To develop recommendations for clinical trial reporting that address the unique efficacy, toxicity, and combination and sequencing aspects of immuno-oncology (IO) treatments.ASCO and the Society for Immunotherapy of Cancer (SITC) convened a working group that consisted of practicing medical oncologists, immunologists, clinical researchers, biostatisticians, and representatives from industry and government to develop Trial Reporting in Immuno-Oncology (TRIO) recommendations. These recommendations are based on expert consensus, given that existing data to support evidence-based recommendations are limited.The TRIO recommendations are intended to improve the reporting of IO clinical trials and thus provide more complete evidence on the relative benefits and risks of an IO therapeutic approach. Given the rapid expansion of the number of IO clinical trials and ongoing improvements to the evidence base supporting the use of IO treatments in clinical care, these recommendations will likely need regular revision as the IO field develops. Cancer immunotherapies are treatments in which the primary mechanism of action is the generation of an immune response against cancer. The US Food and Drug Administration has approved multiple cancer immunotherapies for treating different types of cancer. These cancer immunotherapies or immuno-oncology (IO) treatments include but are not limited to cytokines, interferons, and immune checkpoint blocking agents, such as anti-cytotoxic T-lymphocyte antigen 4 , anti-programmed cell death protein 1 , and anti-programmed death-ligand 1  agents and genetically modified T-cell therapies . Several novel IO treatments are also being explored, such as modified cytokines, cell-based products, oncolytic viruses, CD3 bispecific antibodies, and various vaccine platforms. Clinical trials have been essential to advancing the use of immunotherapies in cancer treatment, and bio-medical journals have been a key mechanism for disseminating this knowledge.To improve the quality of scientific publications, biomedical journals have adopted guidelines for reporting the design, conduct, analysis, and results of clinical trials , tumor marker studies , and other study designs –3. The pIn fall 2016, ASCO and the Society for Immunotherapy of Cancer (SITC) convened a working group to develop clinical trial reporting recommendations that address the unique efficacy, toxicity, and combination and sequencing aspects of IO treatments. The working group consisted of practicing medical oncologists, immunologists, clinical researchers, biostatisticians, and representatives of industry and government. It met via a series of conference calls and held an in-person meeting in February 2017.This ASCO-SITC statement presents 12 specific reporting recommendations—Trial Reporting in Immuno-Oncology (TRIO)— to improve the interpretation and comparison of efficacy and toxicity end points and the combination and sequencing of treatments in IO clinical trials  are used to evaluate antitumor activity of oncology drugs in most clinical trials, including most IO trials . These cMost IO trials display efficacy as a waterfall plot. However, waterfall plots are inherently limited because only the patients’ best overall response is reported. They do not capture the temporal relationship of response after treatment is administered  nor do they provide any information about the duration of response. Thus, in addition to waterfall plots, investigators should report the kinetics of change in tumor burden using spider plots or swimmer plots to display more meaningful information about efficacy over time.Spider plots Fig.  typicallInvestigators often collect and report nonstandardized end points in clinical trials using terms such as “disease control” and “clinical benefit.” These terms may include patients with objective response  and patients with stable disease, usually for a prespecified minimum number of months . When such composite end points are reported, they should be clearly defined, and the frequency and method of assessment of their components should be clearly described. For time-to-event end points, investigators should provide a clear definition of baseline (time 0) as well as what occurrences are considered censored and what are considered events.Although the importance of clearly defining time-to-event end points is not unique to IO trials, it is especially relevant in the current IO trial landscape because of the vast number of nonrandomized IO trials investigating new agents alone and in various combinations. The controversial issue surrounds patients who are determined to have stable disease by standard imaging criteria. For example, a patient with stable disease for 6 months on an experimental IO therapy may have met a trial’s clinical benefit or disease control designation, but if that patient had a cancer with an indolent underlying disease biology, there may have been no significant drug benefit from the intervention. Nonrandomized studies are inherently limited in demonstrating true clinical benefit or disease control of an experimental approach because of the absence of a comparator arm, which is necessary to interpret time-to-event end points.Historically, cancer clinical trials have required cessation of experimental therapy upon determination of disease progression. IO clinical trials, however, often allow patients to continue therapy beyond objective determination of progressive disease because of the recognition of a phenomenon known as pseudo-progression. Pseudo-progression is an event that can occur with IO therapies that denotes the appearance of new lesions  or an initial increase in index lesion(s) with subsequent index lesion response by clinical or radiographic assessment , 12, 16.Common criteria for treating beyond progression are that patients have stable or improved clinical condition, no severe laboratory abnormalities or adverse events associated with IO therapy, and lack of clinically significant additional progression on confirmatory subsequent imaging. However, there are no universally accepted criteria for defining what may happen in an IO clinical trial after disease progression. Thus, the specific criteria used in a trial to permit treatment beyond progression should be reported. This information should include the timing of the required confirmatory imaging. Investigators should also report whether alternative treatments were discussed with the patients and whether patients signed a consent form at the time of initial documentation of disease progression.It is critical to distinguish patients with pseudo-progression from true progression to avoid exposing the latter group to ineffective treatment that may be associated with adverse events . It is aInvestigators should report progression free survival and overall survival using Kaplan-Meier analyses in IO trials. The landmark methodology may be used for the correlation of progression-free survival or overall survival with response status . By usinInvestigators should also report whether patients were censored atthe time they started a new therapy. The date of earliest evidence of disease progression should be reported as the date of disease progression for patients who continued treatment beyond progression but were confirmed to have true progression on a subsequent assessment.Toxicities that patients experience from IO therapies (IO toxicity) differ from those with other cancer therapies, such as chemotherapy and molecularly targeted therapy. Immune check-point blockade, for example, is associated with organ-specific adverse events, such as hepatitis, colitis, dermatitis, pneumonitis, and endocrinopathies such as hypophysitis . The advDespite the critical importance of knowing the safety profiles of cancer treatments, there is evidence that toxicity reporting is suboptimal in clinical trials –33, inclClinical diagnoses of IO toxicity often describe a constellation of specific signs and symptoms that afflict a patient. For example, a patient receiving immunotherapy may have individual symptoms such as headache and fatigue. The clinical diagnosis for these symptoms, however, may be immunotherapy-related hypophysitis. Similarly, a patient who reports abdominal pain and diarrhea may ultimately be diagnosed with colitis. Both symptoms and diagnoses are reported in clinical trials, often describing the same event and resulting in confusion regarding the precise incidence of particular toxicities . To avoiThe terms “immune-related adverse events” (irAE) or “adverse events of special interest” (AEOSI) are often used to describe clinical diagnoses of IO toxicity . Many clinical trials distinguish adverse events belonging to these categories from other toxicities and separately report them. When investigators use predetermined categories of toxicities, such as irAE or AEOSI, they should explain how and why certain toxicities were selected for inclusion within these categories and what criteria were met to attribute a toxicity to one of these terms .Clinical management of IO therapies would be better informed by increased granularity of toxicity grading data. Investigators usually report all grade toxicity and grade 3 to 4 toxicity separately to highlight the incidence of serious (grade 3 to 4) toxicities relative to the total number of toxicities observed. Given the nature of IO toxicities, investigators should report the specific grade of each toxicity collected in a clinical trial. There are clinically meaningful differences between grade 1 and 2 toxicities as well as between grade 3 and 4 toxicities. For example, grade 1 pneumonitis is defined in the CTCAE as asymptomatic and based on clinical or diagnostic observations, whereas grade 2 pneumonitis indicates symptoms interfering with instrumental activities of daily living for which medical intervention is indicated. Grade 3 pneumonitis is associated with oxygen initiation, whereas grade 4 toxicity is life threatening and accompanied by intubation. Grade 2 or higher immune-related pneumonitis usually requires that IO treatment be withheld and that steroids be initiated. When only grade 3 to 4 toxicity is reported in IO trials, grade 2 events are not clearly captured despite being clinically relevant. Reporting toxicity by each specific grade  will optimize the value of the available information on the toxicity profile of IO therapies.Table In addition to capturing the clinical management of adverse events resulting from IO therapies, Table An increasing number of clinical trials are testing immunotherapies in binary, ternary, and more complex combinations and sequences. These studies are evaluating IO therapies with each other and with targeted therapies, chemotherapy, and radiation therapy . There aInvestigators should report preclinical and/or clinical data that support the hypothesis that a combination or sequencing of therapies in an IO trial is likely to have additive or synergistic antitumor activity and a favorable therapeutic index . They shIn conclusion, the 12 reporting recommendations presented in this statement are intended to improve the reporting of IO clinical trials and thus provide more complete evidence on the relative benefits and risks of an IO therapeutic approach. Although some of these recommendations may be relevant for other types of cancer clinical trials, the goal of these recommendations is to address the unique efficacy, toxicity, and combination and sequencing characteristics of IO treatments. Given the rapid expansion of the number of IO clinical trials and ongoing improvements to the evidence base that supports the use of IO treatments in clinical care, these recommendations will likely need regular revision as the IO field develops. This iterative process will be critical to ensuring that IO clinical trial data are interpreted appropriately and are ultimately useful to improving the care of patients with cancer.


As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine.Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived  Despite significant progress made in understanding and treating cancer in the last decades, this disease is still one of the leading causes of mortality worldwide . ProgresPatient-derived cell (PDC) models generated from surgical biopsy specimens have recently emerged as desired pre-clinical tools. PDC models are characterized by increasing complexity and biological relevance as they retain patient-specific features and are amenable to a variety of experimental applications . Thus thin vitro cell models. Here, we describe the main aspects of this biobanking strategy detailing processes such as the acquisition of PDCs, and the generation, annotation, characterization and distribution of resulting cell models. We discuss in detail how clinical tissue biorepositories can be advanced to enable functional studies and personalized oncology and relate to our experience in developing a living cell biobank alongside the diagnostic workup.In the Department of Pathology and Molecular Pathology at the University Hospital Zürich, we have developed a comprehensive biobanking platform that has evolved from our extensive routine sample collection of biospecimens from malignant human tumors and includes the generation of patient-derived Hundreds of clinical samples are obtained every day in routine pathology practice where they are processed for diagnostic purposes. Generally, a large proportion of these samples originates from surgical resections of tumors. These tissue specimens are routinely archived as FFPE blocks, the gold standard material for histological and immunohistochemistry analysis and simultaneously assembled into biobanks that are stored for many years . These pin vitro cell models in order to promote cancer research and precision medicine.The retrospective analysis of these high-quality and readily available repositories that are linked with relevant clinical and pathological information allows the definition of cohorts for example for the creation of tissue microarrays or libraries of high quality nucleic acids for high-throughput molecular analysis . Thus, t3. Even though establishing cell models from smaller specimens can be successful, this ensures that some of the original material can be viably frozen in parallel with cell culture derivation. In daily practice, the size of suitable tumor specimens usually ranges between 0.5 and 8 cm3 , pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Other tumor types have been considered but still play a minor role for our biobanking activities and will not be further described here. Besides being the tumor type of our interest, specimens entering the living cell biobank should ideally have a minimum size of about 0.5 cmnd 8 cm3 . Upon find 8 cm3 .in vitro models of CRC or PDAC, we continue to rely on monolayer cell culture systems for models derived from human RCC tissue.If sufficient tissue was available, suitable pieces were selected for living cell biobanking by a board-certified pathologist and subsequently collected by the biobanking team in cold, sterile RPMI medium. Subsequently, all specimens were subjected to mechanical disaggregation and afterwards further processed according to previously described methods . Tumor oIn vitro cell models could be established from 60% of the RCC, 46% of the CRC and 42% of the PDAC tissue samples  inhibitor Noggin)  were subject to project-specific considerations that we undertook in collaboration with the principle investigators and which preceded any research project involving PDC models from our living cell biobank. In the last years, 3D tumor organoids have been usually maintained under serum-free conditions with growth factors that mimic the  Noggin) . Cells d Noggin) . These g Noggin) . Growth During the propagation phase, morphologies of PDC models were continuously assessed by bright-field microscopy and typical phenotypes like the glandular structures displayed by many CRC-derived organoids were recorded . In addiin vitro cell models match but additionally, details about subclonal composition and their dynamics can be deducted from the large-scale sequencing datasets. This potentially allows grouping cases according to mutational signatures or even unveils information about treatment targets (VHL tumor suppressor gene (80–90% of ccRCCs) that can be easily analyzed by targeted sequencing  of the corresponding PDC cultures by Sanger sequencing. Similarly, we assessed activating mutations in KRAS that are common in CRC (30–40% of cases) and almost ubiquitous in PDAC . Starting at the tissue collection procedure, control of pre-analytic parameters  is important because these variables can potentially impact on the outcome of analytical results. Careful annotation of these parameters may be integrated by the Sample PREanalytical Code (SPREC), a comprehensive tool to document pre-analytical details of biospecimens . AnotherFurthermore, the procurement of patient biospecimens is subject to stringent legal and ethical regulations. Despite the legislative frameworks that apply to biobanking activities varying considerably among the European countries , a numbeBesides the organizational aspects connected to the ethical implications, documentation of all procedures concerning origin, management and storage of samples is a key factor for successfully biobanking and distribution of well-annotated specimens. At the time when we started to develop our next-generation biobanking strategy, a suitable documentation tool for many aspects of living cell biobanking was not readily available and therefore we developed a dedicated, comprehensive biobanking IT infrastructure . This inFinally, it is fundamental to our tissue and living cell biobank that specimens and data that we collect and store are effectively utilized to support basic and translational biomedical research. In order to demonstrate such value, our ongoing endeavors are directed toward sustaining and expanding our living cell biobank workflow alongside the traditional tumor tissue biorepository for future cancer research and clinical application.in vitro models amenable for functional assays will likely be a major enabling factor for the discovery and development of new diagnostic tools and precision treatment options.Advances in molecular medicine have significantly increased our understanding of cancer biology in the last two decades and are spawning a paradigm shift in patient care toward personalized treatment strategies. To realize the promise of precision therapy in the clinics, a strong link between pathological data, molecular alterations, phenotypical consequences and patient outcomes will be instrumental. The comprehensive, tripartite biobanking strategy of collecting FFPE, fresh frozen and patient-derived living cells we have described here offers a toolkit for addressing numerous questions in current and future basic, translational and clinical research. Thus, the extension of traditional patient tumor tissue biobanking with viably frozen PDCs and personalized However, next-generation biobanking adds a new layer of complexity to traditional biospecimen collection as it requires an even higher degree of coordination, resources and expertise. For successful living cell biobanking, culture conditions for PDCs from different tumor types have to be established separately by expert scientific staff and each culture needs to be validated and characterized carefully. It is important to note that growth conditions are major influencing factors during propagation of PDC models as they impact on cellular processes such as replication and cell survival as well as on the genotypic and phenotypic heterogeneity of cell subpopulations. Thus far, only few studies have addressed the representation of tumor subclonal cell populations and their dynamics during PDC culture . To whatBased on our five years of experiences in next-generation biobanking we illustrate a targeted but scalable workflow that can be adjusted to accommodate a variety of research needs or adapted in other pathology departments. We strongly believe that in conjunction with traditional tissue biobanking, a living cell biobank will constitute an important tool for functional cancer research and personalized medicine which at the same time has the potential to fulfill the current demand for more reliable, patient-specific cancer models. However, it is important to note that for next-generation biobanking, additional considerations and long-term investments  are required in order to preserve the integrity of these collections for future research. This may necessitate transformational changes in research and infrastructure environments that often go beyond current standards for tissue specimen collection in pathology departments. Nevertheless, next-generation biobanking promises major new opportunities for cancer patients and will most likely play a decisive role in advancing precision medicine.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Kantonale Ethikkommission Zürich. The patients/participants provided their written informed consent to participate in this study.in vitro models. EG, KM, and HB performed all validation experiments and maintained the organizational infrastructure of the living cell biobank. CP reviewed cytopathological and histological slides. HB, SD, and PS established the comprehensive biobanking database. HB wrote the manuscript, while CP, HM, and PS provided conceptual support and assisted in the manuscript writing. All authors discussed the work and commented on the manuscript.HM and PS established the tissue and living cell biobank and provided critical support for all aspects of the biobanking activities. PS and HB devised the main conceptual ideas of this work. SD and PS were responsible for all aspects of collecting fixed and fresh frozen tumor specimens. EG, KM, CP, and HB collected and processed fresh human tumor specimens to generate 2D and 3D patient-derived 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.


Pancreatic ductal adenocarcinoma (PDAC) remains a major health problem because it induces almost systematic mortality. Carcinogenesis begins with genetic aberrations which trigger epigenetic modifications. While genetic mutations initiate tumorigenesis, they are unable to explain the vast heterogeneity observed among PDAC patients. Instead, epigenetic changes drive transcriptomic alterations that can regulate the malignant phenotype. The contribution of factors from the environment and tumor microenvironment defines different epigenetic landscapes that outline two clinical subtypes: basal, with the worst prognosis, and classical. The epigenetic nature of PDAC, as a reversible phenomenon, encouraged several studies to test epidrugs. However, these drugs lack specificity and although there are epigenetic patterns shared by all PDAC tumors, there are others that are specific to each subtype. Molecular characterization of the epigenetic mechanisms underlying PDAC heterogeneity could be an invaluable tool to predict personalized therapies, stratify patients and search for novel therapies with more specific phenotype-based targets. Novel therapeutic strategies using current anticancer compounds or existing drugs used in other pathologies, alone or in combination, could be used to kill tumor cells or convert aggressive tumors into a more benign phenotype. Approximately 350,000 people worldwide die every year due to pancreatic ductal adenocarcinoma (PDAC), making it the most lethal cancer . DespiteUncovering the mechanisms underlying tumoral heterogeneity has become a hallmark in cancer research , and we Epigenetics is defined as all the changes in phenotype and gene expression which are not due to alterations in the DNA sequence . These cSome of the epigenetic regulators found to be upregulated in PDAC are the H3K4 methyltransferases MLL2 and SETD3, and the H3K acetyltransferase KAT2A, all of which activate transcription . AnotherHistone deacetylase 1 (HDAC1) is another epigenetic modifier that is over-expressed in PDAC samples and may deregulate the histone acetylation pattern . In partNon-coding RNA (ncRNA) transcripts play a role as epigenetic modifiers regulating gene expression by different mechanisms, including interacting with histone modifying complexes or with DNMTs . These mThe DNA methylation is an epigenetic mark that causes gene silencing by retaining DNA in a transcriptionally quiescent state. In tumoral cells transcription of one of the five members DNMTs, the DNMT1 is increased, and higher levels of this enzyme correlate to poor clinical outcome , 26. TraThe development of epidrugs faces several challenges, one of the most important being the lack of specificity: all epigenetic therapies affect the total genome . A deepePart of the heterogeneity observed among PDAC patients can be explained by the classification into two different subgroups based on clinical outcome and therapeutic responses, named basal and classical subtypes. Patients with the basal subtype have a worse prognosis, but they respond better to adjuvant therapy compared to patients with the classical subtype. We know now that these PDAC phenotypes are defined by distinct epigenetic landscapes, and particularly by DNA methylation patterns, which are transduced at the transcriptional level and alter the interaction between the tumor and its stroma . HoweverAs described above, the origin of PDAC subtypes cannot be explained by the common genetic aberrations observed in this pathology. Nicolle et al.  demonstrRemarkably, aberrant DNA methylation patterns and/or deregulation of miRNA epigenetic modifications correlate with alterations in subtype-specific pathways, further supporting the hypothesis that epigenetics drives the PDAC phenotypes , 46. An These observations are in agreement with several previous studies which have successfully classified PDAC tumors into specific subtypes based on tumoral gene expression profiles, indicating that unlike genetic aberrations, the transcriptome as a reflection of the methylome, correlates to therapeutic response and clinical outcome. Collisson et al.  describeRecent data from multi-omic approaches , 7 highlA great advantage of stratifying patients according to epigenetic marks instead of RNA expression is that DNA is much more stable. It is known that solid tumors release cell-free DNA into the circulation and its DNA methylation pattern mirrors the tumoral profile. Aberrant circulating methylated DNA has already been studied for most types of cancers because of its clinical implications: it can be analyzed directly from plasma or serum samples without the need for any surgical or invasive procedures . In addiImmunodeficient mice xenografts established from patient biopsies can be useful for the molecular characterization of tumor samples; however, graft growth can take many months which is not a clinically compatible timeframe. An alternative method to the xenograft is the culture of organoids, and in the last few years this has become a helpful technique for translational medicine in cancer research. This 3D system recapitulates different aspects of tumoral cells such as tissue architecture, cell-cell interactions, polarity, and cellular heterogeneity by conserving the mutational profile, while 2D models do not. Another advantage is that they can be produced not only from resectable PDAC macro-biopsies but also from fine-needle aspiration (FNA) micro-biopsies . MoreoveA recent study found that analysis of the gene expression profile of PDAC organoids is a powerful tool for molecular pharmacotyping . Gene exUnderstanding heterogeneity in PDAC and the epigenetic mechanisms underlying it, brings with it the possibility to search for new potential therapies with more specific targets based on tumor phenotype. However, due to their particular microenvironment, pancreatic tumor cells adapted to survive under intense metabolic pressure . The extIn addition, new strategies could focus not only on the difficult mission of eliminating all cancer cells, but also on converting the phenotypes with the worst outcomes to another phenotype with a better prognosis. With this aim Lomberk et al.  inactivaOne of the most important problems facing the treatment of PDAC is its great heterogeneity. This heterogeneity has an epigenetic origin; it is not the result of genetic aberrations as had previously been thought. We can continue trying to develop new and more efficient drugs that save all PDAC patients, but this is a utopia. Instead, according to the tumor phenotype we can select the most suitable treatment for each patient or group of patients, using drugs already available. Epigenetic characterization of the tumor using circulating DNA or primary culture organoids could allow us to predict clinical outcome and select a therapeutic strategy: current anticancer drugs or existing drugs used in other pathologies, alone or in combination, could be used to eliminate the tumor, convert it to a less aggressive phenotype or increase its sensitivity to chemotherapy.NJ, JI, and ND conceived, wrote the manuscript, and create the figure.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 order to optimally refine the multiple emerging drug targets for hepatitis B virus (HBV), it is vital to evaluate virological and immunological changes at the site of infection. Traditionally liver biopsy has been the mainstay of HBV disease assessment, but with the emergence of non-invasive markers of liver fibrosis, there has been a move away from tissue sampling. Here we argue that liver biopsy remains an important tool, not only for the clinical assessment of HBV but also for research progress and evaluation of novel agents. The importance of liver sampling has been underscored by recent findings of specialised subsets of tissue-resident immune subsets capable of efficient pathogen surveillance, compartmentalised in the liver and not sampled in the blood. Importantly, the assessment of virological parameters, such as cccDNA quantitation, also requires access to liver tissue. We discuss strategies to maximise information obtained from the site of infection and disease pathology. Fine needle aspirates of the liver may allow longitudinal sampling of the local virus/host landscape. The careful utilisation of liver tissue and aspirates in conjunction with blood will provide critical information in the assessment of new therapeutics for the functional cure of HBV. Viral hepatitis remains the seventh most common cause of death worldwide, with mortality increasing sharply in the last decade to levels on the scale of HIV, tuberculosis or malaria.HBV is an hepatotropic virus, so the site of viral replication and disease pathogenesis is within the liver. Traditionally, a mainstay of management has therefore included assessment of liver histology using tissue obtained by a needle core biopsy. This remains the most accurate way of definitively assessing the degree of liver fibrosis for initial HBV disease staging and of ruling out other causes of liver disease. There is a strong case for the continued assessment of liver histology in combination with serological markers for treatment decisions, particularly in ambiguous cases. In addition, their use may instigate other clinical investigations , in cases where advanced liver disease is detected. Liver biopsy is, however, no longer the standard of care in a number of clinics due to the advent of non-invasive methods of fibrosis assessment.TE, using FibroScan, allows rapid assessment of liver stiffness by employing a shear-wave to measure vibration-velocity, a technique previously used in the food industry to gauge the maturity of cheese.BoxPercutaneous biopsy:Palpation/percussion guided or image-guided; transthoracic approach most common, subcostal approach may be used*Transvenous biopsy:Transjugular/transfemoral approach. Required in patients with clotting abnormalities and ascites and for indications where free/wedge hepatic vein pressure measurements are required.Laparoscopic/surgical biopsy:May be performed at time of surgical intervention. Liver can be directly visualised thus tissue yield is good, but general anaesthesia is required.Coaxial biopsy:Needle puncture device through which smaller needle biopsies are performed. Multiple passes through this approach can be performed.*Percutaneous biopsies can be performed with core aspiration needles; Menghini, Jamshidi or Klatskin. Usually performed with 16G needle which provides better cores, 18G needles may be used which may be better accepted by patients, but cores may be more fragmented. Sheath cutting needles may also be used (Tru-cut), usually smaller gauge, but sample may be less fragmented.In this article, we argue that liver biopsy remains an important tool in the majority of patients with HBV infection, for clinical disease assessment and for research progress and evaluation of novel agents . Even inMost studies of HBV immune responses in humans have relied solely on blood sampling; many clear-cut changes have been detectable and useful insights into disease pathogenesis have been made. In a few studies, responses studied thoroughly in the periphery have been compared with those within intrahepatic lymphocytes by paired sampling of the liver in a small subset of donors.Similarly, we were able to detect an expansion of granulocytic myeloid-derived suppressor cells correlating with disease activity in the circulation of patients with CHB, although in the liver this population was further expanded, expressing a more immunoregulatory phenotype .The study of covalently closed circular DNA (cccDNA), the episomal viral genome of HBV within hepatocyte nuclei, remains of critical importance as extensive efforts are made to develop therapeutic approaches to degrade or silence it. A major barrier to progress in the field has been the inability to accurately measure cccDNA in blood and assays for its determination in the periphery are likely to face many hurdles.More recently, hepatitis B core-related antigen (HBcrAg) has emerged as a potentially more accurate surrogate for cccDNA than qHBsAg. Assays for ‘core-related antigen’ measure denatured HBeAg, HBcAg and the pre-core protein (aa28 to aa15)HBV RNA can also be measured in the serum. It is packaged within nucleocapsids and both full-length and truncated RNA forms can circulate in patients.Histology provides a more detailed assessment of the liver, reflecting cumulative damage over time in addition to ongoing inflammation. A recent meta-analysis of 683 patients has shown that even with slight increases in alanine aminotransferase (ALT), a substantial number of patients up to 50%) have significant fibrosis.% have siValidated histological scoring systems are used in HBV; Ishak, Knodell and METAVIR being routinely used to assess disease activity.Beyond diagnostic evaluation, additional immunohistochemical staining can be undertaken, providing novel insights into HBV immunopathogenesis. Combining histochemical analysis of liver injury with specialised stains of paraffin-embedded sections for immune cell distribution can provide valuable insights into HBV pathogenesis. For example, interleukin (IL)-17-producing CD4 T cells were seen to accumulate in the lobular and portal areas of livers with HBV-related injury, suggesting a role in disease pathogenesis.The liver has an immunological composition which is distinct from blood or other lymphoid and non-lymphoid organs. Its inherent cellular composition is strikingly different, and even when circulating cell types pass through the liver they may be differentially shaped by the local milieu of cytokines, nutrients and hypoxia; these influences are likely to be exaggerated by the sluggish blood flow through the narrow-lumen vascular bed of the sinusoids.The intrahepatic lymphocyte population is very different from blood ; for exaRM) that can persist long term in the human HBV-infected liver and do not recirculate. These CD8 T cells have specialised adaptations for maintaining antiviral surveillance within the tolerogenic liver environment. We find liver-resident CD8 T cells,loEomesloBlimp-1hiHobitlo; RM. Despite this, CD8 TRM are able to produce antiviral cytokines rapidly on restimulation, perhaps as a result of their high cell-autonomous IL-2,RM have lower granzyme expression than other non-resident intrahepatic CD8 T cells, suggesting they may be adapted to favour non-cytolytic over cytolytic antiviral function in order to preserve organ integrity.We have characterised a novel population of tissue-resident memory T cells  that we found could impose the liver-resident signature on peripheral T cells in vitro, will have therapeutic applicability.The finding that a population of HBV-specific CD8 T cells with TRM. Tissue-resident memory CD4 T cells are also well-described in other settings and may well play a role in CHB and there are likely other subsets of liver-resident cells yet to be defined. Another liver-resident population that has come to light recently is a subset of NK cells expressing high levels of CXCR6,loEomeshi , it should ultimately be possible to identify fingerprints within the peripheral immune response that can be used as indirect biomarkers of treatment efficacy within an organ. This approach was exemplified by a recent study predicting tumour responses to PD-1 blockade using blood-based immune profiling combined with tumour imaging.Another solution to facilitate longitudinal assessment of novel therapies is the use of fine-needle aspirates (FNA) of the liver in place of biopsies. This technique was first shown to allow monitoring of immune parameters of CHB in 2005,While FNA may prove very useful for selective immune-monitoring of CHB treatment trials, it does not allow histological staining to examine responses in situ and better elucidate HBV pathogenesis. For this, better use could be made of stored paraffin-embedded sections from the era when liver biopsies were more common in CHB. New multiplexed immunostaining, allowing automated quantitative assessment of multiple proteins on a single slide, will open up the opportunity for much more comprehensive visualisation of the topography of HBV viral particles and antigens in combination with immune infiltrates. This is vital in order to image the spatial relationship between infected hepatocytes and immune cells to better illuminate host-pathogen interactions. At present, even the proportion and distribution of infected hepatocytes and their relationship to immune infiltrates has not been defined for different phases of infection. In addition to staining for HBsAg and HBcAg, it is now possible to use highly sensitive probes for in site hybridisation of HBV RNA.In summary, a wave of new agents to treat HBV will be reaching clinical trials in the next few years. In order to capitalise on the opportunity to learn from these initial attempts at functional cure, and to select optimal combinations, it is imperative that we intensify efforts to sample the liver as well as blood. This need has been reinforced by recent studies highlighting unique immune responses compartmentalised within the liver, along with the hallmarks of persistent HBV: integrated and cccDNA. Available liver tissue or aspirates must be used in carefully planned virological and immunological studies, aided by new techniques that allow simultaneous assessment of many parameters. Studies including liver samples will provide vital clues to modify and combine the array of new antiviral and immunomodulatory therapies on the horizon for HBV.


Postural control requires regulating the neural circuitry of musculoskeletal mechanics to maintain and change body spatial orientation to fulfill changing demands specific to the task and the environment. Knowledge progression has closely followed our understanding of the dynamic interplay between organism, task, and environment. Initially, Magnus  focused Le Goic et al. examine subject-specific geometry and inertial parameters. Intrinsic properties of the lower limb, stiffness and damping, were the earliest influences; however, early muscle activity was insufficient to counteract the external forces. The head was the last segment to move; thus, active correction for a fall could not be initiated by vestibular and visual inputs. The authors conclude that proprioception serves as the sole source of information for up to 300 ms following onset of unexpected falls. Rather than improved anticipation with experience, they argue for improved efficiency of reactive behaviors.To discern when multimodal sensory information impacts recovery of upright balance, Rasman et al. agree that active maintenance of the upright position requires sensorimotor control. Since the relative contribution of each sensory system is determined by its sensor dynamics and the coordinate system to which it links, they argue that active control is required to maintain balance. Thus, sensory contributions to postural control can only be fully interpreted by combining two common protocols: external perturbations and manipulating the balance control loop. Results from a multisegmental robotic balance system that responds to manipulation of sensor dynamics are presented as basic principles underlying standing balance. Alternatively, Peterka et al. provide detailed methods for use and interpretation of a commercially available device to assess central sensorimotor integration. They argue that applying an external balance perturbation clarifies the cause and effect relationships between sensory processing, motor action, and body sway. Data from 40 subjects and prior results from individuals with vestibular deficit are presented to support this conclusion.Lhomond et al. explore whether re-calibration of sensorimotor mechanisms in the postural control loop occurs during both movement preparation and execution. Facilitating transmission of cutaneous inputs during the planning phase of gait initiation produced increased somatosensory evoked potentials in primary sensorimotor areas; the neural response decreased when standing still. Premotor cortex, specifically supplementary motor area and superior parietal lobe, was concluded to be the putative source of efferent signals that update current body representation by increasing tactile sensitivity. In a review paper, Sienko et al. argue that long-term training with sensory augmentation devices allow time for the nervous system to develop optimal combinations and weights of sensory cues. They report that individuals with vestibular deficit as well as healthy older adults use real-time sensory augmented cues to reduce sway when the initial stance position, support surface, or visual inputs are modified during a static balance task.Patel et al. identified increased activation in dorsolateral prefrontal cortex, superior parietal lobule, inferior occipital gyrus, and lingual gyrus following slip-perturbation training while walking. Imagined slipping increased activity compared to resting state in supplementary motor area, parietal regions, parahippocampal gyrus, and cingulate gyrus. Thus, higher-level processing is required for the timing and sequencing of an effective balance response.Neuroimaging advances have provided new tools to decipher higher order cortical processing relevant to postural adaptability underlying motor learning and rehabilitation. Using functional magnetic resonance imaging (fMRI) and mental imagery, Adkin and Carpenter who have studied the emotional effects of height-induced threat on human postural control. They argue that threat-related postural changes promote a greater physical safety margin while maintaining upright stance. Their critical review of the static balance research literature highlights the need to recognize the potential contributions of psychological and physiological factors on balance deficits associated with age or pathology.The importance of cortical contributions to postural control is highlighted in the perspective paper by Dakin and Bolton provide a critical review of research methods and progress in the understanding of anticipatory postural behaviors to expose the role of prediction in postural control. Anticipatory behaviors can be facilitated by changes in the state, or set, of the nervous system. They argue that cortical expansion has improved prediction, termed “foresight,” for preparing to interact with the changing environment. Internal models allow for the fine tuning and priming associated with motor affordances, while learning can be implemented in the cortical, basal ganglia, and cerebellar networks.Li et al. distinguishes the neural circuits involved in cognitive or motor performance, and asserts that dual task interference should be greatest when the cognitive and motor tasks engage the same neural circuits in keeping with the principle of neural overlap. The literature reveals age-related differences in neural substrates underlying cognition and the degree to which the age-related decline of sensory systems  contribute to cognitive load. Findings support focused cognitive training, exercise, and multimodal training of older adults to improve postural and gait outcomes.Recent research has targeted cognitive and postural interactions to inform about functional behavior in a complex environment. Dual task paradigms are used extensively to reveal the contributions of cognition and attention to postural control, particularly in the aging population. The focused review by Stins and Roerdink assert that maintaining quiet upright stance shifts between postural reflexes and higher  centers in accordance with the theory of “intermittent control.” This involves a rapid succession of brief periods of postural stability, during which the body dwells relatively motionless in a particular posture, and postural instability, during which the body rapidly transitions to a new stable point. They hypothesize that exerting ballistic control consumes more attention than stiffness control, using variations in reaction time as the index of attention load. Evidence of attentional fluctuations in the control of quiet upright standing is provided by mapping stimulus-response intervals to local COP parameters.Chow et al. corroborates the detrimental effect of directing too much conscious attention toward postural control in young but not old subjects. Using an electroencephalography (EEG) method previously identified as an objective indicator of conscious movement control, they assess neural coherence between T3  and Fz (motor-planning) regions of the brain during a challenging balance task with and without directing attention internally to movement production. Increased EEG T3-Fz coherence in conjunction with increased sway path during the internal focus condition is only observed in young subjects. They caution that the observations may not readily translate between populations and are not robust to age-related changes.The original research of Bargiotas et al. demonstrates that alterations in executive function and attention can lead to postural deficits. Their RIL patients presented isolated dysexecutive syndrome without clinically detectable gait or posture impairment. Postural and visual measurements were made during the ecological task of reading a recipe while cooking. The main finding is that horizontal and vertical eye movements, as well as the average duration of the saccades and fixations, can predict postural deficits in RIL patients. They conclude that increased balance impairment is coupled with a reduced focusing capacity in ecological tasks.By comparing healthy subjects to patients with Radiation-Induced Leukoencephalopathy (RIL), Pantall et al. investigated the relationship between cognitive decline and postural dysfunction in individuals with PD. Longitudinal assessments of cognition and postural dynamics were performed. Postural measures were positively correlated with lower cognitive function and increased geriatric depression scores and negatively associated with a qualitative measure of balance confidence. The positive association between motor and non-motor features of PD reflects the potential for shared neural correlates, potentially the subcortical nuclei, between posture and depression.Parkinson's Disease (PD) has often been used as a model for understanding control mechanisms of posture because of the role of basal ganglia in motor planning and intention. Olson et al. Individuals with PD can learn to execute a new motor plan, but have difficulty with rapidly and flexibly switching between plans; thus, their motor plans do not become implicit. It was concluded that individuals with PD may continuously require explicit information and augmented sensory information to create new motor plans. Wright  examined motor adaptation in individuals with PD by manipulating somatosensory input. Support surface incline was changed during prolonged periods of quiet stance. Young healthy controls and aged-matched older adults exhibited long-term aftereffects of this incline, but those with PD did not. These results suggest that the basal ganglia play a role in tonic postural adaptation.If the sub-cortical nuclei and pre-supplementary motor areas are complicit in the motor dysfunction observed with PD, then we might expect impairments in motor planning and learning rather than motor production. The ability of individuals with PD to adapt to new task demands was reviewed by Fino et al. examine cognitive-locomotor interference using dual-task paradigms during the gait cycle. Dual tasking interfered with the duration of late swing and from foot contact to weight transference, both of which require higher-order cortical processing for planning and postural adjustments. Fling et al. measured spatial and temporal gait asymmetry in age-matched healthy and individuals with PD while in the levodopa off state. Individuals with PD exhibited significantly more temporal and spatial gait asymmetry than healthy controls, and changes in transcallosal fiber tract integrity of the pre-supplementary motor area (pre-SMA) and S1 was associated with their greater step length asymmetry.Cortical control for postural demands during walking is particularly challenging for people with PD. Jacobs et al. propose that the mechanisms underlying low back pain are similar to those of PD. Evidence suggests that both low back pain and PD are associated with impaired proprioceptive function, sensory orientation during standing balance, anticipatory postural adjustments, automatic postural responses, and striatal-cortical function. A review of the data, however, indicated that although both health conditions can be associated with altered striatal-cortical function, the nature of the altered structure or function was different for PD and low back pain.We learn from this special issue that the human organism is a dynamic system where all components, be they sensory, motor, mechanical, or cognitive, are operating in an interactive and continuous fashion within environmental and task constraints. Although it functions most efficiently as an automatic process, postural control is a motor task that needs to be learned and practiced throughout the lifespan to best serve the motor and cognitive demands presented to the individual performer.EK and JF were co-editors of the special issue and shared all editorial responsibilities and contributions to the writing of this editorial.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.


Caenorhabditis elegans lives in a complex habitat in which they routinely experience large fluctuations in temperature, and encounter physical obstacles that vary in size and composition. Their habitat is shared by other nematodes, by beneficial and harmful bacteria, and nematode-trapping fungi. Not surprisingly, these nematodes can detect and discriminate among diverse environmental cues, and exhibit sensory-evoked behaviors that are readily quantifiable in the laboratory at high resolution. Their ability to perform these behaviors depends on <100 sensory neurons, and this compact sensory nervous system together with powerful molecular genetic tools has allowed individual neuron types to be linked to specific sensory responses. Here, we describe the sensory neurons and molecules that enable C. elegans to sense and respond to physical stimuli. We focus primarily on the pathways that allow sensation of mechanical and thermal stimuli, and briefly consider this animal’s ability to sense magnetic and electrical fields, light, and relative humidity. As the study of sensory transduction is critically dependent upon the techniques for stimulus delivery, we also include a section on appropriate laboratory methods for such studies. This chapter summarizes current knowledge about the sensitivity and response dynamics of individual classes of C. elegans mechano- and thermosensory neurons from in vivo calcium imaging and whole-cell patch-clamp electrophysiology studies. We also describe the roles of conserved molecules and signaling pathways in mediating the remarkably sensitive responses of these nematodes to mechanical and thermal cues. These studies have shown that the protein partners that form mechanotransduction channels are drawn from multiple superfamilies of ion channel proteins, and that signal transduction pathways responsible for temperature sensing in C. elegans share many features with those responsible for phototransduction in vertebrates. THE ability of animals to detect mechanical, thermal, and other physical stimuli is conserved across phyla and plays a key role in their navigation of variable and harsh environmental conditions. These senses enable animals to mate, find food, and avoid danger, and depend on the functions of neurons specialized to detect these stimuli. Transduction of these stimuli in sensory neurons is mediated via signaling pathways that converge on ion channels, thereby converting physical stimuli into electrical signals that propagate through the nervous system to trigger appropriate behavioral responses. Animals use diverse receptors and signaling pathways to reliably sense and respond to physical cues. An intriguing feature of sensory transduction is the convergent and divergent evolution of sensory molecules and mechanisms allowing optimization of animal survival in specialized ecological niches. For instance, a signaling pathway used in a sensory modality in one species may evolve to detect a different cue in another. Similarly, the same cue may be sensed via distinct mechanisms in different species. Thus, a complete understanding of mechano- and thermosensation requires identification and comparison of transduction mechanisms across animals.Caenorhabditis elegans nematodes. Research on this topic is enabled by the ability of researchers to combine genetic discovery with quantitative analyses of behaviors and physiological measurements of sensory neuron responses in living animals. Neuroanatomical studies presciently predicted the identities of mechanosensory and thermosensory neurons, which were confirmed by cell ablation followed by behavioral analyses , or body movement (proprioception). The proper function of internal organs also depends on feedback from mechanoreceptor neurons (interoreception). Mechanoreceptor neurons differ in their anatomy and intimate association with skin, muscle, and internal organs, but share the vital function of performing mechanotransduction. Studies in ensation  and the C. elegans hermaphrodites have 45, and males have an additional 42, putative mechanoreceptor neurons .The integration of mechanosensory neurons into neural circuits, and how behavioral responses are linked to external and self-generated mechanical cues are described elsewhere e.g., . In reviC. elegans hermaphrodites  mutants have been critical tools for advancing our understanding of mechanical senses more generally.The gentle touch receptor neurons (TRNs) innervate the body surface and are required for touch-evoked avoidance behaviors. Optogenetic activation of these neurons is sufficient to evoke avoidance behaviors (hrodites . In turnF1/2), depends on body stiffness, indicating that a complex relationship exists between applied force and touch-evoked behavior. Indeed, touch sensitivity is more directly related to body indentation than it is to applied force. An indentation of only 100 nm is sufficient to evoke an avoidance response and ∼450 nm is the indentation required to evoke a half-maximal response  for forces exceeding 2 µN  and staggered to fill the length of the neurite . TRN micthe TRNs  and havehim-4 hemicentin mutants  lie next to muscle cells. As animals mature and epidermal cells expand, TRN neurites separate from the muscle. This process is disrupted in  mutants  and in me mantle . Two adde mantle . A long e mantle .C. elegans muscles  showed that calcium transients increase with probe displacement  at both the onset and withdrawal of mechanical loads . These rIn the TRNs, mechanoreceptor currents depend on extracellular sodium ions and are inhibited by the diuretic drug, amiloride. These properties are shared by channels formed by members of the superfamily of DEG/ENaC/ASIC channel proteins  in the CEP neurons . The chadeg-1 DEG/ENaC/ASIC gene. Unexpectedly, MRCs are unaffected by loss of the osm-9 or the ocr-2 TRPV channel genes alone or in combination  are placed at a temperature T > cT, worms migrate toward cooler temperatures on the gradient (negative thermotaxis)   . Converssotherms . The exaT±∼2°, the temperature changes detected by the sinusoidal oscillation of their heads is translated via as yet uncharacterized mechanisms to suppress turns and maintain extended periods of forward movement   in the bilateral amphid sense organs of the head have been implicated in regulating thermosensory navigation behaviors . Of thesin vivo. Calcium levels in AFD rise as temperatures warm and fall when temperatures drop  provides a measure of the temperature-dependence of a reaction  is also cT-correlated  and negative  thermotaxis behaviors  corresponding shift in AFDT*  upon T downshift  are expressed exclusively in AFD and are localized to their sensory endings  enzymes catalyze the production of cGMP from GTP . Of the cT  and long (hours) timescale upon T upshift allowing these neurons to both retain responsiveness to small temperature changes across a broad temperature range, as well as to precisely shift their operating range in a cT-correlated manner. However, behavioral adaptation to cT is only observed upon a hours-long exposure to a higher T, suggesting that additional mechanisms downstream of AFDT* adaptation—likely acting to modulate AFD synaptic output—must account for the altered behavioral operating range.As described above, AFDT* adapt on both a fast and slow timescale? The fast timescale of minutes precludes transcription-dependent mechanisms. Calcium buffering slows the timescale of this fast adaptation suggesting that calcium modulates this process  is not temperature-regulated, resulting in a decreased magnitude of AFDT* adaptation and lower final AFDT* .Precise adaptation of pression . In cmk-al T*AFD . AlthougAFDT* and AFD synaptic output can be partially decoupled molecularly. Indeed, mutations in the dgk-3 diacylglycerol kinase and pkc-1 nPKCε genes alter the rate of behavioral adaptation and AFD synaptic output without affecting AFDT* adaptation , and that this asymmetry is determined in a stochastic manner  levels of these same stimuli. As in other animals, ion channels play key roles in sensory transduction; mechanical cues activate ion channels directly, while thermal fluctuations act indirectly through regulating the synthesis and degradation of soluble molecules . One theme that emerges from this work is that molecular pathways and signaling principles are remarkably conserved, although certain pathways can be deployed in different sensory contexts, and signaling principles may be preserved in the absence of conserved signal transduction pathways. For instance, the thermotransduction pathway used by AFD to detect thermal fluctuations of as little as <1° is remarkably similar to the one that enables vertebrate photoreceptors to detect single photons. Similarly, the response dynamics and frequency selectivity of the TRNs are reminiscent of those that govern the function of Pacinian corpuscles in mammals. The ASH, CEP, and PVD neurons share these properties, but rely on distinct genes to form ion channels activated by mechanical stimuli. Another theme that arises is that many of the sensory neurons in C. elegans are polymodal, responding to multiple types of physical and chemical stimuli (Genetic dissection of sensory-guided behaviors and the responses of sensory neurons in  stimuli . The pol stimuli . The ASH stimuli . The PVD stimuli .C. elegans nervous system, a more detailed understanding of sensory transduction principles in this organism will allow us to derive general principles by which an animal navigates and thrives in its complex sensory environment.Future work will address several important challenges. An important unsolved issue is to obtain a biophysical understanding of sensory transduction. We do not yet understand how small temperature changes are sensed by the rGCs expressed in AFD to alter their enzymatic properties, nor is it clear how mechanical stress is transmitted across the skin to activate MeT channels in mechanoreceptor neurons. Another important issue is to decode whether the signaling pathways mediating responses to different sensory stimuli in a single sensory neuron type are compartmentalized according to the nature of the stimulus or whether they are integrated, enabling the neuron to deliver information regarding the coexistence of multiple sensory stimuli. Together with ongoing work on circuit computations in the


Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear.The mitochondrial unfolded protein response  study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function.The purpose of this study was to: 1) identify conditions that activate the UPRCultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis.mt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction .Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.These results identify the induction of the UPR  The mammalian heart has the highest oxygen consumption rate of any organ, which may transiently increase several-fold during physiological exercise to support the increased cardiac workload. Chronic increases in cardiac workload resulting from hemodynamic overload or neurohumoral activation eventually lead to heart failure. Approximately 26 million patients worldwide are estimated to have heart failure, and the condition imposes substantial morbidity and mortality despite the use of therapies that target neurohumoral activation and cardiac rhythm disturbance Mitochondria are crucial for cardiac function through oxidative ATP generation to support muscle contraction and relaxation, the metabolism of nucleotides, amino acids and lipids, intracellular calcium buffering, and the regulation of cardiomyocyte survival. Mitochondrial dysfunction is a central feature of heart failure by contributing to energetic dysfunction, oxidative stress, calcium dysregulation, and cardiomyocyte death, and is considered a potential therapeutic target mt) is evolutionarily conserved and evoked when the mitochondrial protein folding environment is compromised and there is an accumulation of misfolded proteins mt is a mitochondrial-to-nuclear signal transduction pathway that leads to increased transcription of numerous mitochondrial protective genes, notably a repertoire of molecular chaperones, proteases, and antioxidant enzymes located primarily in the mitochondrial matrix mt serves to restore mitochondrial protein homeostasis, function, and cell survival. The UPRmt has been extensively characterized in C. elegansmt in the heart is unclear. Here, we identify the induction of the UPRmt in cardiomyocytes subjected to neurohumoral or mitochondrial stress and in the hemodynamically overloaded murine and human heart. We show that pharmacological enhancement of UPRmt with small-molecule agents ameliorates mitochondrial and contractile dysfunction in the overloaded murine heart, and in patients with aortic stenosis, elevated levels of UPRmt indexes correlate with reduced plasma biomarkers of cardiac damage and dysfunction. The UPRmt, therefore, has an important homeostatic role in the pathologically stressed mammalian heart and may be a potential therapeutic target in heart failure.The mitochondrial unfolded protein response , 2-way ANOVA, or repeated measures ANOVA as appropriate, followed by Bonferroni post-hoc testing. A p value <0.05 was considered significant.Data are presented as mean ± SEM, unless otherwise stated. Comparisons were made by Student’s mt involves the transcription factors Atf5 and CCAT-enhancer-binding protein homologous protein (CHOP), which increase the expression of mitochondrial chaperones and proteases The induction of the UPRmt induction in cardiomyocytes occurs as a process distinct from the endoplasmic reticulum unfolded protein response (ER stress) or cytosolic stress responses, we assessed the protein levels of KDEL sequence-containing markers of ER stress  and mRNA levels of the cytosolic chaperones Hsp70 and Hsp90 under these stress conditions. Protein levels of KDEL sequence-containing proteins were unaltered in cardiomyocytes treated with isoproterenol, paraquat, or Δ-OTC overexpression, whereas stimulation with tunicamycin, which induces ER stress, robustly increased the levels of these proteins  or indirectly via the pharmacological inhibition of polyADP-ribose polymerase (PARP), boosts the UPRmtPrevious studies showed that the enhancement of cellular NADmt in stressed cardiomyocytes, we compared the effects of Iso  stimulation on mitochondrial respiration with and without pre-treatment with NR or olaparib to enhance the UPRmt. Iso stimulation resulted in a significant decrease in mitochondrial maximal respiration, which was markedly improved when the UPRmt was boosted with NR , a clinical model of chronic pressure overload. The clinical and demographic characteristics of these patients are shown in mt markers among the AS group compared with control. Notably, approximately 50% of the AS subjects had substantial increases in the expression levels of these transcripts  based on their myocardial mRNA levels of UPRmt markers being higher or lower than the upper limit of “normality” (ULN). ULN was set as 2 SDs above the mean for each transcript in the control group. Subgroup A comprised AS patients with mRNA levels of UPRmt markers within the normal range, and subgroup B comprised those with mRNA levels of at least 3 UPRmt markers above the upper limit. Subjects in subgroup B in fact demonstrated significant elevation of all the UPRmt markers compared with subjects in subgroup A or control, indicating a significant activation of UPRmt in this cohort , activated the UPRproteins . We usedmt has been viewed as a cytoprotective response that promotes organelle recovery mt markers was typically transient. We therefore hypothesized that enhancing the UPRmt might elicit cytoprotective effects. Recent studies have identified agents that activate the UPRmt, thereby facilitating characterization of its functional impact under stress conditions. Notably, the augmentation of cellular NAD+ pools leads to the induction of the UPRmt, secondary to activation of NAD+-dependent deacetylases, and was shown to improve the outcome under conditions of mitochondrial stress+ pools can be achieved via dietary supplementation with NAD+ precursors such as NR, or through the inhibition of NAD+-consuming enzymes such as PARP. We confirmed that agents that augment the NAD+ pools, in particular NR, enhance the UPRmt in cardiomyocytes and that this occurs through the actions of Atf5. We found that the augmentation of the UPRmt led to significant improvement in mitochondrial dysfunction in stressed cardiomyocytes. Importantly, these effects were dependent on Atf5, indicating that they were likely to involve the UPRmt rather than other NAD+-dependent actions in the cell. To extend these data to the in vivo setting, we tested the effects of NR supplementation in mice subjected to TAC. We found that NR treatment markedly improved mitochondrial respiration, cardiomyocyte survival, and cardiac contractile function. These data provide proof-of-concept that enhancement of the UPRmt in the pathologically stressed heart may have therapeutic potential.Activation of the UPRmt and reduced indexes of ongoing myocardial damage. We found that AS patients who had an elevation of UPRmt markers had lower plasma levels of hs-TnT and N-terminal pro–B-type natriuretic peptide, both strong predictors of future adverse outcome independent of the resting LV function mt markers. These findings are consistent with the postulate from our experimental studies that increased activation of the UPRmt is beneficial by preserving mitochondrial function. In the human AS setting, a chronic improvement in mitochondrial function may manifest as a lower rate of cardiomyocyte death, with concomitant lower levels of fibrosis in the myocardium. Unlike the TAC setting, AS subjects with elevated UPRmt markers had similar cardiac function to those with low UPRmt levels, which may reflect the very slowly-progressive pathology of human AS compared with surgically induced TAC in mouse models mt in the pressure-overloaded human heart and suggest that increased activation of the UPRmt may be beneficial.To extend these findings to the human setting, we studied myocardial tissue from patients with severe AS undergoing valve replacement. A comprehensive molecular and histological analysis of myocardial tissue, clinical characteristics, and blood biomarkers revealed a striking relationship between enhanced activation of the UPRmt, may also have beneficial effects. It should be noted, however, that prolonged or dysregulated induction of the UPRmt could be detrimental, for example, contributing to an accumulation of defective mitochondria Activation of the ER stress response has been demonstrated to be an important pathophysiological mechanism in many human diseases mt activation and in different disease settings to better understand the therapeutic potential and/or toxicity of such interventions. Another limitation of our study is the fact that the human data are by necessity correlative and that the control subjects were not perfectly matched with the AS patients. However, there was a very good matching of most clinical characteristics between AS patients in the two subgroups.Future studies will need to investigate the effects of different durations of UPRmt in response to diverse in vitro and in vivo stresses in the mammalian myocardium. We also show that pharmacological boosting of the UPRmt preserves mitochondrial function and cardiomyocyte viability under pathological stress conditions relevant to heart failure. Taken together, the results suggest that the targeting of the UPRmt merits further investigation as a therapeutic strategy for heart failure.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: The detrimental consequences of chronic hemodynamic overload or neurohumoral stress in the heart include mitochondrial dysfunction and contractile depression. Activation of the UPRmt is able to mitigate these effects by restoring mitochondrial homeostasis.TRANSLATIONAL OUTLOOK: The UPRmt can be activated by small molecules that increase NAD+ levels. Additional studies are required to determine whether long-term enhancement of the UPRmt is beneficial in chronic hemodynamic overload.In summary, this is the first study to identify activation of the UPR


Hydrogels represent 3D polymeric networks specially designed for various medical applications. Due to their porous structure, they are able to swollen and to entrap large amounts of therapeutic agents and other molecules. In addition, their biocompatibility and biodegradability properties, together with a controlled release profile, make hydrogels a potential drug delivery system. In vivo studies have demonstrated their effectiveness as curing platforms for various diseases and affections. In addition, the results of the clinical trials are very encouraging and promising for the use of hydrogels as future target therapy strategies. Hydrogels are three-dimensional 3D) polymeric networks, whose hydrophilic structure allows the absorption of large amounts of water (thousands of times their dry weight) . HydrogeD polymerTheir unique properties, including reliable biocompatibility, tunable mechanical and degradation features, sensitivity to various stimuli and the ability to be easily conjugated with hydrophilic and hydrophobic therapeutic compounds  has madeIn the therapeutic area, drug delivery approaches require outstanding improvements in obtaining safe transport systems in order to achieve the desired therapeutic effect and to avoid the side effects. Biomimetic strategies involving polymers describe innovative industrial products orientated to target therapy and controlled release. Hydrogels inspired design offer favorable conditions for therapeutic compounds encapsulation and protection. Furthermore, they are becoming biological responsive structures which ensure adequate biocompatibility and biodegradability.Hydrogel synthesis is an essential step in developing new structures with beneficial properties for drug delivery action. The hydrogel structure is defined by the hydration of hydrophilic groups and domains of the polymers involved. Therefore, these groups and their interconnected chains create three-dimensional networks via crosslinking, avoiding their dissolution in the aqueous phase ,11. The standard synthesis procedures imply polymerization and crosslinking. These techniques can happen in parallel in one step, or one after the other in multiple steps . Polymerization process is part of the gelation process. The structure and the conformation of the starting material influence the formation of soluble branched polymer networks . The staHydrophilic polymers are often used for hydrogel synthesis due to their biocompatibility ,22 in aqThe swelling property of a hydrogel is significant for further use in medical applications because most of the body tissues are composed of large amounts of water . SwellinThe crosslinker agent plays a significant role in hydrogel swelling  and degrOn the other hand, there are factors involved in altering hydrogels assembly . ChemicaSmart polymers can form self-assembly structures through two significant procedures: using peptide/protein materials or using hybrid systems containing minimum a synthetic macromolecule and a peptide/protein motif . Their sHydrogels can be classified considering different parameters like: polymer origin, ionic charge and biodegradability .According to the polymer origin, the hydrogel composition can be synthetic, natural or it can contain both synthetic and natural polymers and is called hybrid . Lately,Ionic charge influences the uptake of the charged drug molecules. A positively charged hydrogel will embed and release negatively charged low molecular weight molecules . FurtherBiodegradable hydrogels need to meet some specific features as biocompatibility and adaptability to various media conditions and preparation procedures. Such hydrogels are potential candidates for drug delivery applications because of their flexibility. Biodegradation process describes a chemical decomposition of a complex hydrogel into a simple structure through solubilizing or hydrolysis dictated by various external stimuli like pH, ionic strength, temperature or enzymes . TherefoOtherwise, non-biodegradable hydrogels offer multiple possibilities for tissue engineering applications by encapsulating growth factors, proteins . Other mCrosslinking synthesis methods make possible the functionalization with drugs and other therapeutic agents in order to develop new delivery systems . The hydWong and Dodou, 2017, synthesized poly(ethylene oxide) hydrogels cross-linked with pentaerythritol tetra-acrylate using ultraviolet light for cross-linking reaction. These hydrogel films were loaded with various drugs  using post-loading and in situ loading methods. The study results revealed that in situ loading procedure was more successful regarding drug encapsulation .co-lactide) hydrogels functionalization with celecoxib. PEG’s length affects hydrogel loading capacity and 2000 g/mol was found to be the optimal length for drug delivery applications [Prince et al., 2019, used in situ loading technique for thermoresponsive poly(ϵ-caprolactone-co-lactide)-block-poly(ethylene glycol)-block-poly. An extensive review of morphological characterization of hydrogels has been recently published , and it While SEM does provide valuable qualitative information, alternatives do exist and can potentially provide complementary information to the limited capacity of regular SEM of generating two-dimensional projections. Laser scanning confocal microscopy (LSCM), used in combination with fluorescent dyes is capable of generating Z-stacks to evaluate similar aspects  to SEM ,148. Whe2S [Functional analysis is paramount to understanding the effects that solute/external stimuli have on the hydrogel. The most important ones seem to be: absorption capacity and rate, absorbency under load, lowest solute, and extra monomer level, pH, and photostability . Drug re2S  or varioThe controlled release can be achieved through various strategies based on hydrogel composition and properties. For instance, there are some molecules that can initiate and enhance the degradation rate ,160. MatIn addition, hydrogel mechanical deformation using magnetic field or ultrasounds is a very common procedure. In this regard, Liu et al., 2006, proposed an intelligent magnetic hydrogel loaded with drug molecules capable of tunable controlled release profile time dependent . UltrasoMany mathematical models are used for predicting the drug delivery efficiency of polymeric hydrogels. These determinations are based on mesh size, mechanical, mass-transport and diffusion behavior using hydrodynamics, obstruction, free volume and combined theories ,170.In order to compare and investigate the effects of hydrogels in vivo, it is necessary to evaluate its biocompatibility, since living organisms are prone to develop inflammatory reactions which are facilitated by the degradation of the synthetic polymers . As statAs mentioned above, a complex problem regarding the administration of various biomaterials in vivo reflects the ability of the material to conserve and promote a biologically safe environment for the subjected animals. In vivo testing is challenging because there still is a need for an established and reliable animal model in order to achieve biomechanical restoration. Following these standards, different injectable hydrogels protocols were developed and optimized, which because the body very well tolerated them, are ideal candidates for performing in vivo testing on rodents . Given tOne of the most efficient methods to evaluate the response to the therapy and assessing the toxic response in vivo in mice is performing a subcutaneous injection. Since the subcutaneous area is vascularized, the implanted hydrogels or other biomaterials are immune privileged, so a mild reaction to foreign moieties is to be expected . Bare poco-glycolic acid hydrogel degradation have been shown to affect healthy metabolism [Presence of digestive enzymes could lead to denaturation;Low permeability through the epithelial membrane into the bloodstream;Superior and inferior digestive systems can represent potential therapeutic targets .The oral delivery route represents by far the most convenient solution under adjustable parameters and patient compliance . Orally tabolism , leadingRectal administration offers plenty of advantages such as rapid absorption of the compound, avoidance of the gastrointestinal tract, limited adverse reactions of the therapeutics, and provides controlled release of the compound in the context of the stable environmental conditions encountered in the rectum. Based on an earlier study that showed excellent biocompatibility on the digestive tract , rectallHydrogel therapy is suitable for the topical and transdermal approach. Nowadays, the general concept about nanosystems in the context of skin penetration is that the skin barrier restricts the delivery via epidermis and dermis. In truth, hydrogel penetration through the skin seems unlikely as the macroscale mesh-like structure would prohibit penetration and has been shown, in the case of collagen hydrogels, to not even affect burn models . VariousAdministration of hydrogels via intratumoral injection is a practical approach, which requires the release of the nanostructure system loaded in a hydrogel matrix. These hydrogel structures are so-called “macroscopic gels” that are coming with the imperative need to decrease potential damages of associated tissues during an injection . Qinjie Intraperitoneal administration of hydrogel systems shows promising results and is considered to be a noninvasive option, as well as an optimal formulation for various pharmaceutical agents. One of these structures was prepared by Chih-Hao Chen et al., in 2018 and was successfully incorporated in the mouse peritoneum achieving dual action: drug delivery and postoperative anti-adhesiveness . SimilarFormulations for ocular delivery are complicated to develop; several implications are creating limitations like inferior bioavailability and absorption . Eye topInnovative biocompatible and biodegradable injectable hydrogels have been synthesized for cartilage and bone tissue , vasculaThe injectable systems have the flexibility to fill irregular-shaped defects and offer the possibility to reduce surgical invasion . SignifiAmong these, stem cell delivery systems are focusing on microfluidics to avoid microenvironment interactions. Various hydrogel constructs like microspheres were fromulated for stem cells encapsulation. For example, Zhao et al., 2016, synthesized light responsive hydrogel microspheres made by gelatin and methacryloyl chloride. Bone marrow-derived mesenchymal stem cells were encapsulated using post-loading procedures and then tested in vivo on rabbit femoral condyle animal model following orthotopic implantation protocol. This microfluidic approach demonstrates the feasibility of this method in osteogenesis . In situ gel forming tissue implants are suitable for transplantation during invasive surgeries directly into defects. The difficult regeneration of articular cartilage represents a very challenging problem. Due to the fact that this tissue is not presenting vascularization, the immune response is absent and it allows the use of allogenic constructs in the context of tissue engineering matter. Perrier-Groult et al., 2019, implemented a protocol for subcutaneous administration of agarose hydrogels embedded with human chondrocytes in order to promote their differentiation to cartilage matrix. In this regard, humanized mouse models are preferred for the evaluation of such implants acceptance . Considering the aspects mentioned above, when choosing the experimental design for in vivo studies using murine models, the age of the animal should be considered because it can influence the outcomes through various metabolic pathways involved in hydrogels degradation and clearance . Regarding hazard assessment, many different hydrogel systems aim to deliver the therapeutic agent in a controlled manner for better effects at the target site . In a geBringing together the advantages of a biocompatible hydrogel with the therapeutic effect of various molecules provides new efficient configurations adapted for medical devices development. Polymer science is holding up the attention of scientists because of the promising properties of biomedical clinical applications. ClinicalTrials.gov, over 300 completed studies are presenting encouraging results for hydrogel use with significant applicability in visual disorders and dermatological affections. In the last decade, an increasing number of clinical trials investigated the use of hydrogel structures for drug delivery systems. According to In the frame of current work, hydrogels aim to become feasible tools for microfluidics, biosensing and tissue regeneration engineering . Future proposed designs highlight the control over structural and morphological properties in order to improve their biomimetic capabilities. Furthermore, hydrogel usage in generating complex 3D multi-cell models offers promise in reducing the number of animals used for testing and while at the same time provides medium that more closely resembles aspects of the human body. All of these considerations will inevitably lead to accelerating pharmaceutical formulation development and deployment on the market. It is truly difficult to exemplify the role and applications that hydrogels have in biomedical research mainly due to their usage not being restricted to the topics described in this review. It is, however, easy to observe the capacity that this technology has to transcend a certain niche and in a very timely manner be implemented in a completely new field of work. The most difficult hurdles for medical and biological research seem to have been overcome and hydrogels have become a common day item in not only researchers’ lives but the general public’s lives too. Recently, electronics progress relieves the use of hydrogel technology for healthcare oriented applications. Lab-on-a-chip microfluidic devices are considered practical and compelling for diagnostic and drug screening investigations. The generation of wearable sensors represents an innovative platform able to monitor pathological particularities of the human subject. Such devices can also perform a controlled release of pharmaceutical formulations according to the monitored parameters.What is clear is the fact that hydrogels have been and are continuing to be relevant “scaffolds” in biomedicine and biomedical research. When it comes to these constructs we have entered an era of fine-tuning already existing structures and technology, where novel hydrogels are quick to reach practical applications in the real world  and more exotic ones hold potential in a variety of fields such as robotics, aerospace engineering, solar cells and photoreactors, environmental research and sports science.


It is generally believed that this improvement is due to the stimulating effect of GH on oocyte quality. However, studies are emerging that show possible positive effect of GH administration on endometrial receptivity, thus suggesting an additional potential benefit at the level of the uterus, especially among women with recurrent implantation failure, thin endometrium, and older normal responders. This review summarizes recent data on GH co-treatment effects on endometrium and endometrial receptivity among infertile women undergoing IVF, and proposes possible mechanisms of GH actions in the endometrium.Administration of growth hormone (GH) during ovarian stimulation has shown beneficial effects on  Receptive endometrium is an absolute prerequisite for a successful embryo implantation, being defined by a limited time-frame when the endometrium is favorable for embryo adhesion and the subsequent attachment and invasion processes .Endometrial receptivity is a complex process that is orchestrated by the synergistic actions of main reproductive hormones estrogen and progesterone, as well as plead of other endocrine, paracrine and autocrine factors , 3. ImpaThe role of growth hormone (GH) in female reproduction has gained renewed interest and has become a heated topic over the last decade. The local GH production in the reproductive tissues themselves exert an important autocrine/intracrine effects on those tissues, in addition to the pituitary production of GH . MoreoveGH is a peptide hormone secreted by the anterior pituitary gland, having important role in cell growth and metabolism throughout the body. GH together with its receptor, GHR, and related growth factors including IGF-1, is expressed in the endometrium of rats and human –31. The Apart from the effects of circulating GH and locally produced GH on endometrium, there is a proposed indirect effect of ovarian GH on endometrial function, namely its involvement in the function and maintenance of the corpus luteum , 38. WhiInitial reports on the use of GH in clinical practice come from cases of hypogonadotropic hypogonadism or panhypopituitarism . Subsequin vitro fertilization (IVF)/embryo transfer cycles  and good quality embryo transferred. Patients with recurrent implantation failure (RIF), having undergone three or more embryo transfer cycles after IVF treatment without a clinical pregnancy, are among the most difficult patients to treat, with no proven standard treatment. Impaired endometrial maturation is suggested as a common cause for RIF –58, makiThese are the first two studies assessing GH effects on endometrium in RIF patients, and, although the findings are promising, clearly more studies on larger patient population, as well as randomized clinical trials (RCTs), are needed for any clinically meaningful conclusions. It is well-accepted that endometrial thickness does not necessarily mean that the endometrium is receptive, yet it is considered as a measure of endometrial maturity, and optimal growth of the endometrium (>7 mm) is required for a successful embryo implantation –61.Infertile women with thin endometrium represent another potential patient population that could benefit from the GH administration. All studies on GH co-treatment during treatment of infertile women with thin endometrium were conducted in frozen embryo transfer (FET) cycles, where GH was administered during the endometrial preparation for FET , 43, 54 Women with poor ovarian response in ART is another patient group where GH co-treatment in stimulation protocols have been studied. All these studies see  have beeThus far, the largest group of infertile patients involved in studies on GH administration during IVF has been the normal responders . The firThe next studies analyzed 1,114  and 1,56To conclude, research on the effects of GH co-treatment in ART among normal responders has been performed on sufficiently powered studies in terms of the sample size, nevertheless as all these studies were not randomized controlled trials, further well-designed research is needed to objectively assess the GH effect on ART outcomes in (young) women with normal ovarian reserve and normal response to ovarian stimulation.Further studies are warranted in order to determine the optimal dose, time, and duration of GH administration and to investigate the long-term safety of GH for patients and their offspring. The dosage and treatment duration of GH differed among conducted studies see . BecauseAnother crucial part is to define the appropriate patient population that would truly benefit from GH treatment for improving their uterine lining quality in terms of thickness and/or receptivity. GH seems to promote endometrial growth, and its use could be considered in women whose endometrium does not grow and/or mature sufficiently with standard treatment protocols. In addition, the current review concludes that even normal responders could potentially benefit from the GH administration in IVF programs, however, the improved pregnancy rates in some of the studies utilizing fresh IVF cycles could not be separated from improved embryo quality. While endometrial thickness and pattern upon GH administration has been recorded and reported, evaluation of endometrial receptivity is not as simple. Future studies need to focus on the molecular level in order to evaluate the endometrial transcriptome/proteome/secretome , with emSample size and objectively designed studies  is a delicate topic in ART as strict double-blind, placebo-controlled, RCTs are difficult to accomplish . It is eThe current review summarizes the recent data on GH co-treatment effects on endometrial parameters in assisted reproduction and proposes possible mechanisms of GH actions in the endometrium. Studies are indicating that co-treatment with GH could improve the endometrial thickness, and possibly receptivity among infertile women. This effect might occur through increasing endometrial blood perfusion and the expression of genes and proteins related to endometrial receptivity such as VEGF and ITGB3 together with IGF-1, however the exact mechanisms in the endometrium remain to be clarified.Whether GH administration during IVF is useful and which patient groups could benefit from it needs further investigation, but the preliminary data suggest that women suffering RIF, patients with thin endometrium and older normo-responders could benefit from GH treatment when undergoing ART. Still, carefully designed and sufficiently powered cohort studies, RCTs, are required in the field in order to establish the most suitable therapeutic regimen for these patients and to clarify the confusion arisen from various studies that have shown either inconsistent or conflicting findings, used small patient cohorts and/or have been poorly designed with no blinding or placebo controls.SA and LA equally contributed to the review idea and manuscript writing.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.