Despite recent clinical trial advances and improvements in clinical care, kidney disease due to diabetes remains the most common cause of chronic kidney failure worldwide. In the search for new treatments, recent attentions have turned to drug repurposing opportunities, including study of the histone deacetylase (HDAC) inhibitor class of agents. HDACs are a group of enzymes that remove functional acetyl groups from histone and non-histone proteins and they can affect cellular function through both epigenetic and non-epigenetic means. Over the past decade, several HDAC inhibitors have been adopted into clinical practice, primarily for the treatment of hematological malignancy, whereas other existing therapies  have been found to have HDAC inhibitory effects. Here we review the current HDAC inhibitors in the clinic and under development; the literature evidence supporting the renoprotective effects of HDAC inhibitors in experimental diabetic kidney disease; and the adverse effect profiles that may prevent existing therapies from entering the clinic for this indication. Whereas recent research efforts have shed light on the fundamental actions of HDACs in the diabetic kidney, whether these efforts will translate into novel therapies for patients will require more specific and better-tolerated therapies. Kidney disease due to diabetes is the most common cause of chronic kidney failure across the globe . When kiThe historical textbook definition of kidney disease in diabetes will refer to the condition as “diabetic nephropathy” which, along with “diabetic retinopathy” and “diabetic neuropathy” is one of the three classical microvascular complications of diabetes. The term “diabetic nephropathy” denotes a chronic condition, originally described in the 1980s , that isDiabetes affects over 451 million people worldwide  and diab2; initiation of renal replacement therapy; or death from renal disease)  [Current treatments for diabetic kidney disease are aimed at slowing the progression of renal decline and include control of blood glucose levels and control of blood pressure, particularly in the latter case, with agents that block the renin angiotensin system (RAS) ,18,19,20< 0.001) . Despite< 0.001) ) and new< 0.001) . Among t2+ for their enzymatic effects. Class I HDACs include HDAC isoforms HDAC1, 2, 3, and 8; Class II HDACs are subdivided into Class IIa  and Class IIb (HDACs 6 and 10). Class III HDACs do not require Zn2+, but use NAD+ as a co-factor. They are known as the sirtuins and, in mammals, they comprise at least seven members (SIRTs 1-7). Because of their different mechanism of action, the sirtuins are not inhibited by known Class I and Class II HDAC inhibitors [HDAC enzymes catalyze the removal of acetyl groups from ε-amino-acetylated lysine residues on both histone and non-histone proteins. There are at least 18 different HDAC enzymes and they are categorized into four classes according to their homology to yeast HDACs, in which they were first discovered  Figure . The “clhibitors . HDAC11 N-CoR) or silencing mediator for retinoid and thyroid receptors (SMRT) [There are several different ways through which HDACs may exert their biological effects. These can be considered as epigenetic effects (which occur in the nucleus) and non-epigenetic effects (which occur in the cytosol or mitochondria) . HDACs cs (SMRT) ). Despite their name, HDACs do not solely deacetylate histone proteins. Indeed, some HDACs  exist almost exclusively in the cytoplasm . Numerou2+ domain and thus commonly are effective in inhibiting multiple different HDAC isoforms, although are ineffective against the sirtuins. Because of their different mode of action, sirtuin inhibitors are not considered further in this review. Amongst the HDAC inhibitors that do target Zn2+-dependent isoforms, agents may have broad-spectrum effects , class effects or isoform-specific effects. There are five classes of HDAC inhibitor compounds, classified according to their chemical structure: hydroxamic acid derivates ; short chain fatty  acids ; cyclic peptides ; benzamides ; and sirtuin inhibitors   26,37,3,39. MostThe hydroxamic acid derivates were the first HDAC inhibitor class to receive regulatory authority approval. These agents generally have poor isoform-specificity. Trichostatin A is one of the first hydroxamic acid HDAC inhibitors to have been discovered and it is not used clinically because of unfavorable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Vorinostat  was identified as an HDAC inhibitor based upon its structural similarity to trichostatin A  and, likIn addition to purpose-developed HDAC inhibitors, several other pharmacological agents have been discovered to have HDAC inhibitory effects and are now undergoing investigation for this mode of action. Perhaps the best example is valproate. Valproate is a branched short chain fatty acid that is used in the treatment of epilepsy and bipolar disorder and in migraine prevention. Even though valproate has been used clinically since 1962 , its preThe development of HDAC inhibitors for other indications and their successful adoption into clinical practice has encouraged exploration of their potential beneficial effects in diabetic kidney disease as a repurposing opportunity. Our group, and likely other investigators, became interested in exploring the possibility of repurposing HDAC inhibitors for the treatment of diabetic kidney disease because their integration into clinical care roughly coincided with the identification of epigenetic processes as important mediators of diabetes complications. More specifically, the long-term follow-up of participants of landmark clinical trials of glucose-lowering in diabetes revealed that individuals who were initially randomized to intensive glycemic control experienced a persistent reduction in the risk of diabetes complications (including kidney disease) for many years after the completion of the initial trials ,50, a paThe area in which HDAC inhibitors have been most extensively studied has been in the field of cancer. In comparison to non-cancerous cells, cancer cells exhibit a relative sensitivity to HDAC inhibitors where the small molecules induce cell cycle arrest, differentiation, and programmed cell death . This moThe first evidence that HDAC inhibitors may have beneficial effects on the kidney came in 2003 when investigators reported that the pan-HDAC inhibitor, trichostatin A reduced proteinuria in a murine model of lupus nephritis . Since t2O2) in NRK-52E cells [One of the first hints that HDAC inhibitors may show efficacy in diabetic kidney disease came with data presented in a review article published in 2007. In that study, investigators reported that the broad-spectrum HDAC inhibitor, trichostatin A attenuated the upregulation of both α-smooth muscle actin (α-SMA) and fibronectin and downregulation of E-cadherin induced by hydrogen peroxide  mice [In a separate study, we treated STZ-diabetic mice with vorinostat (50 mg/kg/day) for 18 weeks, observing an attenuation in the development of albuminuria and a reduction in both glomerular collagen IV deposition and mesangial matrix accumulation ,86. We oThe other major class of HDAC inhibitor that has been studied in the context of diabetic kidney disease is the short chain fatty acid class, studies reporting the effects of both valproate and sodium butyrate.As already described, valproate  has been employed clinically for the treatment of epilepsy, migraine, and bipolar disorders for many years, whereas it was more recently reported to be an inhibitor of Class I and Class II HDACs ,87. KhanThe renoprotective effects of valproate were also reported by a separate group of investigators in 2016 . These i−/− mice with sodium butyrate (5 g/kg/day in chow) for 20 weeks and observed that sodium butyrate attenuated diabetes-associated oxidative damage, inflammation, programmed cell death, fibrosis, and albuminuria in wildtype mice, but not Nrf2−/− animals [Two articles have reported the effects of HDAC inhibition with sodium butyrate in diabetic rats. The first was, again, conducted by Khan and co-workers  and perf animals . Further animals .Finally, aside from the effects of previously recognized HDAC inhibitors, it is plausible that some of the actions of other agents may be mediated by their interference with HDAC activity. For instance, the renoprotective actions of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitor, atorvastatin have been attributed to HDAC inhibiting effects of the statin .As reviewed above, over the past decade a small body of literature has emerged purporting the renoprotective benefits of broad-spectrum HDAC inhibitors in diabetic rats and mice. All of the studies reporting the effects of HDAC inhibitors in experimental diabetic kidney disease to date have employed the STZ-model, which most likely reflects the comparative simplicity of diabetes induction with this approach. However, in the absence of studies employing STZ-independent models of diabetes caution should be exercised before extrapolating these findings more broadly. The reported benefits of HDAC inhibition largely relate to early renal changes of diabetes including nephromegaly, proteinuria and the overelaboration of profibrotic factors and matrix proteins. This may be a consequence of the fact that most rodent models of diabetes do not develop late-stage fibrotic changes that are typically associated with GFR decline . That beEven if HDAC inhibitors do provide clinical benefit for the treatment of diabetic kidney disease, however, this would be far from the sole requirement for a therapy to be adopted into clinical practice. Not least, novel therapies must exhibit a safety and tolerability profile that render them suitable for the long-term treatment of complex chronic diseases. In this respect, tolerability may be where the currently available HDAC inhibitors would likely fall short for this indication even if their efficacy were proven. Beyond tolerability, given the breadth of action of HDACs, isoform-selectivity is likely to be an important determinant of the success of HDAC inhibition if it is to be adopted into clinical practice for the treatment of diabetic kidney disease. Some class-specific HDAC inhibitors are currently undergoing clinical trial evaluation   109,110110, wherIn summary, despite improvements in clinical care, the evolving diabetes pandemic has ensured that kidney disease due to diabetes remains the most common cause of chronic kidney failure. In the search for new treatments, various repurposing opportunities have been explored, including the repurposing of HDAC inhibitors, initially developed for their use in hematological oncology. Over the past decade, a number of reports have emerged that describe a renoprotective effect of HDAC inhibitors in rodent models of diabetic kidney disease. In the non-diabetic setting, a comparable body of literature has reported potential anti-fibrotic renal effects of HDAC inhibition and HDAC inhibitors have also been reported to offer other metabolic benefits in diabetes. These findings have served as useful lenses through which the effects of epigenetic mechanisms in diabetes complications and kidney disease may be observed. Clinically approved HDAC inhibitors exhibit an adverse effect profile that would likely hinder their use for this indication. However, the development of better-tolerated agents and the discovery of coincident HDAC inhibitory effects of existing therapies  may offer an alternative route to the development of new treatments that reduce the occurrence of diabetic kidney disease through HDAC inhibition.


Accurate nitrogen (N) management is crucial for the economic and environmental sustainability of cropping systems. Different methods have been developed to increase the efficiency of N fertilizations. However, their costs and/or low usability have often prevented their adoption in operational contexts. We developed a diagnostic system to support topdressing N fertilization based on the use of smart apps to derive a N nutritional index . The system was tested on paddy rice via dedicated field experiments, where the smart apps PocketLAI and PocketN were used to estimate, respectively, critical (from leaf area index) and actual plant N content. Results highlighted the system’s capability to correctly detect the conditions of N stress (NNI < 1) and N surplus (NNI > 1), thereby effectively supporting topdressing fertilizations. A resource-efficient methodology to derive PocketN calibration curves for different varieties—needed to extend the system to new contexts—was also developed and successfully evaluated on 43 widely grown European varieties. The widespread availability of smartphones and the possibility to integrate NNI and remote sensing technologies to derive variable rate fertilization maps generate new opportunities for supporting N management under real farming conditions. Given the rising food demand and the need for reducing the environmental impact of cropping systems, increasing nitrogen (N) use efficiency is one of the greatest challenges the agricultural sector is facing . After pLong- and short-term strategies were proposed to increase nitrogen use efficiency. The former refers to the development of cultivars specifically improved for their effectiveness in uptaking and using N by exploiting traits related to root growth and activity, shoot N storage/remobilization, and leaf photosynthetic rates ,8. AmongIn order to retrieve VR fertilization maps based on actual N nutritional status, spatially-distributed information on both plant N content  and the variables needed to estimate Ncrit  can be derived from satellite data, by means of empirical relationships between these variables and vegetation indices ,20. ThisDifferent methods are available for indirect PNC estimates, ranging from complex instruments based on leaf spectral response, like Dualex  and SPAD , to simple approaches based on the visual evaluation of leaf colour (leaf colour charts ). All thBesides technological aspects, an open issue for PNC estimates, and thus for N nutritional status determination, is that all indirect methods for PNC require dedicated calibration curves to convert readings (different types of indices according to the specific instruments) in PNC values, and these curves should account for genotypic variability, even within the same species . Many auDeveloping a smart app-based diagnostic system for supporting N fertilization via geo-referenced estimates of N nutritional status obtained by integrating apps for LAI (PocketLAI ) and PNCEvaluating the system’s capability to estimate N nutritional status and to support N management through dedicated field experiments;Proposing an effective procedure to derive calibration curves for indirect methods for PNC estimates, and demonstrating its suitability in a case study with PocketN and 43 rice cultivars widely grown in Europe. This procedure would allow extending the diagnostic system to production districts where other varieties are grown.The objectives of this study were:Two field experiments were carried out in northern Italy in 2014 and 2015 with the aim of developing and testing the smart app-based diagnostic system to support rice topdressing N fertilization . A third experiment was performed in 2017 in the same rice district to derive PocketN calibration curves for 43 widely grown European rice cultivars (experiment 3).Experiments 1 and 2 were carried out, respectively, in Rosasco  in 2014 and in Gaggiano  in 2015.2) and flooded at the 5th leaf stage, whereas in Gaggiano (experiment 2) cultivar Volano was scatter seeded (200 plants m2) on 6 May 2015 and grown under continuous flooding conditions. Rice was harvested on 16 September in Rosasco and on 11 September in Gaggiano. Soil was sandy-loam (USDA texture classification) in Rosasco, with low organic matter content (slightly above 1%) and low cation exchange capacity (CEC), whereas it was silt loam (USDA) in Gaggiano, with medium-high values for organic matter content (2.8%) and CEC. In both cases, available P and exchangeable K were not limiting, and pH was subacid. For both experiments, crop management allowed preventing water and nutrient (other than N) stresses and keeping the field pest-, disease-, and weed-free. Season 2014 was characterized by cold temperatures during summer and rainy conditions in late spring, which led to lower yields compared to the average  and 1.38 [plots 15 and 16]) instead revealed symptoms of luxury consumption (PNC was decidedly higher than Ncrit), with similar yields regardless of the amount of N received at the second topdressing event.The analysis of yield values for the plots with the same fertilization history until panicle initiation , likely because the overall N stress occurred at the beginning of the season partly compromised the crop. This is clearly shown by plots 1–4, which presented NNI values around or lower than 1 before the two topdressing events. For these plots, crop potential was limited by insufficient N availability during the post-emergence and tillering phases, and—regardless of the N amount received at panicle initiation—final yields were decidedly lower than the field mean. This suggests that marked stresses during early stages can hardly be recovered with massive N fertilizations at panicle initiation, which in most cases would result in wasting N. In experiment 1, this is further demonstrated by the relationship between yields and total N received , with the highest productivity achieved by the plots that were less stressed during the first part of their cycle.No relationship was found between yield and N applied at the second topdressing fertilization in experiment 1 .The variability in N treatments had a clear effect on rice productivity in experiment 1, with large differences in final yields among plots . The linear relationships used to convert the PocketN index into PNC were significant for most cultivars, with p-value <0.05 in 67% of the cases. For the cultivars for which the p-value was higher than 0.05, other regression models were tested, but no improvement in R2 and in significance level was obtained. Cultivar-specific calibration curves were largely heterogeneous  could not be included in any of the clusters without a marked worsening of the relationship between PNC and PocketN readings. This suggested that we should adopt—for these cultivars—dedicated (cultivar-specific) calibration curves. As expected, the pronounced heterogeneity in cultivar-specific calibration curves turned into a large variability among cluster-specific ones  allowed testing the diagnostic system under a wide range of nutritional conditions, with rice experiencing different degrees of N stress in the first part of the season in experiment 1 and, instead, experiencing almost unlimited N availability in experiment 2.In experiment 1, N stress was likely due to a combination of N leaching because of the sandy soil, low soil organic matter content, and the time lag between pre-sowing N application and the moment when the crop actually started uptaking N (the autotrophic phase). The retention capability of soils, indeed, can markedly vary according to physical and chemical soil properties . For theThe peculiar design adopted for experiments 1 and 2 reflects the need to generate variability in N nutrition to test the diagnostic system in a range of conditions representative of operational contexts. For this reason, and because the aim was not to derive general conclusions on different products or fertilization strategies, we did not adopt a replicated standard design like those normally used to test the effect of factors like, e.g., alternative N fertilizers, doses, or timings. The aim was to test whether NNI values derived using smart apps were useful to make diagnoses of crop N status and to analyze them in light of the conditions affecting crop growth rates and productivity. Environmental  and management (timings and amounts of fertilizers) conditions should be carefully considered, to properly interpret NNI values and effectively support in-season N management . Our resCalibration curves were derived in this study by exploiting the natural dilution of PNC along the crop cycle due to the relative increase of N-poor plant tissues  and to the reallocation of N-rich compounds driven by leaf self-shading and senescence ,55. ThisGiven the role of the specific PocketN background panel in minimizing the impact of differences in light conditions, and the experimental design that allowed exploring a large variability in PNC and leaf greenness, the calibration curves obtained for the 43 varieties can be considered sufficiently robust for operational conditions. Although factors affecting leaf thickness may have an impact on the relationship between actual PNC and estimates from diagnostic tools based on leaf greenness , such anOur results also underlined, once more, the within-species genotypic variability in terms of leaf properties e.g., ,58), thu, thu58])www.progettosaturno.it), aimed at providing farmers in Lomellina  N prescription maps. The within-field variability map was generated from the 6 July 2018 NDRE  Sentinel 2 image using k-means clustering according to Nutini et al. [The possibility to estimate NNI using GPS-equipped devices that can be connected to back-end services allows the development of workflows that effectively exploit the integration of satellite data and the smart scouting-driven collection of agronomic information. i et al. . This clWe developed a diagnostic system for supporting topdressing N fertilization and evaluated its reliability for paddy rice during two dedicated experiments. The system—estimating actual N requirements via NNI determination—targets operational farming contexts and is based on two smartphone applications for non-destructive estimates of Ncrit and actual PNC. The possibility of estimating NNI using inexpensive and highly portable tools that provide real-time information is expected to promote the adoption of the system within farming communities. According to the authors’ knowledge, this is the first time NNI has been derived and evaluated without using expensive and/or time-consuming methods, which could limit the potential for transferring NNI to operational farming contexts ,50. This2 very close to one, in most cases. Targeting the adoption of the system in operational contexts, calibration curves to convert PocketN readings in actual PNC values were derived for 43 popular European rice cultivars. According to the authors, this is the first time calibration curves were derived for a number of genotypes large enough to guarantee the applicability of non-destructive methods for PNC estimates in a production district. However, new tests could be needed for the varieties not included in any cluster, to provide further guarantees on the robustness of their calibration curves. The resource-efficient methodology used to derive the curves demonstrated the method’s effectiveness and could be exploited for other indirect methods  and cereal crops.PocketN confirmed its value as a diagnostic tool for non-destructive rice PNC estimates, regardless of the cultivar. Linear relationships between PocketN readings and PNC always explained a large part of the variance of reference values, with RThe obtained results and system applicability make this study an important step forward towards more mindful N management and, therefore, towards the improvement of the economic and environmental sustainability of farming activities.


In recent years, imaging has given a fundamental contribution to our understanding of the pathophysiology of acute lung diseases. Several methods have been developed based on computed tomography (CT), positron emission tomography (PET), and magnetic resonance (MR) imaging that allow regional, in vivo measurement of variables such as lung strain, alveolar size, metabolic activity of inflammatory cells, ventilation, and perfusion. Because several of these methods are noninvasive, they can be successfully translated from animal models to patients. The aim of this paper is to review the advances in knowledge that have been accrued with these imaging modalities on the pathophysiology of acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), asthma and chronic obstructive pulmonary disease (COPD). Since the first chest X-ray, lung imaging has focused, for the most part, on detecting structural alterations of lung anatomy due to pulmonary diseases, usually inferred from abnormalities in the distribution of lung density, or changes in lung metabolism, predominantly glucose metabolism, in the diagnosis and monitoring of lung cancer with positron emission tomography (PET).In recent years, however, there has been a tremendous growth of methods that leverage imaging to elucidate pathophysiologic mechanisms of acute lung disease. Because several of these imaging methods are noninvasive, many have been successfully translated from initial studies in animals to humans. This has allowed for a better understanding of how certain acute lung conditions develop as well as identification of which animal species, size, and injury models are most appropriate for translatability of the experimental results to the human scenario, depending on the specific condition or mechanism being investigated.The aim of this review is to highlight a sample of acute pulmonary conditions for which imaging of experimental models has enabled substantial progress in the understanding of their pathophysiology, with a specific focus on the contributions of PET. Major relative strengths and weaknesses of the three imaging modalities discussed in this review are presented in Because one of the defining features of ARDS is bilateral radiographical lung opacities, it is not surprising that imaging has played a major role in elucidating the pathophysiology of this syndrome. The current prevailing theory is that these opacities result from two main factors: (a) surfactant dysfunction , leadingImaging studies performed in these animal models, mainly employing computed tomography (CT), supported the concept that, in the heterogeneously inflated ARDS lung, there is a range of opening pressures for derecruited  alveoli. Such pressure depends on the position of the alveolus along the gravitational axis , on the 13N]nitrogen (13N2), by gating frame acquisition of the equilibrated tracer concentration with the end-inspiratory and end-expiratory phases. When a region of the lung expands, its gas content and hence the 13N2 concentration measured by PET increase. Thus, the change in regional tracer concentration is related to regional strain fluoro-2-deoxy-D-glucose ([18F]FDG) as a means to measure metabolic activation of inflammatory cells induced by mechanical ventilation [18F]FDG phosphorylation rate (commonly denoted as k3) [18F]FDG uptake rate (commonly denoted as Ki) was increased in dependent regions of the surfactant depleted lungs, which are the regions expected to undergo repetitive collapse and reexpansion with tidal breathing [18F] FDG uptake both in dense regions, where atelectasis and inflammation due to the primary etiology of ARDS are expected to predominate, and in aerated regions exposed to the iatrogenic strain of mechanical ventilation either because of tidal overdistension or cyclical alveolar recruitment and derecruitment [18F]FDG uptake on PET and/or CT abnormalities consistent with alterations of regional mechanical properties also reveal gene expression patterns indicative of activation of specific inflammatory pathways FDG uptake has also been used to detect inflammation due to sepsis and smoke inhalation, conditions that are often associated with ARDS. Studies in large animal models of sepsis using endotoxin infusion have demonstrated that inflammation in regions of high mechanical ventilatory strain is amplified by endotoxin [18F]FDG uptake in such regions [18F]FDG uptake  and expression of genes regulated by this transcription factor at 24 and 48 hours after acute smoke inhalation guanine ([18F]FHBG). [18F]FHBG has two properties that make it suited for PET imaging: (1) it is a high-affinity substrate for the HSV1-TK enzyme but has relatively low affinity for mammalian thymidine kinase, resulting in improved detection sensitivity and reduced background noise [κB activation. This PET imaging technique thus holds promise for elucidating the sequence of molecular and genetic events that lead to the inflammatory process of ARDS and VILI noninvasively and in vivo because the same subject can be studied at multiple time points over the evolution of the acute lung condition.Most recently, a transgenic PET reporter mouse model of smoke inhalation has been used to demonstrate activation of nuclear factor-kappa B  and noise ; (2) onc15O-water (H215O) as the radiotracer [215O is freely diffusible in the lung, it rapidly equilibrates between the pulmonary blood and tissue such that the concentration of tracer in the pulmonary venous blood that flows out of a region is equal to the concentration of tracer in the tissue divided by the tissue-to-blood partition coefficient of the tracer . By measuring regional lung activity with PET during H215O infusion and the subsequent equilibration phase, it is possible to calculate regional pulmonary blood flow from the equation that describes the one-compartment mathematical model of tracer distribution [11C- or 15O-carbon monoxide, which binds to hemoglobin with high affinity. Extravascular lung water can then be obtained by subtracting intravascular water from regional water. This technique thus allows determination of regional pulmonary blood flow and extravascular lung water . In conditions characterized by increased pulmonary vascular permeability, such as ARDS, extravascular lung water is expected to increase [68Ga-transferrin or 11C-methylalbumin, between the intravascular and the extravascular space [In addition to development of inflammation and alterations in the distribution of lung density, ARDS is characterized by alterations in the regional distribution of perfusion. Several techniques have been developed to measure regional perfusion with PET in animal models and patients with ARDS. One technique is based on the intravenous administration of iotracer . Becauseribution . Regionaincrease . A speciar space .215O technique, Gust et al. [215O technique have also revealed lack of perfusion redistribution away from edematous regions [68Ga labeled microspheres to measure regional pulmonary perfusion in rats [Using the Ht et al.  demonstr regions , suggest in rats .13N2 in saline solution. A bolus of 13N2 gas dissolved in 20–30 ml of saline solution is infused intravenously at the beginning of a 30- to 60-second apnea while the pulmonary kinetics of 13N2 is measured by sequential PET frames. Because of the low solubility of nitrogen in blood and tissues (partition coefficient between water and air is 0.015), virtually all infused 13N2 diffuses into the alveolar airspace of aerated alveoli at first pass, where it accumulates in proportion to regional perfusion [13N2 kinetics shows an early peak of tracer activity, reflecting perfusion to that region, followed by an exponential decrease toward a plateau for the remainder of apnea. The magnitude of this decrease is proportional to regional shunt  can be calculated from the washout rate of kinetics . In cont13N2 that allowed accurate estimation of blood gases in animal models of asthma by inhaled methacholine. The bimodality of these distributions during bronchoconstriction reflected hypoventilation of large contiguous regions of the lung [13N2 washout kinetics) is substantial, in contrast to the predominant single compartment behavior of normal lungs [Using this multicompartmental model, Vidal Melo et al.  were ablthe lung , a findithe lung . In thesal lungs .13N2 retention on PET frames acquired at the end of the washout phase. This characteristic has been leveraged to demonstrate that the prone position is effective in reducing areas of gas trapping in asthmatic subjects with induced bronchoconstriction [The extreme case of hypoventilation is represented by airway closure leading to gas trapping distal to the occluded airway. Gas trapping regions will appear as regions of triction .Combined measurements of regional perfusion and ventilation in patients with COPD showed that both ventilation and perfusion are more heterogeneously distributed in COPD than in normal subjects. However, the heterogeneity of perfusion was greater than expected from the increase in ventilation heterogeneity and occurred predominantly at large length scales, suggesting that perfusion heterogeneity could serve as an early biomarker of pulmonary vascular involvement in COPD .In recent years, pulmonary structural and functional imaging techniques based on CT, PET, and MR have been applied to animal models of ARDS , sepsis (endotoxin infusion), VILI , and asthma , yielding fundamental progress in our understanding of the pathophysiology of these conditions. Because of their noninvasive nature, several of these techniques can be translated to the corresponding human condition, thus enhancing the clinical relevance of these animal studies.


This serial cross-sectional study examines whether US state Medicaid expansion is associated with county-level counts of opioid overdose deaths. Is state Medicaid expansion associated with county-level opioid-involved overdose deaths in the United States?In this serial cross-sectional study of 3109 counties within 49 states and the District of Columbia from 2001 to 2017, Medicaid expansion was associated with reductions in total opioid overdose deaths and deaths involving heroin and synthetic opioids other than methadone. Expansion was associated with increased mortality involving methadone.The findings suggest that expanding eligibility for Medicaid may help to mitigate the opioid overdose epidemic. The Patient Protection and Affordable Care Act (ACA) permits states to expand Medicaid coverage for most low-income adults to 138% of the federal poverty level and requires the provision of mental health and substance use disorder services on parity with other medical and surgical services. Uptake of substance use disorder services with medications for opioid use disorder has increased more in Medicaid expansion states than in nonexpansion states, but whether ACA-related Medicaid expansion is associated with county-level opioid overdose mortality has not been examined.To examine whether Medicaid expansion is associated with county × year counts of opioid overdose deaths overall and by class of opioid.This serial cross-sectional study used data from 3109 counties within 49 states and the District of Columbia from January 1, 2001, to December 31, 2017 (N = 3109 counties × 17 years = 52 853 county-years). Overdose deaths were modeled using hierarchical Bayesian Poisson models. Analyses were performed from April 1, 2018, to July 31, 2019.The primary exposure was state adoption of Medicaid expansion under the ACA, measured as the proportion of each calendar year during which a given state had Medicaid expansion in effect. By the end of study observation in 2017, a total of 32 states and the District of Columbia had expanded Medicaid eligibility.The outcomes of interest were annual county-level mortality from overdoses involving any opioid, natural and semisynthetic opioids, methadone, heroin, and synthetic opioids other than methadone, derived from the National Vital Statistics System multiple-cause-of-death files. A secondary analysis examined fatal overdoses involving all drugs.There were 383 091 opioid overdose fatalities across observed US counties during the study period, with a mean (SD) of 7.25 (27.45) deaths per county . Adoption of Medicaid expansion was associated with a 6% lower rate of total opioid overdose deaths compared with the rate in nonexpansion states . Counties in expansion states had an 11% lower rate of death involving heroin  and a 10% lower rate of death involving synthetic opioids other than methadone  compared with counties in nonexpansion states. An 11% increase was observed in methadone-related overdose mortality in expansion states . An association between Medicaid expansion and deaths involving natural and semisynthetic opioids was not well supported .Medicaid expansion was associated with reductions in total opioid overdose deaths, particularly deaths involving heroin and synthetic opioids other than methadone, but increases in methadone-related mortality. As states invest more resources in addressing the opioid overdose epidemic, attention should be paid to the role that Medicaid expansion may play in reducing opioid overdose mortality, in part through greater access to medications for opioid use disorder. Fatal drug overdoses have increased markedly during the past 2 decades in large part because of overdoses involving opioids, including prescription opioids and illegal opioids, such as heroin and illicitly manufactured fentanyl. Between 2001 and 2017, the age-adjusted mortality rate for opioid-related overdoses more than quadrupled, from 3.3 to 14.9 per 100 000 standard population. In 2017, more than two-thirds of all drug overdose fatalities (47 600 deaths) involved an opioid.2 The law also requires that individuals who receive coverage through the expansion be provided with mental health and substance use disorder (SUD) services on parity with other medical and surgical services.3 From the beginning of Medicaid expansion in 2014 to the end of study observation in 2017, a total of 32 states and the District of Columbia opted to expand Medicaid eligibility.4The 2010 Patient Protection and Affordable Care Act (ACA) was signed into law during the rise in overdose deaths. Designed to increase access to and improve the quality of health insurance coverage, the ACA permits states to expand Medicaid coverage to essentially all non–Medicare-eligible people younger than 65 years with incomes at or below 138% of the federal poverty level .5 has led some observers to question whether Medicaid expansion will contribute to additional opioid-related harms. To the contrary, recent studies8 have found that although Medicaid expansion was associated with an increased rate of overall Medicaid-reimbursed prescriptions, changes in prescriptions for OPRs before vs after the expansion were not significantly different in expansion vs nonexpansion states.Medicaid provides essential health care access to millions of low-income people and, by extension, greater access to low-cost prescription medications, including opioid pain relievers (OPRs). Such increased access to OPRs, particularly among a patient population with higher rates of chronic disease and disability compared with non-Medicaid recipients,11 These medications  have been linked to improvements in treatment retention and OUD remission as well as reductions, in some cases as high as 50%, in all-cause and overdose-related mortality.13 Medicaid-reimbursed prescriptions for the opioid overdose reversal medication naloxone have also increased significantly more in expansion states compared with nonexpansion states.14 Early Medicaid expansions in Arizona, Maine, and New York in 2001 and 2002,15 along with more recent expansions in state Medicaid-eligibility thresholds for parents,16 have been associated with fewer drug overdose deaths. However, to our knowledge, with only 1 recent exception,17 no study has examined the association of ACA-related Medicaid expansion with opioid-related overdose mortality more specifically.Furthermore, Medicaid expansion has been an important source of coverage for SUD treatment, including for people with opioid use disorder (OUD). Previous research suggests that uptake of medications for opioid use disorder (MOUDs), including methadone, buprenorphine, and extended-release naltrexone, has increased more in expansion states compared with nonexpansion states.17 of the association of Medicaid expansion with fatal overdoses have been conducted at the state level. Although the most appropriate spatial scale for this association remains unclear, state-level analyses may not adequately reflect local (within-state) variation in the level and rate of growth of overdose deaths or differences in policy implementation, such as local disparities in the capacity for or accessibility of SUD treatment. Using overdose mortality and related covariates measured at the county rather than the state level, this study aimed to provide improved estimates of the association between Medicaid expansion under the ACA and fatal opioid-involved overdoses from 2001 to 2017. We examined this association for county × year counts of total opioid overdose deaths and separately by class of opioid . For comparison with prior research, we also examined all drug overdose deaths as a secondary outcome.Previous studiesSTROBE) reporting guideline.This serial, cross-sectional study used data from 3109 counties in 49 states and the District of Columbia from January 1, 2001, to December 31, 2017. We organized this information into a series of space-time observations, with each observation referring to 1 year of data per county for a total of 52 853 county-years (3109 counties × 17 years). Analyses excluded Alaska because of substantial changes in the size and shape of counties within the state during the study period. Individual data were aggregated to the county level. This study and was approved by the institutional review board of the University of California, Davis. No informed consent was required because this was a retrospective review of existing mortality data. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology  external cause of injury codes X40 to 44 , X60 to 64 (suicide), X85 (homicide), and Y10 to 14 (undetermined). Among deaths with drug overdose as the underlying cause, we used the following ICD-10 specific drug codes to identify our outcomes: all opioids, T40.0-T40.4 and T40.6; natural and semisynthetic opioids, T40.2; methadone, T40.3; heroin, T40.1; and synthetic opioids other than methadone, T40.4. Deaths involving more than 1 class of opioid were included in the counts for each opioid subcategory; thus, opioid subcategories are not mutually exclusive.We determined annual, county-level counts of opioid overdose deaths from the restricted-use version of the National Vital Statistics System multiple-cause-of-death files.4 We created an indicator of the proportion of each calendar year during which a given state had Medicaid expansion in effect; states that expanded Medicaid were assigned a value of 0 in years before Medicaid expansion, a value between 0 and 1 in the year in which Medicaid expansion went into effect (according to the policy effective month), and a value of 1 in all subsequent years, whereas states that did not expand Medicaid by the end of the study period were assigned a value of 0 in all years. Of the 32 states (including the District of Columbia) in our study population that opted to expand Medicaid eligibility, 26 did so on January 1, 2014, then 2 additional states did so later that same year, followed by 2 states in 2015 and 2 states in 2016 ; percentage male; percentages non-Hispanic white, non-Hispanic Black, and Hispanic; percentage of families living in poverty; median household income (per $10 000); percentage unemployed; population density (1000 residents per square mile); and overall mortality rate (per 1000 people). We also considered the presence of co-occurring state policies, which have been associated in prior research23 and for comparison with prior research, we conducted a secondary analysis with all drug overdose deaths as the outcome.We examined the association between state Medicaid expansion status and county-level risk of fatal opioid overdoses overall and by class of opioid using Bayesian hierarchical Poisson models, with overdose deaths assumed to be distributed proportionally to the population of each county (aged ≥12 years). We introduced a 1-year lag between overdose rates and Medicaid expansion to address the possibility of temporal bias and to allow time for changes in Medicaid coverage, services, and related behaviors to materialize. Analyses with Medicaid expansion instead measured concurrently with overdose rates produced similar results  and minimize the influence of large outlying rates in low-population counties by allowing each area to borrow strength from neighboring areas. All models also included fixed and random effects by county for Medicaid expansion to account for local variation in policy implementation across counties within states. We modeled secular trends in overdose using fixed linear and quadratic time trends and included annual, county-level sociodemographic covariates measured concurrently with overdose and co-occurring state policies with 1-year time lags.24 from April 1, 2018, to July 31, 2019. Integrated nested Laplace approximation is an alternative to standard Markov chain Monte Carlo methods for estimating the integral of a posterior (probability) distribution. Whereas Markov chain Monte Carlo samples from the posterior distribution of model parameters, integrated nested Laplace approximation returns comparable approximations to the posterior marginals in considerably less time.26 Results are reported as median relative rates (RRs) from the posterior marginal distribution and 95% credible intervals (CrIs) indicating a range of values that is expected to contain the true RR with 95% probability .Analyses were implemented using the Integrated Nested Laplace Approximation method in R software, version 3.4.3 There was a total of 383 091 opioid overdose fatalities across observed US counties for the study period of January 1, 2001, through December 31, 2017, with a mean (SD) of 7.25 (27.45) deaths per county  . The oveThe estimated associations of 1-year lagged Medicaid expansion with RRs of opioid overdose deaths, overall and by class of opioid, are presented in 23 produced substantively similar results as those in the primary analyses . Additional sensitivity analyses excluding 4 states with high levels of underreporting of specific drugs 27 examining Medicaid expansion and mortality from other causes, we found decreased rates of opioid overdose deaths associated with the adoption of Medicaid expansion. In particular, given 82 228 opioid-related deaths from 2015 to 2017 in the 32 states that expanded Medicaid between 2014 and 2016, our findings suggest that these states would have had between 83 906 and 90 360 deaths in the absence of the expansion, implying that Medicaid expansion may have prevented between 1678 and 8132 deaths in these states during those years.In this nationwide, population-based study of the association of Medicaid expansion under the ACA with county-level rates of opioid overdose mortality, we found empirical support for adopting and sustaining health coverage expansions as a potential tool for reducing opioid overdose deaths in the United States. Consistent with prior analyses28 We also did not find support for an association between ACA-related Medicaid expansion and natural and semisynthetic opioid overdose mortality.In analyses differentiated by class of opioid, we found a more substantial decreased risk associated with overdose deaths involving heroin and synthetic opioids other than methadone, which have been associated with continued increases in opioid-related deaths in recent years. These findings align with previous research that indicates that implementation of the ACA was associated with 40% decreased odds of being uninsured among persons with heroin use disorders, primarily because of Medicaid expansion, whereas no changes in insurance coverage were detected among persons with prescription OUDs.29 Recent evidence demonstrates that compared with nonexpansion states, Medicaid expansion states experienced increases in overall prescriptions for, Medicaid-covered prescriptions for, and Medicaid spending on both MOUDs, particularly buprenorphine and naltrexone, and the opioid overdose reversal medication naloxone.35The observed association between Medicaid expansion and decreased total opioid overdose deaths and deaths involving heroin and synthetic opioids other than methadone is likely in part attributable to the ACA’s inclusion of mental health and SUD services as essential health benefits. Expanded Medicaid eligibility has substantially increased access to these services among the low-income population.16 have found associations between income eligibility expansions for Medicaid and reductions in SUD-related deaths, and a recent study17 assessed changes in opioid-related deaths in Medicaid expansion vs nonexpansion states. Whereas the last study17 found that Medicaid expansion was associated with larger increases in opioid overdose mortality, particularly in 2015 and 2016, analyses were conducted only at the state level. This approach may have masked within-state variation in the level and rate of growth of opioid overdoses, as well as differences in local policy implementation. To our knowledge, ours is the first study to quantify the association between ACA-related Medicaid expansion and opioid-related deaths at the county level.Two prior studies32 On the basis of this evidence, in combination with our findings for heroin and synthetic opioids other than methadone, increased access to MOUDs likely not did not contribute to the observed increase in methadone mortality associated with Medicaid expansion. In contrast, past research has found high rates of methadone use to treat pain (rather than to treat OUD) among Medicaid beneficiaries and that the drug is disproportionately associated with overdose deaths among individuals in this population,34 underscoring the importance of ongoing local, state, and federal actions to address safety concerns associated with methadone for pain in tandem with Medicaid expansion.8Although the rate of methadone-related mortality is relatively low compared with other opioid classes, our finding that Medicaid expansion was associated with increased methadone overdose deaths deserves further investigation. At the individual level, treatment of OUD with methadone has been rigorously studied and found to be equally and, in some cases, more effective than other MOUDs in suppressing illicit opioid use, particularly heroin use, and retaining persons in treatment.ICD-10 coding of death certificate data, which may not reliably identify the specific drugs involved in fatal overdoses and may lead to an underestimation or misclassification of opioid overdose mortality.23 However, a secondary analysis that examined overdose deaths involving all drugs and sensitivity analyses excluding states with high levels of underreporting of specific drugs produced similar results as those in our primary models. Second, we included deaths from opioid overdoses across the entire population, not just among Medicaid enrollees, which may understate the estimated outcomes of Medicaid expansion for those individuals most directly affected. Third, although we controlled for various county-level sociodemographic characteristics and state-level co-occurring policies, unmeasured confounding is still a possibility. Fourth, we did not examine the specific provisions of Medicaid expansion that may be associated with changes in opioid-related deaths . In addition, this study focused on the association of Medicaid expansion with fatal overdoses only. Future studies should consider the association of expansion with the spectrum of opioid-related harms, including prevention of SUD and nonfatal overdoses. Also, future studies should explicitly examine possible mediators and moderators of the association between Medicaid expansion and opioid overdose risk, including access to and use of OPRs, MOUDs, and naloxone; local SUD treatment capacity; and the extent to which the association of Medicaid expansion with overdoses varies by individual sociodemographic characteristics and contextual conditions.This study has limitations. First, we relied on This study found that Medicaid expansion was associated with reductions in opioid overdose deaths, particularly deaths involving heroin and synthetic opioids other than methadone, but with increases in methadone-related mortality. These findings add to the emerging body of evidence that Medicaid expansion under the ACA may be a critical component of state efforts to address the continuing opioid overdose epidemic in the United States. As states invest more resources in such efforts, attention should be paid to the role that health coverage expansions can play in reducing opioid overdose mortality, potentially through greater access to MOUDs.


Thyroid hormone levels are reduced in cardiovascular diseases and this phenomenon is associated with worse outcomes. It is unclear whether the changes in thyroid hormone bioavailability to the affected myocardium are beneficial or if this is a maladaptive response. Experimental studies from animal models of acute myocardial infarction (AMI) suggest that thyroid hormone treatment may be beneficial. There is limited data available on the use of thyroid hormones in patients with AMI and heart failure and this suggests that treatment to normalise thyroid hormone levels may be safe and potentially efficacious. Similarly, evidence of thyroid hormone therapy in patients undergoing cardiac surgery or during cardiac transplantation is limited. It is therefore difficult to draw any firm conclusions about benefits or risks of thyroid hormone treatment in these conditions. Large scale clinical trials of thyroid hormones in patients with cardiac conditions are required to confirm safety and evaluate efficacy. Furthermore, it needs to be elucidated which hormone to administer (thyroxine or triiodothyronine), when in the disease pathway to treat, dose of thyroid hormone to administer, and which parameters to utilise to assess safety and efficacy. Until these important questions are answered thyroid hormone therapy in cardiovascular diseases must remain within the research domain. Over the course of the last few decades changes in lifestyle have led to increases in atherosclerotic conditions, such as obesity, hypertension, and type 2 diabetes mellitus. These lifestyle-related morbidities together with an increased lifespan has caused a steep rise in the incidence and prevalence of cardiovascular diseases (CVD). CVD, of which ischaemic heart disease is the predominant condition, affects hundreds of millions of individuals across the world . BehavioThyroid hormones regulate development and metabolism of various organs with the cardiovascular system being a major target for its actions . Minor cThyroid extract was the first hormonal preparation introduced into clinical medicine more than a century ago. In 1891, George Murray, a pathologist in Newcastle, England, injected sheep thyroid extract subcutaneously to a 46-year-old woman with typical clinical features of myxoedema . The cliWhen the striking effects of thyroid hormones on serum cholesterol levels became apparent, cholesterol determination and measurement of basal metabolic rate were utilised as indirect diagnostic tests of thyroid function, particularly in the absence of specific hormonal assays of thyroid function . Furtherd-thyroxine might be superior to l-thyroxine  in decreasing serum cholesterol levels without increasing myocardial oxygen demand [d-thyroxine compared to the group treated with placebo [d-thyroxine group experienced remarkably more proarrhythmic effects. This worrying side-effect was probably due to the fact that the dose of d-thyroxine administered (6 mg/day) was equivalent to more than 300 mg of l-thyroxine [d-thyroxine preparation used in the trial was contaminated with a high level of active l-thyroxine [Enthusiasm for the use of thyroid hormone therapy in CVD was however curbed when it was observed that patients with angina often experienced an increase in symptoms even when physiological doses of thyroid extract were used, especially if therapy was started abruptly. Therefore, it became standard clinical practice to start with low doses and to increase the hormone gradually. It was also believed that synthetic isomer n demand . But, a  placebo . It was hyroxine . In addihyroxine . Thus, tMore recently, data from experimental studies and epidemiological observations have shown that thyroid dysfunction is associated with higher cardiovascular morbidity and mortality –21. FurtThe two main thyroid hormones T4 and T3 are secreted by the thyroid gland in response to stimulation by thyroid stimulating hormone (TSH). Both T4 and T3 have biological actions but T3 is considered the active hormone. Thyroid hormone metabolism is regulated by enzymes called deiodinases. Type I (DIO1) and type II iodothyronine deiodinase (DIO2) lead to extrathyroidal T3 production from the precursor T4. DIO1, which is active in the liver and kidney, produces 15–20% of total circulating T3, whereas DIO2 activity located in the pituitary, brain, brown adipose tissue, and heart is responsible for the remaining two-thirds of T3 production –24. DIO3Thyroid hormones have wide-ranging effects on the cardiovascular system see Fig. . ThyroidIn the nucleus of the cardiomyocyte, T3 binds to thyroid hormone receptors (TR), which in turn bind to thyroid hormone response elements in the regulatory regions of target genes to control transcription. The two main TRs are TRα, which is highly expressed in cardiomyocytes, and TRβ. TRs can bind to thyroid hormone response elements even in the absence of thyroid hormones, leading to repression of transcription of target genes. Thus, gene regulation in the cardiomyocyte is dependent on the availability of thyroid hormones . ThyroidIn cardiac diseases, such as heart failure or acute myocardial infarction, the intracellular cardiomyocyte environment is affected by hypoxia, which in turn leads to inflammation . The hypThere are remarkable similarities between developmental processes and changes occurring in the damaged myocardium, which has refocused research on the role of thyroid hormones in the pathophysiology of cardiac disease. Disparate stressful processes such as pressure overload, hypoxia, ischaemia, inflammation, and metabolic disturbances result in the predominance of the foetal gene programming. This genetic developmental switch of the diseased myocardium is not well understood . HoweverThyroid hormones have multiple effects on the cardiovascular system, ranging from molecular and cellular effects to consequent hemodynamic alterations. There is increasing evidence that suggests an adverse relationship between thyroid dysfunction and heart failure Table . In patiReduced activity of DIO1 and DIO2: a decreased extrathyroidal conversion of T4 to T3 due to diminished activity of peripheral deiodinases, associated with a decreased transport of T4 into tissues , 36.Increased DIO3 activity: an increase in peripheral thyroid hormone break-down due to an ectopic induction of DIO3 activity in peripheral tissues leading to increased reverse T3 levels I .Central hypothyroidism: decreased plasma levels of TSH and by the concomitant abnormalities in thyrotropin releasing hormone .The physiological reasons for the development of a low T3 state in patients with severe illness and, in particular, with heart failure, are not clearly understood. There are at least three potential mechanisms that may explain the development of the low circulating T3 levels in patients with moderate and severe heart failure:The increased levels inflammatory proteins such as interleukins and cytokines  observed in patients with severe heart failure is associated in both experimental models and clinical scenarios with a pattern similar to low T3 state –42. ThisThe increased cardiac risk in patients with heart failure and low T3 syndrome offers a mechanistic basis for thyroid hormone therapy in patients with left ventricular dysfunction and low/borderline levels of serum T3. In this group of patients, restoration of normal thyroid hormone levels could positively re-establish myocardial gene expression, improve cardiac function and haemodynamics, and thus alleviate morbidity and mortality. A few clinical trials of thyroid hormones have been conducted in patients with heart failure. Moruzzi et al. demonstrated that short- and medium-term levothyroxine therapy in a small number of patients with idiopathic dilated cardiomyopathy (NYHA class II–IV) improved cardiac function and exercise performance, without any significant side effects , 47. In Despite these results, the small number of patients studied, the relatively short duration of treatment, and differences in the route and dose of T3 used, limit interpretation of results. Nonetheless, these results suggest that T3 therapy in patients with stable chronic heart failure may be beneficial. Larger multicentre trials for longer periods that also provide information on hard clinical outcomes such as mortality, arrhythmias, and hospitalisations are required before thyroid hormone therapy can be routinely prescribed in patients with heart failure as part of clinical practice.Thyroid hormone action is important for myocardial contractility, acts as a vasodilator and has a direct positive action on myocardial mitochondrial function. Furthermore, antiaptotic and antifibrotic effects of thyroid hormones may also attenuate maladaptive cardiac remodelling and improve myocardial function post-AMI. Reduction in concentration of the active thyroid hormone T3 in ischaemic cardiomyocytes has also been linked to the pathogenesis of ischaemia-reperfusion injury . In rodeThyroid hormones have several effects on the cardiovascular system that are relevant to the pathophysiology of the injured cardiomyocyte after an AMI. T3 plays a critical role in regulating mitochondrial function and morphology, modulating antifibrotic and proangiogenic effect, and also by impacting on cell regeneration and repair , 57. ThiAntiapoptotic and mitochondrial protection by modulating mitochondrial permeability transition pore (MPTP) opening –60.Positive effects on the thyroid hormone deactivating enzyme DIO3, MCT-10 (thyroid hormone transporter) and foetal gene re-expression, and thus restoring cardiomyocyte T3 levels .Attenuates infarct fibrotic area and reverses foetal gene expression .Improved myocyte preservation in the peri-infarct zone .Facilitate recovery of stunned myocardium and left ventricular function with positive effects on alpha- and beta-myosin heavy chain (MHC) and SERCA/Phospholamban ratios , 64.Thyroid hormones are essential in the regulation of molecular mechanisms of angiogenesis, cardioprotection, cardiac metabolism, and myocyte regeneration—processes that can modulate recovery from injury and left ventricular remodelling. Based on experimental studies, the protective effect of thyroid hormone on reducing ischaemic reperfusion injury and improving cardiomyocyte remodelling and function is considered to be mediated by:There have been a number of studies in patients with AMI that have evaluated T3 levels with morbidity and mortality . Most stThere is little data on the safety and efficacy of T3 in AMI patients apart from a recent phase II trial . In thisThere are several trials of thyroid hormones, particularly T3, in other cardiac conditions that provide useful information and help to draw valuable conclusions. T3 administration in patients undergoing cardiac surgery such as coronary artery bypass grafting, a procedure in which myocardial tissue is subjected to ischaemia-reperfusion injury, reduces troponin release and improves post-surgery haemodynamics . SimilarThyroid hormone use can have important implications for organ selection and cardiac function before and after transplantation. Experimental studies demonstrated a surge of adrenaline levels after the induction of brain death in a baboon, which was associated with rapid declines in plasma levels of various hormones including T4 and T3 . The redCurrent experimental and clinical evidence suggests that thyroid hormone treatment may be beneficial in improving cardiac function and that risks of arrhythmias appear to be low. However, despite encouraging indications from experimental studies, large adequately-powered interventional trials of thyroid hormones in patients with various cardiac conditions have not been conducted. One explanation for this is the common belief of physicians that exogenous thyroid hormone  therapy in cardiac patients could precipitate development of arrhythmia, myocardial ischaemia/ infarction, or worsening of congestive heart failure.


Imaging modalities aid clinicians in diagnosis, function assessment, gauging response to treatment and assisting pre-/intra-/postoperatively in surgery.The primary goal of pelvic cancer surgery is to perform radical resection to achieve complete tumour clearance (R0), leading to a significant increase in survival. Proper patient evaluation and surgical planning are necessary to achieve these goals. A wide range of pre-operative planning techniques can be applied, some of which allow surgeons to practice and refine the procedure, improving efficiency in the operating room, shortening operative time, and reducing incidence of iatrogenic complications.Magnetic resonance imaging (MRI) is an important diagnostic tool in identifying relevant anatomy and staging in rectal cancer. Findings on MRI confer major implications for diagnosis and management, including surgical planning, prediction of adverse surgical outcomes, selecting patients for neoadjuvant therapy and eligibility for surgery. MRI is also accurate at identifying invasion of cancer into adjacent pelvic compartments and structures . ExistinThe functional anatomy of the pelvic organs and muscles work synergistically to maintain continence. Dysfunction and disruption of these intricate inter-related structures through disease or surgical intervention result in debilitating conditions such as faecal/urinary incontinence, obstructive defecation syndrome and prolapse.Three-dimensional 3D) imaging can be used to construct ‘virtual’ 3D objects that are visualised and manipulated on a computer screen or Head-Mounted Display (HMD), whilst 3D printing can be used to create 3D objects under computer control using successive layers of material. First described in the 1980s, 3D printing technology has established commercial and research applications using materials such as metal, plastic, or ceramic , 3. HoweD imagingRecently, Sahnan et al.  publisheDespite these rapid advances in the operative application of 3D technology, surgical simulation and planning based on 3D models are not yet routinely used. There are several reasons for its slow adoption. A crucial consideration is the segmentation process , which is typically time consuming and highly operator dependent, yet is the initial step in 3D model generation.Working closely with radiologists, we have established rigorous and reproducible segmentation criteria of male and female pelvic anatomy, and pelvic compartments from MRI scans that streamline the process and ensure the reliability of the resulting 3D models, paving the way for building a substantial database and for the development of fully automated, data-driven segmentation tools. Particular emphasis was given to colorectal cancer (CRC), anatomy, and the boundaries and contents of the central compartment , due to its medical and surgical importance, specifically in total mesorectal excision (TME) Fig. .A total of ten pelvic MRIs from healthy volunteers  have been successfully segmented with our segmentation criteria Fig. , using wWe believe that the use of 3D models can provide doctors with more and better informed choices for surgical planning and consulting patients. Carefully constructed from imaging data, they offer a true-size, detailed representation for surgeons to use, making pre-operative planning more direct and accurate. 3D models can be used as an additional tool in the surgical planning for organ/compartment preservation, where fibrosis or disease post-chemoradiotherapy cannot be adequately estimated with current MRI models. They may also improve surgical efficacy and safety through intraoperative augmented reality and can support the simulation of pelvic organs and their function, with the potential for clinical, educational and engagement (clinicians and patients) applications in the future.The work is very timely from both a CRC and technology perspective. CRC prognosis and survival are based on accurate initial staging. The use of 3D models as an additional tool in staging will improve accuracy and surgical planning. The innovation leverages recent advances in 3D graphics and visualisation, 3D printing and virtual/augmented reality. In the short term, we expect the work to result in adopted guidelines for the building and routine use of 3D models of pelvic anatomy. This will be followed by medium-term technological developments enabling automated 3D model generation, large-scale 3D pelvic anatomy databases, application of machine learning approaches, clinical translation into CRC surgical planning and dynamic function simulation.


Background: Effective hypertrophy-oriented resistance training (RT) should comprise a combination of mechanical tension and metabolic stress. Regarding training variables, the most effective values are widely described in the literature. However, there is still a lack of consensus regarding the efficiency of advanced RT techniques and methods in comparison to traditional approaches. Methods: MEDLINE and SPORTDiscus databases were searched from 1996 to September 2019 for all studies investigating the effects of advanced RT techniques and methods on muscle hypertrophy and training variables. Thirty articles met the inclusion criteria and were consequently included for the quality assessment and data extraction. Results: Concerning the time-efficiency of training, the use of agonist–antagonist, upper–lower body supersets, drop and cluster sets, sarcoplasma stimulating training, employment of fast, but controlled duration of eccentric contractions (~2s), and high-load RT supplemented with low-load RT under blood flow restriction may provide an additional stimulus and an advantage to traditional training protocols. With regard to the higher degree of mechanical tension, the use of accentuated eccentric loading in RT should be considered. Implementation of drop sets, sarcoplasma stimulating training, low-load RT in conjunction with low-load RT under blood flow restriction could provide time-efficient solutions to increased metabolic stress. Conclusions: Due to insufficient evidence, it is difficult to provide specific guidelines for volume, intensity of effort, and frequency of previously mentioned RT techniques and methods. However, well-trained athletes may integrate advanced RT techniques and methods into their routines as an additional stimulus to break through plateaus and to prevent training monotony. Resistance training (RT) is a primary exercise intervention used to develop strength and stimulate muscle hypertrophy. Increases in muscle mass constitute key components of conditioning in various sports due to the correlation between muscle cross-sectional area and muscle strength ,2. AdditMuscle hypertrophy occurs when muscle protein synthesis exceeds muscle protein breakdown and results in positive net protein balance in cumulative periods . This coThe purpose of the present paper was to provide an objective and critical review related to advanced RT methods and techniques influencing skeletal muscle, which may contribute to maximizing muscle hypertrophy in both recreational and competitive athletes.MEDLINE and SPORTDiscus databases were searched from 1996 to September 2019 for all studies investigating the effects of advanced resistance training techniques and methods on muscle hypertrophy and training variables. The search was performed using the following keyword combinations: (‘strength training’ OR ‘resistance training’ OR ‘hypertrophy training’ OR ‘muscle’) AND . The present review includes studies that (1) presented original research data on healthy adult participants in an age range of 19−44 years old; (2) were published in peer-reviewed journals; and (3) were published in the English language. No sex restrictions were imposed during the search stage.Research studies investigating the effects of advanced resistance training techniques and methods on muscle hypertrophy and training variables were the primary focus of the literature search. Early screening of the articles was based on titles and abstracts. A total of 1088 studies were initially identified for further scrutiny.The next step was to select studies with respect to their internal validity: (1) comparison of different advanced RT techniques and methods with the RT programs performed in traditional training protocols, (2) muscle hypertrophy and/or muscle strength and/or training volume were assessed pre- and post-intervention; for muscle hypertrophy both muscle cross-sectional area changes  and changes in muscle thickness (ultrasound imaging) were considered, while for muscle strength, tests with a repetition maximum (RM) component  were considered; for training volume changes in the number of repetitions, total load and time under tension to muscular failure were considered. The researchers conducted the literature review independently based on inclusion and exclusion criteria. In total, 30 studies met the inclusion criteria for the review .Experimental details of the studies included in the review .Three major factors are emphasized in the conventional hypertrophy model: mechanical tension, metabolic stress, and muscle damage . These fFurthermore, implementation of advanced RT techniques could provide an additional stimulus to break through plateaus for trained subjects  and prevOne of the advanced RT techniques is based on a prolonged duration of the eccentric phase of the movement. The duration of each repetition can be identified by movement tempo, which is determined by four digits  corresponding to the duration (in seconds) of particular phases of movement  . ChangesHowever, studies indicate a wide range of manipulation of the duration of the eccentric phase of movement can be employed if the primary goal of training is to maximize muscle hypertrophy ,60. AlthAnother useful method that can be used during RT, based on eccentric contractions includes accentuated eccentric loading (AEL). This training strategy is based on the muscles’ ability to generate greater force during maximal eccentric (~20−60%) versus other types of contraction. The use of weight releasers allow for overloading the muscles during the eccentric phase of movement due to its specific construction. The weight can be unloaded in the transition from the eccentric to the concentric phase of movement. The use of high-loads during the eccentric phase of movement is associated with significant exercise induced muscle damage and mechanical tension, which have been associated with a hypertrophic response . FurtherFurthermore, due to the greater mechanical tension, it could provide an additional hypertrophic stimulus ,33,65. AAnother RT method that allows for the avoidance of high mechanical stress associated with high-load RT and the high training volumes required when exercising with low-loads to volitional fatigue is to combine RT under blood flow restriction (BFR) ,66,67. TThe most frequently and evidence-based repetition and set scheme involves 30 repetitions in the first set followed by three sets of 15 repetitions with 30 s rests in between with 20−40% 1RM and pressure, which contribute to 40−80% of arterial occlusion pressure . HoweverAnother RT technique that partly allows for the balance of both mechanical tension and metabolic stress consists of cluster sets. In a traditional scheme of sets, repetitions, a chosen group of exercises are performed consecutively, with a long inter-set rest interval, are then followed by another set of repetitions. On the contrary, cluster sets include short, inter-set rest intervals (20−60s) with a lower number of repetitions . PreviouSupersets and pre-exhaustion during RT can be defined as a pair of different exercise sets performed without rest. Supersets most commonly consist of two exercises for the same muscle group , agonistThe pre-exhaustion technique is commonly used by bodybuilders seeking to enhance the muscle growth of target muscles. The rationale for this technique is that performing a single-joint exercise first fatigues the agonist in isolation, thereby placing greater stress on the agonist and increasing its activation during multi-joint exercise and potentiating its hypertrophy . AnotherIn conclusion, practitioners aiming to maximize training volume and intensity of effort may be well advised to consider the use of supersets (agonist–antagonist and upper–lower body) in their RT programs. The use of these exercise orders may be more time-efficient than the traditional approach, and especially useful when time limitations exist in the planning of training sessions.Drop sets involve performing a set to volitional fatigue with a given load and then immediately reducing the load  and continuing the exercise until subsequent volitional fatigue . BrieflySimilarly to drop sets, sarcoplasm stimulating training (SST) consists of sets of exercises performed at 70–80% 1RM to volitional fatigue and then repeating this protocol twice more with 20 s rest intervals in between. The next step is to reduce the external load by 20% and perform an additional set with a 4/0/1/0 tempo; following a 20 s rest interval, 20% of the external load is reduced again, and a set with 4/0/1/0 tempo is completed to volitional fatigue. In the last set, the load is further decreased by 20% and after its completion, following a 20 s rest interval, a static hold is performed  to volitional fatigue . AnotherEvidence suggests the beneficial effects of both drop sets and SST in acute increases in triceps brachii muscle thickness  in both The present review has several limitations that should be addressed. The majority of included studies did not control nutritional intake, which can affect the magnitude of muscle adaptations. Another limitation relates to studies that examined the influence of advanced methods and techniques on training variables, but did not analyze hypertrophic responses and/or strength improvements, which would be the basis for explaining their efficiency. In addition, only one study  directlyConsidering the aforementioned studies, effective hypertrophy-oriented training should comprise a combination of mechanical tension and metabolic stress. In summary, foundations for individuals seeking to maximize muscle growth should be hypertrophy-oriented RT consisting of multiple sets (3−6) of six to 12 repetitions with short rest intervals (60 s) and moderate intensity of effort (60−80% 1RM) with subsequent increases in training volume (12–28 sets/muscle/week) . MoreoveFurthermore, persistence in training and diet is essential. Recently, research has shown that muscle hypertrophy that occurs at initial stages of RT (~4 sessions) is mostly attributable to muscle damage induced cell swelling with the majority of strength gains resulting from neural adaptations (8−12 sessions). Within the latter phase of RT (6−10 weeks), muscle growth begins to become the dominant factor .


Brain injury in the asphyxic newborn infant may be exacerbated by delayed restoration of cardiac output and oxygen delivery. With increasing severity of asphyxia, cerebral autoregulatory responses are compromised. Further brain injury may occur in association with high arterial pressures and cerebral blood flows following the restoration of cardiac output. Initial resuscitation aims to rapidly restore cardiac output and oxygenation whilst mitigating the impact of impaired cerebral autoregulation. Recent animal studies have indicated that the current standard practice of immediate umbilical cord clamping prior to resuscitation may exacerbate injury. Resuscitation prior to umbilical cord clamping confers several haemodynamic advantages. In particular, it retains the low-resistance placental circuit that mitigates the rebound hypertension and cerebrovascular injury. Prolonged cerebral hypoxia–ischaemia is likely to contribute to further perinatal brain injury, while, at the same time, tissue hyperoxia is associated with oxidative stress. Efforts to monitor and target cerebral flow and oxygen kinetics, for example, using near-infrared spectroscopy, are currently being evaluated and may facilitate development of novel resuscitation approaches. Birth asphyxia is the term used to describe the continuum of physiological derangements seen in newborn infants as a result of a prolonged or profound mismatch between oxygen demand and oxygen delivery. It ranges from mild to severe; however, when severe, it can cause irreversible cerebral cell damage and death, leading to a syndrome of hypoxic–ischaemic encephalopathy (HIE). HIE may lead to altered conscious state, autonomic instability, absence of primitive reflexes, seizures, and death. Worldwide, birth asphyxia accounts for 24% of neonatal deaths and around 800,000 deaths annually under the age of five years . For surWhole-body and selective head cooling target the secondary phase of reperfusion injury and energy failure, and must be commenced within six hours of birth . An impoin utero, the foetus has very efficient in-built compensatory mechanisms that allow it to survive with reduced oxygen delivery. Experiments in sheep showed that the foetus responds to hypoxia by ceasing breathing  and body movements and by redistributing cardiac output. While, initially, foetal hypoxia induces a vagal bradycardia, this changes into a sustained tachycardia (within an hour) as the hypoxia continues [2), where cerebral DO2 = cerebral blood flow × blood oxygen content. Birth asphyxia may result from a variety of perinatal events, including placental detachment, placental blood flow disruption, foetal blood loss, compression of the umbilical cord, prolonged labour, maternal hypoxia, and failure of the newborn to initiate pulmonary gas exchange . All of ontinues . These cThe foetal response to continuing asphyxia diverges, depending upon its severity and whether the umbilical cord remains intact. If survivable, the redistribution of cardiac output is sustained eventually (weeks/months), leading to an asymmetric growth restriction, with a significantly higher brain-to-body/weight ratio. Interestingly, despite the sustained hypoxemia, foetal breathing movements and body movements return, indicating a resetting of the chemoreceptor-induced inhibition of breathing to a much lower oxygenation level . The impAs the duration of asphyxia increases, these adaptive mechanisms are overwhelmed by a continued reduction in oxygen levels, which, despite the increase in coronary blood flow, leads to a reduction in cardiac oxygen delivery. As myocardial metabolism is highly oxygen-dependent, the reduction in oxygen delivery combined with the gradual exhaustion of tissue oxygen stores (myoglobin) leads to a loss in myocardial function . As a reIn response to asphyxia, autoregulatory signalling pathways at the endothelial–astrocyte junctions maintain relatively constant cerebral blood flow despite falling oxygen levels . As asph2) of predominantly foetal haemoglobin in normal foetuses is approximately 60% [2) may decrease to 30% (corresponding to a pO2 of 12–15 mmHg) or even lower, with no significant acidosis [2 in normal infants rapidly rises to reach on average 80% by 3–4 min and >90% by 6–7 min [2 to increase may signal inadequate lung aeration and/or elevated pulmonary vascular resistance and a persistence of the foetal circulation. An increase in SpO2 is necessary to avoid persistent pulmonary hypertension and acidosis in light of increasing metabolic demand caused by breathing and thermogenesis [In utero, the preductal arterial oxygen saturation . Crucially, avoidance of a rapid rise in blood pressure during transition is likely to be desirable. This may be achieved by keeping the placenta attached to the systemic circulation until well after the sympathetic and catecholamine-driven surge in cardiac output plateaus. However, it remains unclear when the ideal time is to clamp the cord. There may be a role for monitoring preductal blood pressure in the delivery room. Currently available methods utilise the inflation of a cuff and may even provide continuous blood pressure monitoring [2 signal and may not be acceptable for clinical use during resuscitation. Another possibility that is currently available in some pulse oximeters is monitoring of the “perfusion index”. The perfusion index is the ratio of the pulsatile blood flow to the non-pulsatile or static blood in peripheral tissue, thus representing a non-invasive measure of systemic vascular resistance [Following ROSC, cerebral oxygen delivery (including blood flow) may be carefully titrated to avoid secondary injury. Components of oxygen delivery that may be manipulated in the delivery room are oxygen saturation and cerebral vascular dilatation via control of partial pressure of carbon dioxide  and fractional tissue oxygen extraction (FTOE) using near-infrared spectroscopy (NIRS) during the foetal-to-neonatal transition [2 and/or high absolute level may be a useful marker for an undesirable surge in oxygenation/perfusion to the brain. In preterm lambs, crSO2 was shown to correlate well with cerebral blood flow in conditions of constant arterial oxygen saturation and metabolic demand. Concordance is best in the time domain, where crSO2 correlates well across a moderate range of flow volume changes that are allowed to reach a steady state. When blood flow fluctuates, crSO2 corresponds with low-frequency variations with greater fidelity, and is likely to reflect the time delay in achieving equilibrium of oxygenation in the venous blood pool [Monitoring peripheral arterial oxygen saturation provides little information about the adequacy of oxygen delivery, as it does not account for cerebral blood flow, haemoglobin concentration, or metabolic demand. Prolonged cerebral hypoxia–ischaemia is likely to contribute to perinatal cerebral injury, while hyperoxia is associated with oxidative stress . There iansition ,55,56. Cansition ,58. In tood pool ,60.2 may be challenging in the context of acute recovery from an asphyxial insult, as multiple variables are likely to be rapidly changing. These include SpO2, metabolic demand, pCO2, and haemoglobin (from delayed cord clamping), ductal and foramen ovale shunt direction, and, therefore, left ventricular output [2 readings, but may be concurrently monitored and accounted for by calculating FTOE (FTOE = (SpO2 − crSO2)/SpO2), metabolic demand by monitoring amplitude integrated electroencephalography (aEEG) [2 as a surrogate for PaCO2 [2 and includes a consideration of these multiple parameters seems physiologically logical, but may require automated feedback algorithms and is yet to be evaluated [2 may reflect a drop in PaCO2 from overventilation and subsequent reduction in cerebral blood flow, and would be best addressed by reducing minute ventilation rather than increasing the fraction of inspired oxygen. A limitation, however, is that changes in crSO2 are likely to lag behind these rapidly changing physiological variables, but may be reflective of slower changes in oxygen extraction and consumption at the tissue level. Nevertheless, the core principle stands, in that the pattern of change in crSO2, when measured reliably, provides a global picture of (mis)match between oxygen delivery (which includes blood flow) and metabolic demand. The piglet brain exposed to hypoxia–ischaemia is being used to develop computational models of alterations in blood flow and metabolism that can also help to explain changes at a cellular level [2, and pCO2 targets to reduce ischemia–reperfusion injury in the delivery room. Interpreting crSOr output ,62. Thesy (aEEG) , and endor PaCO2 ,65. An avaluated ,66,67. Far level . NIRS-dear level . It is yMost of the knowledge derived to date is from a mix of preterm and term animal studies. It is unknown how similar or different their responses may be in relation to cerebral autoregulation in the setting of perinatal asphyxia in humans. Studies in neonates are challenging because antenatal monitoring is often non-specific in identifying which infants will be born with signs of asphyxia. Research in the emergency setting of resuscitation of an infant who was not anticipated to be asphyxic at birth will require novel trial methodology and tailored ethical considerations. The benefit of physiologically-based cord clamping is likely to be dependent on the cause of asphyxia. In cases of placental or cord compromise, delaying cord clamping may provide little or no benefit in terms of continued venous return to the neonate. On the other hand, leaving the placental circulation intact may confer protection from rebound hypertension. How long the placental circulation should be left intact is still unknown but may be directed by non-invasive markers that indicate a normalisation in blood pressure for the individual neonate. Consideration will also need to be given to the active management of third stage of labour.Several groups have developed techniques and equipment to facilitate neonatal resuscitation with cord intact. These range from high-end, custom-built devices  ,71 to moHaemodynamic instability during delivery room management of asphyxic infants is increasingly being recognised and may represent a therapeutic target with potential for secondary prevention of injury. Resuscitation with the placental circulation intact is feasible and may attenuate the cerebrovascular hypertension that evolves during recovery from the acute hypoxic insult. Future studies should address both prolonged oxygen deficiency and oxygen-mediated toxicity in the delivery room, and more work is needed to define what parameters are most useful to monitor in this regard.


However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8+ T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors . This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably  Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are significant contributors to the global disease burden with ~36.9 million people living with HIV-1 and at least 71 million people persistently infected with HCV WHO, . Anti-reHIV-1 and HCV are rapidly mutating RNA viruses that exhibit considerable genetic diversity  although the results are yet to be disclosed.DNA vaccines against HCV have been routinely tested in small and large animals including non-human primates  cells residing in the female reproductive tract, the gut, the lung and the liver form a formidable frontline defense against various pathogen infections  maintain a stable and durable population following their formation in tissues even in the absence of cognate antigen encounter following their formation  and appear to be crucial for protection against liver tropic pathogens and pathogens exposed in the vagina and the female reproductive tract compared to circulating memory T cells  amongst the total CD8+ T cell population correlated with partial control of viraemia in Hepatitis B Virus (HBV)-infected patients , protein loaded nanoparticles (NP), adenovirus (Ad) vectors, adeno-associated virus (AAV), and HPV pseudovirus (HPV PsV) have been developed to elicit localized protection and in some instances elicit CD8+ TRM cells in the liver or the vagina  viruses, establish infection following transmission reflecting a strong genetic bottleneck -15 and/or inflammation in the liver can be effective in recruiting circulating effector CD8y routes  , etc. These aspects have been reviewed extensively elsewhere  necessary to home to the liver (Sato et al., + T cells are recruited to the cervicovaginal mucosa or the liver following introduction of a vaccine vector or an agent (+ TRM cells. Several studies have shown that the number of CD8+ TRM cells is a crucial parameter that dictates the collective ability of these cells to confer protection against pathogens exposed in the skin, liver, or the vagina with greater numbers favoring protective outcomes (Cuburu et al., DNA can be used as a vector to prime high numbers of circulating antigen-specific T cells (Gummow et al., an agent  to facil+ TRM cells against these viruses and also reviewed strategies as well as caveats associated with using DNA to elicit localized CD8+ TRM cells as a frontline defense against HIV-1 and HCV.DNA has recently re-emerged as an effective vaccination platform in humans, but its use in developing a T cell-based vaccine will likely rely on its ability to be exploited in a regimen that can elicit robust immunity in the vagina and the gut in the context of HIV-1, or the liver in the context of HCV. In this regard, we have highlighted the importance of eliciting cervicovaginal or intrahepatic CD8DW and ZM conceived the initial drafts of the manuscript. BG-B, MM, AS, CR, RB, AL, and EG revised many parts of the manuscript and contributed to finalize the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.