Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and ...mortalities. Despite the high prevalence of C. parvum, there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C. parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C. parvum in vitro and in vivo. In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro. We tested the inhibitors for anti-Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C. parvum in vitro, with IC50 values of 14.88 and 72.65 μM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C. parvum oocysts in infected immunocompromised mice's feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C. parvum. Together, our findings have unveiled promising anti-Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C. parvum infections.
Preferential repair of bulky DNA adducts from the transcribed genes via nucleotide excision repair is well characterized in mammalian cells. However, definitive evidence is lacking for similar repair ...of oxidized bases, the major endogenous DNA lesions. Here we show that the oxidized base-specific human DNA glycosylase NEIL2 associates with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), both in vitro and in cells. NEIL2 immunocomplexes from cell extracts preferentially repaired the mutagenic cytosine oxidation product 5-hydroxyuracil in the transcribed strand. In a reconstituted system, we also observed NEIL2-initiated transcription-dependent base excision repair of 5-hydroxyuracil in the transcribed strand, with hnRNP-U playing a critical role. Chromatin immunoprecipitation/reimmunoprecipitation studies showed association of NEIL2, RNA polymerase II, and hnRNP-U on transcribed but not on transcriptionally silent genes. Furthermore, NEIL2-depleted cells accumulated more DNA damage in active than in silent genes. These results strongly support the preferential role of NEIL2 in repairing oxidized bases in the transcribed genes of mammalian cells.
The etiology of non-alcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease, is poorly understood. To understand the causal mechanisms underlying NAFLD, we conducted a ...multi-omics, multi-tissue integrative study using the Hybrid Mouse Diversity Panel, consisting of ∼100 strains of mice with various degrees of NAFLD. We identified both tissue-specific biological processes and processes that were shared between adipose and liver tissues. We then used gene network modeling to predict candidate regulatory genes of these NAFLD processes, including Fasn, Thrsp, Pklr, and Chchd6. In vivo knockdown experiments of the candidate genes improved both steatosis and insulin resistance. Further in vitro testing demonstrated that downregulation of both Pklr and Chchd6 lowered mitochondrial respiration and led to a shift toward glycolytic metabolism, thus highlighting mitochondria dysfunction as a key mechanistic driver of NAFLD.
Display omitted
•NAFLD was modeled in a population of ∼100 diverse inbred strains of mice•Network modeling was used to predict key driver genes regulating NAFLD•In vivo knockdown of these genes rescued from steatosis and insulin resistance•In vitro knockdown of Pklr or Chchd6 shifted toward glycolytic metabolism
Chella Krishnan et al. apply integrative genetics approaches to delineate “key driver” genes regulating NAFLD using multi-omics data from ∼100 mouse strains. In vivo modulation of these genes rescued animals from steatosis and insulin resistance. Follow-up bioenergetics studies highlight mitochondrial dysfunction as a key mechanistic driver of NAFLD.
Introduction
Emerging long‐acting (LA) prevention and treatment medicines, technologies and regimens could be game‐changing for the HIV response, helping reach the ambitious goal of halting the ...epidemic by 2030. To attain this goal, the rapid expansion of at‐scale, sustainable, quality‐assured, and affordable supplies of LA HIV prevention and treatment products through accelerated and stronger competition, involving both originator and generic companies, will be essential. To do this, global health stakeholders should take advantage of voluntary licensing of intellectual property (IP) rights, such as through the United Nations‐backed, not‐for‐profit Medicines Patent Pool, as a proven mechanism to support broad access to existing HIV medicines across low‐ and middle‐income countries (LMICs).
Discussion
While voluntary licensing may unlock the possibility for generic competition to take place ahead of patent expiry, there are additional elements—of amplified importance for more complex LA HIV medicines—that need to be taken into consideration. This paper discusses 10 enablers of voluntary licensing of IP rights as a model to rapidly expand at‐scale, sustainable, quality‐assured, and affordable supplies of LA HIV prevention and treatment regimens in LMICs:
Identifying promising LA technology platforms and drug formulations at an early developmental stage and engaging with patent holders
Consolidating a multidisciplinary network and strengthening early‐stage coordination and collaboration to foster innovation
Embedding public health considerations in product design and delivery
Building innovative partnerships for product development and commercialization
Raising awareness of and creating demand for emerging LA products
Estimating the market size, ensuring sufficient competition and protecting sustainability
Using technology transfer and hands‐on technical support to reduce product development timelines and costs
Exploring de‐risking mechanisms and financial incentives to support generic manufacturers
Optimizing strategies for generic product development and regulatory filings
Aligning and coordinating efforts of stakeholders across the value chain.
Conclusions
Rapid access to emerging LA prevention and treatment regimens and technologies can be facilitated by voluntary licensing—catalyzed and supplemented by enabling collaborative and non‐duplicative efforts of various other stakeholders. This can effectively lead to improved—accelerated and cheaper—access to quality‐assured medicines for populations in LMICs.
Abstract
Background
Recent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. ...Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior.
Methods
Mice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression.
Results
Transcriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability.
Conclusions
These findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process.
A prototype nanoparticle biosensor based on localized surface plasmon resonance (LSPR) spectroscopy was developed to detect drug binding to human membrane-bound cytochrome P450 3A4 (CYP3A4). CYP3A4 ...is one of the most important enzymes in drug and xenobiotic metabolism in the human body. Because of the inherent propensity of CYP3A4 to aggregate, it is difficult to study drug binding to this protein in solution and on surfaces. In this paper, we use a soluble nanometer scale membrane bilayer disk (Nanodisk) to functionally stabilize monomeric CYP3A4 on Ag nanoparticle surfaces fabricated by nanosphere lithography. CYP3A4-Nanodiscs have absorption bands in the visible wavelength region, which upon binding certain drugs shift to either shorter (type I) or longer wavelengths (type II). On the basis of the coupling between the LSPR of the Ag nanoparticles and the electronic resonances of the heme chromophore in CYP3A4-Nanodiscs, LSPR spectroscopy is used to detect drug binding with high sensitivity. This paper combines LSPR and Nanodisc techniques to optically sense drug binding to a functionally stable membrane protein, with the goal of integrating this with microfluidics and expanding it into a multiarray format, enabling high-throughput screening.
The human body contains endocannabinoids that elicit similar psychoactive and anti‐nociceptive effects to phytocannabinoids in cannabis. Herein we report on the endogenous production of a previously ...unknown class of ω‐3 PUFA–derived endocannabinoid epoxides that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω‐3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid‐ethanolamide (EEQ‐EA) and epoxydocosapentaenoic acid‐ethanolamide (EDP‐EA), respectively. Both EEQ‐EAs and EDP‐EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω‐ 3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV‐2 microglial cells, and by human CYP2J2 epoxygenase. Neuroinflammation studies revealed that the terminal epoxides 17,18‐EEQ‐EA and 19,20‐EDP‐EA dose‐dependently abated proinflammatory IL‐6 cytokines while increasing anti‐inflammatory IL‐10 cytokines, in part through cannabinoid receptor‐2 and PPAR gamma activation. Furthermore, the ω‐3 endocannabinoid epoxides 17,18‐EEQ‐EA and 19,20‐EDP‐EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω‐3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. Furthermore, we examined the anti‐inflammatory and anti‐apoptotic role of the six different regioisomers of EDP‐EA (19,20, 16,17‐, 13,14‐, 10,11‐, 7,8‐ & 3,4) that showed wide range of activity towards cannabinoid receptors 1 & 2. In a separate studies we show that cannabinoids inhibit metabolism of endocannabinoids by CYPs.
Support or Funding Information
American Heart Association Scientist Development grant 15SDG25760064 National Institute of Health (NIH) R01 grant 1R01GM115584‐01A1 and NIH R03 grant 1R03DA042365‐01A1
Endocannabinoid metabolism by P450s
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega‐3 and omega‐6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the ...effects of N‐terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N‐terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N‐terminus was used to express full length CYP2J2 in E. coli while maintaining wild‐type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N‐terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native‐like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.
Background and Aims: Hepatitis C virus (HCV)‐induced chronic inflammation may induce oxidative stress which could compromise the repair of damaged DNA, rendering cells more susceptible to ...spontaneous or mutagen‐induced alterations, the underlying cause of liver cirrhosis and hepatocellular carcinoma. In the current study we examined the induction of reactive oxygen species (ROS) resulting from HCV infection and evaluated its effect on the host DNA damage and repair machinery.
Methods: HCV infected human hepatoma cells were analyzed to determine (i) ROS, (ii) 8‐oxoG and (iii) DNA glycosylases NEIL1, NEIL2, OGG1. Liver biopsies were analyzed for NEIL1.
Results: Human hepatoma cells infected with HCV JFH‐1 showed 30–60‐fold increases in ROS levels compared to uninfected cells. Levels of the oxidatively modified guanosine base 8‐oxoguanine (8‐oxoG) were significantly increased sixfold in the HCV‐infected cells. Because DNA glycosylases are the enzymes that remove oxidized nucleotides, their expression in HCV‐infected cells was analyzed. NEIL1 but not OGG1 or NEIL2 gene expression was impaired in HCV‐infected cells. In accordance, we found reduced glycosylase (NEIL1‐specific) activity in HCV‐infected cells. The antioxidant N‐acetyl cystein (NAC) efficiently reversed the NEIL1 repression by inhibiting ROS induction by HCV. NEIL1 expression was also partly restored when virus‐infected cells were treated with interferon (IFN). HCV core and to a lesser extent NS3‐4a and NS5A induced ROS, and downregulated NEIL1 expression. Liver biopsy specimens showed significant impairment of NEIL1 levels in HCV‐infected patients with advanced liver disease compared to patients with no disease.
Conclusion: Collectively, the data indicate that HCV induction of ROS and perturbation of NEIL1 expression may be mechanistically involved in progression of liver disease and suggest that antioxidant and antiviral therapies can reverse these deleterious effects of HCV in part by restoring function of the DNA repair enzyme/s.
ObjectiveOphthalmology is the busiest outpatient specialty with demand predicted to rise over 40% in the next 20 years. A significant increase in the number of trainee ophthalmologists is required to ...fill currently vacant consultant posts and meet the UK’s workforce demands by 2038. Our aim was to understand what determines success in ophthalmology training, in order to inform future ophthalmologists, refine recruitment and facilitate workforce planning.Methods and AnalysisThis was a retrospective longitudinal cohort study using routinely collected data available from UK Medical Education Database (UKMED) (https://www.ukmed.ac.uk/). Data were analysed on 1350 candidates who had applied for ophthalmology specialty training (OST) between 2012 and 2018, as well as 495 candidates who had attempted Fellow of the Royal College of Ophthalmologists (FRCOphth) Part 1 between 2013 and 2018. Participants who had not obtained their primary medical qualification from the UK medical schools were excluded. Primary outcome measures included gaining a place on the OST programme and passing the FRCOphth Part 1 examination on first attempt.ResultsHigher education performance measure decile scores at medical school are strongly predictive in securing an OST post and passing the part 1 examination first time (p<0.001). Candidates who attempt FRCOphth Part 1 prior to their ST1 application are more likely to get a place on OST on first attempt. Socioeconomic factors, gender and ethnicity do not influence success in OST entry. Male trainees are more likely to pass FRCOphth Part 1 on their first attempt.ConclusionThis study is the first quantitative assessment of the factors that determine success in OST recruitment and ophthalmology postgraduate examinations in the UK. Similar studies should be undertaken in all other medical and surgical specialties to understand what factors predict success.
PDF
