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Genome editing in human induced pluripotent stem cells (iPSCs) provides the potential for disease modeling and cell therapy. By generating iPSCs with specific mutations, researchers can differentiate the modified cells to their lineage of interest for further investigation. However, the low efficiency of targeting in iPSCs has hampered the application of genome editing. In this study we used a CRISPR-Cas9 system that introduces a specific point substitution into the sequence of the Na + /K + -ATPase subunit ATP1A1. The introduced mutation confers resistance to cardiac glycosides, which can then be used to select successfully targeted cells. Using this system, we introduced different formats of donor DNA for homology-directed repair (HDR), including single-strand DNAs, double-strand DNAs, and plasmid donors. We achieved a 35-fold increase in HDR when using plasmid donor with a 400 bp repair template. We further co-targeted ATP1A1 and a second locus of interest to determine the enrichment of mutagenesis after cardiac glycoside selection. Through this approach, INDEL rate was increased after cardiac glycoside treatment, while HDR enrichment was only observed at certain loci. Collectively, these results suggest that a plasmid donor with a 400 bp repair template is an optimal donor DNA for targeted substitution and co-targeting ATP1A1 with the second locus enriches for mutagenesis events through cardiac glycoside selection in human iPSCs., Competing Interests: Stephen A. Duncan is founder and CEO of Gruthan Bioscience, LLC., (©2020 Liu et al.)

The liver is one of the largest organs in the body and is responsible for a diverse repertoire of metabolic processes. Such processes include the secretion of serum proteins, carbohydrate and lipid metabolism, bile acid and urea synthesis, detoxification of drugs and metabolic waste products, and vitamin and carbohydrate storage. Currently, liver disease is one of the most prevalent causes of mortality in the USA with congenital liver defects contributing to a significant proportion of these deaths. Historically the study of liver disease has been hampered by a shortage of organ donors, the subsequent scarcity of healthy tissue, and the failure of animal models to fully recapitulate human liver function. In vitro culture of hepatocytes has also proven difficult because primary hepatocytes rapidly de-differentiate in culture. Recent advances in stem cell technology have facilitated the generation of induced pluripotent stem cells (iPSCs) from various somatic cell types from patients. Such cells can be differentiated to a liver cell fate, essentially providing a limitless supply of cells with hepatocyte characteristics that can mimic the pathophysiology of liver disease. Furthermore, development of the CRISPR-Cas9 system, as well as advancement of miniaturized differentiation platforms has facilitated the development of high throughput models for the investigation of hepatocyte differentiation and drug discovery. In this review, we will explore the latest advances in iPSC-based disease modeling and drug screening platforms and examine how this technology is being used to identify new pharmacological interventions, and to advance our understanding of liver development and mechanisms of disease. We will cover how iPSC technology is being used to develop predictive models for rare diseases and how information gained from large in vitro screening experiments can be used to directly inform clinical investigation., (Copyright © 2019 Corbett and Duncan.)

Patients with mtDNA depletion syndrome 3 (MTDPS3) often die as children from liver failure caused by severe reduction in mtDNA content. The identification of treatments has been impeded by an inability to culture and manipulate MTDPS3 primary hepatocytes. Here we generated DGUOK-deficient hepatocyte-like cells using induced pluripotent stem cells (iPSCs) and used them to identify drugs that could improve mitochondrial ATP production and mitochondrial function. Nicotinamide adenine dinucleotide (NAD) was found to improve mitochondrial function in DGUOK-deficient hepatocyte-like cells by activating the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). NAD treatment also improved ATP production in MTDPS3-null rats and in hepatocyte-like cells that were deficient in ribonucleoside-diphosphate reductase subunit M2B (RRM2B), suggesting that it could be broadly effective. Our studies reveal that DGUOK-deficient iPSC-derived hepatocytes recapitulate the pathophysiology of MTDPS3 in culture and can be used to identify therapeutics for mtDNA depletion syndromes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metaplasia is the irreversible conversion of one differentiated cell or tissue type into another. Metaplasia usually occurs in tissues that undergo regeneration, and may, in a pathological context, predispose to an increased risk of disease. Studying the conditions leading to the development of metaplasia is therefore of significant clinical interest. In contrast, transdifferentiation (or cellular reprogramming) is a subset of metaplasia that describes the permanent conversion of one differentiated cell type into another, and generally occurs between cells that arise from neighbouring regions of the same germ layer. Transdifferentiation, although rare, has been shown to occur in Nature. New insights into the signalling pathways involved in normal tissue development may be obtained by investigating the cellular and molecular mechanisms in metaplasia and transdifferentiation, and additional identification of key molecular regulators in transdifferentiation and metaplasia could provide new targets for therapeutic treatment of diseases such as cancer, as well as generating cells for transplantation into patients with degenerative disorders. In the present review, we focus on the transdifferentiation of pancreatic cells into hepatocyte-like cells, the development of Barrett's metaplasia in the oesophagus, and the cellular and molecular mechanisms underlying both processes.

This retrospective review assesses the efficacy of darbepoetin alfa for treating anemia after renal transplantation. Patients were evaluated over a 12-week period, and efficacy was based on achieving hemoglobin >12 g/dL. Thirty-six patients were analyzed (53% male, 53% cadaveric allografts, median age 42.5 years). Baseline creatinine clearance ranged from approximately 15 to >100 mL/min. Most patients initiated darbepoetin alfa <3 months (50%) or >12 months (44%) after transplantation, 19% were previously receiving recombinant human erythropoietin (rHuEPO), and 47% were on concomitant ACE inhibitors. The majority of patients received either tacrolimus- (53%) or cyclosporine- (44%) based immunosuppression. Overall, 29 (81%) patients achieved the hemoglobin target with a mean time to response of 4.4 weeks. Neither the time to anemia onset, previous rHuEPO therapy, concomitant ACE inhibitor, allograft source, immunosuppressive regimen, nor degree of renal function affected the proportion of patients achieving the hemoglobin target, time to response or darbepoetin alfa dose requirement. Patients with anemia >12 months post-transplantation or on concomitant ACE inhibitors required a significantly longer duration of therapy. No adverse events associated with darbepoetin alfa therapy were detected. These results demonstrate that darbepoetin alfa is a safe and effective treatment for anemia in renal transplant recipients.

The patient described in this report appears to have had a unique, severe, pure pan-dysautonomia, and has been investigated in sufficient detail to specify precisely the disorders of functions subserved by the autonomic nervous system. Though we cannot rule out the possibility of an unknown autonomic toxin, we have no evidence for it and suggest that our patient's disorder be considered an acute polyneuritis, restricted to the autonomic system. Comment is made on tests of autonomic dysfunction and on the reliance which can be placed upon the results.

1. Plasma catecholamines, plasma renin activity, plasma aldosterone and haematocrit were measured in four subjects with physiologically complete cervical spinal cord transections, before, during and after head-up tilt to 45 degrees for 30 min. Plasma catecholamines were measured in five normal male volunteers in the supine position and after head-up tilt to 45 degrees for 10 min. 2. After 10 min of head-up tilt, the plasma noradrenaline rose 14% in the tetraplegic patients and 115% in the control subjects. These findings indicate a failure of sympathetic activity in response to head-up tilt in the tetraplegic patients, probably caused by interruption of pathways by which the brain normally controls sympathetic outflow. 3. In the tetraplegic patients the resting plasma renin activities were above normal, and rose more quickly and greater on head-up tilt than in published studies of normal subjects. It is likely that the renal baroreceptors are important in the control of renin release. 4. In the tetraplegic patients, there was a late rise in plasma aldosterone which was probably due to the elevation in plasma renin activity.

Blood pressure, heart rate, and plasma catecholamine levels were measured in 16 quadriplegic subjects with physiologically complete cervical spinal cord transections above the level of the sympathetic outflow, and in 15 normal subjects (controls). In the quadriplegics the average resting blood pressure was 107/59 (mean, 75) mmHg, heart rate was 65 beats/min, and plasma norepinephrine (NE) and epinephrine (E) levels were 0.05 and 0.005 ng/ml, respectively. In the controls average resting blood pressure was 117/79 (mean, 92) mmHg, heart rate was 61 beats/min, and resting plasma NE and E levels were 0.20 and 0.06 ng/ml, respectively. Resting blood pressure and plasma NE and E levels were significantly lower in the quadriplegics (P less than 0.01, less than 0.001, less than 0.001, and less than 0.001, less than 0.001, respectively) than in the controls. In the quadriplegics, neurogenic hypertension was induced by bladder and muscle stimulation. This resulted in a marked elevation of both systolic and diastolic blood pressure (from an average of 109/60 (mean, 75) to 168/87 (mean, 114) mmHg) as a result of uninhibited sympathetic nervous activity through the isolated spinal cord. Plasma NE consistently rose, from an average of 0.05 to 0.16 ng/ml (P less than 0.001). There was significant linear relationship between plasma NE and mean blood pressure (P less than 0.001). In the quadriplegics infusion of l-norepinephrine to raise the blood pressure to comparable levels (from 105/58 (mean, 74) to 183/93 (mean, 123) mmHg) resulted in plasma NE levels approximately 21 times higher than during muscle and bladder stimulation. It is possible that the lower resting arterial blood pressure and plasma NE and E levels in the quadriplegics in comparison to normal subjects may reflect diminished resting sympathetic nervous activity. The rise in blood pressure following increased sympathetic nervous activity was accompanied by an elevation in plasma NE. The hypertension was not secondary to the rise in plasma NE. Plasma NE in these subjects appears to be a reliable index of prevailing sympathetic nervous activity.

Strokes are the third major cause of death in developed countries and are probably the major cause of severe chronic disability. Hypertension is the most important condition predisposing to strokes, and treatment of hypertension is of great importance in stroke prevention. The relationship of strokes to the degree of hypertension, and to age, sex, race and drug treatment is discussed.

1. A study has been made of the effects of pinprick and a cold pack applied to the skin above and below the level of denervation, deep inspiration, squeezing the chest, and bladder percussion in non-bedridden patients with chronic, closed, complete, localized transection of the cervical spinal cord. Variables studied were blood pressure, heart rate, calf blood flow, hand blood flow and occluded vein pressure in the hand and foot.2. Cutaneous stimuli above the lesion, mental arithmetic and noise from gunshot produced no response.3. Pinprick and cold applied below the lesion usually produced an increase in mean arterial pressure, little change in pulse pressure or heart rate, clear decreases in calf and hand blood flow, and occasional venoconstriction.4. Deep inspiration produced an initial rise in blood pressure and heart rate which was sometimes followed by a decrease in one or both. Squeezing the chest usually produced an increase in blood pressure and a decrease in heart rate. Both stimuli caused decreases in calf and hand blood flow and venoconstriction, the latter being more common after squeezing the chest.5. Bladder percussion produced contraction of the wall of the bladder and this was regularly associated with increased arterial mean and pulse pressures, a decreased heart rate and calf and hand blood flow, and venoconstriction. The responses occurred whether or not the bladder was full and whether or not a rise of intravesical pressure occurred.

Three patients with severe tetanus had episodes of profound arterial hypotension lasting from minutes to hours. The blood pressure was recorded continuously for 13, 19, and six days respectively by an intra-arterial catheter, and other measurements included heart rate, central venous pressure, cardiac output, and blood gases.The hypotension was distinguished from that of "shock", for there was no clinical evidence of peripheral vasoconstriction and no tachycardia. It could not be attributed to disturbances of salt and water balance. During episodes of hypotension the blood pressure fell as low as 32/16 mm Hg, the heart rate fell from a mild tachycardia to normal values or a mild bradycardia, and the central venous pressure did not rise. The onset and the end of such episodes was often abrupt and the hypotension was often produced in response to a stimulus. In one patient extreme hypotension followed the aspiration of secretions from the trachea. These changes may represent another effect of tetanus on autonomic nervous activity, including impairment of baroreceptor reflexes.