AQP4-null mice also exhibit a reduction in calcium signaling, suggesting that this channel may also be involved in triggering pathological downstream signaling events. Associations with the gap junction protein Cx43 possibly recapitulate its role in edema dissipation within the astroglial syncytium. Other roles ascribed to AQP4 include facilitation of astrocyte migration, glial scar formation, modulation of inflammation and signaling functions. Treatment of ischemic cerebral edema is based on the various mechanisms in which fluid content in different brain compartments can be modified. The identification of modulators and inhibitors of AQP4 offer new therapeutic avenues in the hope of reducing the extent of morbidity and mortality in stroke.Stroke is a complex and devastating neurological condition with limited treatment options. Brain edema is a serious complication of stroke. Early edema formation can significantly contribute to infarct formation and thus represents a promising target. Aquaporin (AQP) water channels contribute to water homeostasis by regulating water transport and are implicated in several disease pathways. At least 7 AQP subtypes have been identified in the rodent brain and the use of transgenic mice has greatly aided our understanding of their functions. AQP4, the most abundant channel in the brain, is up-regulated around the peri-infarct border in transient cerebral ischemia and AQP4 knockout mice demonstrate significantly reduced cerebral edema and improved neurological outcome. In models of vasogenic edema, brain swelling is more pronounced in AQP4-null mice than wild-type providing strong evidence of the dual role of AQP4 in the formation and resolution of both vasogenic and cytotoxic edema. AQP4 is co-localized with inwardly rectifying K Stroke is one of the primary causes of death in many developed countries, with a slow increase in prevalence worldwide  are a family of specialized water channels expressed on heterogeneous cell types, of which at least three major types are found in the central nervous system , blood, and the intracellular and interstitial components of the parenchyma. Fluid movement across vascular, ventricular and parenchymal compartments is controlled by osmotic gradients and hydrostatic pressure differences and is crucial for maintaining normal physiological function  hydrophobic, integral membrane proteins. These are expressed widely in the animal and plant kingdoms with 13 subtypes having been identified so far in mammals. The existence of a channel pore for water was hypothesized before the identification of the protein and was based on observations that red blood cells have higher water permeability than expected for an equivalent surface area of a lipid bilayer membrane. This hypothesis was confirmed in 1992 by Peter Agre and colleagues with the identification of AQP0, the first member of the AQP family of water channels , the brain parenchyma (intracellular and extracellular space) and the intraventricular compartment  or microcrystalline assemblies of AQP4  and wild-type have aided in the elucidation of various roles of AQP4 in ischemia , astrocyte swelling was observed in wild-type but was greatly reduced in AQP4−/− mice. Ca2+ signals were only evoked in wild-type mice. More severe hypo-osmotic stress (30% reduction in osmolarity) produced astrocyte swelling and Ca2+ spikes in both wild-type and AQP4−/− mice, suggesting an association between Ca2+ signaling, water influx via AQP4 and cellular swelling  that mediate them play crucial roles together with water channels in regulatory processes that decrease volume  abundantly expressed in astrocyte end-feet, mediates cell-cell interaction by facilitating ionic and molecular transport  would also ensure efficient removal of K+ from astrocytes into the vasculature , which in turn activate phospholipase C (PLC) and generates inositol 1,4,5- triphosphate (IP3) triggering Ca2+ release from intracellular stores and activates calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS). Group I mGluR also regulate water permeability of AQP4-expressing astrocytes  or GLAST (EEAT1)  of ischemia, and that during later stages of edema (7 days and later), delayed but pronounced AQP4 up-regulation may play a beneficial role by promoting edema resolution and attenuating neuroinflammation by inhibition of microglial activation and astrogliosis  , a clinically safe diuretic, which is thought to block the AQP4 channel and water permeability in 2+ (Morishita et al., Also reported to confer neuroprotective effects in ischemia is erythropoietin. Intracerebroventricular administration 24 h prior to induction of ischemia resulted in a 47% reduction of infarction in mice subjected to MCAO (Bernaudin et al., A potentially viable compound which is postulated to interact with AQP4 and which may be useful in ischemic stroke is edaravone, a free radical scavenger that readily crosses the BBB. In a rat transient focal ischemia model, it significantly reduced the area of infarction and improved neurological scores when administered after 24 h of reperfusion. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. It is postulated that by suppression of AQP4 expression, edaravone exerts its influence to reduce cerebral ischemic injury (Kikuchi et al., Since AQP4 aids in migration of reactive astrocytes toward an injury site, candidate AQP4 blockers may augment neuronal generation by attenuating reactive gliosis and hence mitigate potentially detrimental glial scar formation (Saadoun and Papadopoulos, Agmatine attenuated AQP1 expression in a mouse model of transient ischemia. This translates into decrease of cerebral edema and brain swelling, possibly through reduction of matrix metalloproteinases-2 and -9 (Kim et al., Substantial advancements have been made in the field of AQP studies and their role in several pathophysiologies. Regulation of water transport constitutes an integral part of ischemic stroke amelioration and thus continuous efforts are required in the hope of elucidating further mechanisms that could improve outcomes of this disease. Careful application of various AQP modulators at different phases of stroke has the potential of amplifying favorable outcomes and of attenuating undesirable ones, making them clinically highly relevant. Finally, the development of new tools to identify selective modulators with subtype specificity and affinity for select AQPs is poised for exciting progress in basic life sciences and medicine.JV and MV conceived the work, drafted, designed and revised critically. CZ, GG, and RM acquired and compiled information. All authors wrote and revised 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.


Here, we review the current state (and gaps) of knowledge on N2 fixation associated with coral reef organisms and their ecosystems. By summarizing the existing literature, we show that benthic N2 fixation is an omnipresent process in tropical reef environments. Highest N2 fixation rates are detected in reef-associated cyanobacterial mats and sea grass meadows, clearly showing the significance of these functional groups, if present, to the input of new N in reef ecosystems. Nonetheless, key benthic organisms such as hard corals also importantly contribute to benthic N2 fixation in the reef. Given the usually high coral coverage of healthy reef systems, these results indicate that benthic symbiotic associations may be more important than previously thought. In fact, mutualisms between carbon (C) and N2 fixers have likely evolved that may enable reef communities to mitigate N limitation. We then explore the potential effects of the increasing human interferences on the process of benthic reef N2 fixation via changes in diazotrophic populations, enzymatic activities, or availability of benthic substrates favorable to these microorganisms. Current knowledge indicates positive effects of ocean acidification, warming, and deoxygenation and negative effects of increased ultraviolet radiation on the amount of N fixed in coral reefs. Eutrophication may either boost or suppress N2 fixation, depending on the nutrient becoming limiting. As N2 fixation appears to play a fundamental role in nutrient-limited reef ecosystems, these assumptions need to be expanded and confirmed by future research efforts addressing the knowledge gaps identified in this review.Tropical coral reefs are among the most productive and diverse ecosystems, despite being surrounded by ocean waters where nutrients are in short supply. Benthic dinitrogen (N In the decades to come, life in the ocean will be confronted with a series of environmental conditions that have no parallel in human history  from the burning of fossil fuels is dramatically changing the world's climate (IPCC The marine nitrogen (N) cycle is one of the most important of all biogeochemical cycles, as N is an essential building block in all life forms. The N cycle significantly influences the cycles of other elements and particularly the carbon (C) cycle Fig. , becauseate IPCC .2) fixation, the conversion of N2 to organic N) and N loss . These are particularly important because of the inability of most marine organisms to use elemental N2 . As a consequence, their balance determines the net biologically available N for the biosphere and therefore marine productivity (Arp −1) in the oceanic N2 fixation rate compared to N loss  found N2-fixing microorganisms throughout all marine environments, ranging from deep-sea vents to highly productive shelf areas  compared to the amount of N that is fixed annually by benthic N2 fixers (Gruber μm cell diameter) are likely an equally important source of new N in the open ocean  studies have recently shown that assimilation of both organic and inorganic N sources resulted in rapid incorporation of nitrogen into uric acid crystals, forming temporary N storage sites within the dinoflagellate endosymbionts   associations in coral reef ecosystems is not debated, and we have only begun to investigate the distribution and diversity of diazotrophic populations associated with benthic reef organisms and substrates. Furthermore, the role of diazotrophs in contributing to primary production and growth and their potential susceptibility to climate change still needs to be resolved. As mutualisms  bind different species to a common fate, their breakdown as a result of climate change may enhance biodiversity loss and ecosystem disruption  produced by burning of fossil fuels and deforestation  are rare in warm tropical oceans. It has been suggested that the reduced gas solubility and increased respiration rates in warmer waters make the possession of heterocysts under such conditions disadvantageous  are widespread in benthic environments, so that increases in N2 fixation are likely to appear here as well. Nevertheless, heterocystous cyanobacteria form mats in warm coral reef environments   resulting from global climate change have the potential to increase the amount of N fixed globally  are lost  and seawater pH  resulting in bleaching and productivity loss  remains uncertain  consumption and protecting the enzyme nitrogenase from inactivation  significantly increased since the 1920s, most likely due to the increased input of river-borne nutrients   and dissolved organic matter (DOM) as well as trace metals , are mostly limiting N2 fixation and uptake of organic or inorganic N compounds), their ability to fix N2 is advantageous over non-N2-fixing phytoplankton species, especially under N-limiting conditions  can significantly alter the competition within the phytoplankton community, thereby further threatening the stability and functioning of the whole ecosystem  often fix Nd Capone ). Nevertd Capone , additiop et al.  suggestevel Fig. . Therefo2 content in the World's oceans  in the early afternoon  levels dropped to hypoxic conditions as a consequence of coral spawning events , the microbial community tends to be dominated by SRBs, particularly when nitrate is exhausted  reaching the biosphere  on corals and other reef-associated biota  and UVB (290–320 nm), can alter photosynthesis and growth in cyanobacteria  to investigate the potential effects of global and local environmental stressors on the metabolic activity and stability of these partnerships; and (4) to scale the effects of environmental stressors on the input of fixed N to the entire reef ecosystem. Moreover, careful site selection should be considered, where we can utilize natural habitats which currently undergo similar climate-driven stressor(s) expected for the future, for example, CO2 seeps.This article highlights the paramount role of N2 fixation rates as well as ecological and biogeochemical approaches are necessary for understanding the functioning of such a complex ecosystem.Given the difficulty in isolation and cultivation of microorganisms, including symbiotically associated types, several culture-independent approaches Fig.  are usef


Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general. Shigella are human pathogens that infect the gastro-intestinal tract and cause acute shigellosis. Shigellae are Gram-negative, non-motile, facultative anaerobic pathogens that are closely related to Escherichia coli but have evolved specific traits of pathogenicity, physiology and serology  host cells. Among those bacteria, the invasion mechanism triggered by Shigella has similarities to the one induced by the other Gram-negative bacterium, Salmonella; however, in contrast to Salmonella and Mycobacteria, Shigella rapidly escapes the entry vacuole, moves freely in the host cytosol, and is able to spread from cell to cell, which are properties shared with the Gram-positive bacterium Listeria. Thus, host cell invasion by Shigella has overall unique characteristics, and the use of this bacterium as a model of host-bacteria interaction over the past four decades has considerably increased our understanding of bacterial pathogenesis. In this review, we will provide an overview of some of the most recent progress that was made in cellular microbiology and innate immunity, using Shigella as a model.While studying the mechanisms of Shigella infection is as low as 100 bacteria  -mediated suppression of spa32 and spa33 transcription  Figure . The proAt the distal end of the T3SS is the tip complex composed of IpaB, IpaC, and IpaD  for Rac and RhoA to drive actin remodeling  pathway, the pentose-phosphate pathway (PPP) and the Entner-Doudoroff (ED) pathway. Studies by Payne's group demonstrated the importance of the EMP pathway, which converts glucose to pyruvate through the glucose-6-phosphate/fructose-6-phosphate/glyceraldehyde-3-phosphate intermediates, while the PPP and ED pathways were dispensable for Shigella intracellular growth  and integrated stress response (ISR) pathways represent two major signaling axes in eukaryotic cells that control metabolic stress responses to nutrient limitation. We recently demonstrated that infection by Shigella resulted in potent inhibition of mTOR signaling, as well as induction of the ISR, and that the underlying mechanism was the induction of an amino acid (AA) starvation response in infected host cells  is critical for host defense. Interestingly, Lu et al. recently demonstrated that Shigella used the effector protein OspB to inhibit this process in the first phase of the infection by turning on mTOR complex 1 through the scaffolding protein IQ motif-containing guanosine triphosphate (GTP)ase-activating protein 1  cytosolic maturation, which is a critical step in the control of the assembly of the spliceosome, the molecular machinery responsible for mRNA splicing  and control both mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) proinflammatory signaling -inhibitor of nuclear factor kappa-B kinase (IKK) pathway leading to the production of interleukin 8  sense the extracellular and endosomal environment and drive proinflammatory signaling in response to specific stimulation. This TLR-based detection plays a major role in the response to Shigella, a proinflammatory, caspase-dependent form of cell death known as pyroptosis is induced by bacterial components as well as downstream danger signals coming from dead or dying host cells. The rod protein MxiI and the needle protein MxiH of Shigella T3SS have been shown to stimulate the NLR family CARD domain containing 4 protein (NLRC4) inflammasome after being sensed by NLR family, apoptosis inhibitory protein 2 (NAIP2) and human NAIP (murine NAIP1) respectively .As the pathways for sensing intracellular viral nucleic acids have become more comprehensive, investigation into the intracellular sensors of bacterial nucleic acid has become an area of interest. Studies have reported that the intracellular detector of double-stranded deoxyribonucleic acid (dsDNA) is cyclic GMP-AMP synthase (cGAS), an enzyme that generates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) which then serves as a downstream second messenger to activate stimulator of interferon genes (STING) leading to the generation of type-1 interferons and NF-κB signaling  production, which plays numerous roles in the manipulation of host trafficking such as increasing vacuolar membrane disruption /IKKγ, IpaH4.5 targets p65 subunit of NF-κB, and IpaH0722 targets TNF Receptor-Associated Factor 2 (TRAF2), together degrading important components of the NF-κB pathway, halting pro inflammatory signaling  and p38 through epigenetic modifications /Akt-dependent survival pathways, and phosphorylation of mouse double minute 2 homolog (MDM2) which degrades p53  interacts with and activates neuronal Wiskott-Aldrich Syndrome protein (N-WASP), which recruits Arp2/3 to drive actin polymerization , NOD1 and NOD2, play a critical role in targeting the autophagy machinery through interaction with autophagy related 16 like 1 (ATG16L1) to the site of Shigella invasion at the plasma membrane , and escape  and how that relates to host recognition of cytosolic Shigella.The Shigella leads to the upregulation of metabolic stress pathways, it is unclear how the activation of these pathways contributes to host defense against this pathogen. Similarly, it remains to be seen how the upregulated stress pathway affects the transcriptional landscape of Shigella-infected cells. It has previously been shown that pyruvate is a major carbon source for intracellular Shigella, however these studies have been performed using cancer cells that extensively rely on glycolysis. To advance the understanding further, it will be essential to determine if the same holds true for primary intestinal epithelial cells, which represent a physiological target of Shigella. Shigella infection induces mTOR inhibition and eIF2alpha phosphorylation, but the extent to which these pathways are regulated by specific Shigella effectors remains poorly characterized. Identifying if, in addition to OspB, Shigella has evolved means to manipulate these pathways will be an important question for future investigations. Lastly, it was proposed that Shigella may induce amino acid starvation in host cells as a result of membrane damage, however, it currently remains unclear how host membrane damage triggers amino acid starvation.While it is clear that infection with Shigella effectors manipulate a variety of fundamental processes in the host. The list of Shigella effectors is still growing, along with the host processes that are targeted. The induction of cell death remains one of the fundamental defense mechanisms for host cells to combat infection. Further investigation into the risk/benefit of pyroptosis for Shigella will help understand which situations call for such a proinflammatory method of escape from the host cell. Discovery of the noncanonical inflammasome has raised interest concerning the exact nature of stimuli and sensors used by the major inflammasomes, so research focused on clarifying these pathways is needed. Finally, the identification of the intercellular transfer of protective messengers like cGAMP suggests that other paracrine messengers of infection may also exist. This could represent an effective, proactive defense strategy used by the host to counteract the deleterious consequence of the injection of effectors during invasion by alerting non-infected, neighboring cells.Shigella infection suggests possibilities for the future of therapies and treatment for diseases of a bacterial nature. Perhaps the most apparent treatment strategy would be designing specific inhibitors of the T3SS effectors, to block the action of those effectors that are critical to invasion and cell-to-cell spread. Likewise, after understanding the metabolic changes Shigella undergo during the infection process, limiting access to pyruvate could halt propagation of the bacteria. However, with most therapies that target specific components or processes of a microbe, mutations and selection leading to resistance would be a potential pitfall. With this in mind, an alternative route therapies could take involve strengthening the host responses, especially those that are targeted by the bacterial infection, before the bacteria have a chance to target the cells. This defense happens endogenously as surrounding cells amplify their defense when a neighbor is infected, therefore should also work with exogenous stimuli. Such avenues could include sustaining components of the inflammatory and survival signaling pathways, or limiting bacterial access to essential nutrients like iron in patients who have been infected. Each direction will benefit from further investigation into the molecular mechanisms behind Shigella pathogenesis, and the breadth of host response to infection.Such progress in uncovering the pathogenesis of All authors listed, have made 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.


A workshop on “Clinical Translation of Molecular Imaging Probes and Technology” was held August 2, 2013 in Bethesda, Maryland, organized and supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB). This workshop brought together researchers, clinicians, representatives from pharmaceutical companies, molecular probe developers, and regulatory science experts. Attendees met to talk over current challenges in the discovery, validation, and translation of molecular imaging (MI) probes for key clinical applications. Participants also discussed potential strategies to address these challenges. The workshop consisted of 4 sessions, with 14 presentations and 2 panel discussions. Topics of discussion included (1) challenges and opportunities for clinical research and patient care, (2) advances in molecular probe design, (3) current approaches used by industry and pharmaceutical companies, and (4) clinical translation of MI probes. In the presentations and discussions, there were general agreement that while the barriers for validation and translation of MI probes remain high, there are pressing clinical needs and development opportunities for targets in cardiovascular, cancer, endocrine, neurological, and inflammatory diseases. The strengths of different imaging modalities, and the synergy of multimodality imaging, were highlighted. Participants also underscored the continuing need for close interactions and collaborations between academic and industrial partners, and federal agencies in the imaging probe development process. The goal of this workshop was to better understand gaps between clinical needs and probe development, and between regulatory requirements and the reality of clinical translation, including the lack of commercial incentives for the private sector. This report is intended to summarize information presented at the meeting,Molecular imaging plays an important role in advancing knowledge about disease pathophysiology, and diagnostic tools have been developed based on molecular imaging probes and technology. As such, the National Institutes of Health (NIH) has actively supported the development of molecular imaging probes since 2003 with targeted programs under a roadmap initiative and initiatives led by individual institutes. Under the theme “New Pathways to Discovery and Molecular Libraries and Imaging Implementation,” a technology development program was established to encourage the discovery and design of highly specific and highly sensitive molecular imaging probes for basic research and clinical applications. Since then, a plethora of innovative molecular imaging probes have been developed with NIH funding and applied to improve the understanding of disease mechanisms and in some instances to diagnose and track human disease progression. While few probes and techniques have been successfully translated for human application, many have become routine laboratory tools or are being used in the preclinical arena. Challenges remain to demonstrate reliable, reproducible, and quantifiable The first session of this workshop focused on high-priority clinical needs that could be addressed by the development of suitable molecular imaging probes. The speakers focused on (1) cardiovascular, (2) endocrine and metabolic, (3) neurologic, and 4) inflammatory diseases. Probe development for cancer was not specifically addressed due to the extensive programs in this area already sponsored by the National Cancer Institute. inflammaThere was a consensus that probes are needed for specific, high-risk patient categories in cardiovascular diseases though image-based population screening is not warranted.18F]fluoro-d-glucose (18F-FDG)-PET for inflamed plaque -PiB for the fibrillar Aβ-amyloid deposition and [11C]-DTBZ for the evaluation of nigrostriatal dopaminergic projection terminals -raclopride (and others targeting dopamine receptors) which are used to evaluate the health of endogenous dopamine system and [11C]-carfentanyl to assess endogenous opiate peptide levels and release  EDTA for the BBB disruption [11C]-AMT for inflammatory cellular response [11C]-deprenyl for reactive astrocytes [11C]-PBR28 for mitochondrial translocator protein (TSPO) associated with oxidative stress status -dtrocytes , and -PK11195 -FIAU [125I] or [131I] may have potential to be a theranostic agent. Progression elevated gene-3 promoter (PEG-Prom) can be used to drive imaging reporters to detect micrometastatic disease -FIAU , 83 with disease . ChallenIt was concluded during the discussion after the first two sessions that although a number of PET and SPECT probes have been studied in humans, most molecular imaging probes remain in preclinical or limited investigational use. A number of possible reasons for this state of affairs were discussed. While some of the probes have promising specificity, nonspecific binding  is in most cases still suboptimal for the challenges of regulatory approval and eventual everyday clinical use. For probes with peripheral targets, accumulation in the liver, kidneys, and suboptimal blood clearance continue to present challenges. Some of the speakers noted that while the probe development community had good communication, there needed to be closer cooperation between academic researchers, clinicians, industry, and regulators, such as the cooperation that led from the discovery of PiB to the eventual development and FDA approval of florbetapir.A vigorous discussion centered on the possible advantages of multimodality and multifunctional probes. While the panel participants, upon questioning by members of the audience, reiterated the advantages and challenges originally enumerated, no clear consensus emerged in this area. Multimodality probe development has been pursued and continues unabated, but there a most promising translational path was not identified.The third session addressed clinical needs from the perspective of the challenges faced during pharmaceutical development. In this context, molecular imaging probes can be used as pharmacokinetic indicators to accelerate the screening of promising drug candidates, or they can be used as surrogate biomarkers for pharmaceutical efficacy.e.g., see -MNI-659 targeting PDE10A to study Huntington’s disease using PET and [123I]-MNI-420 targeting adenosine A2a receptor for CNS diseases using SPECT -pyruvate for prostate cancer imaging. It took the University of California at San Francisco (UCSF) researchers 10 years to go from the first technology paper on dynamic nuclear polarization (DNP) to IND approval by the FDA for hyperpolarized [13C]-pyruvate and subsequent NIH funding of a phase I clinical trial in prostate cancer patients [13C]-pyruvate reproducibly and efficiently to humans, (2) development of pharmaceutical compounding standard operating procedures for pharmacy technicians to produce doses routinely and in a clean room, (3) creation of a physiologically compatible 13C MRI radiofrequency (RF) coil with MR imaging protocol and analysis methods [As an example of the successful “bench to bedside” translation of a molecular imaging probe, participants discussed the process and challenges in getting to first-in-human testing of hyperpolarized (HP) [patients . Challen methods , 95, (4)i.e., self-evident/established benefit, reasonably likely to predict benefit, bioactivity/pharmacodynamics).The FDA noted that including imaging in a clinical trial can provide assurance that subjects have the defined condition, assess if patients are likely to respond to the investigational drug, and potentially shortening the time needed to demonstrate efficacy of diagnostic or therapeutic drugs for FDA approval. Imaging standards in clinical trials should include standardized acquisition and interpretation to control potential sources of bias and variability and verification of data quality and integrity. To establish efficacy in clinical studies of imaging drugs, the FDA recommends that the anatomic, functional, or physiological measurements using an imaging agent be compared with those of a reference products or procedures or a known true standard. If no standard of truth is available, the FDA recommends that a clinical trial be conducted in order to determine the clinical usefulness. To establish effectiveness of therapeutic drugs using imaging, applicants should consider endpoint meaningfulness, determine applicable imaging standards, and most importantly consult FDA reviewers as early as possible. In sum, the key points for implementing imaging in phase 3 clinical trials are (1) adequate, well-controlled investigations, (2) well-defined and reliable methods of response assessment, (3) imaging standardization, and (4) an endpoint with consideration in meaningfulness continuum  diagnostic tests are rarely therapeutic; (2) diagnostic testing may expose a patient to specific short- or long-term risks; (3) while potential harm may occur, diagnostic tests accrue benefits and can inform downstream clinical management of the patients (4) balance of risk and harm should consider the acuity and severity of the patient’s presentation; (5) avoidance of unnecessary or futile treatment can improve health outcomes; and (6) Medicare does not consider cost in making national coverage decisions. A preferred road to diagnostic coverage should provide adequate evidence that the incremental information obtained by new diagnostic technology, compared to alternatives, changes physician recommendations. In particular, this should result in changes in therapy that improve clinically meaningful health outcomes in Medicare beneficiaries.In the presentation and discussion sessions, the workshop participants identified pressing clinical needs and development opportunities for molecular imaging probes in cardiovascular, endocrine, neurologic, and inflammatory diseases, which complement the existing vigorous cancer imaging programs. Conventional nuclear medicine probes as well as novel MRI contrast agents, US and multimodality approaches appear promising for different applications. Industry representatives stressed the need for continued collaboration between academic, government, and industry sectors in these efforts, and noted that molecular imaging probe success should be measured both by the collection of probes that serve to help screen for disease-modifying therapies in preclinical settings, as well as the smaller number that emerge as FDA-approved imaging agents. Participants also observed that standardization and validation of protocols in order to obtain regulatory approval for clinical translation of molecular imaging probes and technology is an achievable and desirable goal.


Increased levels of transition metals like Fe, Ni, Cr, Hg and Pb are related to free radical generation, lipid peroxidation, DNA strand breaks and tumor growth in cellular systems. In order to evaluate the correlation to malignant growth in humans we investigated the accumulation of heavy metals in 8 healthy and 20 breast cancer biopsies by means of a standardized AAS methodology. A highly significant concentration of Fe, Ni, Cr, Zn, Cd, Hg and Pb was recorded in the cancer samples when compared with the control group (p ≤ 0.001). As previously reported by us, the high heavy metal levels found in various tumors may be used for therapeutic interventions with ascorbic acid or substituted phenolic mixtures. The autooxidation of vitamin C in the presence of heavy metals strongly increase superoxide and H2O2 concentrations at the tumor site with oxidosis and apoptosis induction.In turn, bioactivation of phenolic compounds in the presence of heavy metals and an overexpressed NADPH- quinone oxidoreductase leads to a significant generation of superoxide and semiquinone radicals with deleterious effects for the metal rich malignant cells. Thus, the accumulation of transition metals in malignant tissue confers both ascorbate and phenolic compounds an increased tumor selectivity.


The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell–cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. The Rho GTPase family belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. Although the Rho GTPase family is further divided into seven subfamilies , the most extensively studied members are RhoA, Rac1, and Cdc42. Multiple studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. In particular, RhoA mediates the formation of stress fibers and focal adhesions, Rac1 induces lamellipodia formation and membrane ruﬄes, and Cdc42 elicits formation of filopodia and microspikes . HoweverFigure 1). Rho family GTPase activation is facilitated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP while inactivation of Rho GTPases is induced by GTPase activating proteins (GAPs) that stimulate the intrinsic ability of Rho GTPases to hydrolyze GTP to GDP. The third class of molecules that regulate Rho GTPase activity, guanine dissociation inhibitors (GDIs), sequester Rho GTPases in a GDP-bound state in the cytosol. The dissociation of GDIs is required for membrane localization and subsequent activation of Rho family GTPases . Rho family GTPases contain an ∼200 amino acid residue Dbl homology (DH) domain and an adjacent, C-terminal, 120 residue pleckstrin homology (PH) domain. The DH domains of Rho GTPases facilitate interaction with GEFs and the subsequent exchange of GTP for GDP while the PH domains bind phosphoinositides to localize Rho GTPases to the plasma membrane  demonstrated a complete loss of F-actin concomitant with the formation of multiple axon-like protrusions, underscoring a role for Rho GTPases in neuronal polarization  receptors increases Rac1 and Cdc42 activities while decreasing RhoA activity to promote dendritic growth and branching .The essential functions of Rho family GTPases in regulating neuronal growth cone formation, neurite outgrowth, and nervous system development suggest that Rho GTPases have an important and conserved function in mediating neuronal survival and death. Indeed, similar to the opposing functions exerted between members of the Rho GTPase family in maintenance of neurite outgrowth and growth cone formation, Rac activation typically promotes neuronal survival while Rho activation elicits neuronal death. In further support of an antagonistic relationship between Rac GTPase and Rho GTPase, recent efforts have identified a key signaling network that maintains neuronal survival downstream of these Rho family GTPases, discussed in detail below . The inhibition of Akt by the Rho/ROCK/PTEN pathway occurs indirectly as PTEN dephosphorylates phosphatidylinositol -triphosphate (PIP3) into phosphatidylinositol -biphosphate (PIP2) to terminate Akt activation  to promote survival of neurons . In neurtivation . Maintaiin vitro neuronal models, including brain neuroblasts, PC12 cells, and primary rat cortical, hippocampal, and CGNs  reductase inhibitors (statins) highlighted a likely role for Rho GTPases in regulating neuronal apoptosis. For example, inhibitors of HMG-CoA reductase decrease the localization of Rho GTPases to the plasma membrane by suppressing the synthesis of mevalonate, a precursor required for the isoprenylation of Rho GTPases. Consequently, these compounds induce apoptosis in human brain explants and in a variety of and CGNs . More rein vitro neuronal models. In particular, we have demonstrated a central pro-survival function for Rac GTPase in CGNs. We have previously shown that Clostridium difficile ToxB, a broad-spectrum inhibitor of Rho, Rac, and Cdc42, induces apoptosis of primary CGNs predominantly through deactivation of Rac1 GTPase  cells  and consIn the nervous system, Rho family GTPases play major and evolutionarily conserved roles in the development, migration, and plasticity of neurons. Rho, Rac, and Cdc42 have all been shown to regulate dendritic growth and arborization, as well as axon guidance . In contThe three mammalian Rac isoforms that exist  are ∼90% homologous . HoweverDrosophila Rac genes result in defects in axonal growth, guidance, and branching  have been identified, the involvement of RhoA in nervous system development is best characterized. Nonetheless, RhoB and RhoC share many effectors with RhoA, suggesting potentially overlapping functions. However, while RhoA and RhoC localize to the cell membrane, RhoB localizes to the membranes of intracellular vesicles. Furthermore, RhoB and RhoC knockout mice do not display any obvious developmental defects while RhoA knockout mice are embryonic lethal . The invConsistent with a role in regulating cytoskeletal dynamics, deletion of RhoA in spinal cord neuroepithelium, midbrain, and forebrain each led to loss of both adherens junctions and apical-basal polarity . DeletioUsing a gene knock-in strategy in which mice express dominant-negative RhoA in all developing neurons, -/- neurons migrated normally when transplanted into wild type cerebral cortex, wild type neurons did not migrate properly when transplanted into RhoA-/- cerebral cortex. Intriguingly, the defects in neuronal migration were not due to diminished RhoA activity in neurons, but were attributed to destabilization of the actin and microtubule cytoskeleton of the radial glia scaffold, which directs migrating neurons  are all reduced in RhoB-null mice. Consistent with the essential role dendritic spines morphogenesis in LTP, spine number was decreased and dendritic branching was increased in hippocampal neurons of RhoB-/- mice .The essential functions of Rac GTPase and Rho GTPase in mediating nervous system development and neuronal survival indicate that dysregulation of these GTPases may play a causative role in the pathology of some forms of neurodegenerative disease. Indeed, dysregulation of Rac and/or Rho GTPase has been reported in a variety of neurodegenerative diseases and neuronal traumas, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), ischemia/reperfusion, amongst many others. Here, we will review recent advances examining dysregulation of these GTPases in various models of neurodegenerative disease  was one of eleven mutations initially identified to be causative in familial ALS. Although the G93A SOD1 mutation remains the best characterized to date, mutations in additional genes such as Alsin (ALS2), Tar DNA-binding protein 43 (TDP43), and fused in sarcoma (FUS) have also been linked to other familial forms of ALS. Recent evidence suggests that dysregulation of Rho GTPases may be a factor underlying both genetic and sporadic ALS.Recessive mutations in a Rac GEF, alsin, have been reported in juvenile forms of ALS, primary lateral sclerosis, and infantile-onset ascending hereditary spastic paralysis . When ovThe critical role of Rac in maintaining neuronal survival is evidenced by the fact that disruptions in Rac signaling have been shown to induce apoptosis in a variety of diverse neuronal cell types, including primary motor neurons . In spinIn addition to mutations that code for alsin and SOD1, recent evidence also suggests that ARHGEF16, a GEF for Rac GTPase, is hypermethylated and downregulated in postmortem sporadic ALS spinal cord samples when compared to samples from neurologically normal controls . Thus, d2O2, the interaction between wild type SOD1 and Rac1 GTPase was uncoupled. Intriguingly, ALS causing mutations in SOD1 prevent the enzyme from redox uncoupling, resulting in enhanced and persistent activation of Rac1 GTPase in microglia cells. Inevitably, aberrant Rac1 GTPase activity leads to increased activation of Nox2, a catalytic subunit of NADPH oxidase, resulting in an overproduction of damaging ROS. In agreement with Rac1-dependent activation of NADPH oxidase as a contributing factor underlying motor neuron degeneration in the G93A mSOD1 mouse, inhibition of, or genetic deletion, of Nox2 slows disease progression and increases survival in this particular mouse model of familial ALS  ATP, enhanced Nox2 activation and the production of ROS in a Rac1-dependent manner when compared to microglia derived from non-transgenic animals. Thus, recent experimental evidence suggests that G93A mSOD1 may stimulate Rac1 GTPase activation in a P2X7-dependent manner to enhance the release of toxic ROS from microglia.In a manner dissimilar to motor neurons, several studies indicate that mutant forms of SOD1 associated with the pathology of ALS have an opposing function in microglia, ultimately resulting in enhanced activation of Rac1 GTPase in a redox-sensitive manner. For example, lial ALS . FurtherRecent studies also implicate aberrant Rho GTPase activity as an underlying factor in the pathology of ALS. For example, although the significance remains unknown, the Rho GEF, RGNEF, displays enhanced cytoplasmic inclusions in spinal motor neurons from both sporadic and familial ALS patients . HoweverAlzheimer’s disease is the most common form of dementia and is characterized by extensive loss of neurons in the hippocampus and cerebral cortex. The onset of AD correlates with an aberrant accumulation of extracellular amyloid-β (Aβ) plaques which are the products of proteolytic cleavage of the membrane protein amyloid precursor protein (APP). Aβ plaques have been demonstrated to be at least partially responsible for the dysregulation of actin polymerization that underlies synaptic dysfunction observed during AD progression . AccumulAlthough diminished Rac1 expression is observed in AD brains obtained from patients early in disease , recent Further evidence suggests that alterations of a Rac1-dependent signaling cascade links expression of toxic Aβ fragments to disruption of actin polymerization and dendritic spines. Indeed, it has previously been reported that expression of the downstream Rac effector, PAK, is decreased in hippocampal sections, while levels of phosphorylated (active) PAK were elevated in intraneural aggregates during AD progression . In humaAlthough these previous studies indicate that enhanced Rac1 activity may contribute to AD progression, the global reduction of Rac activity in the brains of human AD patients reveals that the involvement of Rac GTPase in the progression of AD is complex. In accordance with reduced Rac GTPase activity, it was previously reported that the mRNA and protein expression levels of the Rac GEF, Kalirin-7, are reduced in the hippocampus of human AD brains. Kalirin-7 is known to play a critical role in dendritic spine maintenance. Intriguingly, Kalirin-7 associates with iNOS to downregulate its activity in AtT-20, SH-SY5Y, C6, and Neuro-2A cells. In the human AD hippocampus, elevated expression of iNOS correlates with diminished association with Kalirin-7 . Thus, tIn addition to Rac GTPase, aberrant Rho GTPase activity has also been linked to the pathology of AD. Indeed, RhoA is decreased in human AD brains and in the brains of transgenic mice overexpressing the Swedish double mutant of APP. Moreover, while RhoA was decreased in synapses of AD mice, its expression was increased in degenerating neurites consistent with the involvement of RhoA in neurite retraction . AdditioHuntingtin gene which results in expansion of a CAG nucleotide repeat. Mutant huntington (Htt) forms toxic gain-of-function aggregates that underlie the etiology of HD. Although relatively few studies have examined the involvement of Rho GTPases in the progression of HD, recent efforts have linked dysregulated Rho family GTPase signaling to both the production of toxic ROS and mutant Htt aggregation in the degenerating CNS.Huntington’s disease (HD) is a progressive and fatal autosomal dominant disorder characterized by loss of cells in the striatum and cortex, resulting in involuntary body movements (chorea), cognitive decline, psychiatric conditions, and eventually death. HD is caused by a mutation in the In a similar manner to other neurodegenerative diseases, the production of ROS is believed to play a role in the etiology of HD. Compared to neurologically normal controls, HD patients show enhanced NADPH oxidase activity and an increase in ROS in the cortex and striatum of postmortem brain samples. In the 140Q/140Q mouse model of HD, NADPH oxidase activity in cortex and striatum correlated with enhanced production of ROS and neurite swellings, and was prevented by treatment with NOX inhibitors. Similarly, mutant HD mice bred with NOX2 knockout mice showed enhanced survival concomitant with decreased NADPH oxidase activity and lowered production of ROS . Given tin vitro and in vivo and colocalizes with mutant Htt in human HD brains. Overexpression of PAK1 enhances the aggregation of mutant Htt and Htt-induced toxicity of HeLa cells independent of its kinase activity  is taken up by dopaminergic neurons and induces PD symptoms through inhibition of mitochondrial complex I of the electron transport chain, leading to enhanced ROS production and neuronal apoptosis. When administered to mice, MPTP induces cellular polarization of microglia and leads to the formation of neuron-glia contacts, which precede the phagocytosis of dopaminergic cell bodies. This process may be mediated by ROCK activation as inhibition of ROCK utilizing fasudil prevented microglia motility and activation of microglia in the substantia nigra of MPTP-treated mice . Indeed,Charcot marie-tooth disease (CMTD) is an inherited neurological disorder that presents as progressive muscle weakness and decreased muscle size. The first subtype of CMTD is characterized by demyelination and results in lower nerve conduction velocities while the second subtype is due to a defect within the axons themselves. Recent studies have highlighted that dysregulated Rho family GTPase activity may underlie demyelination and axonal abnormalities. Recently, the T332I mutation was identified in ARHGEF10 in patients exhibiting slowed nerve conduction velocities and thin myelination of peripheral nerves. ARHGEF10 is a GEF for RhoA that is highly expressed in the peripheral nervous system and exhibits enhanced activity when harboring the T332I mutation . ConsistThe spinal cord connects the CNS to the PNS to control sensory and motor functions. Following spinal cord injury (SCI), this connection can be disrupted to elicit neurological deficits due to impaired transmission between the CNS and PNS. The symptoms of SCI vary widely dependent upon the location and severity of injury but neurological defects can range from pain to paralysis. Given the extensive involvement of Rho GTPases in regulating axonal outgrowth and pathfinding, it is not surprising that members of the Rho GTPase family have emerged as key modulators of axonal regeneration following SCI.in vivo models of SCI, administration of either Rho GTPase inhibitors  or ROCK inhibitors  is protective following traumatic nerve injury , and oligodendrocytes-myelin glycoprotein (OMgp). Early studies in CGNs demonstrated that NogoA and MAG mediate their inhibitory effects on neurite outgrowth through disruption of the delicate balance between antagonistic Rho/ROCK and Rac signaling pathways. In particular, the inhibitory proteins enhance activation of Rho/ROCK signaling and diminish activation of Rac1 . Indeed,e injury . In vivo in mice . In a si in mice . Moreovecontrols . Collectin vivo following sciatic nerve axotomy in mice. However, constitutively active Rac1 failed to induce myelin fragmentation on uninjured nerves, indicating that Rac1 is an upstream regulator, but is not sufficient to induce myelin degradation following sciatic nerve injury in mice. In contrast to this prior study indicating Rac1 has an essential role in myelin fragmentation following nerve injury, In opposition to Rho, recent studies indicate that activation of Rac may be a critical feature which promotes axonal regeneration following SCI. Wallerian degeneration occurs in response to axonal transection and involves axonal degeneration and fragmentation of the myelin sheath. Cerebral ischemia and reperfusion (I/R) have devastating effects on the brain and increasing evidence suggests that neuronal cell death may be induced by dysregulation of Rho family GTPase activity. In a similar manner to other neurodegenerative diseases and neuronal traumas, recent data indicate that Rac GTPase activity may be dysregulated following I/R. For example, active Rac1 was decreased while total Rac1 redistributed to retracting neuronal processes in the hippocampus 24 h after rats were exposed to global cerebral ischemia . This stin vivo data in rodents has underscored the Rho/ROCK pathway as a significant determinant of neuronal cell death following I/R. Following focal ischemia in mice, expression of Rho is rapidly upregulated in the ischemic hemisphere, peaking between 12 and 24 h after onset and may contribute to neurodegeneration by inducing profound actin cytoskeletal restructuring (In opposition to Rac, Rho is upregulated in the brains of human stroke patients when compared to controls . Furtheructuring . Interesucturing . Rho appucturing , which lucturing .Recent pre-clinical studies have examined the therapeutic use of ROCK inhibitors for the treatment of I/R. Administration of the ROCK inhibitor fasudil significantly protected against delayed neuronal death induced by ischemic injury in gerbils, even when administered as late as 24 h after the ischemic insult . In a siThe essential functions of Rho family GTPases in regulating neuronal growth cone dynamics, neurite outgrowth, and neuronal development suggest that Rho GTPases have an important and conserved function in mediating neuronal survival and death. Indeed, although neurodegenerative diseases are complex and multifaceted, dysregulated activity of Rho family GTPases has emerged as a common feature underlying the etiology of diverse degenerative disorders of the central and peripheral nervous systems. Future research will be aimed at further elucidating the signaling pathways that are aberrantly activated or disrupted downstream of Rho GTPases in neurodegenerative diseases. Many studies suggest that targeting Rho GTPases, or their downstream effectors, may have beneficial therapeutic effects for the treatment of neurodegenerative diseases.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.


Cohesin facilitates sister chromatid cohesion through the formation of a large ring structure that encircles DNA. Its function relies on two structural maintenance of chromosomes (Smc) proteins, which are found in almost all organisms tested, from bacteria to humans. In accordance with their ubiquity, Smc complexes, such as cohesin, condensin, Smc5-6, and the dosage compensation complex, affect almost all processes of DNA homeostasis. Although their precise molecular mechanism remains enigmatic, here we provide an overview of the architecture of eukaryotic Smc complexes with a particular focus on cohesin, which has seen the most progress recently. Given the evident conservation of many structural features between Smc complexes, it is expected that architecture and topology will have a significant role when deciphering their precise molecular mechanisms. SMC dimeric complexes combine with a subunit of the kleisin family and form ring complexes entrapping DNA. In eukaryotes, cohesins, condensins, and SMC5-6 act on sister chromatid cohesion, chromosome condensation, and DNA repair.Cohesin complexes are dynamic as a result of two opposing activities: a DNA-loading activity mediated by the Scc2-Scc4 complex and a releasing activity elicited by the Pds5-Wapl-Scc3 complex. Crystal structures of these proteins are providing insight into their function. Pds5 and Scc3 have distinct positive functions in cohesion maintenance after S phase. The concept emerging is that different arrangements of these complexes exist in different stages of the cell cycle.SMCs are found in both open V-shaped and closed rod-shaped forms, which likely represent functional states of the complexes. Their formation depends on interactions with DNA and ATP hydrolysis. Every cell division is accompanied by the segregation of two replicated sister genomes. From bacteria to Archaea and up to the most evolved eukaryotic metazoans, equal segregation of the chromosomal complement is mediated and secured by the extended family of Smc proteins The first step of complex formation is mediated by dimerisation through the hinge domains of two Smc proteins, forming a V-shaped dimer A. Each NSmc gene has evolved through duplication and speciation into a range of orthologues and paralogues Caenorhabditis elegans, where the MIX1-DPY27 SMC heterodimer interacts with DPY-26 kleisin In eukaryotes, the primordial Given the overall size and presumed shape of the Smc-kleisin molecules, it became obvious that mechanistic solutions to questions regarding their function would only come from using rigorous structural and biochemical studies. Several crystal structures of SMC-related complexes have been solved recently, providing insight into their function. In this review, we focus on recent structural studies of the eukaryotic Smc1–6 members, their kleisin partners, and their regulators in hope of providing a picture of their mechanisms in chromosome segregation  α-kleisin subunit or its meiotic counterpart Rec8, form cohesin, which holds sister chromatids together from S phase to anaphase. The Smc1–3/Scc1 kleisin interface forms a tripartite structure resembling a ring Completing the cohesin ring, the Scc1 N-terminal domain interacts with the coiled coil emerging from the NBD of Smc3, but not with the bottom surface of the NBD itself in vivo with thiol-specific cross-linking to entrap yeast circular mini-chromosomes Solving the structure of all three interfaces allowed testing of the ring model Cohesin trimeric rings are destined to entrap DNA; however, the establishment and maintenance of sister chromatid cohesion depends on several peripheral proteins acting upon cohesin and eventually determining its residence time on chromatin. Proteins with essential functions in sister chromatid cohesion have been categorised based on the stage of their action and are involved in loading cohesin on DNA, establishing cohesion during S phase, or maintaining cohesion during mitosis . The SccSince not all cohesin loading events will produce sister chromatid cohesion, a process must exist to remove this noncohesive population of rings from chromatin. The discovery of the so-called ‘prophase pathway’ in higher eukaryotes With this in mind, the dynamics of cohesin in the cell appear more complicated than previously anticipated . At certCollectively, these findings converge on the idea that Pds5, Scc3, and Wapl act together to both maintain cohesin on, and release it from, DNA. Thus, all three of these proteins have been the focus of a structural approach aimed at gaining mechanistic insight in these processesAshbya gossypii (AgWapl) and human (HsWapl) were recently solved The above findings established Wapl as a key contributor to DNA-releasing activity. In budding yeast, Wapl (Wpl1) determines the turnover of cohesin on the yeast point kinetochores, the latter being the major cohesin-loading site on every chromosome Despite being crystallised with no interacting proteins, functional analysis assisted by the HsWapl structure revealed important regions for the function of Wapl The most dramatic manifestation of the release of cohesin from DNA is the prophase/prometaphase pathway found in vertebrates One model explaining this phenomenon relies on the finding that phosphorylation of Scc3/SA2 of cohesin, most likely by the mitotic kinase Plk1, destabilises cohesin on chromosomal arms, while the localised action of the Sgo1-PP2A phosphatase complex near kinetochores counteracts the destabilising phosphorylation, protecting centromeric cohesin from being released Zygosaccharomyces rouxiiin vitro and resulted in premature sister chromatid separation in human cells  complex Ashbya gossypiiS. cerevisiaeThe N-terminal part of Scc2, interacting with the full-length Scc4 protein (the NScc2-Scc4 complex) was recently crystallised from the yeasts in vitro using a minimum of six factors, along with DNA: histone octamers, histone chaperones, topoisomerase II, and condensin I From the earliest studies on condensin, its significance as a structural component of eukaryotic chromosomes was clear Bacillus subtilis Smc-ScpA trimer, where the Smc-kleisin trimers appear closed and rod shaped Pyrococcus furiosus Smc hinge with approximately 60 residue-long coiled coils shows differences in the orientation of the emerging coiled coils but an overall side-by-side coiled coil arrangement is still present. Using thiol-specific crosslinking, the close proximity of the yeast Smc2-Smc4 coiled coils was verified in immunoprecipitated yeast condensin complexes.Recent structural work in vitro assay using thiol-specific crosslinking, full-length bacterial Smc molecules abolished their rod shape when both DNA and ATP were present, again making a hinge–NBD allosteric interaction likely in vitro crosslinking experiments might be capturing either artificial or transitory states. The dynamic behaviour of Smc2-Smc4 heterodimers was tracked in real time and conformational transitions, primarily from O- to B-shapes, were observed. Even though the kleisin subunit was not present to determine the behaviour of condensin rings, these experiments do point towards the notion that condensin complexes are likely more flexible and dynamic in nature than previously anticipated.How do these data reconcile with the evidence supporting a ring for cohesin and condensin? It seems likely that distinct conformations exist at different times of the functional cycle of these complexes. For example, in an Recently, the analysis of available structures of the Nse1-Nse3 subunits of the Smc5-6 complex Why does Smc5-6 maintain kite protein interactions whereas cohesin and condensin do not? The interesting hypothesis arising is that Smc5-6 potentially represents the closest eukaryotic relative to a common Smc ancestor. Based on this model, cohesin and condensin eventually lost their kite dimers while Smc5-6 retained them. An essential remaining question is whether the Nse5-Nse6 subunits of Smc5-6 are orthologues of the HEAT repeat subunits of cohesin and condensin . A definitive answer to this question is not possible given the lack of relevant structural information for Nse5-Nse6. Another possibility could be that Nse5-Nse6, which operate as DNA recruiters during DNA repair and form a dimer Irrespectively, an interesting implication of the emergence of the kite family is that the common eukaryotic ancestor likely performed DNA maintenance, cohesion, and condensation functions. Hence, the dedicated and chromatin-related cohesion and condensation processes could represent an evolutionary refinement of the more general entrapment activity. This could also explain why Smc5-6 is not generally essential Recent progress in the structural biology of the Smc complexes, especially in the case of cohesin, has helped us to better understand the distinct roles of different subunits, domains, and various regulators of the interactions of Smc complexes with DNA and chromatin. Over the next few years, we anticipate that cryoEM, cryotomography and single molecule-imaging methods, such as smFRET, will take over as the most important methods for characterising the various holocomplexes B, becausOutstanding QuestionsWhat is the most basic, fundamental function that makes Smc complexes ubiquitous?What is the precise series of events that leads to loading, entrapment, releasing, and stable cohesion?What is the exact role of the NBD domains? How does ATP binding and hydrolysis affect the loading and releasing processes?Are there similar processes, such as loading and releasing activities, in condensin and Smc5-6?What is the precise architecture of the head complex ?Is there any long-range communication between the NBDs and the hinges?What are the structures of the Smc5-6 kleisin complexes? Is Nse4 a bona fide kleisin?How does CTCF direct DNA looping by cohesin and what is the topology of those complexes?Is there a direct mechanistic interplay between different Smc-kleisin complexes in organising chromatin?An even bigger question to answer will be how these machines that shape chromosomes and dictate chromatin functions functionally interact with other factors, such as the CTCF pioneer binding factor in mammals


Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings.Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca The cornea is a transparent, convex, avascular continuation of the sclera covering the front part of the eye. Light enters the eye through the cornea, which provides about 2/3 of the eye's refractive power and forms together with the lens the focusing power of the eye Maurice, . At the The cornea with its smooth, transparent, strong and durable characteristics has several functions in the eye. The transparent convex surface of the cornea acts as the eye's outermost optical element of fixed focal length and enables focusing light onto the retina of the eye, hereby making the cornea act as an important lens. The avascular nature of the cornea and the regular arrangement of the collagen fibrils of the stroma ensure the transparency of the cornea Davson, , which iThe main physiological role of the corneal endothelium is maintenance of corneal transparency by controlling stromal hydration Davson, . Since tIn order to maintain corneal function, regulation of the barrier integrity is crucial. The barrier function of the endothelial monolayer is controlled by the activation of different signaling mechanisms that affect paracellular and transcellular pathways. Although much is known about the identity of ion transport mechanisms in the corneal endothelium Bonanno, , the mec2 at birth and then slowly declines with age  endothelial dysfunction occurs  receptor (IP3R)-mediated release of Ca2+ from the endoplasmic reticulum due to the activation of phospholipase C (PLC)  provide an invaluable tool to analyze the properties of communicating channels, like connexins and pannexins  direct coupling via gap junction channel, allowing the passage of signaling molecules that can mobilize intracellular Ca2+, like IP3, and (ii) indirect coupling via hemichannels, allowing the release of signaling molecules, like ATP, that can trigger intracellular Ca2+ signaling by acting on ionotropic and/or metabotropic receptors, like the P2X and P2Y purinergic receptors  as a trigger for the opening of hemichannels, the knockdown of Cx43 resulted in a more than 60% reduction in EGTA-induced ATP release. Collectively, these data indicate Cx43 as the main connexin isoform responsible for forming hemichannels and for mediating hemichannel-mediated ATP release in BCECs, thereby driving intercellular Ca2+-wave propagation.An important mechanism responsible for the release of ATP seems to be mediated via hemichannels  inhibited Cx43-hemichannel activity in BCECs in response to mechanical stimulation  could be completely prevented by (-)-blebbistatin  by acting as a “safety” mechanism to prevent excessive Cx43-hemichannel opening, which would be detrimental for the cell due to loss of ionic, metabolic and energetic gradients, and (ii) by serving as a “Ca2+-dependent” shutdown mechanism of Cx43 hemichannels, allowing a bimodal regulation of Cx43-hemichannel opening by cytosolic [Ca2+]. The Ca2+-dependent inhibition of Cx43 hemichannels promotes the occurrence of sustained Ca2+ oscillations, leading to increased survival  rises (De Vuyst et al., It also remains to be elucidated whether the concept of loop/tail interactions being critical for the activity of Cx hemichannels is limited to Cx43 or can be a general mechanism operative in other Cx isoforms. In any case, although the “hot spots” for loop/tail interactions in Cx43 seem not highly conserved among other Cx isoforms (see Iyyathurai et al., We hereby show that primary BCECs are a good model system for studying the properties of native Cx43 hemichannels and their regulation by physiological signaling events. These studies ought to be complemented with knockdown experiments and ectopically expressed Cx43 hemichannels, allowing the expression of mutated and truncated Cx43 versions. The integrated approach has led to important discoveries, including the negative regulation of Cx43-hemichannel opening by the actin/myosin contractile system, the essential role of loop/tail interactions for Cx43-hemichannel activity and to the development of novel peptide tools that allow selective Cx43-hemichannel inhibition without affecting Cx43 gap junctions or hemichannels formed by other Cx isoforms.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.


An unresolved and critically important question in skeletal muscle biology is how muscle stem cells initiate and regulate the genetic program during muscle development. Epigenetic dynamics are essential for cellular development and organogenesis in early life and it is becoming increasingly clear that epigenetic remodeling may also be responsible for the cellular adaptations that occur in later life. DNA methylation of cytosine bases within CpG dinucleotide pairs is an important epigenetic modification that reduces gene expression when located within a promoter or enhancer region. Recent advances in the field suggest that epigenetic regulation is essential for skeletal muscle stem cell identity and subsequent cell development. This review summarizes what is currently known about how skeletal muscle stem cells regulate the myogenic program through DNA methylation, discusses a novel role for metabolism in this process, and addresses DNA methylation dynamics in adult skeletal muscle in response to physical activity. The term “epigenetics” literally means “above genetics” and is defined by the NIH Roadmap Epigenomics project as “both heritable changes in gene activity and expression  and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.” Epigenetics underlies the ability of embryonic stem cells  to commit to the three germ layers  during the early stages of development and eventually commit to specific cell fates to generate all the different cell types in an organism, including skeletal muscle. These biological trait variations are not a result of changes in the DNA code, but rather structural modifications to the DNA and/or histones, or posttranscriptional gene silencing via small RNAs  .Considering the interest surrounding epigenetics and in particular DNA methylation, in the regulation of stem cell identity, this review aims to discuss some of the recent findings regarding methylation, with a particular focus on skeletal muscle stem cells . While not discussed in this review, it is worth mentioning that, in addition to direct DNA modifications, structural epigenetic control is conferred at the level of histones. The core histone proteins H2A, H2B, H3, and H4 all contain long N-terminal tails which are highly susceptible to posttranslational modifications including methylation (me), acetylation (ac), phosphorylation (p), SUMOylation (sumo), ubiquitination (ub), ADP-ribosylation (ADP), and citrullination (cit) (reviewed in ).Before discussing the role of DNA methylation in MuSC biology, it is essential to first define the process of methylation. Methylation of DNA is a well-described phenomenon and primarily occurs on the 5′ position of cytosine bases within CpG dinucleotide pairs and leads to the formation of 5-methylcytosine (5mC) and a context specific effect on transcription. DNA methylation within the promoter region of genes is typically linked to transcriptional repression due to recruitment of methyl CpG binding domain (MBD) proteins, which block transcription factor and RNA polymerase access . In cont de novo DNA methylation , 69 Dnmtpatterns . Phgdh; phosphoserine aminotransferase 1, Psat1; phosphoserine phosphatase, Psph; and serine hydroxymethyltransferase, Shmt) and a decrease in the expression of isocitrate dehydrogenase 1 . These changes in the expression level of key metabolic enzymes, coupled with the previously identified changes in Dnmt and Tet  coactivator-1α (Ppargc1a), pyruvate dehydrogenase kinase 4 (Pdk4), and Ppard in skeletal muscle, which corresponded to transient induction of gene expression in a time and intensity dependent manner [It is now accepted that DNA methylation is a dynamic process, and as skeletal muscle is a highly plastic tissue able to rapidly respond to changes in demand, DNA methylation may be a particularly important mediator of these adaptations. Skeletal muscle responds to endurance and resistance training through adaptation of contractile apparatus and metabolic capacity. Barrès and colleagues have previously reported that acute exercise, in humans and mice, is linked to transient DNA demethylation in the promoter region of genes including peroxisome proliferator activated receptor t manner . This dat manner , while lt manner . Ppargc1a, in skeletal muscle of the offspring [ Ppargc1a mRNA levels and downstream gene expression  [ Ppargc1a was abolished in the skeletal muscle of the offspring along with the associated functional consequences [There is also mounting evidence that during perinatal development skeletal muscle is susceptible to insults or stimuli that may alter the epigenetic program, which has consequences for gene transcription and functional outcomes later in life –80. For ffspring . This wand CytC) . Furthernd CytC) . Interesequences . It is t Dnmt and Tet enzymes in regulating the MRF transcriptional program would provide new insight into DNA methylation dynamics and MuSC function and could be extended to investigate periods of muscle adaptation and plasticity. Finally, whole genome sequencing will allow us to take this research beyond the classical muscle-specific genes and also extend our reach to regions of intra- and intergenic DNA methylation in regulating transcriptional programs. These findings will be crucial for furthering our fundamental understanding of stem cell biology and epigenetic regulation and may lead to the development of novel techniques to induce pluripotency in committed cells and unveil new therapeutic targets.Additional studies are warranted to further characterize how DNA methylation and hydroxymethylation differ between MuSCs in different dynamic states and what specifically regulates these methylation events. In doing so, these studies will reveal novel mechanisms to regulate MuSC identity and growth. Furthermore, the identification of isoform-specific roles for


Hypophosphatemic rickets (HR) is a type of hereditary rickets characterized by persistent hypophosphatemia and hyperphosphaturia. The most predominant type is inherited in an X-linked fashion and caused by mutation in the gene encoding the phosphate-regulating endopeptidase homolog, X-linked (PHEX), identified in 1995. The X-linked hypophosphatemic (XLH) rickets is a rare hereditary metabolic disorder with a prevalence of 1 in 20,000 and causes deficient calcification of mineralized structures such as bones and teeth resulting in dental problems in terms of delayed eruption, spontaneous periapical infections, and exfoliation. We report one such unique case of hypophosphatemic vitamin D-resistant rickets in siblings exhibiting classical features of rickets with emphasis on its treatment and dental considerations. Rickets, a disease that occurs in children, is the failure of mineralization of osteoid commonly associated with either vitamin D deficiency or decrease of serum phosphate levels leading to hypophosphatemia. The condition, hypophosphatemic rickets, first described by Albright et al.  in 1937,The case report describes the clinical, radiological, and biochemical features of hypophosphatemic rickets in siblings who came to the clinic with a chief complaint of missing teeth.A 10-year-old girl (Case 1) presented to the department of oral medicine with a chief complaint of missing teeth in the upper front region of the jaw for 7 years. There was history of trauma to the upper front region of jaw at about 3 years of age resulting in loss of deciduous teeth and delay in the eruption of permanent teeth.The patient is the second offspring after a full term normal uneventful pregnancy.General examination (Case 1) revealed the height and weight as 45 inches and 14 kgs, which are less when compared with average height and weight of an age-matched Indian girl . There were frontal bossing, depressed nasal bridge  were advised.Chest radiograph  revealedRadiograph of hip with femur and tibia  revealedWrist radiograph  revealedOPG  revealedRadiographic findings of chest radiograph, radiograph of hip with femur and tibia, and wrist radiograph were interpreted by a general radiologist with 30-year experience.On further investigation, the patient's mother revealed history of consanguineous marriage and that her younger daughter (Case 2), who is 7 years old, also showed similar features.General examination revealed the height and weight as 23 inches and 10 kgs, which are less when compared with average height and weight of an age-matched Indian girl . Further examination revealed the clinical characteristics of base diagnosis given: There was bowing of legs  radiographs were advised.Chest radiograph  revealedRadiograph of hip with femur and tibia  revealedWrist radiograph  revealedIOPA'S  shows thLaboratory investigations of both Cases 1 and 2 are presented in The general management is taking care by using neutral phosphate and calcitriol supplements for both siblings. As the patient's chief complaint was delayed eruption of teeth, Case 1 was given denture  so that The siblings were advised for periodical follow-ups for prevention of formation of abscesses along with fluoride and pit and fissure sealants application. Regular blood investigations were done to rule out the complications of medications used such as secondary hyperparathyroidism and nephrocalcinosis. On long term follow-up, of about 1 year, they showed improvement in gait physical activity and eruption of permanent teeth.The term rickets is derived from an old English word “wrickken,” which means “to twist,” and a Greek word “rachitis,” which means inflammation of the spine. When the demineralized osteoid accumulates at the sites of bone formation, bones gradually soften leading to deformities in association with weight-bearing sites.Initially, environmentally caused rickets was very common; however, when it was treated and prevented by public health measures, the vitamin D-resistant rickets came into picture. Econs and Francis  highlighInorganic phosphate plays a major role in many biological systems, including cell membrane functions, energy metabolism, cell signalling, and oxygen transport. In hypophosphatemic rickets, renal proximal tubular resorption is compromised due to mutations in PHEX gene which is a phosphate-regulating gene. It encodes a zinc metalloendopeptidase which is predominantly expressed in osteoblast and odontoblast , 5. So tFamilial hypophosphatemic rickets, in most patients, appears in a familial line of X-linked, dominant inheritance with the same prevalence in both sexes; however, it may also occur sporadically , 7. HypoHypophosphatemic rickets has been related to several primary and permanent teeth alterations. The most commonly reported ones include normal but thin enamel and globular dentin and enlarged pulp chambers with pulp horns extending up to the dentinoenamel junction . In thisLaboratory findings including low serum phosphate concentration and reduced tubular resorption of phosphate corrected for glomerular filtration rate (TmP/GFR) are characteristic. Serum calcium and 25-hydroxy vitamin D are within the normal range or decreased; parathyroid hormone is normal to slightly elevated. Alkaline phosphatase is characteristically elevated. In Case 2, all findings were positive along with low calcium and vitamin D levels whereas in Case 1 there were elevated alkaline phosphatase activity and decreased serum inorganic phosphorus levels. Additionally, the normal physiologic response of hypophosphatemia with supplementation of vitamin D is absent in these siblings suggestive of vitamin D-resistant hypophosphatemic rickets.A family history of short stature, orthopedic abnormalities, poor dentition, and parental consanguinity may signify inherited rickets and the diagnostic criteria of X-linked hypophosphatemic rickets globally are based on history, clinical examination, and biochemical and radiological evaluation which are significant in these siblings.The treatment of hypophosphatemic rickets is done by the oral administration of phosphorus and vitamin D. Currently, growth hormone  and KRN2In our cases, general management is taking care by using neutral phosphate and calcitriol supplements. Regarding dental complaint Case 1 was given denture  and CaseHypophosphatemic rickets is a rare disorder with dental manifestations seen in spectrum of severity, ranging from very severe, with involvement of nearly the entire dentition, to very mild with normal appearance of the teeth.Being dentists, we hardly come across this rare variety of rickets in our clinics. Dental abscess in clinically normal teeth, early spontaneous exfoliation of clinically intact primary teeth, and the numerous teeth missing were the characteristics of patients with hypophosphatemic rickets.So early detection, treatment, and proper adherence to treatment provide a good dental future to the patients limiting delayed eruption, spontaneous dental necrosis, and dental diseases classically associated with hypophosphatemic rickets.


We show that humpback-whale vocalization behavior is synchronous with peak annual Atlantic herring spawning processes in the Gulf of Maine. With a passive, wide-aperture, densely-sampled, coherent hydrophone array towed north of Georges Bank in a Fall 2006 Ocean Acoustic Waveguide Remote Sensing (OAWRS) experiment, vocalizing whales could be instantaneously detected and localized over most of the Gulf of Maine ecosystem in a roughly 400-km diameter area by introducing array gain, of 18 dB, orders of magnitude higher than previously available in acoustic whale sensing. With humpback-whale vocalizations consistently recorded at roughly 2000/day, we show that vocalizing humpbacks (i) were overwhelmingly distributed along the northern flank of Georges Bank, coinciding with the peak spawning time and location of Atlantic herring, and (ii) their overall vocalization behavior was strongly diurnal, synchronous with the formation of large nocturnal herring shoals, with a call rate roughly ten-times higher at night than during the day. Humpback-whale vocalizations were comprised of (1) highly diurnal non-song calls, suited to hunting and feeding behavior, and (2) songs, which had constant occurrence rate over a diurnal cycle, invariant to diurnal herring shoaling. Before and during OAWRS survey transmissions: (a) no vocalizing whales were found at Stellwagen Bank, which had negligible herring populations, and (b) a constant humpback-whale song occurrence rate indicates the transmissions had no effect on humpback song. These measurements contradict the conclusions of Risch et al. Our analysis indicates that (a) the song occurrence variation reported in Risch et al. is consistent with natural causes other than sonar, (b) the reducing change in song reported in Risch et al. occurred days before the sonar survey began, and (c) the Risch et al. method lacks the statistical significance to draw the conclusions of Risch et al. because it has a 98–100% false-positive rate and lacks any true-positive confirmation. Passive acoustic survey methods employing hydrophones at fixed locations We find that the distribution of the vast majority of vocalizing humpback whales coincided with the primary time and location of Atlantic herring during their peak annual spawning period. During daylight hours, herring were found to be dispersed on the seafloor in deeper waters over wide areas of Georges Bank's northern flank Before and during Ocean Acoustic Waveguide Remote Sensing (OAWRS) survey transmissions Vocalizing humpback whales and spawning herring populations 2), before migration to shallower water (≈50 m) spawning grounds on Georges Bank 2) were found to be widely distributed between water depths of 50 m and 300 m, which include dense shoal formation areas We find that the vast majority of vocalizing humpback whales were spatially distributed in regions coinciding with the primary aggregations of spawning herring during the peak annual herring spawning period We find humpback whale vocalization behavior follows a strong diurnal pattern that is temporally synchronous with the regular herring shoal formation process 2) in deeper waters on the northern flank of Georges Bank  beam-time series by time-domain beamforming With our densely sampled, large-aperture array, multiple vocalizing humpback whale individuals could be tracked in beam-time and compared with the bearings of historic humpback whale habitats in the Gulf of Maine, including the Georges Bank, Stellwagen Bank, Great South Channel, and Northeast Channel as shown in consistently originated or ended at the the same spatial position as song calls, to within our reported position error in Section 3.3, and occurred immediately after or before these co-located song calls, alternating with song calls, it is most likely that the same species and group of whales produced the song and non-song calls we report. Given this and the fact that humpback whales are the only species known to produce song in this region, season and frequency range, it is most likely that the non-song calls we report are also from humpback whales and extremely unlikely that they originate from other species. Furthermore, humpback whales are the most abundant, by 1–2 orders of magnitude, vocalizing whales in the 250–700 Hz frequency range As noted in Section 2.1, both humpback whale song The diurnal humpback whale call rate  time series of To determine the horizontal location of a vocalizing humpback whale, both bearing and range need to be estimated. With our densely sampled, large-aperture horizontal receiver array, bearings of vocalizing humpback whales are determined by time-domain beamforming. Synthetic aperture tracking To evaluate its bias and quantify the impact of this bias, the Risch et al. statistical test of Ref. P in Risch et al. The Risch et al. statistical test M years, and for each year there are N 33-day periods. Let Suppose there are daily humpback whale song occurrence time series over For a given 33-day period over The rate of false positive findings that whales respond to sonar when no sonar is present isWhen the Risch et al. statistical test P values and results reported in the ‘Risch et al. Statement’ column of We apply the Risch et al. statistical test When the Risch et al. statistical test Detectable humpback whale song occurrence for a coherent sensor array can be quantified in terms of local wind-speed-dependent ambient noise for a given spatial distribution of vocalizing humpback whales. The humpback whale song occurrence depends on the presence of at least one singing humpback whale inside the mean wind-dependent detection range of the sensor array. The percentage of time in a day over which a humpback whale is within the mean detection area and is singing corresponds to the measured daily humpback whale song occurrence rate.The detection range A standard parabolic equation model of the US Navy and the scientific community, Range-dependent Acoustic Model (RAM) We find that the humpback whale song source levels measured from more than 4,000 song units recorded during the same 2006 Gulf of Maine experiment discussed here at the same time and at the same location approximately follow a Gaussian distribution and are in the range 155 to 205 dB re 1 µPa and 1 m  with a mThe total humpback whale song occurrence in a day detectable by a sensor in varying wind speeds ise is the e-folding time scale defining the width of the correlation peak, or coherence time scale, within which processes are conventionally taken to be correlated We calculated the normalized autocorrelation function


Heat shock proteins are molecular chaperones with a central role in protein folding and cellular protein homeostasis. They also play major roles in the development of cancer and in recent years have emerged as promising therapeutic targets. In this review, we discuss the known molecular mechanisms of various heat shock protein families and their involvement in cancer and in particular, multiple myeloma. In addition, we address the current progress and challenges in pharmacologically targeting these proteins as anti-cancer therapeutic strategies Heat shock proteins are a group of highly conserved proteins found in both eukaryotic and prokaryotic cells. They are involved in a wide range of cellular processes such as assisting protein folding and degradation of misfolded proteins, intracellular trafficking, modulating signalling pathways and regulating immune responses -5. The mDrosophila melanogaster ..195].The consistent upregulation of Hsp70s following Hsp90 and proteasome inhibition, and their proven anti-apoptotic roles contributing to drug resistance leads to a growing interest in the development of Hsp70 inhibitors to be used as single anti-cancer agents or in combination with conventional or targeted chemotherapies. However, to date few Hsp70 specific inhibitors have been identified. Two Hsp70 specific compounds, Ver-155008 and MAL3-101, have been tested on myeloma in the preclinical setting.in vitro and in vivo, and demonstrated synergy with proteasome and Hsp90 inhibitors [Ver-155008 is an ATP-analogue capable of inducing caspase dependent apoptosis in a panel of myeloma cell lines via the modulation of multiple oncogenic pathways and enhancing Hsp90 inhibition induced cell death , 180. Inhibitors . Althoughibitors .in vitro, illustrates the therapeutic potential of an HSF1 inhibitor in combination with HSP90 inhibition [As an alternative to targeting individual heat shock proteins, there has been an interest in the development of inhibitors against Hsf1, the ‘master regulator’ of heat shock response. Since the inhibition of a single heat shock protein such as Hsp90 inevitably leads to the compensatory upregulation of other heat shock proteins such as Hsp70 and Hsp27, targeting Hsf1 instead of the individual chaperones separately is potentially more therapeutically effective, as inhibition of Hsf1 could in theory abolish the ability of a cancer cell to activate the whole heat shock response during cellular stress. The increased sensitivity of hepatocellular carcinoma and melanoma cell lines to HSP90 inhibition with HSF1 knocked down hibition . While shibition . As the hibition .It is becoming increasingly apparent that targeting individual cellular stress pathways or components may not be sufficient for killing myeloma cells as other compensatory pathways or components can be upregulated. Therefore, targeting multiple oncogenic and signalling pathways simultaneously may be the future of myeloma treatment, and cancer treatment in general.The fact that cancers such as myeloma rely on the protein handling pathway for survival creates a ‘therapeutic window’ for heat shock protein inhibition. Evidence suggests that the inhibition of heat shock proteins affect cancer cells more than normal cells , 182, maChallenges however remain in the effective targeting of these proteins in myeloma. Firstly, the molecular mechanisms of heat shock proteins are still not fully understood, with multiple isoforms of the same heat shock protein playing distinct or compensatory roles. This is exemplified by the consistent upregulation of Hsp72 following Hsc70 inhibition, and inhibition of both isoforms may be required. Understanding the roles of individual heat shock proteins and the effect of combined inhibition of multiple heats shock proteins is the key to developing an effective treatment strategy against myeloma with minimal side effects in patients. Secondly, the development of heat shock protein inhibitors suitable for the clinic remains a major challenge. Apart from Hsp90 inhibitors, inhibitors against Hsp70, Hsp27 and Hsf1 are still in the early phase of development despite strong evidence of their involvement in myeloma survival. Thirdly, preliminary data shows enhanced myeloma cell killing by combining heat shock protein inhibition with inhibition of other pathways such as the proteasome, but the best combination treatment strategies are yet to be established.In conclusion, targeting the heat shock pathway is a promising therapeutic strategy in myeloma as well as in other cancers. Much work is currently ongoing in this area and the results are eagerly awaited.