Combining physical & cognitive training with iPSC-derived dopaminergic neuron transplantation promotes graft integration & better functional outcome in parkinsonian marmosets.

Parkinson's disease (PD) is a relentlessly progressive and currently incurable neurodegenerative disease with significant unmet medical needs. Since PD stems from the degeneration of midbrain dopaminergic (DA) neurons in a defined brain location, PD patients are considered optimal candidates for cell replacement therapy. Clinical trials for cell transplantation in PD are beginning to re-emerge worldwide with a new focus on induced pluripotent stem cells (iPSCs) as a source of DA neurons since they can be derived from adult somatic cells and produced in large quantities under current good manufacturing practices. However, for this therapeutic strategy to be realized as a viable clinical option, fundamental translational challenges need to be addressed including the manufacturing process, purity and efficacy of the cells, the method of delivery, the extent of host reinnervation and the impact of patient-centered adjunctive interventions. In this study we report on the impact of physical and cognitive training (PCT) on functional recovery in the nonhuman primate (NHP) model of PD after cell transplantation. We observed that at 6 months post-transplant, the PCT group returned to normal baseline in their daily activity measured by actigraphy, significantly improved in their sensorimotor and cognitive tasks, and showed enhanced synapse formation between grafted cells and host cells. We also describe a robust, simple, efficient, scalable, and cost-effective manufacturing process of engraftable DA neurons derived from iPSCs. This study suggests that integrating PCT with cell transplantation therapy could promote optimal graft functional integration and better outcome for patients with PD.

LRRK2 Attenuates Antioxidant Response in Familial Parkinson's Disease Derived Neural Stem Cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.

Hepatic artery pseudoaneurysm rupture: A case report.

Hepatic artery pseudoaneurysms are an uncommon complication of blunt hepatic trauma typically presenting in a delayed fashion. A 40-year-old male presented to a trauma centre after a 6-metre fall from a construction site with multiple injuries including a grade IV liver laceration centred around the porta hepatis. This liver injury was managed non-operatively. On day sixteen of admission, he had a sudden cardiac arrest from haemorrhagic shock with a subsequent CT demonstrating a ruptured extrahepatic proper hepatic artery pseudoaneurysm. Despite laparotomy and vessel repair, he died from pulmonary complications of aspiration pneumonia and acute lung injury associated with massive transfusion. The literature demonstrates conflicting views regarding the utility of repeat CT to detect a pseudoaneurysm in asymptomatic, non-operatively managed patients with blunt hepatic trauma. In particular, the literature does not distinguish the utility of repeat routine CT for intrahepatic and extrahepatic hepatic artery pseudoaneurysm, the latter if which is rarer. Current guidelines recommend against it, but there are observational studies suggesting utility, particularly in high grade (≥IV) liver injury. In patients with a high-grade injury extending to the porta hepatis, repeat imaging should be considered to detect possible pseudoaneurysm.

Human Induced Pluripotent Stem Cell Phenotyping and Preclinical Modeling of Familial Parkinson's Disease.

Parkinson's disease (PD) is primarily idiopathic and a highly heterogenous neurodegenerative disease with patients experiencing a wide array of motor and non-motor symptoms. A major challenge for understanding susceptibility to PD is to determine the genetic and environmental factors that influence the mechanisms underlying the variations in disease-associated traits. The pathological hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the brain and post-mortem Lewy pathology, which leads to the loss of projecting axons innervating the striatum and to impaired motor and cognitive functions. While the cause of PD is still largely unknown, genome-wide association studies provide evidence that numerous polymorphic variants in various genes contribute to sporadic PD, and 10 to 15% of all cases are linked to some form of hereditary mutations, either autosomal dominant or recessive. Among the most common mutations observed in PD patients are in the genes LRRK2, SNCA, GBA1, PINK1, PRKN, and PARK7/DJ-1. In this review, we cover these PD-related mutations, the use of induced pluripotent stem cells as a disease in a dish model, and genetic animal models to better understand the diversity in the pathogenesis and long-term outcomes seen in PD patients.

Simulation in Perioperative Liver Transplant Anesthesia: A Systematic Review.

Due to the complexity of liver transplant patients and the variability in exposure to transplantation by anesthesia trainees, simulation is often required as an adjunct to clinical experience. This systematic review identifies current simulation models in the literature that pertain to perioperative liver transplant anesthesia. Data were collected by performing an electronic search of the PubMed and Scopus databases for articles describing simulation in transplant anesthesia. Abstracts were screened using the preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines. Three reviewers analyzed 16 abstracts found in the search and agreed upon articles that met the inclusion criteria for the systematic review. A total of five publications met the inclusion criteria; they could be grouped as cognitive skills and technical skills simulators. Cognitive skills simulators utilized high-fidelity mannequins and animal models combined with traditional educational material to enhance pattern recognition of critical complications during liver transplantation. One manuscript focused on a technical skills acquisition by utilizing transesophageal echocardiography (TEE) to identify intraoperative pathologies. There is a heterogeneity in the exposure to liver transplant care during anesthesia training. Simulation provides low-stakes exposure to the high-stakes skills required in the operating room. Hence, it can be used as an adjunct to improve both cognitive and technical skill acquisition for perioperative transplant anesthesia. The goal of these simulation programs is to improve patient outcomes and produce more capable anesthesiologists.

Dopamine D3 receptor ligand suppresses the expression of levodopa-induced dyskinesia in nonhuman primate model of parkinson's disease.

Parkinson's disease (PD) is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. The incidence of PD increases with aging and the expected burden will continue to escalate with our aging population. Since its discovery in the 1961 levodopa has remained the gold standard pharmacotherapy for PD. However, the progressive nature of the neurodegenerative process in and beyond the nigrostriatal system causes a multitude of side effects, including levodopa-induced dyskinesia within 5 years of therapy. Attenuating dyskinesia has been a significant challenge in the clinical management of PD. We report on a small molecule that eliminates the expression of levodopa-induced dyskinesia and significantly improves PD-like symptoms. The lead compound PD13R we discovered is a dopamine D3 receptor partial agonist with high affinity and selectivity, orally active and with desirable drug-like properties. Future studies are aimed at developing this lead compound for treating PD patients with dyskinesia.

Impact of societal restrictions and lockdown on trauma admissions during the COVID-19 pandemic: a single-centre cross-sectional observational study.

BACKGROUND: Societal restrictions and lockdown during the coronavirus (COVID-19) pandemic have had a significant impact on the volume and nature of trauma admissions. We assessed the impact of COVID-19 related societal restrictions and lockdown on trauma admissions to single level 1 trauma centre in Westmead, Australia. We hypothesized that the number of trauma admissions would decrease and number of admissions due to self-harm and assault (specifically domestic violence) would increase. METHODS: Data was collected from the prospectively maintained Westmead Hospital Trauma Registry. The primary outcome compared was the average number of trauma admissions during March and April during years 2016 to 2020. Analysis of variance was used to analyse means. Pairwise differences among group means were evaluated with Tukey's honestly significant difference test. Secondary outcomes compared were in-hospital interventions and patient outcomes. RESULTS: There was a 23-34% decrease (P = 0.018) in the mean monthly average trauma admissions during March/April 2020 compared with previous years 2016-2019. In addition, there was a 40-52% decrease (P = 0.025) and 13-29% decrease (P = 0.020) in admissions due to road traffic collisions and falls respectively. CONCLUSION: There was a significant decrease in the overall number of trauma-related admissions during the COVID-19-related period of societal restrictions and lockdown. This was due to a decrease in minor traumas, falls and road traffic collisions. There was no difference in the number of admissions secondary to major traumas, self-harm or assault.

Real-time fluorescence imaging with 20 nm axial resolution.

Measuring the nanoscale organization of protein structures near the plasma membrane of live cells is challenging, especially when the structure is dynamic. Here we present the development of a two-wavelength total internal reflection fluorescence method capable of real-time imaging of cellular structure height with nanometre resolution. The method employs a protein of interest tagged with two different fluorophores and imaged to obtain the ratio of emission in the two channels. We use this approach to visualize the nanoscale organization of microtubules and endocytosis of the epidermal growth factor receptor.

Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein.

Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins.

Genetic influences on survival time after severe hemorrhage in inbred rat strains.

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system.

Hepatitis C virus (HCV), which infects 2-3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.

Predicting response to hepatitis C therapy.

Current treatment for chronic hepatitis C is expensive, is often accompanied by burdensome side effects, and, sadly, fails in almost half of cases. The ability to predict such failures prior to treatment could save a great deal of pain and expense for the patient with HCV. In this issue of the JCI, Aurora and colleagues describe the development of genetic markers predictive of treatment response based on a study of viral sequence variation (see the related article beginning on page 225). Genome-wide covariation analyses of pretreatment virus sequences from 94 patients showed distinct patterns of mutations strongly associated with the ultimate success or failure of treatment. Such analyses suggest markers predictive of response to therapy and may lead to new insights into the underlying biology of hepatitis C.

Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (Pinus taeda).

MicroRNAs (miRNAs) are known to regulate plant development, but have not been studied in gymnosperm seed tissues. The presence and characteristics of several miRNAs were examined in zygotic embryos (ZEs) and female gametophytes (FGs) of Pinus taeda (loblolly pine). Evidence for miRNAs was obtained using northern analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mediated with poly(A) polymerase. Partial sequences of two miRNAs were verified. Three regions of putative mRNA targets were analyzed by qRT-PCR to monitor the occurrence of stage-dependent miRNA-mediated cleavage. Five miRNAs were identified in ZEs and FGs along with partial sequences of Pta-miR166 and Pta-miR167. Both miRNAs showed differing degrees of tissue-specific and stage-specific modulation. Analysis of HB15L mRNA (a potential Pta-miR166 target) suggested miRNA-guided cleavage in ZEs and FGs. Analysis of ARF8L mRNA (a potential Pta-miR167 target) implied cleavage in ZEs but not in FGs. Argonaute9-like mRNA (ptAGO9L) showed stage-specific modulation of expression in ZEs that appeared to be inverted in the corresponding FGs. MicroRNAs and argonaute genes varied spatiotemporally during seed development. The peak levels of Pta-miR166 in FGs and ptAGO9L in embryos occurred at stage 9.1, a critical transition point during embryo development and a point where somatic embryo maturation often stops. MicroRNAs identified in FG tissue may play a role in embryogenesis.

Public health model for prevention of liver cancer among Asian Americans.

Asian Americans and Pacific Islanders (AAPIs) account for over half of the 1.3 million chronic hepatitis B cases and for over half of the deaths resulting from chronic hepatitis B infection in United States. There are very few studies published about hepatitis B virus (HBV) data in the Baltimore-Washington metropolitan area. In 2003, the Hepatitis B Initiative-DC (HBI-DC) worked closely with a large Korean church, located in Vienna, Virginia. Their partnership included a pilot-test of a faith-based HBV program, which educates, screens and vaccinates for the HBV. This pilot program was later expanded to include a total of nine Korean and Chinese American churches in this region, plus a Pastor's Conference targeting Asian American pastors from around the United States. During 2003-2006, a total of 1,775 persons were tested for HBV infection through the HBI-DC program. Of all the participants, 2% (n=35) were tested HBV positive (HbsAg+, HbsAb-), 37% (n=651) were HBV negative but protected (HbsAg-, HbsAb+), and 61% (n=1089) were unprotected (HbsAg-, HbsAb-). Most of these unprotected individuals (n=924) received the first vaccination. The proportion of the second vaccination was 88.8% (n=824). About 79% completed 3-shot vaccine series. Our study contributes to the literature by providing an overview of the hepatitis B unprotected rate among Asian American adults. It indicates that culturally integrated liver cancer prevention program will reduce cancer health disparities in high risk immigrant populations.

Expressed sequence tags from loblolly pine embryos reveal similarities with angiosperm embryogenesis.

The process of embryogenesis in gymnosperms differs in significant ways from the more widely studied process in angiosperms. To further our understanding of embryogenesis in gymnosperms, we have generated Expressed Sequence Tags (ESTs) from four cDNA libraries constructed from un-normalized, normalized, and subtracted RNA populations of zygotic and somatic embryos of loblolly pine (Pinus taeda L.). A total of 68,721 ESTs were generated from 68,131 cDNA clones. Following clustering and assembly, these sequences collapsed into 5,274 contigs and 6,880 singleton sequences for a total of 12,154 non-redundant sequences. Searches of a non-identical amino acid database revealed a putative homolog for 9,189 sequences, leaving 2,965 sequences with no known function. More extensive searches of additional plant sequence data sets revealed a putative homolog for all but 1,388 (11.4%) of the sequences. Using gene ontologies, a known function could be assigned for 5,495 of the 12,154 total non-redundant sequences with 13,633 associations in total assigned. When compared to approximately 72,000 sequences in a collated P. taeda transcript assembly derived from >245,000 ESTs derived from root, xylem, stem, needles, pollen cone, and shoot ESTs, 3,458 (28.5%) of the non-redundant embryo sequences were unique and thereby provide a valuable addition to development of a complete loblolly pine transcriptome. To assess similarities between angiosperm and gymnosperm embryo development, we examined our EST collection for putative homologs of angiosperm genes implicated in embryogenesis. Out of 108 angiosperm embryogenesis-related genes, homologs were present for 83 of these genes suggesting that pine contains similar genes for embryogenesis and that our RNA sampling methods were successful. We also identified sequences from the pine embryo transcriptome that have no known function and may contribute to the programming of gene expression and embryo development.

Labile DNA sequences in flax identified by combined sample representational difference analysis (csRDA).

Flax (Linum usitatissimum) has a genome in which changes have been associated with environmental factors. The inbred flax variety, Stormont Cirrus (Pl), served as the parent, and several lines (termed genotrophs) were derived from this parent. The phenotypes of the genotrophs were stable in a number of different growth environments, unlike the original Pl line in which changes associated with environmental factors continued to occur. These genotrophs differed from the original line in a number of characteristics, but the only known phenotypic characteristic that is shared by all the genotrophs and different from the parental, Pl, line is the lack of changes associated with the original environmental factors. However, some of these genotrophs have changed in both phenotype and nuclear DNA subsequent to their original growth and differentiation from Pl. Representational difference analysis (RDA) has been used to identify differences between Pl and all the genotrophs in an attempt to identify the loci controlling these aspects of plasticity. Subtractions between Pl DNA as a tester (target) and one of the genotrophs (individual RDA) or a mixture of different types of genotroph (L6, S6, C2, and LH) DNAs as a driver were done (combined sample RDA; csRDA). In addition, contrary RDA, where of the genotroph DNA was used as a tester and Pl DNA as a driver, was also executed. Three difference clones (163-4-2, 123-5-2, and 163-13), from 74 primary clones obtained after three rounds of subtractions with Pl DNA as tester were further characterized. In addition, 2 difference products (213-r1 and 213-r9) were characterized from contrary RDA. The clones 163-4-2 and 163-13 from the csRDA showed polymorphisms between Pl and all the genotrophs when PCR was done with primers derived from sequences of the clones, but only the clone 163-13 polymorphism was confirmed by Southern blot analysis. Four of 5 clones (163-4-2, 123-5-2, 163-13 and 213-r9) that have been characterized appear to be associated with structural changes in the DNA. From the contrary csRDA, it was observed that no clones could be recovered from subtractions between a mixture of genotrophs as a tester and Pl as a driver, and several possible explanations have been proposed.

Impacts of transcriptional regulation on aging and senescence.

The genetic makeup of the organism appears to dictate the species-specific rate of aging and the maximum life-span potential. The genotype is converted to phenotype through transcriptional and translational regulation. A group of gene regulatory proteins (transcription factors) play critical roles in controlling the rates of transcription of specific genes by directly interacting with regulatory sequences at gene promoters. Here, we review the basic mechanism of transcriptional control and the role of a number of transcription factors whose level and/or activity alter with age. Among these age-dependent transcription factors, many are involved in the regulation of stress and inflammatory responses and are subjected to functional alterations by reactive oxygen species (ROSs). A progressive rise of oxidative stress, impaired ability to cope with stressful stimuli and prolongation of the inflammatory response are some of the hallmarks of the senescent phenotype. Results published to date are supportive of the concept that a species-specific program of the temporal regulation of genes with additional modulation by a number of epigenetic factors, mediates the age-dependent deterioration of physiological functions and development of the senescent phenotype.