For the use of stem cell-derived neurons in clinical applications, improved differentiation efficiency and more careful characterization of resultant cells are needed. The authors refined a procedure for differentiation of mouse embryonic stem cells into functional dopamine neurons. This preparation provides a high yield of dopaminergic cells that are morphologically and functionally similar to cultured midbrain dopamine neurons and can be used as a platform for thorough investigation of the mechanisms of dopaminergic neurotransmission.
The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations.
For the use of stem cell-derived neurons in clinical applications, improved differentiation efficiency and more careful characterization of resultant cells are needed. A procedure has been refined for differentiation of mouse embryonic stem cells into functional dopamine neurons. This preparation provides a high yield of dopaminergic cells that are morphologically and functionally similar to cultured midbrain dopamine neurons and can be used as a platform for thorough investigation of the mechanisms of dopaminergic neurotransmission.
Embryonic stem cells; Dopamine; Electrophysiology; Synaptic vesicle; Quantum dots
The Ser/Thr protein kinase, RSK,
is associated with oncogenesis,
and therefore, there are ongoing efforts to develop RSK inhibitors
that are suitable for use in vivo. SL0101 is a natural
product that demonstrates selectivity for RSK inhibition. However,
SL0101 has a short biological half-life in vivo.
To address this issue we designed a set of eight cyclitol analogues,
which should be resistant to acid catalyzed anomeric bond hydrolysis.
The analogues were synthesized and evaluated for their ability to
selectively inhibit RSK in vitro and in cell-based
assays. All the analogues were prepared using a stereodivergent palladium-catalyzed
glycosylation/cyclitolization for installing the aglycon. The l-cyclitol analogues were found to inhibit RSK2 in in
vitro kinase activity with a similar efficacy to that of
SL0101, however, the analogues were not specific for RSK in cell-based
assays. In contrast, the d-isomers showed no RSK inhibitory
activity in in vitro kinase assay.
Ser/Thr protein kinase; cyclitol; RSK inhibition; SL0101; de novo synthesis
Vertebrate-specific neuronal genes are expected to play a critical role in the diversification and evolution of higher brain functions. Among them, the glycosylphosphatidylinositol (GPI)-anchored netrin-G subfamily members in the UNC6/netrin family are unique in their differential expression patterns in many neuronal circuits, and differential binding ability to their cognate homologous post-synaptic receptors.
To gain insight into the roles of these genes in higher brain functions, we performed comprehensive behavioral batteries using netrin-G knockout mice. We found that two netrin-G paralogs that recently diverged in evolution, netrin-G1 and netrin-G2 (gene symbols: Ntng1 and Ntng2, respectively), were responsible for complementary behavioral functions. Netrin-G2, but not netrin-G1, encoded demanding sensorimotor functions. Both paralogs were responsible for complex vertebrate-specific cognitive functions and fine-scale regulation of basic adaptive behaviors conserved between invertebrates and vertebrates, such as spatial reference and working memory, attention, impulsivity and anxiety etc. Remarkably, netrin-G1 and netrin-G2 encoded a genetic “division of labor” in behavioral regulation, selectively mediating different tasks or even different details of the same task. At the cellular level, netrin-G1 and netrin-G2 differentially regulated the sub-synaptic localization of their cognate receptors and differentiated the properties of postsynaptic scaffold proteins in complementary neural pathways.
Pre-synaptic netrin-G1 and netrin-G2 diversify the complexity of vertebrate behaviors and differentially regulate post-synaptic properties. Our findings constitute the first genetic analysis of the behavioral and synaptic diversification roles of a vertebrate GPI protein and presynaptic adhesion molecule family.
Netrtin-G1; Netrin-G2; Molecular evolution; Cognitive diversification; GPI-protein; Postsynapse; Presynapse
Myelodysplastic syndromes (MDS) are hallmarked by cytopenia and dysplasia of hematopoietic cells, often accompanied by mitochondrial dysfunction and increases of reactive oxygen species (ROS) within affected cells. However, it is not known whether the increase in ROS production is an instigator or a byproduct of the disease. The present investigation shows that mice lacking immediate early responsive gene X-1 (IEX-1) exhibit lineage specific increases in ROS production and abnormal cytology upon radiation in blood cell types commonly identified in MDS. These affected cell lineages chiefly have the bone marrow as a primary site of differentiation and maturation, while cells with extramedullary differentiation and maturation like B- and T-cells remain unaffected. Increased ROS production is likely to contribute significantly to irradiation-induced thrombocytopenia in the absence of IEX-1 as demonstrated by effective reversal of the disorder after mitoquinone (MitoQ) treatment, a mitochondria-specific antioxidant. MitoQ reduced intracellular ROS production within megakaryocytes and platelets. It also normalized mitochondrial membrane potential and superoxide production in platelets in irradiated, IEX-1 deficient mice. The lineage-specific effects of mitochondrial ROS may help us understand the etiology of thrombocytopenia in association with MDS in a subgroup of the patients.
Megakaryocytes; Mitoquinone; Reactive Oxygen Species; Thrombocytopenia
Reactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy. We recently identified Inauhzin (INZ) as a novel non-genotoxic p53-activating compound. To develop INZ into a clinically applicable anticancer drug, we have initiated preclinical toxicity studies. Here, we report our study on determining the maximum tolerated dose (MTD) of INZ analog, Inauhzin-C (INZ (C)), following intraperitoneal (i.p) administration (Phase A) and its toxicity following i.p administration over a period of 5-day dosing plus 2-day recovery (Phase B) in CD-1 mice. The phase A study showed that the MTD of INZ (C) is 200 mg/kg for female and 250 mg/kg for male, respectively. The phase B study showed that the administration of INZ (C) via 5-day consecutive i.p injection is tolerated by female CD-1 mice at all dose levels tested from 50mg/kg to 120 mg/kg without significant changes in biochemical and pathological parameters in the animals. Together, these results indicate that our previously determined effective dose of INZ at 30–60 mg/kg via i.p is quite safe to mice, and imply that this compound have the features worthy for further development into a clinically applicable drug.
Inauhzin; mouse; toxicity study; dose range finding; maximum tolerated dose
Primary cilia are sensory organelles present on most mammalian cells. The assembly and maintenance of primary cilia are facilitated by intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium. Mutations in genes coding for IFT components have been associated with a group of diseases called ciliopathies. These genetic disorders can affect a variety of organs including the retina. Using whole exome sequencing in three families, we identified mutations in Intraflagellar Transport 172 Homolog [IFT172 (Chlamydomonas)] that underlie an isolated retinal degeneration and Bardet–Biedl syndrome. Extensive functional analyses of the identified mutations in cell culture, rat retina and in zebrafish demonstrated their hypomorphic or null nature. It has recently been reported that mutations in IFT172 cause a severe ciliopathy syndrome involving skeletal, renal, hepatic and retinal abnormalities (Jeune and Mainzer-Saldino syndromes). Here, we report for the first time that mutations in this gene can also lead to an isolated form of retinal degeneration. The functional data for the mutations can partially explain milder phenotypes; however, the involvement of modifying alleles in the IFT172-associated phenotypes cannot be excluded. These findings expand the spectrum of disease associated with mutations in IFT172 and suggest that mutations in genes originally reported to be associated with syndromic ciliopathies should also be considered in subjects with non-syndromic retinal dystrophy.
Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities.
conformation analysis; drug design; NMR spectroscopy; RNA
Understanding the processes that influence the structure of biotic communities is one of the major ecological topics, and both stochastic and deterministic processes are expected to be at work simultaneously in most communities. Here, we investigated the vertical distribution patterns of bacterial communities in a 10-m-long soil core taken within permafrost of the Qinghai-Tibet Plateau. To get a better understanding of the forces that govern these patterns, we examined the diversity and structure of bacterial communities, and the change in community composition along the vertical distance (spatial turnover) from both taxonomic and phylogenetic perspectives. Measures of taxonomic and phylogenetic beta diversity revealed that bacterial community composition changed continuously along the soil core, and showed a vertical distance-decay relationship. Multiple stepwise regression analysis suggested that bacterial alpha diversity and phylogenetic structure were strongly correlated with soil conductivity and pH but weakly correlated with depth. There was evidence that deterministic and stochastic processes collectively drived bacterial vertically-structured pattern. Bacterial communities in five soil horizons (two originated from the active layer and three from permafrost) of the permafrost core were phylogenetically random, indicator of stochastic processes. However, we found a stronger effect of deterministic processes related to soil pH, conductivity, and organic carbon content that were structuring the bacterial communities. We therefore conclude that the vertical distribution of bacterial communities was governed primarily by deterministic ecological selection, although stochastic processes were also at work. Furthermore, the strong impact of environmental conditions (for example, soil physicochemical parameters and seasonal freeze-thaw cycles) on these communities underlines the sensitivity of permafrost microorganisms to climate change and potentially subsequent permafrost thaw.
The mixture of volatile organic compounds in the headspace gas of urine may be able to distinguish lung cancer patients from relevant control populations.
Subjects with biopsy confirmed untreated lung cancer, and others at risk for developing lung cancer, provided a urine sample. A colorimetric sensor array was exposed to the headspace gas of neat and pre-treated urine samples. Random forest models were trained from the sensor output of 70 % of the study subjects and were tested against the remaining 30 %. Models were developed to separate cancer and cancer subgroups from control, and to characterize the cancer. An additional model was developed on the largest clinical subgroup.
90 subjects with lung cancer and 55 control subjects participated. The accuracies, reported as C-statistics, for models of cancer or cancer subgroups vs. control ranged from 0.795 – 0.917. A model of lung cancer vs. control built using only subjects from the largest available clinical subgroup (30 subjects) had a C-statistic of 0.970. Models developed and tested to characterize cancer histology, and to compare early to late stage cancer, had C-statistics of 0.849 and 0.922 respectively.
The colorimetric sensor array signature of volatile organic compounds in the urine headspace may be capable of distinguishing lung cancer patients from clinically relevant controls. The incorporation of clinical phenotypes into the development of this biomarker may optimize its accuracy.
Volatile organic compounds; Urine; Biomarker; Lung cancer
It is well-known that ionic surfactant coated single-walled carbon nanotubes (SWNTs) possess higher near-infrared fluorescence (NIRF) quantum yield than nonionic polymer functionalized SWNTs. However, the influence of surface functionalization on the magnetic properties of SWNTs for T2-weighted magnetic resonance imaging (MRI) has not been reported. Here, we demonstrate that SWNTs functionalized by nonionic polymers display superior T2 relaxivity for MRI as compared to those coated by ionic surfactants. This difference may indicate that micelle structures formed by ionic surfactants are sufficiently tight to partially exclude water protons from the iron catalysts attached to the ends of SWNTs. On the basis of the different effects of the two types of suspension agents on NIRF and MRI of functionalized SWNTs, we further explore the competitive surface functionalization between ionic surfactants and nonionic polymers by stepwise replacing ionic surfactant molecules in a nanotube suspension with nonionic polymers. The superior NIRF of ionic surfactant coated SWNTs gradually quenches whereas no improvement on T2 relaxivity is observed during this replacement process. This result may indicate that nonionic polymers wrap around the outside of micelle structures to form small nanotube bundles rather than replacing ionic surfactants in the micelle structures to directly interact with the SWNT surface. Finally, we demonstrate the feasibility of dual-modality NIRF and MRI of nonionic polymer functionalized SWNTs in brain cells.
Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states—or excited conformational states—that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond 13C-1H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.
CRISPR-Cas systems have augmented our ability to produce
precise genome manipulations. Here we demonstrate and characterize
the ability of CRISPR-Cas derived nickases to direct targeted recombination
of both small and large genomic regions flanked by repetitive elements
in Escherichia coli. While CRISPR directed double-stranded DNA breaks are highly lethal
in many bacteria, we show that CRISPR-guided nickase systems can be
programmed to make precise, nonlethal, single-stranded incisions in
targeted genomic regions. This induces recombination events and leads
to targeted deletion. We demonstrate that dual-targeted nicking enables
deletion of 36 and 97 Kb of the genome. Furthermore, multiplex targeting
enables deletion of 133 Kb, accounting for approximately 3% of the
entire E. coli genome. This technology provides a
framework for methods to manipulate bacterial genomes using CRISPR-nickase
systems. We envision this system working synergistically with preexisting
bacterial genome engineering methods.
nickase; genome engineering; chromosome deletion; direct repeats; recombination
Trichophyton rubrum (T.rubrum) caused onychomycosis is the most common nail fungal disease. The common diagnostic methods are direct microscopic examination and fungal culture. In this study we used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study the subungual ultrastructural changes in T. rubrum induced onychomycosis.
Six outpatients with onychomycosis were recruited and T.rubrum infection was confirmed by fungal culture. Six toenail samples were collected and prepared for SEM characterization. The cultured fugal colonies were prepared for SEM and TEM characterization.
1) SEM showed significant structural damages and the formation of a thin layer or a single layer of keratinocytes in all infected nail plates. Hyphae (piercing or penetrating keratinocytes layers), arthrospores and local bacterial aggregation were observed on the ventral surface of the nail plates. 2) SEM of the cultured fungal colony showed relatively straight, highly branched hyphae and microconidias; TEM showed branching hyphae that were composed of double-layer cell walls. Hyphae had nucleus, mitochondria, liposomes, lysosomes, scattered rough endoplasmic reticulum, myeloid bodies and aggregated ribosomes. There were high-density particles outside the hyphae.
SEM showed a large number of hyphae penetrated the keratinocytes layer, suggesting that T. rubrum can cause severe damage to the stratum corneum. TEM showed the ultrastructural features of T. rubrum-induced infection before treatment.
Onychomycosis; Trichophyton rubrum; Scanning electron microscopy; Transmission electron microscopy; Ultrastructure
In an effort to improve
upon the in vivo half-life
of the known ribosomal s6 kinase (RSK) inhibitor SL0101, C4″-amide/C6″-alkyl
substituted analogues of SL0101 were synthesized and evaluated in
cell-based assays. The analogues were prepared using a de novo asymmetric
synthetic approach, which featured Pd-π-allylic catalyzed glycosylation
for the introduction of a C4″-azido group. Surprisingly replacement
of the C4″-acetate with a C4″-amide resulted in analogues
that were no longer specific for RSK in cell-based assays.
Motivation: In chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and other short-read sequencing experiments, a considerable fraction of the short reads align to multiple locations on the reference genome (multi-reads). Inferring the origin of multi-reads is critical for accurately mapping reads to repetitive regions. Current state-of-the-art multi-read allocation algorithms rely on the read counts in the local neighborhood of the alignment locations and ignore the variation in the copy numbers of these regions. Copy-number variation (CNV) can directly affect the read densities and, therefore, bias allocation of multi-reads.
Results: We propose cnvCSEM (CNV-guided ChIP-Seq by expectation-maximization algorithm), a flexible framework that incorporates CNV in multi-read allocation. cnvCSEM eliminates the CNV bias in multi-read allocation by initializing the read allocation algorithm with CNV-aware initial values. Our data-driven simulations illustrate that cnvCSEM leads to higher read coverage with satisfactory accuracy and lower loss in read-depth recovery (estimation). We evaluate the biological relevance of the cnvCSEM-allocated reads and the resultant peaks with the analysis of several ENCODE ChIP-seq datasets.
Availability and implementation: Available at http://www.stat.wisc.edu/∼qizhang/
Contact: firstname.lastname@example.org or email@example.com
Supplementary data are available at Bioinformatics online.
Objective: To investigate the phenotype conversion of epicardial adipocytes and its potential molecular mechanism during the occurrence and development of coronary atherosclerosis. Methods: A total of 30 health male New Zealand white rabbits were used. In experiment group (n=15), rabbits were fed with high fat food to establish atherosclerosis animal model; rabbits in control group (n=15) were fed with normal food. Results: At week 0, UCP-1 and PPARγ mRNA expressions in EAT and sBAT were significantly higher than in eWAT, and leptin mRNA expression lower than (P<0.05). In experiment group, the mRNA expressions of UCP-1 and PPARγ reduced gradually, but leptin mRNA increased progressively in EAT (P<0.05). UCP-1 expression reduced gradually, the newly generated blood vessels reduced significantly, but leptin and RAM11 increased gradually (P<0.05). The adipocyte volume in EAT increased gradually, but the adipocyte number reduced progressively (P<0.05). The number of mitochondria with multiple crests reduced gradually in EAT; IL-6 reduced the mRNA expressions of UCP-1 and PPARγ in adipocytes of BAT in a dose dependent manner, but it increased the mRNA expressions of leptin and STAT3 (P<0.05). In the presence of IL-6, JSI-124 increased the mRNA expressions of UCP-1 and PPAR-γ in adipocytes of BAT in a dose dependent manner, but it reduced the mRNA expressions of leptin and STAT3 (P<0.05). Conclusion: During the progression of atherosclerosis, there is a phenotype conversion of EAT from BAT to WAT, which further promotes the focal occurrence and development of atherosclerosis; IL-6 may activate JAK-STAT3 pathway to induce this conversion.
Epicardiac adipose tissue; atherosclerosis; cellular phenotype; IL-6; STAT
Formaldehyde inhalation exposure, which can occur through occupational exposure, can lead to sensory irritation, neurotoxicity, mood disorders, and learning and memory impairment. However, its influence on olfactory function is unclear.
To investigate the mechanism and the effect of repeated formaldehyde inhalation exposure on olfactory function.
Rats were treated with formaldehyde inhalation (13.5±1.5 ppm, twice 30 minutes/day) for 14 days. Buried food pellet and locomotive activity tests were used to detect olfactory function and locomotion. Western blots were used to evaluate synaptosomal-associated protein 25 (SNAP25) protein levels in the olfactory bulb (OB) lysate and synaptosome, as well as mature and immature olfactory sensory neuron markers, olfactory marker protein (OMP), and Tuj-1. Real-time polymerase chain reaction (PCR) was used to detect SNAP25 mRNA amounts.
Repeated formaldehyde inhalation exposure impaired olfactory function, whereas locomotive activities were unaffected. SNAP25 protein decreased significantly in the OB, but not in the occipital lobe. SNAP25 also decreased in the OB synaptosome when synaptophysin did not change after formaldehyde treatment. mRNA levels of SNAP25A and SNAP25B were unaffected. Mature and immature olfactory sensory neuron marker, OMP, and Tuj-1, did not change after formaldehyde treatment.
Repeated formaldehyde exposure impaired olfactory function by disturbing SNAP25 protein in the OB.
Formaldehyde inhalation; SNAP25; Olfactory function; Synaptosome
Part of the G protein (3094–4170 bp) of spring viremia of carp virus (SVCV) was expressed in Escherichia coli and purified by dialysis in our study. Two clones of monoclonal antibodies (MAbs 1H11 and 4B8) against G protein were generated by fusion of mouse myeloma cell line SP2/0 and spleen lymphocytes from part of G protein (3094–4170 bp) immunized mice. The results of ELISA (enzyme-linked immunosorbent assay), IFA (indirect immunofluorescent assay), and Western blot assay further demonstrated the characterizations of the two MAbs. Both 1H11 and 4B8 were specific to SVCV G protein. Ten pairs of synthesized overlapping peptides were used to identify the epitope of the MAbs. The MAbs are useful in the development of SVCV diagnostic methods.
Malnutrition is found in almost 100% of patients with end stage liver disease (ESLD) awaiting transplantation and malnutrition before transplantation leads to higher rates of post-transplant complications and worse graft survival outcomes. Reasons for protein energy malnutrition include several metabolic alterations such as inadequate intake, malabsorption, and overloaded expenditure. And also, stress from surgery, gastrointestinal reperfusion injury, immunosuppressive therapy and corticosteriods use lead to delayed bowl function recovery and disorder of nutrients absorption. In the pretransplant phase, nutritional goals include optimization of nutritional status and treatment of nutrition-related symptoms induced by hepatic decompensation. During the acute post-transplant phase, adequate nutrition is required to help support metabolic demands, replenish lost stores, prevent infection, arrive at a new immunologic balance, and promote overall recovery. In a word, it is extremely important to identify and correct nutritional deficiencies in this population and provide an adequate nutritional support during all phases of liver transplantation (LT). This study review focuses on prevalence, nutrition support, evaluation, and management of perioperative nutrition disorder in patients with ESLD undergoing LT.
Nutritional management; end stage liver disease (ESLD); liver transplantation (LT)
Due to the proved clinical efficacy, Shuang-Huang-Lian (SHL) has
developed a variety of dosage forms. However, the in-depth
research on targets and pharmacological mechanisms of SHL
preparations was scarce. In the presented study, the
bioinformatics approaches were adopted to integrate relevant data
and biological information. As a result, a PPI network was built
and the common topological parameters were characterized. The
results suggested that the PPI network of SHL exhibited a
scale-free property and modular architecture. The drug target
network of SHL was structured with 21 functional modules.
According to certain modules and pharmacological effects
distribution, an antitumor effect and potential drug targets were
predicted. A biological network which contained 26 subnetworks was
constructed to elucidate the antipneumonia mechanism of SHL. We
also extracted the subnetwork to explicitly display the pathway
where one effective component acts on the pneumonia related
targets. In conclusions, a bioinformatics approach was established
for exploring the drug targets, pharmacological activity
distribution, effective components of SHL, and its mechanism of
antipneumonia. Above all, we identified the effective components
and disclosed the mechanism of SHL from the view of system.
The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D “organoid” system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression and CHD1 loss. Whole exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as of DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.
We investigated whether and to what extent cystatin C was associated with angiographic coronary collateralization in patients with stable coronary artery disease and chronic total occlusion.
Serum levels of cystatin C and high-sensitive C-reactive protein (hsCRP) and glomerular filtration rate (GFR) were determined in 866 patients with stable angina and angiographic total occlusion of at least one major coronary artery. The degree of collaterals supplying the distal aspect of a total occlusion from the contra-lateral vessel was graded as poor (Rentrop score of 0 or 1) or good coronary collateralization (Rentrop score of 2 or 3).
In total, serum cystatin C was higher in patients with poor collateralization than in those with good collateralization (1.08 ± 0.32 mg/L vs. 0.90 ± 0.34 mg/L, P < 0.001), and correlated inversely with Rentrop score (adjusted Spearmen’s r = -0.145, P < 0.001). The prevalence of poor coronary collateralization increased stepwise with increasing cystatin C quartiles (P for trend < 0.001). After adjusting for age, gender, risk factors for coronary artery disease, GFR and hsCRP, serum cystatin C ≥ 0.97 mg/L remained independently associated with poor collateralization (OR 2.374, 95% CI 1.660 ~ 3.396, P < 0.001). The diagnostic value of cystatin C levels for detecting poor coronary collateralization persisted regardless of age, gender, presence or absence of diabetes, hypertension or renal dysfunction.
Serum cystatin C reflects angiographic coronary collateralization in patients with stable coronary artery disease, and cystatin C ≥ 0.97 mg/L indicates a great risk of poor coronary collaterals.
Lanthionine-containing peptides (lanthipeptides)
are a rapidly
growing family of polycyclic peptide natural products belonging to
the large class of ribosomally synthesized and post-translationally
modified peptides (RiPPs). These compounds are widely distributed
in taxonomically distant species, and their biosynthetic systems and
biological activities are diverse. A unique example of lanthipeptide
biosynthesis is the prochlorosin synthetase ProcM from the marine
cyanobacterium Prochlorococcus MIT9313,
which transforms up to 29 different precursor peptides (ProcAs) into
a library of lanthipeptides called prochlorosins (Pcns) with highly
diverse sequences and ring topologies. Here, we show that many ProcM-like
enzymes from a variety of bacteria have the capacity to carry out
post-translational modifications on highly diverse precursor peptides,
providing new examples of natural combinatorial biosynthesis. We also
demonstrate that the leader peptides come from different evolutionary
origins, suggesting that the combinatorial biosynthesis is tied to
the enzyme and not a specific type of leader peptide. For some precursor
peptides encoded in the genomes, the leader peptides apparently have
been truncated at the N-termini, and we show that these N-terminally
truncated peptides are still substrates of the enzymes. Consistent
with this hypothesis, we demonstrate that about two-thirds of the
ProcA N-terminal sequence is not essential for ProcM activity. Our
results also highlight the potential of exploring this class of natural
products by genome mining and bioengineering.