et al.  studied Combining information about the fluorescence of stromal collagen and epithelial NADH, Pandey et al.  reportedApart from the changes in cellular metabolic processes and in the extracellular tissue matrix induced by CIN progression, both scattering and absorption of light in tissues may significantly influence the measured fluorescence spectra modifying the intensity and width of specific peaks. Georgakoudi et al.  suggesteDespite a clinically significant increase in NADH fluorescence and decrease in collagen fluorescence in the spectra measured on dysplastic cervical tissue the age of the patient may affect the fluorescence-based diagnosis of CIN. Some age-related changes of cervical tissue modify the fluorescence spectra in a similar way as dysplasia ,49. Due The accuracy of the detection of CIN lesions with spectral autofluorescence measurements depends on several factors including (i) changes in autofluorescence background, which may influence the quantum yield of fluorophore, (ii) inhomogeneities in the optical properties of tissue, (iii) alterations of the tissue architecture , (iv) the spectral dependence of the absorption of light by non-fluorescent chromophores such as hemoglobin. Weingandt et al.  observedGu et al.  suggesteThe growth of tumor in mice, inoculated with highly tumorigenic TC-1 cells immortalized using HPV type 16 proteins, was studied as a model of cervical cancer by Bae and co-workers . Using aAn intrinsic problem of fluorescence spectroscopy is linked to the fact that both intensity and contrast of autofluorescence in tissue are quite low. Often the spectral difference between normal and pathological tissue can be enhanced by external administration of fluorophores or fluorophore precursors. The preferential accumulation of exogenous fluorophores in abnormal cells  results During the interaction of light with matter a number of different processes may take place: reflection, transmission, absorption, elastic and inelastic scattering of incident radiation. Raman spectroscopy (RS) is an optical technique that relies on inelastic scattering of light. The sample is usually illuminated with a monochromatic laser beam that vibrationally excites molecular chemical bonds. The energy of inelastically scattered light is changed by those vibrations that are strictly related to the structure of molecules. A plot of intensity of inelastically scattered radiation as a function of the difference of its frequency from the frequency of the incident radiation is called Raman spectrum. Consequently, positions, shapes and relative intensities of the peaks in a Raman spectrum carry valuable information about both chemical composition and morphology of the sample. That is why RS performs well as a versatile optical technique for chemical and structural characterization of studied samples in a rapid and non-destructive manner .The biochemical components of tissue  contribute to the measured Raman spectra by superposition of their individual Raman signals. The combinations of these components, which are specific for the different type and physiological status of tissue, produce a unique biochemical “signature” of the sample in the form of particular fingerprint-like spectral features in the Raman spectrum.It suggests that Raman spectroscopy may be used as a tool to detect early biochemical changes at a molecular level that are associated with the precancerous modifications of tissue. During the last years the potential of RS as label-free diagnostics technique for the detection of different types of cancers has been studied by many research groups both in vivo and ex vivo –65.−1) and high wavenumber  spectral regions [In one study of 44 patients Raman spectra were acquired from 356 normal and 120 precancerous sites during the colposcopy in the fingerprint  no specific requirements for sample preparation, (ii) the possibility to use this technique with fiber optics for ex vivo and in vivo measurements, (iii) a high spatial resolution suitable for imaging of subcellular components.−30 cm2·sr−1). So, one of the main difficulties of RS consists in separating the contribution of the weak intensity of the inelastically scattered light from the strong intensity of the Rayleigh scattering signal. Current solution consists in using notch or edge optical filters to cut the contribution of the Raman probing wavelength. To avoid the interference of the Raman signal with fluorescence emission, special attention should be paid to the selection of the laser excitation wavelength.Typically, Raman scattering produces a very weak signal  and coherent anti-Stokes Raman scattering (CARS)) or metal nanoparticles . However, these improvements often increase the time of measurements and the complexity/cost of the instrument, which may hinder clinical applications of Raman spectroscopy. In addition, the spread of diagnostically relevant peaks across the Raman spectra requires the development of efficient classifiers, which can fully explore rich spectral information for accurate and reliable diagnostics. One of the promising applications of RS can be the monitoring of the patients undergoing chemotherapy. A priori knowledge of administrated drugs will help to detect the new Raman peaks. There will be no need for point-by-point scanning. Hence, the time of measurements can be significantly reduced.The optical techniques for CIN diagnostics discussed so far focused on macroscopic imaging or spectral probing of tissue. It is known that CIN lesions are characterized by morphological changes, such as modified tissue architecture, increased size of cell nuclei and increased nuclear/cytoplasmic ratio. The assessment of these morphological changes is currently done through microscopic histological analysis of biopsies (gold-standard diagnostics). Screening and diagnostics can be significantly improved by the high-resolution optical imaging technologies that image subcellular structures in vivo, thus, replacing tissue removal, processing, and examination by pathologists .In vivo confocal microscopy is an optical technology that can non-invasively reconstruct three-dimensional cell structures from successive microscopic images taken at different depths (around 300–400 µm) within a thick tissue . A point illumination and a pinhole placed at the optically conjugate image plane in front of the detector isolate light reflected or fluorescent from a finite volume and block scattered and out-of-focus light. This increases optical resolution and image contrast compared to conventional optical microscopy. The sample plane is scanned by focused laser beam and confocal images are built up point-by-point. The fluorescence scanning confocal microscopy is typically used for imaging in the majority of biological applications –70. The Confocal microscopy has been extensively used in different branches of medicine –72. Due CIN lesions were characterized by increased nuclear density and size, and the presence of atypical cells. Examination of a CIN 3 lesion with a confocal endomicroscopic imaging probe (site marked by the asterisk in the colposcopy image (A low-cost high-resolution microendoscope (HRME) was developed and used for the direct visualization of neoplastic biomarkers  during colposcopy –79. GranA set of images from over 60 patients obtained by fluorescence confocal endomicroscopy was used for ex vivo and in vivo studies of four types of cervical tissue relevant for the diagnostics: normal columnar epithelium, normal and precancerous squamous epithelium, and stromal tissue . ResearcThe rapid progress of nanotechnology had an important impact on cancer management research. The variety of new nanoscale platforms  are used for cancer theranostics, which means the simultaneous diagnosis and treatment of diseases –86. ThesIt is known that progression of CIN from mild dysplasia to invasive cancer is accompanied by the increase in level of epidermal growth factor receptor (EGFR). The overexpression of EGFR has been correlated to uncontrolled cell growth and inhibition of cell apoptosis. Hence, EGFR can be used as a unique molecular tumor marker .The contrast agents consisting of a targeting agent conjugated with optically active labels  can be used for in vivo imaging of this biomarker. Sokolov et al  reportedIn recent years many research groups explored the potential of using quantum dots (QDs) as inorganic fluorophores for cellular imaging ,85,90–93There is an emerging set of optical techniques based on the detection of changes in the polarization of light instead of (or together with) intensity measurements. Apart from the intensity and wavelength of probing light its polarization can carry important information about the sample. Many research groups work in the field of biomedical applications of polarized light –103.) and orthogonally  to the polarization state of the illuminating light. The OSC parameter is then calculated from these two measurements as . The diagnostic utility of this optical technique relies on the fact that polarized light loses its polarization when undergoing multiple scattering events within biological tissue. The part of backscattered light that preserves its polarization was most probably scattered only once or reflected at the sample surface. Thus, the differential signal removes the contribution of light that has been diffused deeply within tissue and keeps the contribution of the superficial layer at which epithelial cancer starts.Even the simplest orthogonal state contrast (OSC) polarimetric techniques provide data about the polarimetric characteristics of the sample. Typically, the sample is illuminated with linearly or circularly polarized white light. Two set of measurements are performed, detecting the intensity of signal after interaction with sample through another linear or circular polarizer, set parallely  to the illumination with polarized light in the absence of non-linear effects. So, using the Stokes–Mueller formalism has proven to be necessary when dealing with biological samples. Rich polarimetric information about the sample properties is contained in the coefficients of the Mueller matrix. Currently, the phenomenological approach based on polar decomposition of the Mueller matrix by the Lu–Chipman algorithm [103M is decomposed into the product of three matrices: M = MΔMRMD, where MΔ, MR, and MD are the Mueller matrices of depolarizer, retarder and diattenuator, respectively. Finally, the scalar values of depolarization, retardance and diattenuation, as well as the orientation of the optical axes of the retarder and the diattenuator can be obtained from the matrices MΔ, MR, and MD. Strictly speaking, these parameters represent a set of “effective” optical markers of tissue. Lu–Chipman decomposition implies a sequential order of elementary polarimetric properties along the trajectory of the probing beam, whereas these polarimetric properties can be mixed within the volume of tissue. Nevertheless, these effective values of depolarization and retardance are found to be the important parameters for the polarimetric analysis.where ectively . The linlgorithm  is widelrithm 103,109–110 Shukla et al.  obtainedDuring in vivo clinical studies linear OSC images of healthy uterine cervices acquired during colposcopy demonstrated a strong change of OSC contrasts with a 90° periodicity  when the azimuth of the polarizer was varied . ContrarThis effect was attributed to the degeneration of stromal collagen beneath the precancerous epithelial lesions –32. The At first glance the trends in depolarization look contradictory to the results of Shukla et al. . HoweverEx vivo studies of 17 fixed cervical specimens performed with a multi-spectral Muller imaging polarimeter  showed oThis suggests that Mueller polarimetry as wide-field imaging technique can greatly enhance colposcopy performance for the detection of CIN zones provided the raw data are processed by properly chosen algorithms. Finally, since polarimetric imaging is sensitive to the overall conditions of the collagen in the extracellular matrix , it may also be relevant for the optical diagnostic of various gynecological pathologies involving connective tissues  is an optical technique for non-invasive cross-sectional imaging of biological tissue. This technique makes use of low-coherence interferometry with a near-infrared light source to create two-dimensional images of tissue cross-sections by exploring elastic light scattering from internal tissue microstructures . OCT proNowadays OCT is the reference technique in ophthalmology –121 and Normal squamous cervical tissue exhibits a well-organized three-layer architecture . Prior sIn the OCT image of healthy squamous cervical tissue a basement membrane (BM) is not resolved because of the lack of OCT resolution . HoweverStudies on using OCT for the detection of CIN and invasive carcinomas were carried by Gallwas and co-workers . During A study with 299 women on using OCT as adjunct to colposcopy for improving its sensitivity and specificity in a real-time clinical evaluation were conducted by Liu and co-workers . They deGallwas et al.  suggestePolarization-sensitive OCT (PS-OCT) combines the spatial information on the polarization state of light scattered from tissue with the recorded intensity of interference fringes . Depth-rS3/S0, the slope of axial decay of DOCP signal near the cervical epithelium was determined by a linear fitting procedure. Using the abovementioned slope as parameter for CIN diagnostics a sensitivity of 94.7% and a specificity of 71.2% was obtained for a slope threshold value of 1.8 mm−1.Lee et al.  examinedSeveral studies reported using the combination of different optical  techniques to improve the performance of CIN diagnostics. Georgakoudi et al.  developeFreeberg et al.  reportedWeber et al.  measuredA multimodal hyperspectroscopy (MHS) instrument that combines fluorescence and reflectance spectroscopy was tested in 1607 women at risk for cervical dysplasia . The senThe comparative studies of diffuse reflectance and Raman spectroscopic measurements performed in vivo with a fiber-optic probe on 22 patients  showed a slightly better diagnostic accuracy of Raman spectroscopy . The senA hybrid optical imaging modality that explores photoacoustic effect was used by Peng et al.  for the The efficacy and advantages/disadvantages of new alternative technologies or technologies adjunct to colposcopy using multimodal hyperspectroscopy, dynamic spectral imaging, OCT, confocal microcolposcopy, electrical impedance spectroscopy and combined optical/electrical instruments are discussed in –147.The use of all functionalities of smartphones for the wide-field imaging of the uterine cervix with white and green light sources and magnification lens for an enhanced visualization is suggested and being tested by MobileODT . AnotherCurrent programs for cervical cancer screening still rely on Pap/HPV tests for the primary screening and on colposcopy for the diagnostics and guiding biopsies, if necessary. With more countries introducing HPV vaccination programs the prevalence of HSIL is expected to drop. The challenges for standard colposcopy will grow, since correct diagnosis depends to a large extent on the experience of the operator trained to recognize the high-grade cervical dysplasia on a sheer number of cases.To maintain the satisfactory level of sensitivity and specificity of HSIL diagnostics we need to improve the instruments and advance the screening procedures. The innovative biomedical optical imaging and spectroscopic techniques provide clinicians the possibility to inspect the epithelial volume and underlying tissue non-invasively and to extract an accurate information regarding tissue morphological and biochemical states. The choice of the most appropriate optical technique will always involve a trade-off between the technical parameters  and medical diagnostic outcome (specificity and sensitivity) that can be achieved.Currently, new optical instruments and contrast enhancement techniques for the accurate and reliable diagnostics of cervical neoplasia are still at an exploratory stage and have not yet been widely accepted for routine screening and diagnostics. Commercially available instruments are being tested by medical practitioners in real-life settings, while other devices still undergo clinical trials for the confirmation and optimization of their diagnostic performance.Modern trends in biomedical optical instrumentation require the development of portable and cost-effective versions of medical devices. In particular, these needs are driven by the necessity to support global transition, namely, to deploy and use these instruments in low-resource countries. This is the problem of paramount importance for the screening, diagnosis and treatment of cervical cancer. Ideally the wide-field imaging (polarimetric or fluorescent) should be combined with optical point-probe measurements  and different contrast-enhancing techniques to perform the optical biopsy of tissue. Such instruments might be considered for the first-line screening and triage by optical means during the same medical visit, thus, significantly reducing the cost of cervical cancer prevention programs.


Rhizobia form symbiotic nitrogen-fixing nodules on leguminous plants, which provides an important source of fixed nitrogen input into the soil ecosystem. The improvement of symbiotic nitrogen fixation is one of the main challenges facing agriculture research. Doing so will reduce the usage of chemical nitrogen fertilizer, contributing to the development of sustainable agriculture practices to deal with the increasing global human population. Sociomicrobiological studies of rhizobia have become a model for the study of the evolution of mutualistic interactions. The exploitation of the wide range of social interactions rhizobia establish among themselves, with the soil and root microbiota, and with the host plant, could constitute a great advantage in the development of a new generation of highly effective rhizobia inoculants. Here, we provide a brief overview of the current knowledge on three main aspects of rhizobia interaction: trade of fixed nitrogen with the plant; diplomacy in terms of communication and possible synergistic effects; and warfare, as antagonism and plant control over symbiosis. Then, we propose new areas of investigation and the selection of strains based on the combination of the genetic determinants for the relevant rhizobia symbiotic behavioral phenotypes. Microorganisms have become popular models for addressing sociobiological questions related to the evolution of multicellularity, communication, and to the exchange of nutrients. Indeed, microbial systems are suitable for the study of mutualistic interactions due to their readiness of manipulation, their short generation times, and the simple methods for tracking of resources . MoreoveProteobacteria, with all rhizobia identified to date belonging to two proteobacterial classes: Alphaproteobacteria and Betaproteobacteria. Rhizobia live as free bacteria in soil or plants , and may form symbiotic nitrogen fixing associations with compatible and leguminous plants. Fabaceae (Leguminosae) is the third largest family of Angiosperms and includes more than 20,000 species , the rhizobia–plant mutualism evolved the capacity for partner discrimination. Molecularly, the “entry visa” is based on the lipochitooligosaccharide molecule known as Nod Factor (NF) and NF receptors . Dependir choice . HoweverFigure 1). Among the most studied examples is the apparatus involved in quorum sensing. This apparatus is related to phytohormone modulating genes, such as those for auxin production and ACC deaminase that are potentially related to modulation of the rhizobia–legume interaction . Moreover, signaling is strongly dependant on the context where strains are growing, and can be influenced by co-resident microbes. Thus signaling cannot be fully examined using conventional microbiological investigations based on pure culture. Reconstructed root microbiotas and easy-to-use gene reporter systems coupled with genome-wide assessments of transcriptional activation during the various stages of symbiosis  could beSinorhizobium , spanning from bacteriocin transporter genes to bacteriocin protection genes. We hypothesize that such bacteriocins may promote antagonistic interactions among members of the same species both in the rhizosphere and within the nodule.In order for rhizobia to enter into a symbiotic nitrogen fixing relationship with a host plant, they have to effectively compete for rhizosphere colonization with other rhizobia and non-rhizobia. From the bacterial point of view, competition implies preventing the colonization of the niche by other strains. This can take two forms: exploitative (indirect), and interference (direct). Exploitative competition involves more effectively utilizing a common limiting nutrient. As examples, rhizobia can produce siderophores that sequester all available iron for use by themselves, limiting the ability of competitors to grow , or straIn several host species , plant–rhizobia interaction involves the tight control of bacterial cell proliferation and differentiation based on antimicrobial peptides [nodule cysteine-rich peptides (NCRs)], as well as the suppression of the host immune response mediated through NF . The evoad hoc rhizobia inoculant strains  by shuffling secondary replicons from different donors. We can imagine the development of an approach to promote horizontal gene transfer of such genetic determinants between strains in a sort of “pangenome-assisted selection,” conceptually analogous to the marker-assisted selection in crops (The genomes of rhizobia are extremely variable , and amoplasmids . Rhizobiplasmids . For rhiplasmids . Additioplasmids . These pin crops . In thisAM conceived the study. AC drafted the manuscript. GD and MB contributed discussing the ideas and contributed in drafting the manuscript. All authors read and approved the final version of the manuscript.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.


The shape of the cardiac action potential (AP) is determined by the contributions of numerous ion channels. Any dysfunction in the proper function or expression of these ion channels can result in a change in effective refractory period (ERP) and lead to arrhythmia. The processes underlying the correct targeting of ion channels to the plasma membrane are complex, and have not been fully characterized in cardiac myocytes. Emerging evidence highlights ion channel trafficking as a potential causative factor in certain acquired and inherited arrhythmias, and therapies which target trafficking as opposed to pore block are starting to receive attention. In this review we present the current evidence for the mechanisms which underlie precise control of cardiac ion channel trafficking and targeting. The function of the heart is governed by the electrical and mechanical activity of myocytes. The functional expression of several different types of ion channels in the myocyte sarcolemma determines the shape and duration of the action potential (AP), and therefore controls the effective refractory period (ERP) of the myocardium. The ERP is a protective mechanism that keeps the heart rate in check and thus prevents arrhythmias. Any prolongation or shortening of the ERP is therefore potentially arrhythmogenic. Most genetic arrhythmias are caused by mutations which alter the biophysical properties of ion channels. However, the proper functional expression of ion channels can be disrupted at several points including at the transcriptional, translational, and post-translational levels. In the last two decades, studies have emerged in which mutations carried by ion channels have been shown to be linked to trafficking defects, resulting in retention and/or degradation of the channel early in the trafficking process.The density of active ion channels in specific membrane domains is a dynamic process resulting from the concomitant and antagonistic action of anterograde  and retrograde  pathways. Targeting and stabilization of these channels by anchoring partners in specialized domains of the sarcolemma also dynamically regulate the electrical activity of the cell  exit defects, leading to targeting of misfolded channels to degradation by the ERAD system . Such mutations have been identified for via ion channel trafficking. Although trafficking defects have been reported for certain ion channel mutations, and are associated with retention in intracellular organelles, the characterization of intracellular trafficking in control and disease conditions are largely lacking in native cardiomyocytes. The fact that these cells are highly structurally and functionally specialized suggests that trafficking and targeting of cardiac ion channels may involve unique and specific pathways. Furthermore, the propagation of electrical activation within the myocardium is a complex process controlled by the spatial/differential distribution of ion channels within single cardiomyocytes that eventually establishes the anisotropic ratio. The underlying mechanisms regulating ion channel targeting to sarcolemmal subdomains, and tethering into macromolecular complexes remains a largely unanswered, yet important question.Trafficking and targeting of integral membrane proteins, including ion channels and receptors, have been explored in other cell types such as epithelial cells and neurons. Although these studies have yielded great insights, these mechanisms have not been studied extensively in cardiomyocytes. Such research may provide insight into the regulation of cardiac excitability In acquired arrhythmias such as heart failure and atrial fibrillation, ion channel dysfunction and electrical remodeling are often associated with tissue remodeling including hypertrophy/dilatation, replacement and interstitial fibrosis, and gap junction disorganization  in the heart , which predisposes individuals to life-threatening arrhythmias. HERG mutations often disrupt the forward trafficking of hERG , and subsequently result in decreased IKr  conditions. They showed that lowering extracellular potassium drastically accelerated hERG internalization and degradation both in overexpression systems and native Cardiac excitability can be regulated by cellular lipid content. Free cholesterol is a major lipid class shown to regulate membrane fluidity, curvature, and stiffness  increased the number of functional KV1.5 channels in the sarcolemma of atrial myocytes, also reducing their mobility as shown by Fluorescence recovery After Photobleaching  and relies on an intact microtubule system. We also found that the response was dysregulated in a model of chronic hemodynamic overload. Hypertrophied atrial myocytes had reduced Kv1.5 expression, despite an increase in basal IKur. The response of these cardiomyocytes to shear stress was reduced, and the kinetics altered. Our results suggested chronically increased mechanical stress over activates the integrin signaling pathway, resulting in an increased IKur, AP shortening and a reduction in the capability of cells to respond to shear stress  in the HL-1 cell line and in dissociated neonatal mouse myocytes. The effect of quinidine was stereospecific as quinine has no effect on KV1.5 channel surface density (despite exerting the pore-block effect of its stereoisomer) and subunit-dependent, as the KV1.5-related channels KV2.1 or KV4.2 did not show internalization at the same time points and doses. In addition, quinidine-induced KV1.5 channel internalization followed the same endocytotic pathway as constitutive endocytosis , the AP is conducted along the myocyte at the lateral membrane (LM).via the microtubule plus-end-tracking protein EB1 , NaV1.5 channels at the LM show a lower density , desmosomal (plakophilin-2), actin cytoskeleton-binding (ankyrin-G), and the MAGUK protein SAP97 at the ID and syntrophin at the LM , anti-serotoninergic (Cisapride) and antiarrhythmics , that are difficult to manipulate as they can also block the channel and worsen the disease. However, certain pore-blocking drugs, such as fexofenadine, have been shown to rescue hERG trafficking of some mutant channels at concentrations far below the IC50 for pore-block (Rajamani et al., Although only proven All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Natator depressus) are endemic to Australia and are the only marine turtle species with an exclusive neritic development. To explain the lack of oceanic dispersal of this species, we predicted the dispersal of post-hatchlings in the Great Barrier Reef (GBR), Australia, using oceanographic advection-dispersal models. We included directional swimming in our models and calibrated them against the observed distribution of post-hatchling and adult turtles. We simulated the dispersal of green and loggerhead turtles since they also breed in the same region. Our study suggests that the neritic distribution of flatback post-hatchlings is favoured by the inshore distribution of nesting beaches, the local water circulation and directional swimming during their early dispersal. This combination of factors is important because, under the conditions tested, if flatback post-hatchlings were entirely passively transported, they would be advected into oceanic habitats after 40 days. Our results reinforce the importance of oceanography and directional swimming in the early life stages and their influence on the distribution of a marine turtle species.The ability of individuals to actively control their movements, especially during the early life stages, can significantly influence the distribution of their population. Most marine turtle species develop oceanic foraging habitats during different life stages. However, flatback turtles ( In thMarine turtles begin their life at sea, after emerging from nests on land, with one to several days of hyperactive swimming, in which hatchlings constantly swim away from the coastline to reach offshore waters –13. FollChelonia mydas) and loggerhead (Caretta caretta) turtles originating from this region have an oceanic dispersal and are not commonly found in the GBR Lagoon .,69.27,69figure 2 turtles –72.−1) and time spent swimming (more than 75% of the day) are influential parameters that increase the inshore dispersal success. In addition, our simulations show that accounting for directionality causes substantial differences in the dispersal outputs . The scenario with a south heading (DS-3) also favoured a coastal retention of s-flatbacks, leading them to southern areas where reports of wild post-hatchlings occur but are scarce. The results of our study are based on hypothetical swimming parameters and provide valuable baseline criteria to experimentally test the orientation and swimming efforts of flatback turtles in the GBR.In particular, s-flatbacks with active and directed swimming behaviour during the first months after entering the sea are more likely to disperse into neritic habitats. The range of horizontal swimming that favours s-turtle dispersal within the GBR   is unlik5.We provide a potential explanation to the differences observed historically in the dispersal strategies (oceanic versus neritic) of coexisting marine turtle species in the GBR. The neritic distribution of s-flatback turtles was favoured by the inshore location of nesting beaches. In contrast, the outer location of reef cays and beaches, where s-greens and s-loggerheads nest, favoured a swift dispersal of these species into oceanic waters. The local water circulation in the different regions (Broad Sound and Capricornia) also seems to play an important role in the retention/advection of flatback post-hatchlings, especially during the first month of dispersal. S-turtles released during spring tides displayed a nearshore dispersal during the first week, which could have potential effects on their survival rates. Finally, under typical oceanographic conditions the inshore dispersal is further favoured if directional swimming of s-turtles is added to the models. In particular, our models suggest that high swimming efforts (in terms of speed and sustained swimming) and directionality could be important parameters to consider in future studies of flatback turtle dispersal. Our results highlight the value of integrating knowledge and applications from biological and physical sciences to improve knowledge of animal behaviour. High-resolution hydrodynamic models are a valuable tool to understand the influences of physical processes on the distribution of cryptic marine species and to infer potential pathways of dispersal ,29,46,48


We report the case of an 80-year-old woman, presented to our department for an Intestinal obstruction due to postoperative adhesions from an anterior exploratory laparotomy. Preoperative imaging showed a downward elongation of the liver, ending at the level of the iliac crest. Originally reported in 1888 by a German Surgeon named Carl Ludwig Riedel in seven female patients who had palpable masses, they were all confirmed by surgical exploration. The potential complications are rare and include torsion, mass effect morbidity and laparoscopic associated difficulty during different surgical procedures. Unnecessary imaging can be avoided with the knowledge of this normal variant of the right lobe of the liver.


The recently published paper by Yang and colleagues on the comparison of frequently used severity-assessment scores in patients with hypertriglyceridemia-induced acute pancreatitis (HP) provided important information and represented the largest cohort with such focus in this population . We woulOur primary concern is that one of the outcomes (severity of HP) was defined by the scores calculated . The authors then tested each individual score's performance in predicting the outcome. Such an assessment introduced incorporation bias, which occurs when components of an index test also are incorporated in the outcome. Incorporation bias is expected to overestimate test accuracy . A similOne potential limitation of directly applying the existing scoring systems for acute pancreatitis to HP is the lack of consideration for concurrent diabetic ketoacidosis, which is highly prevalent in this population. As Quintanilla-Flores et al. demonstrated in their cohort of 55 HP cases, 14.5% patients (8 cases) had concurrent DKA . The triAccording to the Revised Atlanta Classification of Acute Pancreatitis , moderatIn conclusion, we congratulate Yang and colleagues on their study, which provided valuable information in regard to frequently used scoring systems to determine the severity of HP. However, concerns of incorporation bias and the lack of consideration of diabetic ketoacidosis in existing scoring systems complicated the interpretation of their findings. Therefore, we propose that it is necessary to design a HP-specific scoring system to better characterize and predict the clinical courses and outcomes of HP.


A plausible and congruent clinicopathological and epidemiological paradigm is suggested.Recent findings suggest that infection (and sepsis) stand alone as the only plausible mechanism of causation of sudden infant death syndrome (SIDS) and accordingly achieves congruence with all clinicopathological and epidemiological findings. This review examines the role of infection in the pathogenesis of SIDS in the context of the major risk factor of prone sleep position. The study explores how sleep position could interact with the immune system and inflammatory response  In a recent review of the role of infection in sudden infant death syndrome (SIDS) , it was This study considers the role of infection in the etiopathogenesis of SIDS. Of all areas of SIDS etiological research, infection remains the only one that is completely congruent with the clinicopathological and epidemiological findings . PreviouStaphylococcus aureus and coliforms) in a notable number of SIDS cases (vide infra). Potential source of infection in SIDS could logically arise from either the gut or the respiratory tract. In favor of a gut origin was the finding of non-Shiga toxin verotoxigenic Escherichia coli belonging to a restricted range of serogroups not seen in live matched control babies  has been shown not to be a SIDS risk factor , 18. WhiE. coli/coliforms in lung tissue .” It should not be forgotten that many SIDS cases have died in supine or lateral positions. The underlying reasons for the increased risk of prone sleeping in babies undergoing an overt infectious process requires deeper enquiry and discussion (vide infra).A further clue in regard to the role of infection especially in relation to prone sleeping was revealed in a landmark study by Ponsonby et al. . The papWhile our previous publications , 16, 26 Understanding of the role of the sympathetic and parasympathetic nervous systems in relation to immunological status has increased in recent times. Neurotransmitters such as noradrenaline, acetylcholine, histamine, vasoactive intestinal peptide, and substance P demonstrably play important immunological roles . In addiThe two-way communication between the gut microbiota and the brain is essential in immune homeostasis and the main connection between brain and gut (and spleen) (the brain–gut axis) involves the vagus nerve. The gut bacterial microbiota provides important peptides and short-chain fatty acids that direct the development and maintenance of a healthy immune system . It is tIt has been shown in an animal model that neurochemical and other effects are abolished by vagotomy showing that the vagus acts as the major modulatory constitutive communication pathway between the gut microbiota and the brain . The gutvia the vagus nerve inflammatory reflex can prevent cytokine-induced tissue damage and death. Vagal stimulation has been shown in animal models to prevent cytokine release and damage during sepsis, endotoxemia, ischemia/reperfusion injury, and hypotensive shock (+) from the spleen produced increased levels of pro-inflammatory cytokines, e.g., TNF and IFN-gamma. Following administration of nicotine and the treatment of non-vagotomized animals with a nicotinic receptor antagonist, control levels of cellular responses were restored, mimicking the effect of vagotomy  has expanded. Cytokines are key contributors to health and disease. Their production ve shock . Moreoveve shock . Indeed,vagotomy .The question remains in regard to prone positioning: how does this affect vagal neurophysiology in the context of SIDS? A possible clue comes from a study of familial pattern overactivity of the vagus nerve indicating a possible link with risk of sudden death . Involvep = 0.011) and predicted mortality assessed at day 14  contribute to SUDI/SIDS but for the wrong reasons. These include prolonged breath holding, failure of arousal, and laryngeal reflex apnea potentiated by upper airway infection and failure of brainstem-mediated autoresuscitation . All of via spinal neurones ] . This reA] . ThisS. aureus and E. coli)  , 12, 13 E. coli) . A complWork pertaining to some sections of the paper was supported by the Foundation for the Study of Infant Death, United Kingdom.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The high prevalence of obesity in patients suffering from psychosis is a major concern as it dramatically increases the mortality rates of such patients in the long term. The mechanisms by which these patients develop overweight are poorly understood. It has been suggested that exposure to Childhood Trauma (CT) may play a role in the risk for obesity; however, whether this is the case for Early Psychosis (EP) patients and independently of the impact of medication has yet to be investigated. In addition, it is unknown whether the age at the time of exposure to CT can modulate the link between CT and obesity in EP patients.136 EP patients aged 18–35 were recruited from the Treatment and Early Intervention in Psychosis Program (TIPP-Lausanne). Body Mass Index (BMI), Weight Gain (WG) and Waist Circumference (WC) were measured and monitored prospectively after psychotropic prescription during a follow-up period of 1 year . Patients were classified into Early-Trauma if they had faced at least one experience of abuse  or neglect  before age 12, and Late-Trauma if the exposure had occurred between ages 12 and 16. Linear Mixed effect models with a random intercept were used to investigate the impact of Trauma (early or late) on the metabolic parameters longitudinally, Marko Chain Monte Carlo (MCMC) method was used to adjust these models with sufficiently large number of MCMC iterations. Models were adjusted for age, socioeconomic status, baseline BMI, medication intake prior to the first assessment and during the treatment phase, and by the diagnosis of depression.Patients were more likely to have a diagnosis of Schizophrenia , they had a mean age of 26 at the time of first assessment, and exposure to 1 or more forms of traumatic experiences before 16 years of age was present in 32% of the sample. No differences between groups were found at baseline in terms of BMI or WC. Late-Trauma patients, when compared to Non-Trauma patients showed greater WCs during the follow-up (p=0.012). No differences between Early or Late-Trauma patients and Non-Trauma patients were found in any of the other outcome measures during the follow up. Baseline BMI and treatment duration were significantly associated with the level of BMI and WC during the follow up. None of the other potential confounding factors were significantly associated with the outcome measures during the follow up.Exposition to trauma during adolescence in EP patients is associated with a higher risk of greater WC during the early phase of the disease, independently of the medication intake, depression and other confounding factors. Specific preventive measures should be addressed in these patients in order to reduce the risk of obesity. Depending on the timing of traumatic exposure, different developmental mechanisms may underlie this differential possible impact on WC. Further studies on interactions between central consequences of traumatism and metabolic syndrome are warranted.


Physical activity is recommended to prevent age-related bone loss. However, the proximal femur mechanoresponse is variable, possibly because of a muscle-dependant mechanoresponse. We compared the proximal femur response with the femoral strain pattern generated by the hip extensor muscles. A healthy participant underwent a six-month unilateral training of the hip extensor muscles using a resistance weight regularly adjusted to the 80% of the one-repetition maximum weight. DXA-based measurements of the areal Bone Mineral Density (aBMD) in the exercise leg were adjusted for changes in the control leg. The biomechanical stimulus for bone adaptation (BS) was calculated using published models of the musculoskeletal system and the average hip extension moment in elderly participants. Volumetric (ΔvBMD) and areal (ΔaBMD) BMD changes were calculated. The measured and calculated BMD changes consistently showed a positive and negative effect of exercise in the femoral neck  and the trochanter region , respectively. The 17% of the femoral neck exceeded the 75th percentile of the spatially heterogeneous BS distribution. Hip extensor exercises may be beneficial in the proximal femoral neck but not in the trochanteric region. DXA-based measurements may not capture significant aBMD local changes. Muscle forces contribute to maintaining the bone mass by modulating bone adaptation . PhysicaThe femoral neck is characterised by a variable cortical thickness reaching its thinnest region in the proximal neck where fracture is most likely to occur , 9. SeveIt has been inherently difficult to quantify the bone response and the mechanical stimulus in the proximal femur generated by specific muscle groups, mostly because of 1) the time required for bone adaptation to take place i.e., ≥6 months) [ months , (2) a g the timeThe aim of the current study was to compare the bone response in the proximal femur to intense contractions of the hip extensor muscles with the deformation pattern generated by the same muscle group. The femoral response to hip extensor exercises was measured using common DXA procedures and compared to the mechanical stimulus calculated using bone adaptation theory.A healthy premenopausal Caucasian woman of average weight and height  underwent a longitudinal unilateral training of the hip extensor muscles informed by the work of Bailey and Brooke-Wavell [http://www.abs.gov.au/).Recruitment of the participant was conducted using the University of Melbourne staff newsletter. Eligibility criteria were (a) ability to perform intense impact activity; (b) no reported medical problems potentially altering bone metabolism, lower limb, or back pain problems during the previous 12 months; (c) adequate calcium from dairy products; (d) regular menstrual cycles; (e) not having been pregnant nor having given birth or lactated; (f) sedentary lifestyle during the same previous 12 months, meaning no participation in high-impact or weight-bearing exercises for more than one hour per week. The participant was matched to the average weight and height of Caucasian women in Australia  and the second DXA was taken at training completion (t2 = 6 months). The DXA machine was routinely recalibrated at six-month intervals, therefore minimizing the eventual long-term drift of DXA measurements [The exercise intervention was a unilateral longitudinal trial involving a six-month training targeting the hip extensor muscles for no less than four sessions per week. The participant was instructed in the correct execution of the exercise and supervised by the gym staff over the entire training. Each training session involved a gentle warm-up and mobilisation exercises followed by 5 sets of 10 unilateral exercises on a leg-press machine  available at the gym . The participant chose the leg to be exercised for the entire study duration and was instructed to sit comfortably in the machine, to use a medium foot position, to place feet as high as possible on the foot plate, to push through the heels, and to use full range of motion from nearly reaching the chest with the knees to full leg extension . The parurements .Compliance was determined by calculating the percentage ratio between the amount of exercise executed and the total amount in the training protocol. The Body Mass Index at training commencement and completion was calculated using the body weight (kg) and height (m) listed in the DXA reports. The aBMD changes in the femoral neck and trochanteric and intertrochanteric regions were extracted from the DXA reports taken at training commencement and completion. The aBMD changes in the exercise leg were adjusted for changes in the control leg , 17. The−2, one standard deviation below the average 85-year-old Caucasian woman. In summary, the musculoskeletal model was a 12-segment, 18-degrees-of-freedom articulated system, actuated by 82 Hill-type muscle-tendon units. The hip and the knee were modeled using ideal ball-and-socket and hinge joints. The hip center was defined as the center of the sphere that best fits the femoral head surface. The knee axis was assumed to pass through the femoral epicondyles. The hip extensor muscles comprised the gluteus maximus, semimembranosus, semitendinosus, and biceps femoris long head. The gluteus maximus was modelled using three separated lines of action representing the anterior, intermediate, and posterior fibre bundles while the semimembranosus, semitendinosus, and the biceps femoris long head were modeled using a single line of action. The attachment points were defined as the mean point of the digitized attachment area during dissection and the muscle path was defined using the digitized superficial muscle fiber path as reference [με and a slope of 1.15 [The computational model utilized in our study included a lower-limb musculoskeletal model and a finite-element model of the right femur from an average sized female donor  with no reported history of musculoskeletal diseases prior to death , 14. Theeference . The fineference . CorrelaThe peak muscle force of the hip extensor muscles was uniformly scaled to match, at full activation, the average quasi-static isokinetic peak hip extension torque (105.1 Nm) measured on a cohort of healthy elderly controls (54 to 73 years of age) reported by Steinhilber et al. (2011) . The isoW) pattern in sedentary volunteers [HE is the element-by-element strain energy induced by the hip extensor (HE) muscles averaged over the studied hip flexion angles and SEW is the element-by-element strain energy averaged over the stance phase of walking taken from our previous work [j represents the specific region of interest ; Vj is the volume of the jth region of interest, k is a constant scaling factor representing the bone mechanosensitivity in the respective region, that is, the amount of bone that is deposited or resorbed based on a positive ((SEHE − SEW) > 0) or negative ((SEHE − SEW) < 0) biomechanical stimulus; k was assumed to be the same in all three regions and calculated based on the assumption that vBMD changes were equal to the adjusted aBMD changes in the intertrochanteric region where small to negligible BMD changes are expected [k was then used to calculate vBMD changes in the femoral neck and trochanteric region using . The biomechanical stimulus (BS) for bone adaption was calculated according to Frost's mechanostat theory , which slunteers . The bioous work . The proexpected . The facus work [ΔvBMDj=k×http://www.abs.gov.au). The participant compliance, Body Mass Index (BMI), the average subjective indicator of the exercise difficulty, and the adjusted aBMD changes in the femoral neck, trochanteric, and intertrochanteric region were calculated. The measured aBMD changes were compared with the short-term CV of the DXA machine. The magnitude of the hip contact force, the peak tensile and compressive strain, strain energy density, and von Mises stress over physiological hip flexion angles were plotted against the studied hip flexion angles. The spatial distribution of the calculated biomechanical stimulus for bone adaptation was plotted and the volume fraction exceeding the 75th percentile of the biomechanical stimulus distribution in the femoral neck, trochanter, and intertrochanteric region was calculated. The calculated vBMD changes were compared with the adjusted aBMD changes . No signs of anatomical or bone density abnormalities were evident from the DXA report. Participant compliance was 97%. The resistance weight increased from 10 kg to 90 kg after nineteen weeks of training, when a two-week interruption was necessary due to the insurgence of back-pain for unrelated causes. For safety reasons, training was then completed using a reduced 70 kg resistance weight  who matched the average height and weight in the Australian population  and a BMD decrease in the trochanter . Although the moderate aBMD increase measured in the femoral neck may not be biological significant because it is smaller than the short-term CV (1.5%) of the utilized DXA device , the relTo the best of the authors' knowledge this is the first study combining DXA measurements of bone response in the proximal femur with computer models for describing the distribution of the biomechanical stimulus during isolated contraction of the hip extensor muscles. The present findings confirm the capability of hip extensor muscles to induce high localized loads in the femoral neck  and provThere are several limitations to the present study. Firstly, the present study was based on a single subject limiting the generality of the conclusions. More research is necessary to understand the interaction between anatomical and physiological factors in determining the bone response to specific loading scenarios across individuals. Secondly, the exercise was conducted by a 40-year-old healthy woman while older postmenopausal woman, most at risk for hip fractures, may experience a far slower bone adaptation response. While exercising earlier in life can prevent excessive bone loss later in life, the optimal age-dependent training length needs to be determined. Thirdly, exercising did not prevent bone loss as appears evident from the reported absolute aBMD changes , thus raGiven the above limitations, the current results provide a general understanding of the mechanical effect of the hip extensor muscles in the proximal femur. The present study provides a theoretical foundation supporting the observed bone response in the proximal femur during training of the hip extensor muscle  and it pIn conclusion, training of the hip extensor muscles induces spatially heterogeneous bone changes in the proximal femur and may help contrasting the age-related bone loss in the very proximal neck, a region of critical importance to hip fragility fractures , 9. Stan