Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses. Oncolytic viruses (OVs) are a family of viruses characterized by their preferential replication in—and subsequent lysis of—tumor cells following in vivo injection/trafficking to the site of the tumor. Early studies in animal models showed that OV treatment effectively inhibited tumor growth and prolonged survival ,2. HypotPoxviruses are large enveloped viruses with a double-stranded linear DNA genome ranging in size from 130 to 375 kbp and containing hundreds of genes . While tOrthopoxviruses are capable of infecting humans, the best known example being the variola virus, the causative agent of smallpox. Vaccinia virus (VV), another orthopoxvirus was first utilized by Edward Jenner in developing the smallpox vaccine, considered the first recorded instance of vaccination  . When caThese early successes and discoveries regarding VV led to its continued development and research for cancer treatment, and the isolation of several different VV strains. One particular strain of growing clinical interest is the Modified Vaccinia Ankara (MVA) strain. MVA was developed initially as an attenuated smallpox vaccine by passaging VV hundreds of times in chicken embryo fibroblasts, leading to the deletion and mutation of large sections of the genome . These cThe various VV strains developed for clinical use have pronounced cancer tropism due to deletions and mutations in various virulence genes, making VV well suited for OV therapy ,26,27,28While normally restricted to replication in birds, members of the avipoxvirus genus, specifically the canarypox virus (CV) and fowlpox virus (FV), have also been studied for human therapeutic applications. Because of their limited host range and inability to replicate in non-avian species, avipoxviruses are considered promising vectors for vaccination and gene therapy based on their maintained infectivity in mammals . They beThe mechanisms by which poxvirus can function as an OV therapy are multimodal, complex and still under investigation . VV cleaThe TME is a term used to describe the local tissue area within and surrounding a tumor, including recruited immune cells, and surrounding stroma and vasculature. The TME has been extensively studied—especially with regards to how the tumor alters and controls its environment—and researchers have shown that cells and molecules within the TME can impact the local and by extension systemic immune response to tumor . Tumors One direct way that OVs may impact the immune microenvironment and immunization is via viral modulation of cytokine activity. Poxviruses have evolved defense and escape mechanisms to evade the host immune system that include the production of proteins that mimic the structure of cytokines and chemokines (virokines), and their receptors (viroceptors) ,49,50,51As mentioned previously, early trials with wild type VV in the 1960s showed signs of it being capable of inducing a systemic tumor-specific adaptive immune response . This waIn early OV therapy trials using wild-type VV, however, the in situ vaccination effect was modest at best (possibly due to the suppressive effects of the TME) and occurring only in a subset of patients ,37. ThesTo maximize effectiveness of OV therapies and overcome immunosuppression within the TME, various strategies have been developed and tested. Taking advantage of the ability to encode OVs with immunostimulatory molecules and/or TAAs, we hypothesized that the best chance for success would be found in combining the viral lysis with pro-immune cytokines and, where possible, identified TAAs.The ability to encode genes for both cell surface and soluble immune modulating molecules and, when using poxvirus, the ability to encode multiple genes without compromising infectivity, makes poxvirus an ideal candidate for modulating the TME. The production of these virus-encoded immune-regulatory factors by the virus-infected cells within the TME would have the ability to enhance the anti-tumor immune response potentially primed by viral lysis. Potential immune-enhancing molecules tested include chemokines ,57, reguGM-CSF has a number of functions, primarily in the stimulation of the development of hematopoietic cells . Of partOur group was the first to engineer vaccinia virus to encode human GM-CSF (VV-GMCSF) and to show that it could be safely used to treat melanoma patients . In thatBuilding on the success of this study, the recombinant VV developed by our group has been licensed and continues to be developed under the name pexastimogene devacirepvec  ,73. ActiWhile not a poxvirus, another successful oncolytic DNA virus that should be mentioned is Talimogene Laherparepvec (T-VEC), a recombinant herpes simplex virus that also encodes GM-CSF . T-VEC iWhile the use of GM-CSF encoding OV can lead to DC recruitment and overall immune activation, it should be noted that GM-CSF has also been shown to activate and recruit MDSCs, potentially countering its positive effects in stimulating adaptive immune responses ,85. GM-CAs a means of further enhancing the generation of systemic tumor antigen specific adaptive responses, we have taken the approach of combining genes for immune regulating molecules such as GM-CSF with encoding TAA.lacZ gene with a readout of antibody response to the beta-galactosidase protein. These studies showed that immunizing within the IL10 high TME skewed the response to IgG1 (TH2) while immunizing in the contralateral flank resulted in a IgG2 (TH1) response [In early studies of immune regulation within the tumor microenvironment, we found that the immune regulating cytokine IL10 was overexpressed in biopsies of melanoma lesions in patients . In dissresponse  thus demresponse  led us tresponse  while taWhile OVs encoding immunostimulatory molecules or TAAs have shown some clinical benefits, we hypothesized that combining them would prove more therapeutically effective. To investigate the role of the TME on OV therapy, our group conducted several studies using rVV constructs encoding GM-CSF and various TAAs. Utilizing our MB49 model, we showed that tumor-induced IL-10 production by immune cells in the TME inhibited the ability of DCs to induce an immune response . IntratuWe have found similar results using a second model (orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) developed in our lab) where there was an expansion of MDSC in the TME. We found that i.t. injection of a combination of VV-GMCSF and VV-neu, but not VV-GMCSF alone, induced a systemic anti-neu CD8+ T-cell response a, led toOV cancer therapy has progressed in the past decades, as recombinant technology enabled the deliberate expression of immunostimulatory molecules and TAAs. Prime-boost strategies combining different viral species avoid the downsides of anti-viral immune responses while maximizing anti-tumor responses, and injecting i.t. directly disrupts the inhibitory effects of the TME. Many clinical trials studying OVs have been conducted and are underway, with the first OV regimen gaining FDA approval last year. While OV therapy often is capable of great clinical benefit as a monotherapy, addition of chemotherapeutic agents may be of benefit. Several trials with various agents have shown synergistic potential ,94,95,96Decades of research into poxvirus OV therapies have developed several successful clinical treatments and many more in preclinical studies. With the first OV therapy obtaining FDA approval, and increased interest in immunotherapy, the time has never been better for OVs. Poxviruses in particular, with their ability to express multiple recombinant molecules, disrupt the TME and overcome immune evasion, are excellent vectors for OV immunotherapy. Combining poxvirus OVs with next generation targeted agents and checkpoint inhibitors will likely lead to further successful treatments and improved patient outcomes in the future.


The immune system and metabolism are highly integrated and multilevel interactions between metabolic system and T lymphocyte signaling and fate exist. Accumulating evidence indicates that the regulation of nutrient uptake and utilization in T cells is critically important for the control of their differentiation and manipulating metabolic pathways in these cells can shape their function and survival. This review will discuss some potential cell metabolism pathways involved in shaping T lymphocyte function and differentiation. It will also describe show subsets of T cells have specific metabolic requirements and signaling pathways that contribute to their respective function. Examples showing the apparent similarity between cancer cell metabolism and T cells during activation are illustrated and finally some mechanisms being used by tumor microenvironment to orchestrate T-cell metabolic dysregulation and the subsequent emergence of immune suppression are discussed. We believe that targeting T-cell metabolism may provide an additional opportunity to manipulate T-cell function in the development of novel therapeutics. It is well admitted that one of the mechanisms by which immune cells integrate the signals required for their proliferation, migration, differentiation, and effector functions is through the modulation of their metabolic activity . In thisNevertheless, how metabolism regulates immune T-cell differentiation, function, and plasticity remains very challenging and how immune cells function in terms of their intracellular metabolism and how these metabolic pathways affect the phenotype and activation of immune cells is attracting a lot of attention at present. Tumor progression is characterized by a tangled network of relationships among different cell types that collectively exploit a metabolic reprogramming and mutually influence their functionality and, in particular, T-cell functions. Our recent knowledge of T-cell molecules involved in the regulation of antitumor T-cell responses has led to the development of several monoclonal antibody-based therapies, against molecules like cytotoxic T-lymphocyte antigen (CTLA-4) or programmed death-1 (PD-1) . AlthougIt should be noted that the dynamic and reciprocal interactions between tumor cells, metabolites, and a variety of cells including immune cells from the tumor microenvironment orchestrate several events, which are critical for tumor evolution toward metastasis. In this context, many cellular and molecular elements of the tumor ecosystem are emerging as attractive targets for therapeutic approaches. Among these targets, hypoxia, which is a hallmark of solid tumors, is strongly associated with advanced disease stage and poor clinical outcome. This is, in part, due to inappropriate local immune reaction and resistance of hypoxic tumor cells to cytotoxic treatments. In fact, most human tumors develop a pathophysiological microenvironment during growth, characterized by an irregular microvascular network and regions of chronically and transiently hypoxic cells. We and others provided evidence that hypoxia plays a crucial role in tumor promotion and immune escape by conferring tumor resistance  immunosuMetabolism is the process whereby cells can either break down molecules to generate energy in the form of adenosine triphosphate (ATP) or synthesize several macromolecules. Metabolism could be divided into two complex pathways: the catabolic processes, critical for cellular proliferation and functions and the anabolic process, important for cellular growth.Consistent studies focused on the molecular mechanisms that dictate metabolic reprogramming in the immune cells . It is neff)  ligation and binding with costimulatory molecules induce metabolic remodeling , 19. In eff) , 21  .After pathogen clearance, most T cells go through apoptosis while few of them remain as long-lived memory cells responsible for enhanced immunity against upcoming pathogens or tumors re-exposure .m) cells (T lymphocytes (T cells) that undergo an immune response constitute an ideal system to study the rapid shift from quiescent to active state that belongs to growth, proliferation, and differentiation into largely heterogeneous T-cell subsets. Emerging concepts in immunology suggest that lymphocyte activation is intricately linked to metabolic reprogramming –26. In fm) cells .eff could be divided into cytolytic T cells, secreting granzyme B, perforin, interferon-γ (IFN-γ), into helper T cells (Th) including the type-1 (Th1), type-2 (Th2), and type-17 (Th17) producing characteristic cytokines or into regulatory T cells (Treg)  , 29. Tef cancers . After epathogen , 32. Thepathogen .eff subtypes switch their metabolic program to robust aerobic glycolysis, but increased glycolytic rates occurred much higher in Th1, Th2, and Th17 cells than in Treg cells (reg cells sustain enhanced fatty acid oxidation (FAO) metabolism as a major source of energy to maintain their survival  as a quiescent T-cell population share common metabolic requirements with other nonproliferating cells. Tm cells maintain catabolic profile with lower nutrient uptake and biomass synthesis and predominantly engage mitochondrial OXPHOS and FAO metabolism for long-term persistence, ATP production and the capacity to vigorously respond to antigen stimulation  cycle related to OXPHOS metabolism. However, their detailed metabolic profiles remain to be explored  –50. Incrproteins , 52. Recproteins . The fir activation ], has been found to critical during activation  , 128.Myc is a transcription factor upregulated in tumors and considered as master regulator of normoxic cancer cell reprogramming . Indeed,Upon rapid proliferation, hypoxia becomes a key mediator of the Warburg effect and a common feature of human tumors. Extensive studies have provided evidence that cancer cells utilize hypoxia as physiological adaptation pathway that promotes metabolic changes in fast growing tumors . Indeed,Furthermore, PI3K/Akt/mTOR is one of the most frequently altered signaling pathway known to play an important role in glycolysis, cancer metabolism and cancer cell proliferation , 138 Fi. In addiImmuno-metabolism plays a key role of adaptive immunity and is particularly central to effective antitumor T-cell responses. T cells, following the metabolic strategies of growing tumors, have to start their effector programs. However, most of human tumors proliferate in spite of the presence of tumor associated antigen-specific T cells. In fact, tumor microenvironment may impose several limitations to dampen T-cell immunity  and depleff in tumors are severely influenced by the tumor microenvironment context , allows the tumor to evade immune system by inhibiting T-cell function , 169. Reeff and favors Treg expansion   or immunefficacy .Therefore, the use of metabolism-targeting drugs working with checkpoint inhibitors might not only change the activation and differentiation program of tumor-specific T cells but also prohibit the generation of exhausted T cells. Currently, there is a lack of data taking into consideration the metabolic consequences occurring in T cells and/or tumor cells by targeting these immune checkpoint pathways. Nevertheless, combined immunotherapeutic strategies would be exciting and show promise to improve the anticancer efficacy of immunotherapy in the future.2 is a master regulator of the CD8+ T-cell response and T lymphocytes face pathologically low O2 tensions within the tumor bed at which they will have to function. It has become clear that tumor-imposed metabolic restrictions may result in an impairment of T-cell function and that either some programmed changes or pathologic manifestations can inhibit the required energy essential for their several functions. Accordingly, attempts are made to identify approaches aiming at manipulating the reprogramming of T-cell metabolic pathways for therapeutic purposes, in particular, antitumor immunity.It is widely admitted that tumors are not autonomous masses of cells but function as organs composed of many interdependent cells supporting malignant cell survival, growth and progression. To ensure tumor growth and immune evasion, the tumor stromal components undergo numerous metabolic adaptations, reprogramming the mode of energy generation. T cells play key role in the orchestration of the immune response and T-cell metabolic adaptation acts as crucial checkpoint hijacked by tumors to dampen antitumor immunity as T cells are rendered dysfunctional, unable to carry out their effector functions. Accumulating evidence indicate that the diverse functions of the immune system require several bioenergetic processes and that T-cell metabolic reprograming relies upon the activation of distinct transcriptional and signaling pathways. In the context of tumor microenvironment, tumors impose several limitations to dampen T-cell immunity as T cells, experiencing the metabolic framework of growing tumors, fail to activate distinct pathways to accomplish their functional requirements. Tumor microenvironmental hypoxia is in this regard a relevant example demonstrating how the tumor microenvironment of a tumor can paralyze and neutralize T-cell functions. In fact, OAll 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.


In vitro maturation (IVM) is emerging as a popular technology at the forefront offertility treatment and preservation. However, standard in vitro culture (IVC) conditions usually increase reactive oxygen species (ROS), which have been implicated as one of the major causes for reduced embryonic development. It is well-knownthat higher than physiological levels of ROS trigger granulosa cell apoptosis andthereby reduce the transfer of nutrients and survival factors to oocytes, which leadsto apoptosis. ROS are neutralized by an elaborate defense system that consists ofenzymatic and non-enzymatic antioxidants. The balance between ROS levels andantioxidants within IVM media are important for maintenance of oocytes that develop to the blastocyst stage. The effects of antioxidant supplementation of IVMmedia have been studied in various mammalian species. Therefore, this article reviews andsummarizes the effects of ROS on oocyte quality and the use of antioxidant supplementations for IVM, in addition to its effects on maturation rates andfurther embryo development. In vitro embryo production (IVEP) allows the productionof a high and inexpensive number of embryosto conduct basic research and apply emergingbiotechnologies such as cloning and transgenesis.IVEP is a three-step methodology that comprises thefollowing procedures: i. In vitro maturation (IVM)of oocytes recovered directly from follicles, ii. Invitro fertilization (IVF) or co-incubation of capacitatedspermatozoa with in vitro matured oocytes,and iii. In vitro culture (IVC) of zygotes up to theblastocyst stage. According to reports, IVM is thekey factor that determines the proportion of oocyteswhich develop to the blastocyst stage.in vitro advancement of an oocytefrom the diplotene stage of prophase I [germinalvesicle (GV)] to metaphase II (MII), along with cytoplasmicmaturation that encloses a broad set ofstill ill-defined cellular events are essential for fertilizationand early development of the embryo . The generationof pro-oxidants such as reactive oxygen species(ROS) is an invariable phenomenon in the culturecondition. It is possible that OS also influences oocytedevelopment in vitro. On the other hand, ROSare considered signal molecules in oocyte physiology and their impact on maturation promoting factor (MPF) destabilization has recently been reported  on developmental competence of oocytes in vitro have been reported in mice  into O2 and HO2O2 while CAT converts HO2O2 to O2 and H2O. The enzyme SOD exists as three isoenzymes , catalase (CAT), various peroxidases and peroxiredoxins (PRDXs), including glutathione peroxidases (GPXs), which can convert peroxides to water and alcohol . SOD enzoenzymes : SOD1, Soenzymes . GSH is oenzymes .Vitamin C (ascorbic acid) is a known redox catalyst that can reduce and neutralize ROS . Based oVitamin E (α-tocopherol) is a lipid soluble vitamin with antioxidant activity. It consists of eight tocopherols and tocotrienols. Vitamin E may directly destroy free radicals such as peroxyl and alkoxyl (ROO•) generated during ferrous ascorbate-induced lipid peroxidation (LPO), thus it is suggested as a major chain breaking antioxidant . Hyaluroin vitro.Hyaluronan, an essential component of the extracellular matrix and non-sulfated glycosaminoglycan may play an important role in meiotic resumption of oocytes . The horThe addition of enzymatic antioxidants such as SOD, CAT, and thioredoxin are effective for pre-embryo development as scavengers of ROS and serving embryos a low OS condition in mice , 62, porA series of non-enzymatic antioxidants protect oocytes against ROS damage during oocyte maturation. GSH is one of the naturally synthesized antioxidants that protect cells from ROS toxicity and regulate the intracellular redox balance . The intCysteamine supplementation during IVM reportedly improved nuclear maturation rates in canines , mice 7, goats , and porAscorbate is concentrated in granulosa cells, theca cells, luteal cells, and oocytes . Choi etIt is well-known that high levels of ROS beyond the physiological range could induce MPF destabilization, reduce survival factors, and trigger apoptosis in oocytes of several mammalian species. Antioxidants are the main defense factors against OS induced by ROS. Many reports suggest that antioxidant supplementation of IVM media improves cytoplasmic maturation by alleviating OS during oocyte maturation via increasing GSH storage, and contributes to further protect the embryo against oxidative aggressions during its early developmental stages. On the other hand, supplementation by antioxidants during IVC improves oocyte quality by reducing ROS levels and apoptotic factors. However, some of the non-antioxidants such as ascorbic acid and 2, 4, 5-trihydroxybutrophenone do not improve oocyte maturation; rather, they inhibit spontaneous resumption of meiosis. Improvements to culture conditions are complex challenges that depend not only on the choice of an antioxidant but also on its concentration, the medium and its components, the species, and the dynamic changes of the specific requirements of the oocyte according to its developmental stage. Future efforts should be placed on understanding the involvement of ROS in oocyte apoptosis and for guiding antioxidant-based strategies to selectively control ROS-induced damage without compromising the physiological functions of these species.


Nowadays, dental implants have become more common treatment for replacing missing teeth and aim to improve chewing efficiency, physical health, and esthetics. The favorable clinical performance of dental implants has been attributed to their firm osseointegration, as introduced by Brånemark in 1965. Although the survival rate of dental implants over a 10-year observation has been reported to be higher than 90% in totally edentulous jaws, the clinical outcome of implant treatment is challenged in compromised (bone) conditions, as are frequently present in elderly people. The biomechanical characteristics of bone in aged patients do not offer proper stability to implants, being similar to type-IV bone (Lekholm & Zarb classification), in which a decreased clinical fixation of implants has been clearly demonstrated. However, the search for improved osseointegration has continued forward for the new evolution of modern dental implants. This represents a continuum of developments spanning more than 20 years of research on implant related-factors including surgical techniques, implant design, and surface properties. The methods to enhance osseointegration of dental implants in low quality (type-IV) bone are described in a general manner in this review. Dental implants have become a more common treatment for replacing missing teeth . ConsequIn 1965, Brånemark introduced the term “osseointegration” to describe the successful outcome of bone-to-implant integration . ClinicaAlthough the survival rate of dental implants over a 10-year observation has been reported to be higher than 90% in totally edentulous jaws , dental To explain the above-mentioned problem better, the remedies to enhance bone–implant integration in low quality (Type IV) bone are described in a general manner in this review. Over the last few decades, several implant-design concepts have been developed and are commercially available. Dental implants mostly possess a threaded cylindrical- or conical(root)-design . The desDespite an important role of biomechanics in the establishment of osseointegration, implant-design concepts are not expected to promote advantageous functions by specific control of bone cells and tissues interactions at the implant interfaces.Conventionally, the placement of dental implants sacrifices much bone tissue during the drilling procedure, which is performed with a consecutive series of surgical drills to prepare an implant bed fits the implant exactly. Consequently, several modalities of implantation techniques have been proposed to optimize a high degree of implant stability without removing additional bone, especially in situations where limited bone density  is available. For example, a surgical technique has been introduced that compresses the bone tissue laterally and apically using an osteotome spreader . Additio2) particles, and is followed commonly by acid-etching to homogenize the micro-profile of the implant surface and to remove as much of the residual blasting particles as possible. Acid-etching is often performed using hydrofluoric, nitric, sulfuric acid or combinations thereof [The osseointegration process relates to the all biological interactions between the host bone and implant surface. In view of this, implant surface modification is considered as an important approach to favor this process . Implant thereof . Recentl thereof . HoweverIn contrast to the physical modifications, the deposition of bioactive coatings onto the implant surface by means of several biochemical deposition techniques have been explored ,21, amonAdditionally, new coating strategies to improve implant osseointegration involve the development of a dedicated drug-loading ability to locally target bone disorders around dental implants more effectively . For insFrom the aforementioned findings, it appears that there are many methods to modify implant performance by improving the bone response physically, chemically, or therapeutically. However, the exact underlying biological mechanisms of these methods have not been fully characterized. Consequently, research has to focus on the in vivo investigation of existing implant surface modifications in order to achieve the desired biological responses, especially in compromised conditions. Long-term in vivo preclinical research is also necessary to investigate instructive as well as therapeutic capabilities of an implant surface coating using osteoporotic animal models.For the investigation of the osseointegration of bone implants with a newly developed surface modification, animal experiments are of fundamental significance. Several animal models are commonly used to study osseointegration  . HoweverThe developments regarding implant surface modifications seem critical for bone healing and improving osseointegration at sites that lack sufficient quantity or quality of bone. Thus, the research efforts at present are attempting to overcome the problems associated with implant complications that might arise in challenged bone conditions through the development of the surface-coated bone implants. Additionally, in vivo experiments that successfully addressed the instructive/therapeutic capabilities of implant surface coatings should be continued.


Primary emphasis in this review is given to comorbid aging and cognitive aging, though other phenotypes  are also discussed in relation to comorbid aging and cognitive aging.This article critically reviews the utility of “phenotypes” as behavioral descriptors in aging/HIV research that inform biological underpinnings and treatment development. We adopt a phenotypic redefinition of aging conceptualized within a broader context of HIV infection and of aging. Phenotypes are defined as The main findings that emerged from this review are as follows: (1) the phenotypes, comorbid aging and cognitive aging, are distinct from each other, yet overlapping; (2) associative relationships are the rule in HIV for comorbid and cognitive aging phenotypes; and (3) HIV behavioral interventions for both comorbid aging and cognitive aging have been limited.Three paths for research progress are identified for phenotype-defined aging/HIV research , and some important research questions are suggested within each of these research paths. During this era of unprecedented antiretroviral therapeutic efficacy, there is hope for successfully treating people aging with HIV infection, but this has been compromised by the changing clinical pattern of HIV infection with the emerging aging comorbidities and other manifestations . This has provided new challenges relating to the care and management of aging patients. In the aging person, these new challenges are frequently expressed as “phenotypes” and are most often viewed within the now well-accepted notion of HIV infection as a chronic disease. Before we can reach deeper mechanistic insights or develop more effective interventions in aging/HIV-infected individuals, it is essential to rethink how we define aging in this context,by itself may provide limited, and sometimes misleading, information due to variability of the aging process both within and between populations. Aging is a heterogeneous process and spans various domains including inflammatory aging, immunosenescence, metabolic aging, cognitive aging, and psychosocial aging. Research studies on aging/HIV should go beyond using chronological age alone because the process of HIV infection, like the process of aging, is shaped by the individual’s social, physical, cultural, and economic setting in addition to biological senescence. In this context, aging is a developmental biological concept within an integrative lifespan framework, which promotes understanding of how and why individuals change [In aging research, use of the categorical variable chronological age s change , 2.Other than aging as a biological process separate from chronological age, it has been noted  that thedimensions of behavior, closely related to fundamental mechanisms, and, thus, may be more informative than chronological age, especially in HIV research. Phenotypes have been used to identify the vulnerable subsets of the aging population—frailty [The phenotype construct offers multiple advantages in HIV/aging research. “Phenotypes” are —frailty , comorbi—frailty , 6, and —frailty . Future This article critically reviews the phenotype construct within the context of aging and HIV infection. Phenotypes have been studied far more often in gerontology  than in “Phenotypes” are used as behavioral descriptors of aging/HIV that may have different disease courses, biological underpinnings, and treatment responses. The major aging/HIV-related phenotypes are defined in Table Phenotypes are defined as subsets of biological factors that may define sub-groups of aging individuals . We use “endophenotypes” to provide stratifications by sub-group , to optimize intervention targeting and inform clinical trials addressing abnormalities in key biological systems. We did not cover phenotypes of biological aging, such as the immune risk phenotype, reflecting immunosenescence associated with markers of immune aging like the depletion of naïve CD4 cells  and the Comorbidities in HIV infection, in the broadest sense, consist of a spectrum of conditions including medical , neuropsychiatric comorbidities , and social comorbidities  . The extThe largest epidemiological study  addressing the prevalence of HANA comorbidities in HIV indicated a prevalence of 60–63% . Some inMajor depressive disorder (MDD) is the most common psychiatric diagnosis in HIV/AIDS at a prevalence over twice the rates seen in the general population , 27. HowSubstance use disorder comorbidities are also common in older patients and put them at higher risk for HIV infection. Substance use accounts for over 16% of new HIV infection in people aged 50 and older . Unlike Approximately 50% of persons living with HIV suffer from impaired cognition, a frequency that increases with age . MoreoveWe briefly discuss other aging phenotypes that have not been studied in the same depth in HIV infection as comorbid and cognitive aging, the focus of our review. Accepted definitions for all HIV-related aging phenotypes are presented in Table disability  among older HIV-infected patients are independently associated with older age and HIV disease burden  [Both primary types of al load) . Over thal load) . Disabilal load) .frailty phenotype is considered the most well-operationalized and characterized [Among all the aging phenotypes, the cterized , 43. Reccterized , 44. Mulaccelerated aging phenotype suggests that a more rapid aging process is taking place in HIV infection, and there is debate as to whether the outcomes examined represent an acceleration of the typical aging process or a greater accentuated risk for age-related declines [Research on the declines . Intervesuccessful aging phenotype in people with HIV has been scarce. Few HIV-infected persons, let alone those with AIDS, would meet the definition of successful aging proposed by Rowe and Kahn [Although aging with HIV infection is often reportedly accompanied by increases in patience, contentment, moderation, wisdom, and a greater respect for health and life  as well and Kahn , i.e., aand Kahn . Thus, “and Kahn .While aging-related phenotypes  may represent distinct clinical entities and have their own unique challenges, there are considerable inter-relationships among phenotypes . ConsideMedical comorbidities occur earlier and more frequently in older persons living with HIV infection; some of its consequences include neurocognitive and functional impairment, frailty, organ system failure, and increased hospitalizations . The higMost of the empirical overlapping relationships between comorbid and cognitive aging are associative, with few studies demonstrating temporal relationships Table . These cComorbid aging or cognitive aging also overlap with other phenotypes , but they are distinctive in several respects. Correlated associative phenotypic findings are widespread, and few studies have explored their temporal likelihood of developing or how they arise and evolve Table . The onlAn issue of concern for HIV phenotype research is that these aging phenotypes have been identified, by and large, in non-HIV-infected populations, so that they must now be reassessed and evaluated in vulnerable HIV-infected populations . This limits generalizability to other aging HIV-infected populations. To date, most studies examining comorbid and/or cognitive aging in the HIV-infected population have been cross-sectional in nature. The temporal relationship between aging-related phenotypes is not clearly understood, and longitudinal data are needed to better describe the trajectory of cognitive decline and how specific comorbidities contribute to other aging-related phenotypes. Finally, it is unclear from the current results how long-term cART may affect gait speed decline. This is an important question, as nearly all of the aging/HIV-infected individuals studied were receiving cART, and the majority were virologically suppressed—characteristics that are likely to be similar in most populations aging with HIV infection.Typically, HIV behavioral interventions for older adults have employed chronological age to identify this sub-population. To date, little attention has been paid to interventions targeting HIV-related aging phenotypes .Until recently, the older HIV-infected population with comorbid conditions has been largely ignored in intervention research because patients with significant comorbid disease have frequently been excluded. Clinical practice guidelines are more often single-disease focused, usually based on results from clinical trials without significant comorbid disease . ConsequBehavioral interventions that target comorbidities in the context of HIV have been limited to only one research group, employing tele-therapy or coping improvement and interpersonal support randomized controlled trial to relieve HIV-associated depressive symptomatology –90. ThisHAND confers an increased risk for early mortality, independent of medical predictors . As peopBecause cognitive functions begin to decline in middle age or even earlier , it willHIV behavioral interventions for cognitive aging have been limited and mostly focused on cognitive rehabilitation of the speed of information processing , 105. ThWe reviewed evidence for the main premise that chronological age alone is insufficient to represent aging effects in HIV research across the spectrum of aging. The phenotype concept was featured as a way to differentiate among relevant subsets of older adults with HIV infection.The comorbid and cognitive aging phenotypes are distinct, yet overlapping. A distinct profile has been confirmed by cluster analyses and other statistical differentiation methods. However, the commonalities and interactions are many. This is similar to the conceptual debate about frailty as a single or multiple phenotype with different clusters of risk factors and etiologies [iologies . The numiologies .Associative relationships are the rule for HIV relationships between comorbid and cognitive aging and for comorbid or cognitive aging with other aging phenotypes. While cognitive aging is frequently associated with comorbid aging in HIV infection, an aging-specific cognitive phenotype that is distinguishable from a variety of comorbid conditions has been reported in non-HIV/AIDS geriatric volunteers [lunteers . TherefoHIV behavioral interventions for both comorbid aging and cognitive aging have been limited. Group tele-therapy focused on coping skills enhancement has been beneficial for comorbid aging and remediation training for cognitive aging. For these phenotypes, research is needed on other intervention modalities and should be expanded by different research groups. Broad replication is required for these studies to be considered representative and applicable to other settings. The mechanism by which these phenotypic-based effects may be exerted on older HIV-infected populations is also uncertain, although inflammatory mechanisms through a variety of processes are probably involved for comorbid and cognitive aging and cognitive reserve processes for cognitive aging. An important gap in phenotype-targeted interventions is research along each step of the HIV care continuum  to optimize the likelihood of viral suppression in older adults living with HIV/AIDS.clinical and behavioral specification, biological mechanisms, and intervention targets.Taken together, the reviewed findings on HIV/aging phenotypes suggest at least three paths for research progress: clinical and behavioral specification, has the goal of obtaining more precise definitions and improving specificity within the aging population to spawn advances in diagnosis, pathophysiology, and prevention/treatment, as well as to improve our understanding of the aging process. Evidence for using distinct phenotypic clusters for improved clinical and diagnostic assessment is suggested by diversity not only between phenotypes but also within a given phenotype . This should provide greater precision in understanding each patient’s clinical features  and for selecting the most salubrious treatment for each individual patient. Thus, the pursuit of the long-term goal of increased homogeneity and specificity in HIV infection with aging is analogous to the Research Domain Criteria initiative to improve neurobiological validity, clinical prediction, and treatment matching [The approach, matching . A corolbiological mechanisms) is potential connections between the clinical realm of declines/dysfunction and the hypothesized biological mechanisms that may underlie these clinical changes. The role of chronic inflammation in HIV infection, aging, and AIDS comorbidities is one of the most important biological areas of research. If it could be further demonstrated that specific aspects of chronic inflammation or other biological factors are predictors of AIDS comorbidities in the elderly, then this would open additional avenues of prevention research to target these predictors (including host genetic predictors) before HIV/AIDS comorbidities emerge. The biological approach may yield genetically more homogeneous endophenotypes based on using different parent phenotypes to define and stratify older HIV-infected adults into clinical sub-groups. Evidence for this approach might be obtained by more narrowly defining the phenotype to identify more genetically similar sub-groups which may in turn increase power to detect biomarkers and genetic factors influencing risk.Given the overlap of clinical domains within given phenotypes , a key research area (i.e., targeted integrated approach simultaneously targets the multiple phenotypes that are associated with one another through a multi-level integrated approach. This is not only more practical and economical than targeting one phenotype at a time but also is consistent with contemporary high-impact prevention such as combination prevention strategies. Both approaches involve similar methodological challenges—delivering a coordinated package of interventions that matches the epidemiologic profile of a target population; delivering that package at the population level; and evaluating safety, acceptability, coverage, and effectiveness [The tiveness . Within With the above discussion of the three paths for research progress, we suggest some questions within each research path Table .In conclusion, we re-emphasize that the multi-determined nature of the aging process provides a framework for physiological organization by which aging is mediated by multi-system dysfunction and the associated manifestation of heterogeneous, yet inter-related and interacting, behavioral phenotypes. The notion of interacting behavioral phenotypes has been similarly proposed for other aspects of the aging process, such as the integration of aging-related biomarkers into regulatory networks  and of iThe following conclusions emerge from this review.


Vascular calcification (VC) as a manifestation of perturbed mineral balance, is associated with aging, diabetes and kidney dysfunction, as well as poorer patient outcomes. Due to the current limited understanding of the pathophysiology of vascular calcification, the development of effective preventative and therapeutic strategies remains a significant clinical challenge. Recent evidence suggests that traditional risk factors for cardiovascular disease, such as left ventricular hypertrophy and dyslipidaemia, fail to account for clinical observations of vascular calcification. Therefore, more complex underlying processes involving physiochemical changes to mineral balance, vascular remodelling and perturbed hormonal responses such as parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) are likely to contribute to VC. In particular, VC resulting from modifications to calcium, phosphate and vitamin D homeostasis has been recently elucidated. Notably, deregulation of vitamin D metabolism, dietary calcium intake and renal mineral handling are associated with imbalances in systemic calcium and phosphate levels and endothelial cell dysfunction, which can modulate both bone and soft tissue calcification. This review addresses the current understanding of VC pathophysiology, with a focus on the pathogenic role of vitamin D that has provided new insights into the mechanisms of VC. Vascular calcification (VC) is a complex disorder of both major and minor blood vessels that is primarily characterised by calcium deposition along vascular walls. These deposits predominantly consist of calcium and phosphate minerals in the form of hydroxyapatite crystals, which incidentally are the same mineral components found in bone ,2. WhileThe theories behind VC have continued to evolve since the first characterisation of calcified vessels. Historically, based on the high calcium and phosphate content derived from these vessels, VC was described as a passive and degenerative process of calcium and phosphate accumulation . FollowiAlthough the underlying mechanism(s) are complex and still remain to be fully elucidated, the scientific literature has alluded to ossification as one of the principle active processes which orchestrates VC. Indeed, this idea is supported by the prevalence of regulators of bone formation and bone structural proteins such as bone morphogenic protein (BMP) and osteopontin (OPN) within calcified vascular tissue ,29,30. FAs VC frequently occurs with atherosclerotic and arteriosclerotic diseases, initial thoughts on its risk factors were centred on cardiovascular outcomes. Indeed, early studies of VC focused on its development in association with traditional metabolic risk factors such as age, smoking, alcohol consumption and measures of adiposity . ClinicaRecent evidence indicates that many non-atherosclerotic calcification cases may be a result of modulations in the levels and activities of calcification promoters and inhibitors. Indeed, VSMCs readily secrete such mediators of calcium regulation into their extracellular environment during vascular remodelling, and it has been hypothesised that calcification ensues following net changes in promoter and inhibitor levels respectively. Alkaline phosphatase for example is a major matrix vesicle constituent secreted by de-differentiated VSMCs, that promotes calcification through the degradation of pyrophosphate . ClinicaThere is also growing evidence indicating that within a large number of VC cases, physiochemical changes to mineral metabolism actively contribute, rather than being a passive outcome of its pathophysiology. Of relevance, perturbed levels of calcium and phosphate have been shown to mediate VC pathogenesis and development . TraditiIn vitro manipulation of calcium and phosphate levels in culture media of VSMCs to hypercalcaemic and hyperphosphatemic conditions respectively leads to the loss of smooth muscle cell markers and upregulation of genes commonly associated with bone formation ,55. NotaConcomitant with VSMC phenotypic switching, increased calcium and phosphate loading also promotes mineralisation through stimulating the secretion of matrix vesicles and apoptotic bodies, which become the primary nucleation sites for calcification through the de novo synthesis of hydroxyapatite crystals ,60,61,62FGF-23 in particular has recently been identified as a risk factor for VC, with several studies demonstrating correlations between elevated levels of circulating FGF-23 and increased calcification scores in CKD patients ,68,69. GIn summary, there is strong evidence suggesting pro-calcifying effects from both calcium and phosphate which are primarily based on their ability to actively stimulate phenotypic transformation of cells within the vasculature, including both vascular smooth muscle calcification and osteogenic matrix mineralisation. Passive accumulation on the other hand may still be a relevant mechanism that either sustains or aggravates calcification by exacerbating the mineralisation process. While most findings emphasise that calcium synergises with phosphate during the onset of calcification, whether or not these risk factors are able to independently promote the process still requires further clarification. Vitamin D is one of the most important steroid hormones in the body. Its predominant function is the regulation of skeletal health and mineral homeostasis through modulating calcium and phosphate metabolism at major physiological organs, including the intestines, liver, bone and kidneys. As such, its deficiency is often associated with bone-mineral disorders such as rickets and osteomalacia. Vitamin D has also been reported to serve non-skeletal functions, the majority of which are highly implicated in cardiovascular outcomes.While it can be supplemented, vitamin D is normally derived either exogenously through dietary sources, such as fatty fish and eggs, or endogenously through synthesis in the skin. The primary source however, is through skin synthesis from exposure to ultraviolet radiation. The inactive precursor, known as cholecalciferol (vitamin D3) that is initially produced promptly undergoes 25-hydroxylation by one of four cytochrome P450 enzymes  in the liver to produce calcifediol (25-hydroxyvitamin D), a pre-hormone generally used as the biomarker to indicate vitamin D status. Calcifediol, which is normally bound to the circulating vitamin D binding protein (DBP), can undergo a subsequent round of hydroxylation by another cytochrome P450 enzyme, 1α-hydroxylase (CYP27B) in the kidneys to produce the active vitamin D metabolite, calcitriol , which ultimately elicits the effects of vitamin D by binding and signalling through the vitamin D receptor (VDR) in target tissues.Perturbation of vitamin D levels can occur, with the primary manifestation being vitamin D deficiency which is predominantly caused by lack of sunlight exposure and therefore synthesis in the skin. Indeed, this point is reinforced by the increased prevalence of deficiency cases during winter and in populations further away from the equator ,74. LackDespite the general consensus on diagnosing vitamin D-related diseases/disorders based on circulating levels, there is still controversy surrounding whether aberrant levels of vitamin D are equivalent to perturbed activity, given the nature of its metabolism and signalling. For example, production of active vitamin D  in the kidneys is tightly regulated through feedback mechanisms on 1α-hydroxylase activity, such as PTH and FGF-23, and therefore cannot be entirely dependent on pre-hormone levels . FurtherThe impact of vitamin D on VC has yet to be fully elucidated and remains controversial to this day. While a large number of studies suggest that vitamin D excess  is associated with extensive calcification, others report that deficiency also promotes calcification, with long-term supplementation providing protective effects. Current evidence from experimental studies suggests a biphasic response of vitamin D activity, with either excess or deficient levels of vitamin D potentially leading to deleterious calcification outcomes ,83.Induction of calcification through hypervitaminosis with vitamin D has been demonstrated and well characterised in multiple animal models, including mice, rats, goats and pigs see . TreatmeAnimal studies consistently observe that vitamin D induced calcification is concomitant with increased serum calcium, phosphate and calcium-phosphate product, which emphasises the idea that physiologically, vitamin D promotes calcification through stimulating free calcium and phosphate levels, which as previously discussed, lead to vascular osteogenesis and mineralisation. On the other hand, calcitriol has been shown to directly induce mineralisation of bovine VSMCs via the VDR in vitro in a dose-dependent manner . Such VD−/−) mice respectively [Several studies have also challenged the view of vitamin D inducing and supporting calcification, with evidence that vitamin D deficiency, or hypovitaminosis D, is actually a stimulus to deleterious calcification outcomes. In uremic rat models, medial calcification in the aorta and ectopic calcification within soft tissue have been detected despite low levels of serum calcium and calcitriol, and instead have been associated with extremely high levels of PTH and inorganic phosphate . Inadequectively ,97. Unfoectively ,103. Infectively ,104. Phyectively ,97. In aectively . The autectively . Indeed,ectively . InteresIn addition to evidence suggesting that inflammation and endothelial dysfunction may be the primary mechanisms by which vitamin D deficiency promotes calcification, reports that vitamin D sterols can downregulate the RUNX2 pathway suggest its potential to directly inhibit the osteogenic differentiation process of VSMCs . FinallyWhile there is considerable evidence supporting that both vitamin D excess and deficiency promote calcification, the important question still remains as to how vitamin D is able to elicit such a biphasic response to ultimately induce calcification outcomes . Indeed Ellam et al. provided critical insights into this concept by demonstrating the biphasic properties of vitamin D on VC development using an apolipoprotein E knockout mouse model . While mFinally, the vitamin D receptor (VDR), which is the actual molecular mediator for active vitamin D signalling, has also been reported to elicit profound effects on VC. Both stimulation and ablation of VDR signalling promotes different types of calcification, such as arterial atherosclerotic calcification and medial calcification, indicating that VDR signalling itself impacts on VC by regulating gene expression programs within target tissues ,100,101.The association between vitamin D and cardiovascular mortality is well established. Deficient vitamin D levels, in the form of calcifediol, has been linked with increased cardiovascular and non-cardiovascular mortality in a number of large-scale clinical and epidemiological studies . The preOn the other hand, while the focus of the impact of vitamin D on calcification has been primarily on vitamin D deficiency, limited as they may be, studies which have independently examined the relationship between hypervitaminosis D and calcification in patients are also scarce. This may be largely due to the fact that hypervitaminosis D is rarely seen in human populations, and also that the induction of vitamin D toxicity in patients is very impractical, given that homeostasis of vitamin D is tightly regulated under physiological conditions, and that over-supplementation may be ethically impractical . FurtherInterestingly, one clinical study performed on a paediatric cohort highlighted the biphasic response of vitamin D with regards to calcification, in which patients with low or high levels of vitamin D exhibited significantly greater carotid intima-media thickness and calcification scores than those with normal levels . FurtherVC is a degenerative vascular disease which typically affects major branches of the arterial tree, and independently contributes to deranged cardiovascular outcomes and increased mortality. The majority of calcified tissues are characterised by not only inert mineral deposits, but also dynamic bone elements indicating that it is an active pathogenic process which mimics some features of ossification. While traditional atherogenic stimuli have been considered to contribute to VC pathogenesis, recent findings indicate more important roles for non-traditional risk factors, such as hormonal perturbations and disturbed mineral metabolism in accounting for the aberrant VC clinical outcomes related to severe cardiovascular events. In particular, deregulated calcium and vitamin D levels facilitate calcification through stimulating both VSMC osteogenic differentiation and mineralisation. Current understanding of vitamin D’s involvement during VC is limited, and primarily relies on evidence provided from experimental studies. Both hypervitaminosis and hypovitaminosis D can contribute to the development of VC via multiple complex mechanisms, indicating the biphasic impact of vitamin D on the vasculature. Although the specific molecular mechanism remains to be fully clarified, understanding how modulations in systemic vitamin D levels changes its metabolic and signalling activity during VC development may offer more insights into explaining the biphasic response. On the other hand, clinical evidence of the biphasic impact of vitamin D on VC in patients is limited, with current guidelines suggesting that tightly regulated levels of vitamin D are ultimately necessary to prevent VC. Further studies are required to elucidate the underlying mechanisms of how the vitamin D hormone, its metabolism and signalling via the VDR interacts with calcification processes and risk factors to clarify therapeutic or detrimental impact in disease pathogenesis and development.


Transplanting bioengineered human lung organoids into mice could lead to a humanized model for pre-clinical studies of lung disease. Related research article Dye BR, Dedhia PH, Miller AJ, Nagy MS, White ES, Shea LD, Spence JR. 2016. A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids. eLife5:e19732. doi: 10.7554/eLife.19732in vitro by taking airway cells from patients and cadavers and growing them in culture. These epithelia have been used for a range of applications, from fundamental cell biology and cancer genetics to clinical diagnosis and pharmacological testing  and, finally, various types of airway epithelial cells. One recent innovation involved the growth of three-dimensional structures called human lung organoids by a group at the University of Michigan  and anot in mice .Figure 1in vivo environment, typically in the kidney capsule or in fatty tissue adjacent to the stomach or epididymis, helps them to mature properly, presumably due to the physiological levels of nutrients, oxygen and circulating factors. Dye et al. started by taking human lung organoids that had been grown in a basement membrane substrate (Matrigel) and implanting them directly into the mice. Although human epithelia were recovered from these initial transplants, they rarely contained lung cells.It has become a standard technique to implant immature organoids (or cells derived from pluripotent stem cells) into well-vascularized sites in mice whose immune system has been compromised. The growth of the cells in this in vivo, the organoids had frequently grown into tubes that resembled the adult airway and, moreover, they contained a wider range of mature cell types than had been produced before , which is used in medical devices and for culturing pancreatic cells . Given td before . Importain vivo system developed by Dye, Spence and co-workers appears to be reproducible across multiple pluripotent cell lines and could be used to develop improved models of adult lung diseases and for pre-clinical drug testing. And if we can learn more about the mechanisms that underlie the maturation process, it might be possible to produce mature epithelia in vitro for high-throughput (drug discovery-type) experiments. It will also be important to assess whether mature alveoli (the lung cells that are involved in the exchange of oxygen and carbon dioxide) can be generated from pluripotent stem cells through similar in vivo transplantation approaches. Further study is also needed to assess how different types of scaffolds affect the maturation of cells in vivo.The


Plant tolerance to insect pests has been indicated to be a unique category of resistance, however, very little information is available on the mechanism of tolerance against insect pests. Tolerance is distinctive in terms of the plant’s ability to withstand or recover from herbivore injury through growth and compensatory physiological processes. Because plant tolerance involves plant compensatory characteristics, the plant is able to harbor large numbers of herbivores without interfering with the insect pest’s physiology or behavior. Some studies have observed that tolerant plants can compensate photosynthetically by avoiding feedback inhibition and impaired electron flow through photosystem II that occurs as a result of insect feeding. Similarly, the up-regulation of peroxidases and other oxidative enzymes during insect feeding, in conjunction with elevated levels of phytohormones can play an important role in providing plant tolerance to insect pests. Hemipteran insects comprise some of the most economically important plant pests , due to their ability to achieve high population growth and their potential to transmit plant viruses. In this review, results from studies on plant tolerance to hemipterans are summarized, and potential models to understand tolerance are presented. Plants are constantly challenged by a diverse array of insect attackers, which can impose significant costs to plant fitness. Accordingly, plants employ multiple strategies to defend against, tolerate or avoid insect herbivory. Plant resistance can be categorized into three categories: antibiosis, antixenosis or non-preference, and tolerance. Antibiotic plant traits negatively impact a pest’s biology through increases in mortality, reduced growth, longevity, and fecundity . AntixenWhen employed in integrated pest management systems, tolerance can potentially reduce yield loss caused by insect feeding and colonization . InsectsTable 1): (1) increased photosynthetic activity (Table 1), it is clear that underlying mechanisms that contribute to plant tolerance are largely unknown.Although tolerance to insect herbivores has received increased attention, detailed characterizations of the underlying mechanisms have remained elusive. Broadly five primary physiological mechanisms have been described by which plants may tolerate herbivory: (1) increased net photosynthetic rate after injury, (2) high relative growth rates, (3) increased branching or tillering after release of apical dominance, (4) pre-existing high levels of carbon storage in roots, and (5) ability to reallocate carbon after injury from roots to shoots . To dateactivity  and (2) activity . As evida and b and carotenoid concentrations on susceptible wheat lines in response to Diuraphis noxia (Russian wheat aphid) feeding, suggesting that D. noxia feeding possibly damages the light harvesting complex II, where chlorophylls a and b and carotenoids are important chromophores. Conversely, chlorophyll concentrations were similar between infested plants and their uninfested counterparts in the aphid-resistant isolines, suggesting that aphid feeding may have less effect on chlorophyll loss in D. noxia resistant wheat lines  cultivars to the western chinch bug (Blissus occiduus). Notably, after prolonged exposure to chinch bugs, the susceptible buffalograss displayed reductions in photochemical quantum yield and photosynthetic electron transport rate; however, those differences were not observed in the tolerant cultivar  wheat lines, exhibited reduced chlorophyll fluorescence yield and were unable to recover, suggesting that D. noxia injury resulted in a disruption of the electron transport system reducing light absorption for photosynthesis in the susceptible but not the tolerant wheat line  that integrates both the short-term (arbitrarily <5 days) and longer term (>10 days) responses that could underlie the tolerant response. Plants have both constitutive and inducible defenses . Most frequently, there is considerable overlap between the short-term and 5 to 10 day responses, but they have been separated  to indicate that many physiological changes are noticed 5–10 days post infestation. Continued ROS mitigation appears to be a hallmark in tolerant plants, suggesting that mechanisms that permit modulation of cellular redox could be potential pathways for understanding the tolerance response. ROS mitigation appears to be linked to resumption of growth, providing another window to look for genes that both transduce and activate these pathways. It is likely that these changes do not become evident until much later (>10 days) during a plant-hemipteran interaction. Unfortunately, longer-term studies are often confounded by physiological changes that occur as plant mature that can make data interpretation more difficult. Nevertheless, detailed investigations using a range of omics strategies in well-defined tolerance systems are likely to provide significant insights about the traits controlling the tolerance response.Based on these findings, a model is suggested  in maize seedlings infested with Rhopalosiphum padi (Bird cherry-oat aphid). Accordingly, aphid infestation also resulted in a significant increase in transcript abundances of genes encoding GSTs in the resistant maize plants, relative to the susceptible variety, suggesting a potential role of GST in limiting the adverse effects of oxidative stress within the resistant maize , CAT , and GRAS  transcripts , radish (Raphanus sativus) and Arabidopsis seedlings infested with the green peach aphid (Myzus persicae), there was a differential regulation of nitrogen metabolism in aphid-infested plants relative to uninfested plants. Infestation led to greater enrichment of 15N in the infested plants, primarily as a result of changes in host N-metabolism. These changes were attributed to increased nitrate reductase activities along with changes in nitrate flux, resulting in greater incorporation of 15N. When coupled to selective removal of 14N by aphids, the net result was increasing levels of 15N in infested plants . How these different processes influence each other at a biochemical level is largely unexplored, and deep transcriptional sequencing of multiple tissues in aphid-tolerant plants is absent. Recovery of leaf, meristem and other plant growth functions are the hallmark of a “tolerant phenotype” in the face of manageable pest populations. Recovery of growth requires overcoming the cellular reprogramming caused by hemipteran pests discussed earlier. However, it is not entirely clear if there is successive or simultaneous rebalancing of chloroplast functions, ROS mitigation and increased delivery of nutrients to sinks to promote growth. It is plausible that integration of these processes might be involved in attenuation of the defense response, maintaining higher levels of ROS mitigating systems, compensation of photosynthates lost due to insect herbivory and renewed growth of the meristems . These features appear to be consistent with much of the literature discussed above.Chloroplasts are critical gatekeepers of leaf health, and altering chloroplast physiology has a significant effect on transcription through retrograde signaling and via shifts in the levels of metabolites such as starch, sugars , and JA among others . Thus, nInterestingly, a recent study on comparing the molecular mechanisms of plant responses to phloem-feeding insects indicate unique and different signaling networks being activated following attack by the generalist and specialist aphids . PredictTyria jacobaeae) is due to the induced production of new capitula on regrowth shoots in ragwort (Altica subplicata) demonstrated genetic variation in tolerance to herbivory in Salix cordata  on Nicotiana attenuata (tobacco), carbon and sugar were allocated to the roots, a less-vulnerable location (M. sexta regurgitant on defoliated tomato accelerated leaf regrowth via responses similar to resource appropriation (Diabrotica virgifera virgifera). Upon below-ground herbivory, maize plants allocated more carbon to above-ground foliage, thickening stem tissues and increasing crown-root growth as a means of compensation (Asclepias) symbiosis with arbuscular mycorrhizal fungi (AMF) enhances tolerance to herbivory though changes in nutrient status, allocation patters, and growth rate  to measure chlorophyll content in leaves, ELISAs to measure virus transmission, and plant metabolite assays . Most baBecause of the tedious nature of these methods, newer techniques of phenotype screening have allowed plants to be measured for specific defensive/tolerant traits. HTP systems quantify a number of traits within plant populations through automated image collection and analysis, effectively streamlining the search process, and contributing further to plant phenomics.Arabidopsis genotypes for resistance to the green peach aphid, M. persicae. As a means to measure tolerance, visual cameras can measure plant growth, architecture, and, chlorosis, and necrosis, all of which can be negatively affected by insect infestation. Fluorescence cameras can also be used to measure chlorophyll fluorescence, which can be indicative of the plant’s photosynthetic activity , rather than a resistance-mechanism  species . It is k species  and a so species . These d species . AnotherFigure 3). In the case of induced-tolerance, infestation elicits a strong response across a spectrum of plant cells and pathways . This strong initial response subsequently reprograms metabolism to counter the negative impact of herbivory, such as, by recalibrating cellular redox, photosynthesis and nutrient acquisition.Based on research reported so far, it is possible to envisage at least two different routes to tolerance, one where tolerance is induced, and the other where tolerance is constitutive , increased transcript/protein and antioxidant abundances for ROS mitigation, and possibly greater photosynthetic capacity . Under insect pressure, there is a more modulated response, potentially resulting in a shorter duration in suppressed growth .In a plant with a constitutive tolerant response, several biochemical/physiological aspects of tolerance can be expected to be present, for example: structural fortification . Identifying underlying mechanisms of tolerance will provide meaningful insight into our understanding of plant-insect interactions and have utility for breeding plants with more durable pest resistance.Graphically, plant responses can be envisaged as shown in Continued focus on the contributions of specific mechanisms underlying plant tolerance to hemipterans will be critical for the development of tolerant germplasm. Additionally, the role of phytohormones in the expression of tolerance to hemipterans presents an appealing avenue of future research. Phytohormones are not only instrumental in regulating plant development, but they are also significantly involved in mediating plant responses to abiotic and biotic stresses. JA and SA in particular have been implicated in defense against pathogens and herbivores alike, however, the life-styles of the stressors determines which pathways are activated. Piercing-sucking insects such as hemipterans are homologous to biotrophic pathogens in the sense that they feed on the plant’s nutrients without killing host cells. It is generally assumed that SA is a crucial signaling molecule required for the plant defense response against biotrophic pathogens and sucking insect pests: in contrast, JA is associated more in the defense against necrotrophic pathogens and chewing insects .D. noxia, and this heightened constitutive expression may help to attenuate stress associated with D. noxia feeding immediately after attack, through adjustments in stomatal opening and root growth. Correspondingly, upregulation of transcripts related to abscisic acid and ET signaling pathways have also been reported in D. noxia-resistant wheat plants suggesting their importance in D. noxia tolerance (However, to date, few studies have investigated the role of phytohormones in plant tolerance to insects. olerance . HoweverPlant tolerance to insect herbivory is a compelling category of resistance, consistent with integrated pest management strategies . BecauseAll authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication. GS developed the figures.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.


Since epidemiological studies first demonstrated a potential positive effect of metformin in reducing cancer incidence and mortality, there has been an increased interest in not only better understanding metformin’s mechanisms of action but also in exploring its potential anti-cancer effects. In this review, we aim to summarise the current evidence exploring a role for metformin in prostate cancer therapy.Preclinical studies have demonstrated a number of antineoplastic biological effects via a range of molecular mechanisms. Data from retrospective epidemiological studies in prostate cancer has been mixed; however, there are several clinical trials currently underway evaluating metformin’s role as an anti-cancer agent. Early studies have shown benefits of metformin to inhibit cancer cell proliferation and improve metabolic syndrome in prostate cancer patients receiving androgen deprivation therapy (ADT).While the body of evidence to support a role for metformin in prostate cancer therapy is rapidly growing, there is still insufficient data from randomised trials, which are currently still ongoing. However, evidence so far suggests metformin could be a useful adjuvant agent, particularly in patients on ADT. Prostate cancer (PCa) remains the most commonly diagnosed male cancer in the Western world and is the second leading cause of cancer-related deaths in men . Over thGalega officinalis (French lilac), a plant reputedly used to treat diabetes-like conditions in medieval Europe [directly by acting on the tumour and indirectly by lowering systemic insulin levels  effect is via inhibition of the mitochondrial electron transport chain (ETC) and consequent activation of the enzyme adenosine mono-phosphate-activated protein kinase (AMPK) [Metformin was originally derived from l Europe . Despitel Europe . It is tels Fig. . The dire (AMPK) . AMPK ise (AMPK) , 14. Acte (AMPK) . mTOR ise (AMPK) . Inhibite (AMPK) –20. Addie (AMPK) . Fatty ae (AMPK)  which maThe indirect (insulin-dependent) actions of metformin occur via inhibition of hepatic gluconeogenesis. Activation of AMPK in the liver leads to inhibition of the transcription of key gluconeogenesis genes, and stimulates glucose uptake in muscle, thus reducing fasting blood glucose and insulin levels , 23. InsWhich mechanism predominates in prostate cancer is unknown but it is likely that both pathways provide anti-cancer benefits. Also, it still remains unclear whether AMPK activation is essential for metformin activity, as its ability to inhibit mTORC1 has also been demonstrated via AMPK-independent pathways . FurtherMetformin’s inhibitory effects on various pro-oncogenic pathways prompted epidemiologist to retrospectively analyse the effect of metformin treatment on type 2 diabetic patients with cancer. Evans and colleagues were the first to show that taking metformin may be associated with reduced risk of cancer in patients with type 2 diabetes, and that the longer the period of exposure the greater the benefit •. A moreAlongside its potential anti-cancer role, clinical trials have also examined a role for metformin in preventing androgen deprivation therapy (ADT)-induced metabolic syndrome in prostate cancer. Whilst ADT is the mainstay of treatment for men with advanced prostate cancer, improving patient’s survival, it can also cause significant morbidity and a decline in quality of life (QOL). Body composition changes, insulin resistance, sexual dysfunction, fatigue, reduced bone mineral density (BMD), hyperlipidaemia, metabolic syndrome, and acute coronary syndrome are all reported adverse effects of ADT as a consequence of reduced levels of circulating testosterone and raised insulin levels , 40. Menn = 22), not placebo controlled, and that the tissue examined was obtained by different methods (biopsy vs prostatectomy); however, it provides strong rationale for further studies to not only examine disease and survival end-points but to also incorporate tissue and serum markers to better elucidate metformin’s actions.Metformin has been shown to inhibit the proliferation of a range cancers, including breast, endometrial, ovarian, colon, and prostate , 46–50. Interestingly, metformin’s biological effects are not limited to inhibiting cancer proliferation, but include effects on epithelial to mesenchymal transition (EMT), bone turnover, the androgen receptor, and cancer stem cells. EMT is an important step in prostate cancer invasion and metastases, conferring upon cancer cells the ability to invade basement membranes and metastasize to distant sites. EMT can be characterised by expression of certain markers by the cell. Epithelial markers include E-cadherin, cytokeratin, and desmoplakin, while mesenchymal markers include vimentin, slug, snail, and twist. Expressions of twist and vimentin have been shown to be predictive of biochemical recurrence in prostate cancer patients after radical prostatectomy for localised disease . In melaProstate cancer most commonly metastasises to the bone leading to skeletal-related events. In combination with this, ADT leads to loss of bone mineral density and increased risk of fracture. Metformin has been shown to enhance osteoblast and inhibit osteoclast differentiation in vitro , 61, andThe androgen receptor (AR) plays a vital role in prostate cancer, regulating a multitude of events including proliferation, apoptosis, migration, invasion, and differentiation. ADT remains the standard treatment for advanced prostate cancer; however, almost all patients treated with ADT will eventually relapse into castration-resistant prostate cancer (CRPC). Studies have demonstrated that persistent AR signalling remains the key driver in the progression to CRPC, and AR down-regulation is considered a preventive strategy for prostate cancer . MetformCancer stem cells (CSCs) are a subset of tumour cells that are resistant to many anti-cancer treatments, and are able to self-renew and regenerate the various cell types that make up a tumour . ProstatIliopoulos et al. found metformin in combination with the chemotherapeutic drug doxorubicin was more effective than either drug alone at blocking breast, prostate, and lung cancer growth and preventing relapse in mouse xenograft models. Interestingly, metformin had a comparable effect on tumour regression and preventing relapse when combined with a fourfold decreased dose of doxorubicin that was not effective as a monotherapy, suggesting a complementary role for metformin, potentially allowing lower doses of toxic drugs to be used, improving their tolerability and side effects . Indeed,Metformin is an attractive anti-cancer agent in view of its well-documented safety record, well-characterised pharmacodynamics profile, and low cost as a generic drug. Over the last 10 years, interest in this field has grown exponentially with a combination of epidemiological, clinical, and preclinical data supporting a potential anti-cancer role for metformin. However, many questions still remain. It is still unclear if metformin will be effective in a non-diabetic population, and if its effects are limited to particular patient populations . Alongside this, much of the preclinical work has used supra-physiological doses of metformin. There are currently a large number of studies worldwide being undertaken examining metformin in a range of cancers; however, many of these are using metformin at the conventional anti-diabetic doses. It awaits to be seen if these doses will be effective or if further phase I and phase II clinical trials will be required to assess metformin’s tolerability and efficacy as an anti-cancer agent at higher doses. Well-designed prospective controlled clinical trial outcomes will be important to provide more definitive answers regarding the efficacy of metformin in prevention and treatment of prostate cancer, and its role as a potential adjuvant therapy. However, data so far is very compelling for metformin’s role in the treatment of prostate cancer, particularly in view of its potential synergy with currently used treatments such as ADT and radiotherapy.


Mild traumatic brain injury (mTBI) is a common, although poorly-defined clinical entity. Despite its initially mild presentation, patients with mTBI can rapidly deteriorate, often due to significant expansion of intracranial hemorrhage. TBI-associated coagulopathy is the topic of significant clinical and basic science research. Unlike trauma-induced coagulopathy (TIC), TBI-associated coagulopathy does not generally follow widespread injury or global hypoperfusion, suggesting a distinct pathogenesis. Although the fundamental mechanisms of TBI-associated coagulopathy are far from clearly elucidated, several candidate molecules (tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), tissue factor (TF), and brain-derived microparticles (BDMP)) have been proposed which might explain how even minor brain injury can induce local and systemic coagulopathy. Here, we review the incidence, proposed mechanisms, and common clinical tests relevant to mTBI-associated coagulopathy and briefly summarize our own institutional experience in addition to identifying areas for further research. Coagulopathy occurs frequently after traumatic brain injury (TBI). Coagulopathy can affect outcome and contribute to sequela after the initial injury. Most study of coagulopathy has been in patients with severe TBI. However, mild TBI (mTBI) accounts for about 70%–90% of TBI patients, depending on one’s definition of mTBI . UnderstmTBI can be defined in a number of different manners. Classical categorization of mTBI has included patients with Glasgow Coma Scale (GCS) of 13–15. Others have broadly characterized mTBI as the sequelae of neurotrauma leading to mild, transient alterations of consciousness without structural abnormality on neuroimaging ,3,4,5. SThe characterization, underlying mechanism(s), and treatment of coagulopathy in trauma patients, or trauma-induced coagulopathy (TIC), remains an elusive target despite intensive research from both civilian and military centers ,21,22,23Coagulopathy related to TBI was first reported nearly 60 years ago  and has Isolated TBI generally does not involve systemic shock unlike that which is often seen in TIC. Therefore, it is unlikely that TBI-induced coagulopathy depends heavily on activated protein C (APC), a commonly-cited potential mechanism for TIC (particularly for early TIC) which is strongly correlated to hypoperfusion in trauma victims ,20,21,29The role of TF in post-TBI coagulopathy should not be entirely discounted, however. Microparticles (MP) are circulating phospholipid vesicles less than 1 μm in diameter which are derived from platelets, endothelial cells, as well as other hematopoietic cells  and, morClinical tests for coagulopathy most commonly include assessments of the coagulation cascade  and platelet function (thromboelastography). We briefly review these clinical laboratory tests and their limitations. Prothrombin time (PT), international normalized ratio  and partial thromboplastin time (PTT) are tests of the coagulation cascade and were initially designed to determine clotting factor deficiencies . While cThromboelastography (TEG), or Rotational thromboelastography (ROTEM), a nearly identical assay which has only minor differences in mechanics, utilizes the same principle of clot formation and renders similar data. It is sometimes studied in tandem with TEG . We collected TEG with platelet mapping (PM) data at the University of Missouri Hospital and Clinics as part of an ongoing quality improvement study evaluating TEG abnormalities in TBI patients . Adult pThus, even mild TBI can be associated with platelet dysfunction. Questions that remain to be answered include whether this level of platelet dysfunction is a risk factor for poor outcomes in TBI patients. If platelet dysfunction does result in poor outcomes, how should the patients be managed?3 /microliter. Mild TBI was defined as GCS 13–15, moderate TBI was GCS 8–12, and severe TBI was GCS 3–7. The limits for determining coagulopathy for PT and PTT were collected and determined by the University of Missouri Hospital laboratory [We also examined traditional tests for coagulopathy, including PT, PTT, and platelet count in 142 of the patients included in our TEG/TBI project at the University of Missouri Hospital and Clinics . Again, boratory . The datWe additionally examined the cohort of patients with isolated TBI who were not taking anti-platelet or anticoagulant medications . ControlAlthough the vast majority of patients with mild TBI have uneventful clinical courses and improve back to their baseline with time, a small subset goes on to develop a feared complication known as delayed neurological deterioration (DND), known colloquially as the “talk-and-die” phenomenon . The exap < 0.02). Twenty-three percent of patients with DND died, all within 12 h of admission, underscoring the importance of anticipation and early recognition in the acute management of these patients.A more recent single-center retrospective study by Choudhry et al.  examinedDespite its recognition even in ancient times, the management of traumatic brain injury is in many ways still in its infancy. Widespread disagreement and controversy exists over even minor classification criteria and treatment protocols. Over the last several decades, however, there have been paradigm-shifting advances in both the basic science and clinical practice of TBI management. There are several areas in particular where more research is needed to guide clinical practice.The fundamental molecular biology underlying TBI-induced is far from fully elucidated. The research into the roles of tPA and BDMPs which are briefly reviewed above offer potential therapeutic targets for prevention and treatment of TBI-induced coagulopathy in addition to deepening our understanding of the underlying mechanisms of platelet activation. The animal studies that are briefly reviewed in this article could form the basis of prospectively collected data from human TBI patients.Prophylactic anticoagulation using subcutaneous unfractionated heparin or low-molecular weight heparin has become routine in American hospitals for prevention of deep venous thrombosis (DVT) and pulmonary embolism (PE). Indeed, pulmonary embolism was found to be a small but significant contributor to late mortality in TBI patients in the San Diego study . OptimalWhat is the appropriate treatment of TBI-associated coagulopathy, and what should the treatment parameters be? The most recent Brain Trauma Foundation guidelines for management of severe TBI do not make any comment regarding therapeutic strategies for TBI-associated coagulopathy . Some reTBI-induced coagulopathy remains a significant and probably under-recognized contributor to early mortality in patients with head injury. The current body of research demonstrates that even mild TBI can cause clinically significant coagulopathy, particularly with regard to platelet dysfunction, while also highlighting the considerable gaps in our basic knowledge of its pathogenesis and molecular biology.


Cytokine storm is a poorly explained clinical entity caused by an undesired and aggrandized immune system response leading to unregulated activation of the proinflammatory cascade, often contributing to multisystem organ failure and even death. Its potentially diverse etiologies and sepsis-like presentation have made it even more challenging to diagnose, and so far, no well-established treatment protocol has been proposed. Its association with certain medications, especially with monoclonal antibodies, has well been reported in literature. To the best of our knowledge, so far, no previous case of cytokine storm associated with imatinib has been reported. We herein present a case report of a 77-year-old male with a past medical history of hypereosinophilic syndrome who developed acute fatal cytokine storm following treatment with imatinib. This study highlights a life-threatening complication of the medication that may be underrecognized. Imatinib is a tyrosine kinase inhibitor that is used in various hematologic malignancies, including idiopathic hypereosinophilic syndrome (IHES) which is a myeloproliferative disorder characterized by sustained, nonreactive, unexplained persistent hypereosinophilia that commonly results in multiorgan dysfunction. This case highlights the development of a cytokine storm with severe uncontrolled systemic inflammatory response with a fatal outcome following the initiation of imatinib in a patient with IHES.Mycobacterium avium complex (MAC) infection on treatment with rifampin, azithromycin, and levofloxacin was sent to the ER from oncology clinic for evaluation of progressive weakness, lethargy, and hyperkalemia.A 77-year-old male with a history of IHES, COPD, CKD stage III, and active The patient had outpatient workup for unexplained hypereosinophilia. He underwent lymph node biopsy which showed no evidence of lymphoma . PeripheKlebsiella pneumoniae and moderate Pseudomonas aeruginosa. A CT abdomen and pelvis showed prominent bowel loops with wall thickening, suggestive of bowel ischemia  classification, hypereosinophilic syndrome (HES) is classified into three distinct subsets caused by different pathogenic mechanisms including chronic eosinophilic leukemia caused by fusion protein FIP1L1-PDGFRA) with tyrosine kinase activities, lymphoproliferative HES caused by clonal IL-5/Th2 lymphocyte-mediated hypereosinophilia, and idiopathic or myeloproliferative HES where no evidence of clonal hypereosinophilia is identified [L1-PDGFRAThe goal of therapy in HES is to prevent eosinophil-induced progressive end-organ damage and irreversible complications of sustained hypereosinophilia. Available therapeutic options include corticosteroids, hydroxyurea, interferon-alpha, bone marrow transplant, and other cytotoxic and immunomodulatory agents , 3. Due Cytokine storm syndrome refers to massive release of cytokines from an undesired activation of the immune system by various factors that lead to uncontrolled systemic inflammatory response causing multiple organ dysfunction and often death. Cytokines are glycoproteins that are produced by leukocytes. They may have autocrine, paracrine, and endocrine actions, which explains both local and systemic response seen in cytokine storm. Under normal circumstances, cytokine causes monitored release of proinflammatory mediators, which not only helps the body mount response to any threat but also prevents any further injury to local tissue and helps in the process of healing. Contrarily, in cytokine storm, unregulated and robust activation of proinflammatory mediators aggrandized immune cellular lineages with enhanced activity, resulting in the unregulated cascade of acute inflammatory response. This all contributes to endothelial cell dysfunction that leads to increased capillary permeability/leak, which has been postulated as a mechanism for parenchymal injury and end-organ damage. As expected, organs susceptible to the worst injuries are the ones with stronger immune defense mechanisms, notably the lungs and gut .Cytokine storm has been attributed to certain medications and treatments. Of note, monoclonal antibodies have long been associated with cytokine storm. In 2006, the first human clinical trial of TGN1412 monoclonal antibody to treat rheumatologic disease was conducted, and it was proposed that all 6 volunteers participated had fatal outcome from TGN1412-induced cytokine storm . AlemtuzImatinib, a monoclonal antibody, which acts as a tyrosine kinase inhibitor is FDA approved for treatment of hematological malignancies. It is considered to be a safe and effective drug and is on the WHO's list of essential medicines. Imatinib has well tolerability with a low incidence of toxicities . Common Given the nonspecific presentation of cytokine storm, possibility of coexisting septicemia cannot be ruled; although the patient had left lower lobe infiltrates and positive respiratory cultures, it is worth mentioning that prior to imatinib administration, there was no clinical or radiological suspicion of the acute infectious process. Moreover, the patient had two blood cultures taken following the onset of distributive shock, and both did not show any bacterial growth. Though negative blood cultures cannot rule out septicemia as a cause of death, the timeline of events and the clinical, laboratory, and radiological findings that developed as a consequence of initiation of imatinib therapy pointed towards a massive cytokine storm .In this case, imatinib caused severe acute distributive shock and multiorgan failure secondary to massive histamine release and cytokine storm evident by the temporal relation with initiation of therapy and dramatic decline of eosinophilic count to zero. Such an observation has not been documented in the past. This study highlights a life-threatening complication of the medication that may be underrecognized. HES remains a therapeutic challenge. The choice of therapy for HES should be carefully guided based on the balance between therapeutic benefit and potential for side effects. More research on safety of use of imatinib on a wide scale of patients with HES is needed.