Sound is ubiquitous in nature. Recent evidence supports the notion that naturally occurring and artificially generated sound waves contribute to plant robustness. New information is emerging about the responses of plants to sound and the associated downstream signaling pathways. Here, beyond chemical triggers which can improve plant health by enhancing plant growth and resistance, we provide an overview of the latest findings, limitations, and potential applications of sound wave treatment as a physical trigger to modulate physiological traits and to confer an adaptive advantage in plants. We believe that sound wave treatment is a new trigger to help protect plants against unfavorable conditions and to maintain plant fitness. Sound is an omnipresent feature throughout the world . The defTo understand how plants respond to sound, we need a new framework beyond chemical compound-based signal initiation and responses in plants. We therefore classified the steps involved in this multi-layered process from the emission of sound by plants to the altered phenotypes observed after the plant has recognized the sound information. This basic knowledge helps us elucidate how sound signals trigger changes in plants in nature.Figure 1A). Since the xylem is a water-transporting system in plants, transpiration, and re-hydration occur in xylem vessels. Transpiration produces tension in xylem vessels, and simultaneously, gas bubbles (cavitation) are produced in xylem vessels during transpiration. Indeed, gas bubbles adhering to vessels may produce sound in plants  encoding mechanosensitive ion channels, which may recognize mechanical signals, was reported to differ between sound-exposed and touch-treated Arabidopsis plants .How can plants perceive sound and thereby respond to specific stress stimuli without a hearing organ? The roots of c system , indicats plants . This sus plants . Here, wald) and Rubisco small sub-unit (rbcS) genes, which play critical roles in photosynthesis, were specifically induced in rice following 125 and 250 Hz sound treatment (Table 1). Continuous exposure to sound is thought to enhance plant growth by promoting CO2 fixation . These results imply that sound vibrations provide a stimulus to plants. More extensive research is needed on the function of the identified genes and the signaling network. Many questions still remain to be answered, such as “which part of the response is specific to sound ?” and “Can plant recognize the sound and other mechanical signals differently?”Although sound is not a visible or chemical stimulus, plants exposed to sound  produce increasing amounts of mRNA , suggestreatment   and gibberellin (GA) 3 and the defense-related hormones salicylic acid (SA) and jasmonic acid (JA) (Chrysanthemum exposed to a 1.4 kHz sound stimulus (Table 1). Although the optimal sound treatment differs depending on the plant species, such sound-induced hormonal changes might increase plant growth and provide strong resistance against biotic or abiotic stress. A recent study reported that plant roots can respond to environmental sound (Table 1). Specifically, Pisum sativum roots locate water by actively growing toward flowing water belowground  . Increasstimulus  . The Ca2+ ions influx the cytosol from outside the plants membrane by 1000 Hz sound exposure. These ions might serve as secondary messengers upon exposure to environmental stress, thereby enhancing plant resistance against microbial pathogens. Arabidopsis calmodulin-like 38 (CML38) gene, which encodes a calcium-binding messenger protein, is upregulated in response to sound treatment in Arabidopsis leaves . Water deficiency is first detected in the plant root, and drought stress signaling is transmitted to the shoot through the xylem. Since membrane architecture changes in response to sound treatment, the plant is better able to absorb water in situations where water is lacking. From a hormonal perspective, crosstalk between ABA and JA regulates the response to drought  elicits representative SA and JA defense signaling pathways similar to those observed in response to different chemical triggers . Meta-ans leaves . In addis leaves . In addis leaves  . The expression of ethylene biosynthesis genes ACS2, ACS4, ACO1, E4, and E8 and ripening-related genes RIN, TAGL1, HB-1, NOR, and CNR was delayed in tomato treated with 1 kHz sound versus the control (Table 1). Although the optimal sound conditions (frequency and decibels) must be determined depending on crop species, the use of sound wave treatment would be a convenient way to delay fruit ripening without the use of chemical preservatives or genetic modification. In addition to delaying fruit ripening, perhaps the quality and yields of post-harvest crops can be improved by sound treatment.Fruit ripening is associated with dramatic increases in ethylene production after harvest. Reducing ethylene production is an important way to delay fruit ripening. We previously showed that sound-treated tomato showed reduced ethylene production and delayed softening compared with the control  . The result would be good to speculate not only on direct cellular mechanisms but also on indirect targets such as hormones and photosynthesis signaling while sound transduction pathway remains to be identified. Additionally, the improvement of plant growth by sound treatment has been studied in many crops such as chrysanthemum, sweet potato, cucumber, lettuce, spinach, cotton, rice, and wheat (Table 1). However, the mechanism underlying how plant growth is improved by treatment with sound waves has not been intensively studied. A simple explanation for this effect is that this treatment alters the levels of plant growth regulatory hormones. As mentioned earlier, sound exposure alters endogenous hormone levels in plants. Increased IAA and decreased ABA levels in response to sound exposure may be the major factors underlying the effect of sound waves on promoting plant growth. Other studies have shown that the levels of soluble proteins and soluble sugars increase in response to sound treatment (Table 1). Soluble sugars can also be a factor in promoting plant growth, as they can serve as an energy source. In addition, although the proper frequency of sound differs depending on plant species, a number of molecular studies support the notion that sound also induces plant growth promotion and seed germination. Of the possible mechanisms underlying the plant growth-promoting effects of sound treatment, the enhancement of photosynthesis represents a strong candidate for further characterization . Increased photosynthetic ability has been observed in strawberry and rice in response to sound treatment (Table 1). Proteomics analysis showed that photosynthesis-related proteins were highly expressed at 8 h after 250 or 500 Hz sound exposure in Arabidopsis (Table 1). Since sound energy induced secondary products can make chemical energy, sound treatment is thought to improve photosynthesis (Sound treatments have been broadly applied to alter plant growth. For example, sound-treated tomato showed 13.2% increased yields compared with the control . In contnd wheat  (Table 1reatment  (Table 1reatment  (Table 1bidopsis  (Table 1ynthesis . These fSound represents a potential new trigger for plant protection . To dateC-MR and JJ designed the review. JJ, S-KK, JK, M-JJ, and C-MR wrote the review.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.


Pneumocystis jiroveci pneumonia. The third patient was treated with cisplatin-based chemotherapy for carcinoma. First two cases revealed foci of diffuse tubular dilatation containing hyaline casts and interstitial inflammatory infiltrate, in addition to globally sclerotic glomeruli with ultrastructural foot process fusion and mesangium expansion. The third case showed acute tubular injury and cast formation of irregular casts composed of amorphous or granular material of low density admixed with scattered high electron-dense globules. Myeloma-like cast nephropathy and true myeloma cast nephropathy pose similar destructive effects on renal parenchyma. This new pattern of HIV-related nephropathy should be considered in HIV patients with MLCN, once monoclonal gammopathy is ruled out.Myeloma cast nephropathy is an obstructing disorder of renal tubules, caused by precipitation of Bence Jones proteins. Myeloma-like cast nephropathy (MLCN) has been reported in the literature to occur in various primary renal and nonrenal diseases. We present a series of three rare cases of cast nephropathy, two of which are HIV patients, and the third patient is receiving cisplatin-based chemotherapy. However, in all three patients plasma cell dyscrasia has been ruled out. A 30-year-old male was admitted to the hospital with facial cellulitis. The second patient is a 31-year-old male who presented with Myeloma cast nephropathy (MCN) is the most common pattern of renal injury found in plasma cell dyscrasia and more specifically multiple myeloma (MM). It commonly presents as acute renal injury, which prompts the physician to perform renal biopsy. The pathophysiology of MCN is precipitation of monoclonal immunoglobulins and Tamm-Horsfall glycoproteins in distal tubules and collecting ducts. The histologic hallmark of MCN is the presence of tubular casts, which appear eosinophilic and brittle and usually instigate an intense inflammatory response (cellular reaction) in kidney leading to acute renal failure (ARF); if untreated it can lead to irreversible damage , 2.Myeloma-like cast nephropathy (MLCN) has been reported in the literature to occur in various primary renal and nonrenal diseases including some neoplastic processes, such as pancreatic carcinoma . Some reHuman immunodeficiency virus associated nephropathy (HIVAN) is the most common disease delineated in biopsy series of patients with HIV infection and renal disease , 6. Two We present three patients with MLCN. Two of them were associated with HIV infection while the third patient was treated with cisplatin-based chemotherapy.A 30-year-old African American male was admitted to the hospital who sustained a laceration a year ago for which he required stitches, and at that time he had some facial swelling. Physician suspected the fungus and antifungal was prescribed at that time. He was doing fine when he presented to our hospital with recurrent painful swelling and yellowish discharge. Comprehensive blood profile along with renal functions tests, CT orbits, and HIV testing were performed. He had an elevated BP and creatinine. HIV was positive and CT showed preseptal swelling. Skin biopsy came positive for coccidiosis. Nephrology consultation for suspected acute renal failure, with mild proteinuria (2.2 g/24 h), was done and a left renal needle biopsy was performed. Pneumocystis carinii pneumonia (PCP). A right renal needle biopsy was performed.A 31-year-old African American male came to ER with complaints of progressive difficulty breathing for 4 months and unintentional weight loss of about 50 lbs. Chest X-ray was done which showed reticular nodular pattern. Infectious disease consultation was sought. Comprehensive blood profile along with renal functions tests and HIV testing were performed. He had an elevated BP and creatinine. HIV was positive. Nephrology consultation was done for an elevated creatinine (17 mg/dl) and proteinuria (3.7 g/24 h) with microscopic hematuria, and hemodialysis was initiated. CT scan showed bilateral ground-glass opacities. Bronchoalveolar lavage was done which confirmedA 65-year-old male with history of squamous cell carcinoma of tongue was being treated with cisplatin and radiation therapy. During the second round of chemotherapy he developed acute renal failure with oliguria and markedly elevated serum creatinine levels. The drug history was negative for nonsteroidal anti-inflammatory drugs (NSAIDs). Emergent renal biopsy was performed and appropriately triaged.HIV status for cases I and II was confirmed by ELISA and Western Blot tests.Myeloma work-up included serum protein electrophoresis and immune-fixation confirmed absence of monoclonal gammopathy.All two cases were diagnosed as myeloma-like cast nephropathy. In HIV positive patients biopsy finding confirmed HIV associated changes in addition to myeloma-like cast nephropathy. In all two cases immunofluorescence microscopy failed to demonstrate monoclonal restrictions.Renal needle biopsy was done showing conventional HIV associated nephropathy with coexisting myeloma-like cast nephropathy. Light microscopy revealed foci of tubular cystic dilatation and diffuse tubular dilatation containing eosinophilic, Periodic Acid–Schiff (PAS) negative casts coupled with segmental collapse of capillary tufts and focal glomerulosclerosis. Multiple casts have intense neutrophilic reaction Figures  and 2. FThe ultrastructural findings include podocytes with extensive foot process effacement . The mesDirect immunofluorescence stains were performed using a panel of ten antisera. It failed to demonstrate monoclonal restriction. All glomeruli were negative for albumin, fibrinogen, C1q, C3, C4. IgA, IgM, kappa, and lambda light chain stains.Renal disease is one of the major causes of morbidity and mortality in HIV infected patients receiving effective antiretroviral treatment. HIVAN, the classic kidney disease of HIV infection and the most common cause of CKD in HIV infected individuals, is mostly seen in African American descent and is consistent with a strong genetic predisposition. HIV mediates dysregulation of glomerular podocytes, the epithelial cells that maintain the glomerular basement membrane, and apoptosis of renal tubular cells. The resulting lesion of HIVAN is a focal glomerulosclerosis (FGS) and microcytic dilation of the tubules filled with PAS positive casts , 11. HIVIt appears that HIVAN occurs in the setting of specific host genes and it has been widely accepted that the gene encoding a non-myosin A heavy chain (MYH9) is associated with FGS, HIVAN, and end-stage kidney disease (ESKD) due to hypertension in individuals of African descent , 24. TheThe MLCN, as described earlier, has similar histomorphological features to MCN and both can lead to renal damage. CD4, a receptor for HIV-1 on lymphocyte and macrophages, is the main culprit involved in the pathogenesis of renal cast formation. The renal lesion, MLCN, caused by HIV-1 bears a similarity to the nephropathy seen in cases of myeloma kidney, and it is also quite similar to the lesions seen in experimental Bence Jones protein nephropathy . The lesCisplatin, a chemotherapeutic agent that has been well recognized as a nephrotoxic agent, primarily causes tubulointerstitial lesions and in spite of extreme efforts of finding less toxic alternatives, cisplatin yet is prescribed widely. The nephrotoxicity related to cisplatin involves multiple pathways including apoptotic cascades activation, endonucleases, and oxidant stress. Most of these pathways share the same mechanisms for the cytotoxic effects of cisplatin on neoplastic cells; hence the strategies to reduce the nephrotoxic effects of cisplatin may reduce the antineoplastic effects of cisplatin . DespiteCisplatin is cleared by the kidney by both glomerular filtration and tubular secretion and its concentrations within the kidney exceed those in blood suggesting an active accumulation of drug by renal parenchymal cells. It damages the proximal tubules, specifically the S3 segment of the outer medullary stripe, while the mitochondrial swelling and nuclear pallor occur in the distal nephron. The glomerulus has no obvious morphologic changes. The site of injury involves both the distal tubule and collecting ducts or the proximal and distal tubules. The predominant lesion in patients with acute renal failure is acute necrosis and is located mostly in the proximal convoluted tubules. The severity of necrosis is dependent on dose, concentration, and duration. Patients with chronic nephrotoxicity have focal acute tubular necrosis characterized by cystic dilated tubules lined by a flattened epithelium showing atypical nuclei and atypical mitotic figures with hyaline casts. Long-term cisplatin treatment and injury may cause cyst formation and interstitial fibrosis. Two different membrane transporters capable of transporting cisplatin are Ctr1, a copper transporter, and OCT2, an organic cation transporter. Downregulation of Ctr1 expression in kidney decreases both cisplatin uptake and cytotoxicity , while lThe hallmark of MLCN histologically is the presence of tubular cast associated with intense inflammatory response. Although this is not a typical presentation of an acute renal failure caused by HIV infection, MLCN could be a form of renal injury seen on HIV patients. Based on the histologic evaluation of the two cases we presented, we noticed remarkable similarities in the histologic features of typical myeloma cast nephropathy and myeloma-like cast nephropathy, despite using the standard diagnostic tools to rule out the presence of monoclonal gammopathy or any other condition that could cause MLCN. The cause of the underlying pathology that is causing kidney damage could be obscured, and an incorrect diagnosis of MLCN can set the treating physician on the wrong path seeking diagnosis. Accordingly, having MLCN on the differential diagnosis list of typical myeloma cast nephropathy is advisable.


Atherosclerotic diseases account for nearly half of all deaths and are leading causes of adult disability. Our understanding of how atherosclerosis leads to cardiovascular disease events has evolved: from a concept of progressive luminal narrowing, to that of sudden rupture and thrombosis of biologically active atheroma. In concert with this conceptual shift, contemporary imaging techniques now allow imaging of biological processes that associate with plaque instability: active calcification and plaque inflammation. This review focuses on opportunities provided by positron emission tomography/computed tomography, to identify these high-risk biological features of atherosclerosis.The online version of this article (doi:10.1007/s12350-016-0766-y) contains supplementary material, which is available to authorized users. Despite advances in cardiovascular therapeutics, atherosclerotic events, such as myocardial infarction (MI) and stroke, continue to account for nearly half of all deaths and are a leading cause of adult disability. Decades’ worth of scientific studies refined our understanding of how atherosclerotic disease results in atherothrombotic events: from a concept of progressive luminal narrowing to that of sudden rupture and thrombosis of biologically active atheroma. In concert with this conceptual shift, the approach to imaging atherosclerotic milieu has moved beyond lumenological imaging and now incorporates identification of high-risk features of the arterial wall, including structural features, along with key biological processes .Atherosclerotic plaques are biologically active tissues. Oxidized low-density lipoprotein (LDL) particles induce endothelial cells to express leukocyte adhesion molecules . These adhesion molecules prompt recruitment of circulating monocytes and T-lymphocytes that in turn release cytokines and magnify the inflammatory cascade. Monocytes differentiate into resident macrophages, which phagocytose oxidized LDL and subsequently evolve into foam cells. As inflammatory cells and lipids accumulate within the atheroma, the plaque enlarges. Macrophages and smooth muscle cells undergo apoptosis, and their discarded lipids add to the evolving lipid-rich necrotic core. Neovascularization is triggered by hypoxia and is considered a marker of ongoing disease activity and indicative of a high-risk plaque. These new microvessels, originating from the adventitial vasa vasorum, are immature and leaky resulting in local intra-plaque hemorrhage. Active calcium deposition represents another common feature of vulnerable plaque. Eventually, the production of matrix metalloproteinases by activated leukocytes leads to enzymatic degradation of the plaque’s fibrous cap. This disruption can prompt plaque rupture, leading to atherothrombotic consequences. Accordingly, atherosclerotic plaques provide several attractive targets for imaging biological activity  has been validated as a tracer for imaging vascular inflammation. The radio-labeled glucose analogue accumulates in glycolytically active cells and is used in clinical PET imaging to identify metabolically active tissue such as tumors and inflammatory foci. Both pre-clinical and human studies show that the uptake of FDG within the artery wall correlates with macrophage accumulation in arterial plaques. Several studies have shown that FDG-PET/CT imaging of arterial inflammation provides potent prognostic information to predict the risk of developing cardiovascular disease (CVD) events. Figueroa et al. demonstrated in cancer-free patients that aortic FDG uptake predicts incident CVD beyond traditional risk factors and coronary calcium scores. Furthermore, in patients with recent stroke secondary to carotid atherosclerosis, carotid inflammation independently predicts recurrent stroke. , lipoprotein-associated phospholipase A2 inhibitor (rilapladib), and P-38 MAP kinase antagonists (BMS-582949 and losmapimod) each failed to significantly reduce arterial inflammation in PET/CT trials (with the notable exception of post hoc exploratory endpoints). Congruently, large clinical endpoint trials have shown that compounds in the same drug class so far also have failed to reduce CVD events. Accordingly, FDG-PET/CT trials have the potential to quantify therapeutic efficacy for novel drug discovery and treatment strategies.Vascular calcification is another important feature of atherosclerotic disease. Importantly, the associated risk attributable to vascular calcification diverges according to the size of the calcification. Microcalcification  occurs predominantly in the fibrous cap and may contribute to mechanical plaque destabilization while macrocalcification may confer plaque stability18F-sodium fluoride (NaF) is a PET/CT radiotracer used in oncology to detect metastatic bone spread, where 18F-fluoride replaces the hydroxyl groups in hydroxyapatite. Within atheroma, microcalcification is largely felt to be an active process driven by osteoblast-like cells derived from vascular smooth muscle, though passive deposition of calcium phosphate deposits resulting from phosphate release from necrotic cells may also play a role. It is believed that NaF is incorporated into hydroxyapatite at the sites of active microcalcification sites in a similar manner to that seen in its oncology applications. This process is likely accelerated by inflammation, with plaque macrophage burden strongly associated with osteoblastic activity in the aorta of hyperlipidemic mice.Several studies have demonstrated NaF-PET/CT’s utility to detect microcalcification. Such studies show that NaF localizes to sites of active microcalcification . Clinical studies have shown NaF uptake (measured by tissue-to-background ratios) is higher in symptomatic and high-risk atheroma compared to asymptomatic atheroma in both coronary and carotid arteries Figure .Figure 418NaF uptake are more likely to have a clinical diagnosis of coronary artery disease and higher Framingham risk scores, with a correlation between NaF uptake and coronary artery calcium score, leading the authors to suggest NaF uptake could be used to refine CVD risk.The development of techniques capable of imaging microcalcification has the potential to improve CVD risk, prognosis and guide the identification of new therapeutic strategies. Dweck et al. demonstrated that patients with higher vascular 68Ga-DOTATATE targets the somatostatin receptor2, which is over-expressed in activated macrophages and endothelial cells responsible for inflammation and angiogenesis. This novel tracer has advantages over FDG for imaging coronary atheroma, since it is not taken up by myocardium. Additional tracers that target macrophages include 11C-PK11195 (targeting TSPO receptor), and 18F-FMCH (targeting macrophages cell membrane) have been shown in pre-clinical studies to localize to areas of active inflammation. Hypoxia within the plaque stimulates new vessel formation, increases LDL oxidation and promotes a pro-inflammatory response in macrophages. Hypoxia can be identified using 18F-fluoromisonidazole (18F-FMISO), a new tracer that identified cellular hypoxia as demonstrated by increased aortic uptake in an animal model fed an atherogenic diet. Neoangiogenesis can also be detected, using a tracer targeting integrin αvβ3expression .Several newer tracers have been evaluated for imaging high-risk biological features of atherosclerosis. As new therapies are developed , there will be a greater need for enhanced risk stratification. With such theoretical expansion of treatment options, there will be a need to better-match an individual’s risk against the cost and side effects of the new treatments. Additionally, imaging may be needed in order to assess whether the dose of the new therapies should be changed, or switched altogether, depending on changes seen on serial imaging, much the same way such decisions are made in oncologic diseases. Imaging biological features of atherosclerosis may contribute importantly to such enhanced risk assessment.PET/CT imaging provides outstanding opportunities to assess atherosclerotic plaque biology non-invasively. Current use of FDG and NaF enables the early detection of atherosclerotic plaque inflammation and microcalcification. Such an approach enhances our assessment of individual risk, and may help identify new therapies. Additional studies are needed to delineate the potential clinical role of such approaches especially as armamentarium to treat atherosclerotic disease growth, placing demands on better delineation of risk and a better understanding of treatment response.Supplementary material 1 (PPTX 4009 kb)Below is the link to the electronic supplementary material.


We have recently shown that in individuals with the minor rs17228616 allele, this SNP interrupts AChE suppression by microRNA (miRNA)-608, leading to cortical elevation of brain AChE and reduced cortisol and the miRNA-608 target GABAergic modulator CDC42, all stress-associated. To examine whether this SNP has effects on PTSS and threat-related brain circuits, we exposed 76 healthy Israel Defense Forces soldiers who experienced chronic military stress to a functional magnetic resonance imaging task of emotional and neutral visual stimuli. Minor allele individuals predictably reacted to emotional stimuli by hyperactivated amygdala, a hallmark of PTSS and a predisposing factor of posttraumatic stress disorder (PTSD). Despite this, minor allele individuals showed no difference in PTSS levels. Mediation analyses indicated that the potentiated amygdala reactivity in minor allele soldiers promoted enhanced vmPFC recruitment that was associated with their limited PTSS. Furthermore, we found interrelated expression levels of several miRNA-608 targets including CD44, CDC42 and interleukin 6 in human amygdala samples (N=7). Our findings suggest that miRNA-608/AChE interaction is involved in the threat circuitry and PTSS and support a model where greater vmPFC regulatory activity compensates for amygdala hyperactivation in minor allele individuals to neutralize their PTSS susceptibility.Trauma causes variable risk of posttraumatic stress symptoms (PTSS) owing to yet-unknown genome–neuronal interactions. Here, we report co-intensified amygdala and ventromedial prefrontal cortex (vmPFC) emotional responses that may overcome PTSS in individuals with the single-nucleotide polymorphism (SNP)  The variable responses to stressful situations manifest in healthy stress-exposed individuals as subthreshold posttraumatic stress symptoms (PTSS), known to increase the risk for PTSD4 and cause impairments in daily life situation,6 including difficulties in emotion regulation.7 Although several neural and genetic risk factors for PTSS are known,11 the molecular mechanisms underlying the variable intensity of such symptoms remain elusive.Posttraumatic stress disorder (PTSD) is a severely debilitating psychiatric condition, especially in combat soldiers at high risk for trauma or chronic stress exposure.13 However, the great majority of genetic polymorphisms are located in noncoding regulatory regions.14 One of these is the single-nucleotide polymorphism (SNP rs17228616) C2098A substitution in the ‘seed' domain of miRNA-608 binding site, at the 3′-untranslated region of the acetylcholinesterase (AChE) gene. We have recently shown that individuals with the minor A-allele demonstrate weakened interaction between miRNA-608 and the AChE gene.15 These individuals also exhibit reduced cortisol and their cortical tissues present excessive brain AChE hydrolytic activity, accompanied by decreases in other predicted neuronal protein targets of miRNA-608, including the Rho GTPase CDC42 involved in GABAergic signaling and the inflammation-regulating cytokine interleukin 6 (IL-6). These effects were attributed to weakened AChE interaction with the primate-specific regulatory miRNA-608,16 and are all stress-associated and predicting PTSS and PTSD.18 Intriguingly, a recent genome-wide association study has reported significant association between the rs78011900 SNP, which is in full-linkage disequilibrium with rs17228616, and hypertension19 that is also related to PTSD.20 As the predicted cholinergic/GABAergic and inflammatory imbalance may modify the inherited risk or resiliency to develop stress-related symptoms following stressful challenges, we initiated a study of the functional consequences of this genetic variation on the development of PTSS symptoms that indicate a risk of PTSD and the relation of the rs78011900 SNP to the threat neural circuitry.Most imaging genetic studies in PTSS and PTSD focused on polymorphisms in coding regions of candidate genes.22 and receive high levels of cholinergic and miRNA inputs.24 Rodent studies show that pharmacological and molecularly induced suppression of hippocampal AChE activity associate with increased anxiety- and depression-like behaviors and decreased resilience to repeated stress.25 This plausibly involves stress-impaired hippocampal inhibitory feedback to the amygdala,26 which may lead to enhanced amygdala reactivity, a hallmark and a predisposing factor of PTSD.29 Excessive acetylcholine (ACh) release in the amygdala strengthens the associations between environmental stimuli and stressful events, potentially contributing to maladaptive learning and underlying affective disorders.30 Human studies indeed demonstrate that increases in ACh signaling contribute to stress-related illnesses, such as major depression.31 As the rs17228616 polymorphism is related to excessive brain AChE hydrolytic activity,15 we surmised that the AChE gene is close to its maximal expression capacity in individuals with the minor allele. This would limit their ability to react to a changing environment by overproducing AChE to suppress the induced stress. We expected that this would be manifested by enhanced neural response of the threat circuit, including the amygdala which is rich in both GABAergic synapses and AChE.32 In addition, animal studies have demonstrated involvement of inherited tendencies of AChE increases in response to stress,33 miRNA suppression of brain AChE (by miRNA-132) which limits stress-induced cognitive impairments,34 and CDC42 suppression leading to anxiogenic reaction.15 Therefore, we predicted modulated PTSS reactions in individuals with the minor allele of the rs17228616 SNP.The amygdala, hippocampus and prefrontal cortex have interrelated roles in PTSS and PTSD pathophysiology36 Under normal conditions, suppressing messages by the vmPFC lead to amygdala downregulation in response to negative stimuli, whereas PTSD patients show less activity in the vmPFC than healthy volunteers when experiencing or regulating emotions.37 This indicates vmPFC involvement in the process of regulation and thus resilience to trauma. In the current study, we used functional magnetic resonance imaging (fMRI) to examine how the rs17228616 polymorphism in the AChE gene impacts PTSS following chronic military stress exposure; specifically, we asked whether it serves as a vulnerability, resilience or an inert factor, and what are its functional influences on the human amygdala and vmPFC responses to stressful challenges.How individuals compensate for risk-associated genotypes is a challenging, scarcely studied issue. A neural candidate for coping with exaggerated amygdala activity is the ventromedial prefrontal cortex (vmPFC). This region is activated when a negative emotional response is suppressed, in a manner related to the elimination of previously learned associations of threat-conditioned signals.38 and may impact the development of stress-related symptoms.2 The fMRI data of Group A was already published by our group.29 As there were no differences in amygdala and vmPFC activity or in PTSS between paramedics and soldiers in training , all 76 volunteers were considered as a single group. All participants had no reported history of psychiatric or neurological disorders, no current use of psychoactive drugs, no family history of major psychiatric disorders and no incidence of childhood abuse or potentially traumatic events before military enrollment.The participants were 76 Israel Defense Forces soldiers  who experienced 1 year of chronic military stress during their mandatory service. Of those, 29 participants (Group A) served as combat paramedics and 47 (Group B) were recruited after 1 year of intensive and advanced combat training. During military training, the participants were exposed to a wide variety of stressful physical and psychological demands, including sleep restrictions, prolonged periods of physical survival challenges, face-to-face combat training and counter-terrorism combat course, that have been shown to affect well-beingrs17228616 (C2098A) versus the C-allele  were assigned to the minor allele group (N=13), whereas C homozygotes (CC) were assigned to the major allele group (N=63).DNA was extracted from saliva samples collected with an OrageneTM DNA Self-Collection Kit in Group A and from blood samples in group B, using commercial kits. Genotyping of the A-allele of C-allele  was perfThe participants viewed backward-masked colored photographed pictures of three contents  presented for either 33 or 83 ms in a block design and were asked to indicate whether they recognized a person or an object as described in ref. 39 In Group B, the symptoms were evaluated with the military version of the 17-item PTSD checklist,40 which specifically asks about symptoms related to stressful military experiences. As each of these questionnaires uses different scales, we calculated z-scores for each group, enabling to integrate data from both groups. Five participants did not complete the PTSS questionnaires, and thus were excluded from analyses concerning this variable.The PTSS levels in Group A were evaluated using the posttraumatic stress diagnostic scale questionnaire (PDS).41 which tests indirect effects using a bootstrap approach. The indirect effect was considered to be significant if its 95% bootstrap confidence intervals from 10 000 iterations did not include zero at P=0.05. In all indirect effect analyses, A-allele carriers were coded as 1.The mediation indirect effects were tested using the PROCESS macro in SPSS,n=7, three males, four females) were provided by the Netherlands Brain Bank  formed the minor A-allele group  and 63 participants (82.9%) with the CC genotype formed the major allele group . The ethnic differences in the prevalence of this SNP are compatible with evolution studies of miRNAs in population migrations45 that should be considered in functional studies of the brain-related implications of this and other polymorphisms.The le group . This di46 and AChE,47 suggesting that their activity may be modulated by the cholinergic/GABAergic imbalance imposed by the rs17228616 polymorphism in the AChE gene. Compatible with this prediction, genotypes with the minor allele  were associated with greater amygdala reactivity to emotional stimuli compared with other volunteers . Moreover, despite their neurogenetic vulnerability,15 PTSS levels did not differ between minor allele and major allele individuals , and its occurrence in healthy adults is associated with multiple anxiety- and inflammation-related changes: elevated brain AChE activity and correspondingly reduced brain levels of the GABAergic-related Rho GTPase CDC42, suppression of which induces anxiety, and the inflammation-regulating cytokine IL-6.15 MiRNA-608 has recently been identified as a tumor-suppressive miRNA,55 and has been proposed as a potential novel therapeutic target for treating osteoarthritis.56 Yet, because miRNA-608 is primate-specific,45 previous animal experiments of miRNA/AChE interactions and stress34 cannot be reproduced here to determine a causal link to stress. Despite this difficulty, the current study points to the involvement of miRNA-608 in stress-related neural reactions. We demonstrated that following chronic stress, individuals with the rs17228616 minor allele enhance neural activity in the amygdala accompanied by non-pathological PTSS levels. This finding was supplemented with a marginal increase in vmPFC response. Our model predicts multileveled effects of this polymorphism on the neural response to emotional stimuli within the threat circuit and its association with PTSS, as is schematically summarized in The rs17228616 polymorphism and PTSS by demonstrating that individuals with the rs17228616 minor allele show heightened amygdala response to emotional stimuli. This is compatible with earlier functional neuroimaging studies reporting exaggerated amygdala activation in patients with PTSD compared with control subjects28 and support the notion that amygdala hyperactivation is a predisposing factor for developing stress-related disorders.29 The finding that minor allele individuals show hyperactive amygdala but do not present elevated PTSS has led us to jointly investigate how they cope with stressful challenges.Although PTSS were not directly related to the polymorphism, our fMRI results provide support for the relation between the rs17228616 minor allele group showed a trend of increased vmPFC activation compared with the major allele group. This finding was further strengthened by a significant indirect path analysis showing that soldiers with the minor A-allele also exhibited higher levels of amygdala activity compared with those with the major C-allele, and that the minor allele was involved in increased recruitment of the vmPFC in these volunteers. The results of this mediation analysis suggest that minor allele individuals may recruit the vmPFC to downregulate their elevated amygdala reactivity more effectively than the major allele group. An additional mediation analysis suggested that the elevated vmPFC activity has a clinically protective role in A-allele volunteers, such that they attained lower than predicted levels of PTSS compared to major allele carriers by increasing the recruitment of their vmPFC. However, other, yet-unknown explanations for these fMRI results may exist. To validate or exclude this hypothesis, future studies could test whether parallel vmPFC hyperactivation occurs in other cases where the inherited tendency to develop PTSS is compensated for, and whether PTSD patients with the A-allele lack this compensatory mechanism and thus develop and maintain high levels of symptoms.The 57  gene, which strengthens an existing miR-189 target site, supports neurite growth and is involved in Tourette's syndrome and attention-deficit hyperactivity disorder.60 Also, college students homozygous for the ancestral A variant of an A>G polymorphism, which changes miRNA-96 regulation of the aggressive behavior-associated HTR1B gene, exhibit more conduct-disorder behaviors than individuals with the G allele.61 Our findings, in comparison implicate the ability to cope with miRNA dysregulation in brain-related diseases. The genetic variations evident in the miRNA-608 gene itself (the 'seed' region) may alter its binding and thus activity.62 Consequently, in addition to the 3′-untranslated region polymorphism, miRNA polymorphisms can potentially serve as additional biomarkers for PTSS, which warrants investigation.The noncoding SNPs affecting higher brain functions and chronic disease include a polymorphism in the 337 The currently observed effect thus reflects differential increases in the reactivity of the amygdala following prolonged exposure to stress and trauma.63 However, the genotype effect may also reflect pre-trauma vulnerability factors rather than consequences of stressful situations.29 The prospective studies examining the relation between the rs17228616 polymorphism and amygdala activity before and after stress insults of variable impact and length are required to disentangle this question. In addition, the currently studied population consisted of healthy Israel Defense Forces soldiers selected by extensive mental and physical screenings, and the rs17228616 polymorphism may have a different effect in the general population; genuine highly anxious populations should be tested before the results can be generalized.64 However, the use of a relatively healthy sample minimizes confounding factors such as comorbidities and medication use, thus representing a good approach for studying PTSS as a continuum. Nevertheless, the use of a small homogenous sample may have reduced PTSS variability, weakening the statistical power to detect all statistical effects, thus warranting additional studies on larger and more variable trauma-prone populations, such as paramedics or firefighters. Further, the sample size of amygdala tissues and soldiers participating in the fMRI experiment may be too small to detect all the associations. It is also possible that the results of this study may be only applicable to chronic cases and not to acute stress symptoms.There are several limitations to this study. The neurocircuitry models posit that in PTSD, the vmPFC fails to inhibit the amygdala, which leads to exaggerated threat responses and emotion regulation deficits.rs17228616 gene variant influences neural responses to emotional stimuli; but that in the context of trauma load, its effect on PTSS is limited, plausibly due to intensified recruitment of the vmPFC. Importantly, our findings support the concept that noncoding RNAs may be involved in PTSS, and should be integrated into a neural model of the disorder. This study highlights the effectiveness of combined genomic and neuroimaging methods in the identification of intermediate phenotypes for PTSS and other psychiatric disorders and provides a further step towards personalized medicine in psychiatry by identifying previously neglected genetic variants of disease risks that may contribute to its vulnerability. Notably, our current findings point to one possible PTSS risk pathway, in which a risk genotype predisposes individuals to increased fear reactivity. This involves exaggerated amygdala responses that are supposedly downregulated by the vmPFC to avoid the development of PTSS. These findings also point to potential new treatment avenues by neuromodulation techniques targeting the amygdala or amygdala–vmPFC interactions.To conclude, using a unique population at high risk for chronic stress exposure, we found that the


Proximal interphalangeal (PIP) fracture dislocations remain a complex injury pattern to treat. There are several treatment methods available aimed to restore stability, preserve range of motion, and reconstitute the articular surface. This study looked at the mid-term clinical and radiographic results of open reduction internal fixation through a shotgun approach of comminuted PIP fracture dislocations.A retrospective review was conducted of all PIP fracture dislocations treated through a volar, shotgun approach at a single institution over a 15-year period. Patients identified were contacted and asked to return to the office for clinical and radiographic evaluation. Patient reported outcomes were assessed with the Michigan hand questionnaire (MHQ) and visual analog scale (VAS) for pain.5 patients returned to the office for further evaluation with average follow-up of 69 months . 3 patients were found to have post traumatic arthritis on radiographs. 1 case had recurrent instability and one case had a deep infection, both necessitating further surgical intervention. Average PIP arc of motion was found to be 79°. Average VAS score of 0 and MHQ result of 95 (out of a possible score of 100) indicating no residual pain and excellent functionality of the affected hand.Open reduction internal fixation of comminuted PIP fracture dislocations utilizing the volar, shotgun approach provides excellent mid-term functional results despite the high incidence of post traumatic arthritis. If suboptimally treated, this injury complex can be potentially debilitating secondary to subsequent stiffness, pain, development of arthritis, and limited function of the affected digit.Dorsal fracture dislocations are more common than volar dislocations and are considered unstable when the fracture fragment of the middle phalanx base is over 50% of the articular surface or more than 30° of flexion is required to maintain a reduced joint . As descA number of surgical options have been described for the treatment of unstable fractures including, external fixation, volar plate arthroplasty, hemihammate autograft, and open reduction internal fixation (ORIF) with mini screws, plates, and Kirschner wires. ORIF with mini-screws has been traditionally reserved for fractures with minimal comminution and articular impactionFew studies have investigated the long term outcomes of ORIF treatment of dorsal fracture-dislocations of the PIP joint. This study reviews our experience with ORIF of PIP dorsal fracture-dislocations treated through a volar, shotgun approach with mini-screw fixation. The aim of this study is to determine the mid-term objective and subjective outcomes of patients who underwent this surgical procedure for PIP dorsal fracture-dislocations.2After obtaining approval from the institutional review board, a search was conducted using CPT codes  over a 15-year period (2000-2015).Medical charts and radiographs were reviewed to identify patient demographics, injury complex, surgical procedure. The identified patients meeting inclusion criteria were asked to complete the Michigan Hand questionnaire, a validated instrument used to measure patient reported outcomes . These pAll surgeries were performed at a single institution. The surgical technique performed is as follows. A volar Brunner incision was utilized in most cases. The radial and ulnar neurovascular bundles were identified and protected. An incision was made through the A3 pulley. The A2-A4 were preserved. The volar plate was identified and usually found to be attached to the middle phalanx base fracture fragment.The check-rein ligaments were carefully released. The middle phalanx was then hyperextended, into a shotgun position, exposing the entire articular surface. Articular comminution and impaction was visualized. Fracture fragments were reduced, held with small caliber K-wires and fixed with mini-screws. Once, the articular surface was restored, the volar plate was repaired with 3-0 vicryl suture in horizontal mattress fashion.31.12 patients with PIP dorsal fracture dislocations that underwent ORIF through a volar approach with mini-screw fixation were identified. These 12 patients were contacted and 5 patients returned to clinic for radiographic and clinical evaluation Average follow-up was 69.4 months (33-133 months). The demographics of these patients are seen in Table 4 out of 5 patients underwent surgical fixation within 7 days from injury. 1 patient attempted a short course of conservative treatment before undergoing surgical intervention 16 days after her injury.4 out of the 5 injuries sustained their injury as the result of blunt trauma. One patient’s injury was the result of a circular saw. This injury complex, unlike the other four included patients, involved transection of the ulnar and radial neurovascular bundles, the flexor and extensor tendons. In addition to open reduction internal fixation through a modified volar approach (utilizing the traumatic laceration), revascularization and tendon repairs were also performed.2).  demonstrate a patient with restoration of the articular surface, while  show the development of post-traumatic arthritis in another patient.Radiographic evidence of arthritis, range of motion, and subjective outcomes are shown in  indicating a highly functional PIP joint.There are limitations to this study. This is a small retrospective case series and only 5 of 12 patients were successfully recruited to return to clinic for radiographic and clinical evaluation. With this small cohort, statistical comparison across studies was not feasible. However, despite small numbers we were able to achieve a mid-term follow-up with an average of 69 months after surgery and utilized a validated patient reported outcome tool in the Michigan Hand Questionnaire.Our findings would indicate that the volar approach to complex, comminuted PIP fracture dislocations treated with mini-screw internal fixation without supplemental fixation provides comparable objective outcomes to other treatment options for this injury pattern. As such, even amongst a number of newer techniques and fixation strategies, we believe this procedure remains a useful tool in the treatment armamentarium of these injuries. Despite a high incidence of post-traumatic arthritis, patients report excellent outcomes and no significant dysfunction with activities of daily living.


Lmna H222P, N195K mutations, and LMNA knockout on cardiac function, in terms of hemodynamic and electrical signal propagation; certain strategies targeted on stress-related MAPK are mentioned. We will also discuss human iPSC cardiomyocytes serving as a platform to reveal the underlying mechanisms, such as the altered mechanical sensation in electrical coupling of the heart conduction system and ion channel alternation in relation to altered nuclear architecture, and furthermore to enable screening of drugs that can attenuate this cardiac premature aging phenotype by inhibition of prelamin misfolding and oxidative stress, and also enhancement of autophagy protein clearance and cardiac-protective microRNA.Laminopathy is a disease closely related to deficiency of the nuclear matrix protein lamin A/C or failure in prelamin A processing, and leads to accumulation of the misfold protein causing progeria. The resultant disrupted lamin function is highly associated with abnormal nuclear architecture, cell senescence, apoptosis, and unstable genome integrity. To date, the effects of loss in nuclear integrity on the susceptible organ, striated muscle, have been commonly associated with muscular dystrophy, dilated cardiac myopathy (DCM), and conduction defeats, but have not been studied intensively. In this review, we aim to summarize recent breakthroughs in an in vivo laminopathy model and in vitro study using patient-specific human induced pluripotent stem cells (iPSCs) that reproduce the pathophysiological phenotype for further drug screening. We describe several in-vivo transgenic mouse models to elucidate the effects of  LMNA gene locates in the long branch of chromosome 1, producing two main isoforms by alternative splicing . These isoforms are the intermediate filaments and constitute the major components of the nuclear lamina [LMNA have been shown to cause a wide range of human diseases, collectively referred to as “laminopathies” [LMNA gene, resulting in the generation of progerin, muscular dystrophy, and familial dilated cardiomyopathy (DCM). The mutations may also affect muscle, fat, bone, nerve, and skin tissues and lead to inherited neuromuscular disease with multiple phenotypic expressions such as Emery–Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy 1B (LGMD1B), Dunnigan-type familial partial lipodystrophy, a recessive axonal form of Charcot–Marie–Tooth neuropathy, and mandibuloacral dysplasia. However, there is a lack of understanding about the underlying mechanisms concerning lamin insufficiency or misfolding of such protein in cardiac disease progression. Current existing platforms for cardiolaminopathy modeling rely on transgenic mice to determine gene dose effects of the heterogeneous and homogeneous mutation system, the animal replicated clinical phenotypes with muscle dystrophy, premature DCM syndromes, as well as atrioventricular (AV) block. Although rodent systems allow studies of whole heart function, the cardiac physiological makeup is deviated from the human condition. Recent breakthroughs in generation of human induced pluripotent stem cell (iPSC) technologies allow access to patient-specific materials  that recapitulate the disease phenotype in a culture system. Recently, scientists have relied on such a system for electrophysiological study at a single cell level, as a platform to determine deterioration of nuclear architecture due to premature cell senescence, and also to determine energy synthesis dynamics. More importantly, the human cardiac cell would allow pilot drug-screening studies on targeting oxidative stress signaling in cardiac laminopathy, clearance of misfolded lamin proteins, delay in the rate of producing toxic farnesylated lamin, arising from mutation at cleavage sites of prelamin A/C protein, the blockade of stress-related MEK1–Erk1/2, JNK, and p38-mediated MAPK pathways, or even the cardiac protective microRNA (miR) that reduces prelamin A accumulation. More recently, the breakthroughs in gene editing technologies allow allogeneic cell therapies or generation of isogenic control. The use of iPSC derivatives could be used as a critical and powerful tool for standardized and comparative pharmacological studies.The pathies” –4. TheseLMNA), that cause monogenic DCM [LMNA-related DCM is characterized by early onset of atrial fibrillation, conduction system disease, and subsequent progression to sudden cardiac death and premature heart failure [TTN). LMNA mutations are the second most common cause of familial DCM, responsible for 5–10 % of overall familial DCM and up to 30–45 % of families with DCM and conduction system disease [LMNA-related DCM ranges from the first to sixth decade of life, the laminopathy-mediated cardiac defeats are always progressive and almost all patients become symptomatic after age 60 [LMNA-related DCM, especially that associated with conductive system diseases, has a more malignant clinical course than other familial DCM because of the high rates of progressive heart failure and sudden cardiac death due to ventricular tachyarrhythmias, and the ultimate treatment would rely on heart transplantation [LMNA-related DCM, the mechanisms of the disease as well as therapeutic strategies to prevent its onset and progression remain unclear. Early clinical manifestations are often apparent in the conduction system and specifically lead to sick sinus syndrome, and AV block or bundle branch block with approximately 28 % of affected patients requiring permanent pacemaker implantation [LMNA gene mutation suggested that cardiomyopathy due to LMNA mutations indicates a high probability of sudden death [Various genetic causes have been identified that play a vital role in the formation of DCM, although in most cases the underlying mechanism remains unknown. More than 60 genes have been identified, including the lamin A/C gene  (dominant negative) [LMNA knockout (KO) (haploinsufficiency) transgene presented apart from DCM phenotypes [−/−) was established by Sullivan and colleagues [lmna, Bonne and colleagues introduced an H222P mutation in LMNA in a mouse model, which displayed typical cardiac conduction defects, chamber dilation, and increased fibrosis but showed a lack of hypertrophy [Lmna N195K mice [To provide initial insight into the pathophysiology of enerated –21. Eithegative)  or LMNA enotypes , 21, butlleagues , 23. In ertrophy . In fact95K mice  that rec95K mice , 27.LMNA null mouse (LMNA GT–/–) by inserting a promoter in intron 2 of LMNA, resulting in a LMNA-β-geo fusion allele. This model combined the LMNA gene KO with LMNA-driven reporter, and thus enabled in-vivo study of the effect of conditional lamin A/C ablations during early postnatal development. In these KO mice, hindered growth, postnatal cardiomyocyte hypertrophy, skeletal muscle dystrophy, and metabolic defects were observed in the first 2 weeks after birth. Premature fatal events were commonly observed in mice before weaning. Similar results were later observed in a conditional LMNA KO mouse model created by Kim and Zheng [LoxP sites flanking the LMNA exon 2, which were further crossed with CMV-Cre mice to create a conditional KO driven by LMNA expression. The generated LMNA–/– mice exhibited growth delay from the first 12 days and died between postnatal days 16 and 18. It was also suggested that loss of function in muscle was due to the decreased skeletal myofibril size, similar to observations in Lmna GT−/− mice [In 2011, Kubben et al.  developend Zheng , with th−/− mice . OverallLMNA-related DCM, the mechanism of the disease as well as therapeutic strategies to prevent its onset and progression remain unclear. DCM with Lmna mutation is always very aggressive. Common clinical manifestations are related to development of heart failure and sudden cardiac death, for which ultimate treatment/prevention relies on cardiac transplantation [LMNA-related cardiomyopathy. A mechanistic understanding of the physiopathological basis of such disease is necessary to develop more specific and efficacious therapeutic strategies.Despite our increasing awareness of the importance of antation . Althoug. [LMNA mutation necessitated implantation of a cardioverter defibrillator instead of a pacemaker. In 2007, in order to investigate the pathogenesis of LMNA cardiomyopathy, Muchir et al. implemented a genome-wide transcriptome analysis of hearts isolated from Lmna H222P mice. Significant differences were noted in the expression of gene encoding proteins in stress-activated MAPK and mTOR signaling pathways in the mutated mice. Their work clearly verified an abnormal increase in both MAPK and mTOR activity in heart tissue from Lmna H222P mice [LMNA-induced cardiac disorders, Muchir et al. also treated Lmna H222P mice with daily intraperitoneal injections of the MEK1/2 inhibitor (Selumetinib). Selumetinib treatment resulted in left ventricular (LV) end-systolic dilatation, increased ejection fraction, and blocked molecular cardiac remodeling , with consequent improved cardiac function compared with placebo-treated mice. Since cardiac fibrosis is a common manifestation in end-stage DCM, and particularly in LMNA cardiomyopathy, cardiac fibrosis was also examined in this experiment. The Selumetinib-treated group had a lower degree of cardiac fibrosis than the placebo group. The same research group also revealed that germline deletion of ERK1 in the same mutant mice resulted in enhanced heart function at an early age (16 weeks old) [Lmna H222P mice. After cardiac ERK2 activity was blocked with Selumetinib, the ejection fraction at 20 weeks was significantly enhanced, implying that the increased ERK2 activity compensated for the ERK1 ablation and resulted in deteriorated heart function in the Lmna H222P mice that lacked ERK1 activity. They also found that inhibiting JNK (SP600125) [Lmna H222P mice hearts [Lmna H222P/Erk1 null mice compared with control LMNA-WT/Erk1 null mice when LV function started to change. We have previously reported the benefits of inhibiting JNK (SP600125) [LMNA mutation.In recent decades, the incidence of fatal tachyarrhythmia has been greatly reduced by prophylactic implantation of a cardioverter defibrillator . Anselme.  reportedeks old) , althougP600125) , 34 or pe hearts . TherefoP600125) , 34 or pLmna H222P mice for 2 weeks with a mTOR inhibitor, Temsirolimu, for clearance of waste protein generated by autophagy [After the experiment with MAPK inhibitors, Muchir’s team treated the utophagy . SimilarLMNA knockout mice restricted the possibility of chronic whole animal study. In addition, differences in cardiac electrophysiological behavior between humans and rodents may hinder the feasibility of translating pathophysiological discoveries into clinical practice. The mechanisms by which different LMNA mutations cause AV block or DCM remain uncertain. An in-vitro platform of human cardiomyocytes derived from patients with different LMNA mutations would be extremely useful for understanding disease mechanisms under stress conditions such as electrical field stimulation and mechanical stretch, as well as a hypoxic environment, and hence developing patient-specific therapies.The high mortality of these The recent breakthrough of human iPSCs generated from adult somatic tissues , 38 provLMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. The level of progerin accumulates with ages and leads to various ageing-associated nuclear defects including disorganization of the nuclear lamina and loss of heterochromatin. The reversible suppression of progerin expression by reprogramming was resumed upon differentiation with ageing-associated phenotypic consequences. The HGPS-iPSCs derived from skin fibroblasts showed an absence of progerin and more importantly lacked the nuclear envelope and epigenetic alterations normally associated with premature ageing. Nevertheless, the appearance of premature senescence phenotypes in HGPS-iPSC-derived smooth muscle cells (SMCs) was associated with vascular ageing. Additionally, they identified a DNA-dependent protein kinase catalytic subunit  as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlated with premature as well as physiological ageing. Others have reported the use of a human iPSC platform to model the disease phenotypes of HGPS in mesenchymal lineages and SMCs [LMNA [In 2011, Liu et al.  began toand SMCs , 43. Ho Cs [LMNA , 44. TheCs [LMNA . The invLMNA gene, R225X. Although no clear nuclear phenotype was observed in iPSCs from the DCM patient with the LMNA mutation, several cellular phenotypes were observed in the human iPSC-derived cardiomyocytes, including nuclear morphology abnormality (blebbing), slow proliferation, improved cellular senescence, and increased incidence of apoptosis under electrical stimulation. Under field electrical stimulation to mimic the native cardiac environment, the percentage of LMNA‐mutated iPSC cardiomyocytes that exhibited nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA, resembling the halploinsufficiency situation of the R225X mutant, replicated those phenotypes of the mutated LMNA field electrical stress. We also demonstrated the central role of the MAPK–extracellular signal-regulated kinase-1 (MEK1) pathway in governing susceptibility to cardiac cell stress-response. Blockage of the extracellular signal-regulated kinase (ERK) pathway by MEK1 inhibitors attenuated the electrical stimulation-induced proapoptotic phenotypes of DCM iPSC cardiomyocytes [LMNA mutation-related disease, there were new breakthroughs in gene editing technologies for correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. However, Liu et al. [LMNA gene was transcriptionally inactive and would impede targeted gene editing. They further explored using helper-dependent adenoviral vectors (HDAdVs) as a robust and highly efficient vehicle for the delivery of gene editing tools. In comparison with the conventional piggybac method, the advantage of this system is the inclusion of a negative selection step by ganciclovir (GNAC) resistance to eliminate random insertion of clones that contain the HSVtk cassette. The resultant corrected HPGS iPSCs were essentially proved to be genetically identical to fibroblasts as well as epigenetically similar to the uncorrected clones. Such a new method would enhance the reliability of gene correction as a therapeutic tool to rescue the disease phenotype for cell therapies or to generate a patient-matched control for disease modeling and further the dissected disease causal target for drug discovery [In 2012, our group subsequently generated another human iPSC platform from a patient bearing a premature termination codon in the myocytes . ERK1/2 myocytes . Most ofu et al.  discoveriscovery .In fact, somatic reprogramming of the progeria patient-specific cell to a human iPSC is not an easy task with the considerable drawback of low efficiency of stem cell clone formation. The stress of premature aged cells was basically due to oxidative stress-related NF-kB activation, which blocks the generation of iPSCs and MSC differentiation. Soria-Valles et al. discovered that NF-kB repression occurred during reprogramming towards a pluripotent state. In contrast, the hyperactivation of NF-kB impaired the process though DOT1L, a histone H3 methyltransferase, which reinforced the senescence signals . In the LMNA.Apart from epigenetic profiling, the tissue-specific expression profile of miR may provide clues for laminopathy therapies. miR-9 was specifically expressed in neuronal cells derived from HGPS patients, which exerted a protective role of the miR specifically to preserve cognitive function . The miRLMNA present varying severity of cardiac laminopathy phenotypes, such as alternation in splice variant maturation causing progerin accumulation and haploid insufficiency. The mutations could cause familial cardiomyopathy, early onset of AV block, and lethal ventricular tachycardia. The findings of translational implication facilitate screening of LMNA mutation which might be beneficial for risk stratification and clinical management of this type of familial cardiomyopathy or arrhythmia. Further studies concerning the effects of different lengths of truncated lamin proteins, such as location in proximity to the prelamin A cleavage site, need to be revealed. The overexpression of the unstable form of truncated proteins would generate an artificial system to extrapolate their prominent role in disease progression or the severe disease phenotype was only based on a reduced level of full-length Lmna. In an animal model, cardiac laminopathy has been found to be closely related to heart block, atrial fibrillation, and DCM. The transgenic animal would allow cardiac hemodynamic functional study and pharmacological testing. However, the direct role of a specific mutation in presentation of different forms of arrhythmia remains unknown. Further in-depth investigation in the human cell environment concerning the role of lamin in ion channel trafficking and the contribution of tight junction protein  in the conduction system to the cell conductance would be necessary. Up to now, it is clear that cardiac defeat mediated by LMNA mutation could be ameliorated by manipulation of the Akt/mTOR pathway by facilitating clearance of accumulated mutant protein through the process of autophagy and MEK1-mediated Erk1/2 by inhibition of apoptotic stress responses. As a consequence, further studies would also rely on a human iPSC model to investigate more clinical relevant outcomes. It would be interesting to explore cardiac-specific presentation of a laminopathy phenotype based on mechanical sensitivity of nuclear lamins coupled to membrane surface receptors.Different types of mutations in In the new era of advances in epigenetic studies, we could use a bioinformatics algorithm as a mathematical model to predict the age of human tissues based on profiles of cytosine-5 methylation within CpG dinucleotides, also known as DNA methylation (DNAm). The use of such an epigenetic clock based on 353 CpG sites could be validated in multiple tissues to predict the research gap in premature aging studies. It is well known that age-related DNA hypomethylation has long been observed in rodents . The aut


Cardiac xenotransplantation (CXTx) is a promising solution to the chronic shortage of donor hearts. Recent advancements in immune suppression have greatly improved the survival of heterotopic CXTx, now extended beyond 2 years, and life-supporting kidney XTx. Advances in donor genetic modification (B4GALNT2 and CMAH mutations) with proven Gal-deficient donors expressing human complement regulatory protein(s) have also accelerated, reducing donor pig organ antigenicity. These advances can now be combined and tested in life-supporting orthotopic preclinical studies in nonhuman primates and immunologically appropriate models confirming their efficacy and safety for a clinical CXTx program. Preclinical studies should also allow for organ rejection to develop xenospecific assays and therapies to reverse rejection. The complexity of future clinical CXTx presents a substantial and unique set of regulatory challenges which must be addressed to avoid delay; however, dependent on these prospective life-supporting preclinical studies in NHPs, it appears that the scientific path forward is well defined and the era of clinical CXTx is approaching. About 5.7 million Americans have heart failure, half of whom will die within 5 years . Organ tTo justify a clinical xenotransplantation (XTx) program, it is necessary to demonstrate transplant efficacy in clinically relevant animal models. The International Society of Heart Lung Transplantation (ISHLT) has suggested that a prospectively defined series of life-supporting cardiac xenotransplants in NHPs, using predefined immune suppression, with “60% survival at 3 months with a minimum of 10 animals surviving for this period,” would be sufficient to consider a clinical trial . The recThe most prominent variables which contribute to XTx efficacy are likely to be immune suppression and donor genetics, but recipient species , viral sin vivo using a Gal polymer, more consistent graft survival, median 35 days, was reported in pig-to-cynomolgus monkey hCXTx Gal β4GlcNAcβ3Gal, the SDa blood group antigen. This is an immunogenic glycan in pig-to-NHP CXTx [Donor genetic modifications have focused on four main categories, antigen reduction , thromboNHP CXTx . Humans NHP CXTx –59. TargNHP CXTx , 60–62. NHP CXTx  and reduNHP CXTx , which iin vitro which are proposed to contribute to donor endothelial cell activation [in vitro analysis of porcine endothelial cells expressing human TBM shows that it alleviates the molecular incompatibility with efficient production of activated human protein C [Interest in transgenic augmentation of thromboregulation stems primarily from recognition of molecular incompatibilities between porcine and human TBM . Secondativation , 66, systivation –69. Sevetivation –72, and rotein C , 73. A lrotein C , 74. Therotein C , 75.α-galactosyltransferase locus [in vitro [in vivo studies in both pig-to-NHP [The common donor modifications, transgenic expression of hCRPs, and the GTKO mutation have been thoroughly analysed and validated. Physiological or supraphysiological vascular expression of one or more human complement regulatory genes, CD59, CD55, or CD46, establishes an intrinsic barrier which regulates the complement cascade –78, reduse locus –81 have in vitro  and in vg-to-NHP –86 and Gg-to-NHP –91. The g-to-NHP , 92. ThiThe vast bulk of CXTx studies have to date utilized an abdominal non-life-supporting heterotopic transplant model where the graft is contractile but does not support the recipient's circulation. In comparison, there have been a limited number of oCXTx studies , 93–99. Orthotopic CXTx studies also unmasked a potential impediment to clinical CXTx which was not apparent in hCXTx studies. Every research group that has performed oCXTx has reported variable perioperative mortality ranging from 40 to 60% within the first 48 hours. Xenograft failure in this time period was not due to hyperacute rejection as the explanted hearts show vascular antibody deposition but otherwise normal myocardial histology . InsteadThe ability to diagnose and treat rejection is a key component of clinical transplantation. The ISHLT has developed pathologic grades (pAMR1–3) of immunopathologic features of endomyocardial biopsies which along with graft dysfunction and levels of donor-specific antibody are used for the diagnosis AMR in cardiac allotransplantation . The mosBased on the high frequency of AMR, the wide diversity of potential polymorphic porcine peptides and the chronic detection of vascular antibody deposition in GTKO donor hearts, it is necessary to establish methods for early diagnosis and effective treatment. It appears that anti-CD40-based immune suppression, which is likely to be used in clinical CXTx, relies heavily on effective costimulation blockade, as withdrawal of anti-CD40 therapy has resulted in the induction of non-Gal IgM and IgG . This hapol gene has been shown to induce widespread PERV proviral deletions [Complete knowledge and risk-free application of clinical CXTx, as with most major advances in medicine, retains elements of uncertainty. The potential for disease transmission has been a significant concern for clinical XTx . Concerneletions , but thiIt is clear that cardiac and renal XTx can benefit patients in need of organ replacement. If continued studies as outlined above are performed, we are optimistic that this technology will soon be ready for clinical testing. These remaining key preclinical studies are required to ensure the efficacy and safety of clinical CXTx. Principally a life-supporting preclinical oCXTx study in NHPs must be performed to demonstrate acceptable perioperative and postoperative recipient survival. This study should optimize organ preservation and utilize immune suppression based on anti-CD40 costimulation blockade. To meet ISHLT suggested standards , this stGenetic engineering has significantly improved CXTx organ survival but ongoing creation of new genetics, in pursuit of the perfect donor, has the potential to delay clinical studies. We believe the initial clinical studies will rely primarily on known systemic immune suppression and genetics and that further optimization of donor genetics is best pursued in response to identified, researched clinical immune and physiological requirements.Dependent on the results of these prospective preclinical oCXTx studies in NHPs, it appears to us that the era of clinical CXTx is approaching. The scientific path forward is demanding but well defined; however, the complexity of any clinical XTx program, including the heart, presents a substantial and unique set of regulatory challenges which need to be addressed expeditiously to avoid delaying the realization of clinical use.


Acute respiratory infections are a leading cause of death worldwide. Clinical data is conflicted regarding whether statins improve outcomes for pneumonia. Potential confounding factors including specific etiology of pneumonia as well as obesity could potentially mask protective benefit. Obesity is a risk factor for high cholesterol, the main target for statin therapy. We demonstrate that statin intervention conferred no protective benefit in the context of wild-type mice regardless of infectious agent. Statin intervention conferred either a protective benefit, during influenza infection, or detrimental effect, in the case of pneumococcal infection, in obese animals. These data suggest etiology of pneumonia in the context of obesity could be dramatically altered by the protective effects of statin therapy during bacterial and viral pneumonia. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) lower circulating lipid levels and decrease risk of cardiovascular disease. Statins also have a number of pleiotropic effects including anti-inflammatory and immunomodulatory properties that may confer benefit during infection  ≥ 25 kg/mAll experiments involving animals were performed with prior approval of and in accordance with guidelines of the St. Jude Institutional Animal Care and Use Committee. The St Jude laboratory animal facilities have been fully accredited by the American Association for Accreditation of Laboratory Animal Care. Laboratory animals are maintained in accordance with the applicable portions of the Animal Welfare Act and the guidelines prescribed in the DHHS publication, Guide for the Care and Use of Laboratory Animals.Streptococcus pneumoniae D39x (serotype 2)  plates supplemented with 3% sheep blood. Strains were then inoculated directly into semisynthetic liquid culture (CY broth) and grown to log phase before being administered to mice. The influenza A virus A/California/04/2009(A/H1N1pdm) generated by reverse genetics  mice were obtained from The Jackson Laboratory, Bar Harbor, ME, USA. ob/ob mice lack the anorexigenic adipokine Leptin which makes them hyperphagic leading to profound obesity. These animals have been used extensively to study both diabetes and obesity phenotypes  beginning at age 4 weeks and continuing for 4 weeks. Mock treated animals received a matched diet lacking simvastatin. Following treatment, serum was collected and high-density lipoprotein (HDL), low-density lipoprotein (LDL), triacylglycerol (TAG), and total cholesterol levels were measured to confirm pharmacological benefit. Mice were fed Mice were placed for 4 weeks on either a high-cholesterol diet (HCD)  or a normal diet (ND), which was identical, except for omission of cocoa butter, cholesterol, and cholate (test diet #95138). Cholesterol levels were measured in serum using the ABX Pentra Cholesterol CP kit  according to manufacturer guidelines.All mice were maintained in BSL2-level, specific pathogen free facilities. All experimental inoculation procedures were conducted under general anesthesia with inhaled isofluorane at 2.5%. Mice were monitored daily for signs of infection.620 of 0.4 and diluted according to a previously determined standard curve. Bacteria were introduced into mice (Jackson Laboratory) via intranasal (IN) administration of 107 CFU of bacteria in PBS (30 μL). Mice were monitored for disease progression and euthanized via CO2 asphyxiation. Blood for titer determination was collected via tail snip at 24 and 48 h post-infection and subsequent serial dilution and plating. For the viral challenge, mice were lightly anesthetized with isofluorane and intranasally inoculated with 102 TCID50 units of influenza A/H1N1pdm in 25 μl PBS. Mice were monitored daily for clinical signs of infection  on Manin Darby Canine Kidney (MDCK) cells  and pneumonia, respiratory infection, influenza, and bacteria. Papers that met this criteria were then further refined by only choosing articles focused on human trials. Reviews were excluded from the search. These papers were then scrutinized for type of statin used, outcomes, presence of obesity or BMI as a factor in the analysis or outcomes as well as clinical diagnosis of etiology.We hypothesized that that the effect of prophylactic statin therapy could have significantly altered benefit depending on the infectious agent, in this case bacterial and viral pneumonia, as well as the obesity status of the host. To test the impact of statins on acute respiratory infection in obesity, wild type and genetically obese mice were given either a statin or a mock diet for 4 weeks. Subsequent serum analysis confirmed a significant reduction in the levels of cholesterol in response to the statin diet, confirming pharmacological benefit Figure . In addiWe next sought to determine if statin intervention would alter the susceptibility of either wild-type or obese mice to respiratory infection. This was initially modeled using an influenza virus challenge. Obese mice were found to demonstrate enhanced susceptibility to influenza infection in concordance with previously published reports (Karlsson and Beck, S. pneumoniae infection with a significant decrease in survival (Figure S. pneumoniae, effect on survival following infection.We next sought to determine whether the metabolic-status dependent protection conferred by statin intervention could be extrapolated to other etiological agents of pneumonia, specifically pneumococcal pneumonia. In agreement with previous studies, no significant protection in terms of mortality was observed in wild-type statin treated animals compared to the mock group Figure . In contl Figure . This all Figure . CollectS. pneumoniae co-infected mice, regardless of metabolic status (Figures S. pneumoniae respectively, may cancel each other out in the context of co-infection.These data also call into the question the potential protective mechanism of statins in the context of influenza-mediated pneumococcal superinfection. It has been established both clinically and murine models that prior influenza infection greatly enhances host susceptibility to secondary bacterial infection, particularly with the pneumococcus through a variety of mechanisms (McCullers,  Figures . These dS. pneumoniae and influenza infection (Figure S. pneumoniae infection. This suggests factors independent of cholesterol levels are likely mediating the differences in susceptibility to infection in the context of statin prophylaxis in the obese murine models.There are several possible mechanisms that could contribute to the differences in statin-mediated alterations in obese but not wild-type animals. First of all, the lipid-altering effects of statins could be contributing to altered disease susceptibility. This is particularly relevant to the pneumococcus, as one of its major virulence factors is a cholesterol dependent cytolysin, pneumolysin, which could potentially be inhibited by high levels of serum cholesterol. To test this hypothesis, mice fed a high cholesterol diet, but which have a normal BMI, were infected with pneumococcus. The pneumococcal infection susceptibility was directly opposite from the obese phenotype with mice fed the high cholesterol diet being extremely susceptible to bacterial infection Figure . In contn Figure . These rn Figure . These rThe pleiotropic effects of statins with potential protective effects extending to pneumonia and sepsis have long been recognized. These effects have primarily been ascribed to their proposed immune modulatory functions, though the clinical data has been conflicted into the potential protective benefit despite large patient cohorts Table . This heThe observation that statins were beneficial for a viral infection but detrimental for a bacterial infection in the context of obesity was an unexpected finding. Obesity is known to impact of number of immunologic processes though the mechanisms underlying these differences are mechanistically complex (Milner and Beck, In contrast to pneumococcal infection, mortality for the influenza infection typically occurred days later indicating this response may be more dependent more upon the adaptive immune response. It has been demonstrated that leptin can enhance for the proliferation and the activation of T-cells, though such stimulation was dependent upon a stimulatory co-factor (Martín-Romero et al., Overall, these results indicate the need for controlling for both infection etiology as well as risk factors such as obesity during epidemiological studies to determine the groups who will benefit or possibly have increased risk of respiratory infection from the use of statins. Further work on the effects of statins on the pathogenesis and immunological responses against primary and co-infection with respiratory pathogens is warranted.EK, SS-C, and JR designed the experiments, interpreted the data, and wrote the manuscript. EK and JR performed the experiments.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.


It combines art and science to help triage and guide more focused and specialized ancillary studies. Unfortunately, the perceived value of histomorphologic analysis in the era of precision medicine is diminishing in recent years due to the emergence of more contemporary and data-rich molecular studies.4 Ironically, this is no different than the scrutiny that the patient history and physical exam have faced, in light of widely available whole-body imaging technologies.7 Some have even proposed that given the exponential decrease in sequencing costs, medical assessment could effectively begin with whole-genome analysis.8 Here, we discuss the current state and the possible future of the H&E stain by highlighting some of its strengths and shortcomings. It may well be that the scrutiny that the H&E microscopic exam has faced in recent years4 is no fault of its own, but the lack of effective approaches to routinely extract more of the rich morphologic information it contains.Accurate interpretation of the hematoxylin and eosin (H&E) slide has remained the foundation of pathological analysis and diagnostic medicine for over a century.The H&E slide continues to be a valuable tool for pathologists and clinicians alike. For example, quite often, surgeons request urgent intra-operative pathological interpretations to help guide surgery. This clinical scenario often necessitates that an accurate diagnosis be rendered within 5–10 min. The outcome usually has huge implications for the trajectory of the remaining surgery . As a result, most surgeons have a strong preference for the expert opinion of highly subspecialized pathologists . Until molecular or alternative analytic approaches become compatible with these acute timeframes, the H&E slide will continue to be an essential tool to help guide surgical care.10 This list of molecular tests, each with their own strengths and weaknesses, continues to grow. However, even with decreasing costs of sequencing, performing routine multi-platform molecular analysis on every specimen will likely not become a time-effective or cost-effective strategy in the foreseeable future. This relatively high cost of multi-omic analysis will continue to necessitate molecular triaging to help narrow testing to those most appropriate for the specific tumor type and clinical scenario. Lastly, the H&E slide still remains one of the most versatile diagnostic tools when only minute amounts of tissue, insufficient for molecular analysis, is available. Similarly, unlike bulk tissue-based molecular tests, microscopic analysis preserves important region-to-region, single-cell-level spatial information that may have significant implications for diagnostic and treatment decisions.12 For example, even for tumors that have been analyzed at the molecular level, treatment regimens can dramatically change when specific microscopic features are noted .14 Therefore, there are many compelling reasons to retain the H&E exam as a non-overlapping and essential tool in our growing precision oncology toolbox.The H&E slide also has a key role in precision oncology in sub-acute settings. Technological advances now allow patients’ tumors to be globally profiled at the genomic, epigenomic, transcriptomic, proteomic, phosphoproteomic, and other -omic levels.16 This diagnostic uncertainty has promoted liberal and widespread use of costly molecular testing to differentiate between seemingly histologically indistinguishable lesions.18 Similarly, in efforts to maintain diagnostic objectivity, other potential prognostic and therapeutic morphologic biomarkers, such as foci of tumor-infiltrating lymphocytes and fibrotic tumor reaction, are often omitted. Indeed, even in the molecular era, the unassisted physician still largely relies on simple decision tree approaches that utilize only a small fraction of available -omics knowledge  known as deep neural networks (DNNs) is proving to be a valuable tool for the generation and implementation of complex multi-parametric decision algorithms.26 With robust clinically and genomically well-annotated training sets, minute differences not apparent to the human observer may even be used to aid classification and predict likelihood of specific molecular alterations and prognosis26  that are time-consuming to identify by the unaided human eye.29Unlike simplified algorithms pathologists are trained to use, DNNs deconstruct images into pixels and sequentially aggregate them to form shapes  and reproducible features that represent distinct diagnostic patterns Fig. . When gis26 Fig. . Such im23 Such studies highlight how machine learning can extract novel and multi-parametric morphologic features not readily accessible by the human eye.30 In addition to refining current diagnostic criteria, a recent study by Yu et al. showed that automated computer-based image analysis of the non-small cell lung cancers found in The Cancer Genome Atlas (TCGA) could identify novel morphologic features that predict survival.26 Integration of DNN-based analyses will likely continue to push the boundaries of clinically relevant morphologic information not found in our current human-derived pathologic criteria.31 More recently, the use of DNNs to scan for metastatic tumor foci in lymph nodes achieved substantially lower false-negative rates when compared to pathologists (from 26.8 to 7.6%).29Digital revitalization of histomorphology is already showing promise. For example, Dong et al. recently showed that upwards of 22 different morphologic nuclear features  could be extracted and used to train classification models to reliably differentiate between neoplastic  and non-neoplastic  breast lesions.22 Such an evolution would help address the shortage of pathologists and improve consistency in reporting.Automation and routine AI-based analysis of the H&E slide to include these milestones would provide immediate benefits to patient care. For example, robustly trained DNNs may assist general pathologists in providing subspecialist-level expertise for intra-operative consultations. Integration of AI/DNNs into laboratory medicine could eventually shift training and the role of future pathologists toward “big-data” information specialists.Similarly, automating AI-assisted histologic analysis in non-emergent settings would likely also improve diagnostic workflow. Currently, there are numerous “practical” delays in rendering diagnoses. There is usually a day lost between generation of an H&E slide and delivery to a pathologist or clinical fellow for review. Once analyzed, the pathologist generates a differential diagnosis and triages appropriate immunohistochemical tests to narrow diagnostic considerations. This usually takes an additional 2–3 days. Only then are appropriate diagnostic and confirmatory molecular tests considered. This linear series of steps, and the need to revisit cases multiple times, creates significant delays in rendering diagnoses and valuable molecular information to clinicians for personalized patient care. Often, a multitude of unnecessary molecular tests are liberally and prematurely ordered in attempts to avoid such delays. Implementation of DNN-based image analysis could allow a preliminary digital diagnosis to be provided and initiation of appropriate immunohistochemical stains and molecular tests before the slides even reach the pathologists’ microscope. Automated selection of lesional regions of interest and initiation of relevant multi-omics testing could even be initiated minutes after the initial slide is generated!12 These valuable and publicly available resources are ideally suited to train DNNs to incorporate follow-up studies into their diagnostic algorithms. Machine-driven decisions could therefore provide improved and optimized immunohistochemical approaches that lead to rapid and cost-effective diagnostic convergence among various other morphologic considerations.Recent large-scale immunohistochemical efforts are also generating massive amounts of protein-based histologic information across different tissue and cancer types.22 Robust training of DNNs with a large number of molecularly confirmed cases, such as those found in TCGA, may also identify subtle morphologic features that better predict presence/absence of molecular alterations.32 This could provide significant cost savings for pathology departments without affecting patient care. Similarly, training DNNs with images from known responders and non-responders to specific treatments  may help to better stratify patients for appropriate future personalized and precision-based clinical trials. For example, the presence of tumor-infiltrating myeloid (TIM) cells are thought to promote invasion and immunosuppression in glioblastomas.33 Their presence in the tumor microenvironment may be an important cause of treatment failure to both conventional and immune-based therapies.35 Prompt and reliable quantification of TIM in gliomas may thus provide vital information to stratify a patient to combined tripartite immune therapies that include colony stimulating factor 1 inhibitors.34 Alternatively, H&E-based deep learning may uncover specific TIM density thresholds that, when exceeded, suggest that immune-based therapies will likely be ineffective and signal prioritization of patients to other therapeutic strategies. Similarly, neutrophilia in glioblastomas have been associated with release of elastase that promotes brain invasion and malignancy.37 High-content image analysis that quantifies these additional immune cell parameters may help predict patients with well-circumscribed tumor borders. Such patients could be good candidates for de-escalation therapy38 following gross total resection. AI-based discovery of robust morphologic surrogates of genetic alterations  may even become objective stand-alone inclusion criteria that could grant patients early enrollment in precision-based clinical trials as they await confirmatory molecular testing  and quality, coupled with the decreasing cost of data storage, are now at a point where some pathology departments are considering archiving their complete slide libraries digitally.48Thankfully, unlike many other emerging molecular technologies, AI-based diagnostics presents a relatively cost-effective addition to the current workflow with a low financial barrier of entry for most institutions. Recent digital pathology educational initiatives have helped equip most pathology departments with whole-slide scanning technologies that can digitize slides in a standardized high-resolution format.52 Introduction of random perturbations to the brightness and color saturation levels of training images allows AI-based algorithms to effectively learn color-invariant features for classification.29 Such simple image-augmentation procedures allow for cross-institutional sharing and pooling of digital resources that could allow DNNs to handle even rare and highly variable tumor types. Similarly, they allow access to freely available and multi-omic data sets  to carry out large-scale morpho-genomic corrrelations.50Similarly, many technical concerns of computer-assisted diagnostics have now also been adequately addressed. For example, there have been concerns that variations in imaging quality resulting from difference in staining protocols, technical handling, and scanner setting could introduce “non-biological” batch effects that can significantly compromise the performance of computer-driven morphologic analysis and classification accuracy. Fortunately, these concerns have now also been largely addressed with quality control algorithms that can carry out automated color normalization to reduce batch-related variance to insignificant levels.25As scanners can now routinely handle batches of 400 slides, it is conceivable that the current throughput of scanning could allow for routine digitization and analysis of every slide produced in a large proportion of pathology departments. Even if additional capital and personnel investment is necessary, image-based analysis is already making a compelling case as a cost-effective tool to reduce pathology workload and excessive confirmatory tests to a more sustainable level. In one study, for example, DNNs could effectively highlight slides with cancer for pathologists and safely exclude 30–40% of slides containing only normal tissue.53 The H&E slide provides many of the same positive sentiments to pathologists and represents the origins of precision-based diagnostics and medicine.1 DNN-based technologies are now poised to revolutionize how pathologists use the H&E slide by helping physicians extract unprecedented and colossal amounts of objective and multiparametric morphologic information. Although recent studies highlighted give reasons for optimism, this field is still in its infancy. Integration of this new technology into diagnostic workflows will require pathologists to continue to practice their mastery of the microscopic exam to oversee and approve machine-based interpretations. Such an evolution will allow the H&E slide to remain a pivotal component to the multi-omics approach to personalized and precision oncology.Despite technological advances and criticisms, the physical exam has endured the test of time to remain a powerful, cost-effective, and adaptable diagnostic tool that contributes to strengthen the patient–physician bond, trust, and ultimately patient care.