Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.
The aim of this study was to screen for retinopathy of prematurity (ROP) in southwestern China and understand the prevalence and risk factors of ROP, which may provide evidence useful in the prevention and treatment of ROP.
1864 preterm infants (gestational age of <37 weeks and birth weight of ≤2500 g) underwent ROP screening from January 2009 to November 2012 in Southwest China. The medical information of infants during perinatal period was reviewed, and risk factors of ROP were determined. A total of 1614 infants were recruited for final analysis.
Incidence of ROP was 12.8%. The first, second, third, and fourth stage of ROP was found in 64.6%, 29.6%, 3.4%, and 0.5% of infants, respectively. No fifth stage of ROP was observed. In addition, 7.7% of infants required surgical intervention. In our Department of Neonatology, the incidence of ROP was 20.0%, which was significantly higher than in non-hospitalized patients (9.9%). The incidence of ROP remained unchanged over the years. Independent risk factors of ROP included low birth weight (p=0.049), low gestational age (p=0.008), days of oxygen supplementation (p=0.008), and myocardial injury after birth (p=0.001).
The prevalence of ROP in preterm infants is relatively high in Southwest China, and low birth weight, low gestational age, days of oxygen supplementation, and myocardial injury after birth are independent risk factors for ROP.
China; Prevalence; Retinopathy of Prematurity; Risk Factors
Silica coated silver (Ag@SiO2) nanoparticles were prepared and characterized by transmission electron microscope (TEM) and UV-vis absorption, and the nanoparticles were used as labels in sandwich-type immunosensor of Escherichia coli O157:H7 (E. coli O157:H7). The labels involved in immunoreaction were dissolved by mixed acid of hydrofluoric acid and nitric acid, and the released Ag+ ions were electrochemical stripping analyzed (via differential pulse voltammetry, DPV) at poly(acrylic acid)/poly(diallyldimethylammonium chloride)/carbon nanotubes (PAA/PDCNT) modified glass carbon electrode (GCE), which obviously enhanced the signal of Ag+ stripping. Then, the number of E. coli O157:H7 could be indirectly reflected by the signal intensity of labeled Ag+. And the results showed that the DPV signals were proportional to the logarithm of the E. coli O157:H7 concentration in the range from 20 cfu/mL to 8.0 × 103 cfu/mL with the detection limit of 13 cfu/mL.
Adipose-resident T-cells (ARTs) regulate metabolic and inflammatory responses in obesity, but ART activation signals are poorly understood. Here, we describe class II major histocompatibility complex (MHCII) as an important component of high-fat diet (HFD)-induced obesity. Microarray analysis of primary adipocytes revealed that multiple genes involved in MHCII antigen processing and presentation increased in obese women. In mice, adipocyte MHCII increased within two weeks HFD, paralleling increases in pro-inflammatory and decreases in anti-inflammatory ART markers, and preceding adipose tissue macrophage (ATM) accumulation and pro-inflammatory M1 polarization. Mouse 3T3-L1 and primary adipocytes activated T-cells in an antigen-specific, contact-dependent manner, indicating adipocyte MHCII is functional. HFD-fed MHCII−/− mice developed less adipose inflammation and insulin resistance than wild-type mice, despite developing similar adiposity. These investigations uncover a mechanism whereby a HFD-induced adipocyte/ART dialogue involving MHCII instigates adipose inflammation and, together with ATM MHCII, escalates its progression.
Obesity; adipose tissue; MHCII; T-cell activation
To construct a new visual acuity measuring function for congenital nystagmus (CN) patients by studying the relationships between acuity, velocities and positions of the eye.
After assessing the relationship between acuity, movement velocities and positions of the eye separately, a new function, which we call the automated nystagmus acuity function (ANAF), was constructed to measure the visual acuity of CN patients. Using a high-speed digital video system working at 500 frames per second, each eye was calibrated during monocular fixation. Twenty-six recorded nystagmus data were selected randomly. Using nystagmus waveforms, the best vision position (foveation period) and visual acuity were analyzed in three groups of subjects, and then all outputs were compared with the well-known expanded nystagmus acuity function (NAFX) and ANAF. Standard descriptive statistics were used to summarize the outputs of the two programs.
Foveation periods were brief intervals in the CN waveform when the image was on or near the fovea and eye velocity was relatively slow. Results showed good visual acuity happened during the period when velocity was low and the eye position was near the zero position, which fitted the foveation periods. The data analyzed with NAFX and ANAF had a correlation coefficient of 0.934276, with an average error of -0.00973.
The results from ANAF and NAFX analyses showed no significant difference. The NAFX manually identifies foveation eye positions and produces accurate measurements. The ANAF, however, can be calculated simply using the factors eye position and velocity, and it automatically calculates the ANAF without the need to manually identify foveation eye positions.
nystagmus; vision acuity; nystagmus acuity function; eye movement measurements
Contemporary views of tumorigenesis regard its inception as a convergence of genetic mutation and developmental context. Glioma is the most common and deadly malignancy in the CNS; therefore, understanding how regulators of glial development contribute to its formation remains a key question. Previously we identified nuclear factor I-A (NFIA) as a key regulator of developmental gliogenesis, while miR-223 has been shown to repress NFIA expression in other systems. Using this relationship as a starting point, we found that miR-223 can suppress glial precursor proliferation via repression of NFIA during chick spinal cord development. This relationship is conserved in glioma, as miR-223 and NFIA expression is negatively correlated in human glioma tumors, and the miR-223/NFIA axis suppresses tumorigenesis in a human glioma cell line. Subsequent analysis of NFIA function revealed that it directly represses p21 and is required for tumorigenesis in a mouse neural stem cell model of glioma. These studies represent the first characterization of miR-223/NFIA axis function in glioma and demonstrate that it is a conserved proliferative mechanism across CNS development and tumorigenesis.
Currently, the main treatment for hepatocellular carcinoma (HCC) involves the surgical removal of tumors or liver transplantation. However, these treatments are often not completely curative, as they are associated with a risk for postoperative recurrence and metastasis. Circulating tumor cells (CTCs) are increasingly recognized as the main source for recurrence and metastasis after radical hepatectomies are performed. Many studies have demonstrated the association between the presence of either pre- or postoperative CTCs and an increased risk for HCC recurrence. To improve the therapeutic outcome of HCC, a personalized, comprehensive and multidisciplinary approach should be considered, involving the application of appropriate diagnostic and therapeutic measures targeting HCC CTCs in different stages throughout the course of treatment. This article proposes some HCC CTC-based strategies for the treatment of HCC, including the monitoring of HCC CTCs before, during and after radical hepatectomy, therapeutic targeting of HCC CTCs, prevention of the generation and colonization of CTCs, as well as the use of CTC indexes for the selection of indications, prediction of prognoses, and planning of individualized therapeutic regimens. Innovation and technological development of therapies targeting CTCs, as well as their translation into clinical practice, will help to effectively reduce postoperative recurrence and metastasis, and significantly prolong the survival of HCC patients.
Hepatocellular carcinoma; Circulating tumor cells; Recurrence and metastasis; Surgical treatment; Individualized treatment
The way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila hemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.
To identify pathogenic mutations responsible for retinal dystrophies (RDs) in three unrelated Chinese families.
Three probands from unrelated families with RDs were recruited. Genomic DNA prepared from leukocytes was analyzed using gene chip–based next-generation sequencing (NGS) to capture and sequence all of the exons of 100 known RD-associated genes. Candidate variants were validated with PCR and Sanger sequencing in the respective families. Thorough ophthalmic examinations including best-corrected visual acuity, funduscopic examination, and full-field electroretinograms were performed in the affected individuals.
We successfully identified causative mutations in patients from the Chinese families with RDs: the known mutation IMPDH1 c.942_944delGAA in a family with retinitis pigmentosa, the novel mutation ABCA4 c.1924T>A in a family with Stargardt disease, and the novel mutation NMNAT1 c.272A>G and known mutation NMNAT1 c.196C>T in a family with Leber congenital amaurosis. All variations segregated with the disease phenotypes in the respective families and were absent from ethnically matched control chromosomes. Prediction analysis demonstrated the two novel missense mutations might be damaging.
The results strongly suggested these mutations were responsible for different RD phenotypes in the Chinese families. NGS technology provides an accurate and economic method for identifying causative genes for RDs.
Comprising 1011 neurons with 1014 synaptic connections the human brain is the ultimate systems biology puzzle. An increasing body of evidence highlights the observation that changes in brain function, both normal and pathological, consistently correlate with dynamic changes in neuronal anatomy. Anatomical changes occur on a full range of scales from the trafficking of individual proteins, to alterations in synaptic morphology both individually and on a systems level, to reductions in long distance connectivity and brain volume. The major sites of contact for synapsing neurons are dendritic spines, which provide an excellent metric for the number and strength of signaling connections between elements of functional neuronal circuits. A comprehensive model of anatomical changes and their functional consequences would be a holy grail for the field of systems neuroscience but its realization appears far on the horizon. Various imaging technologies have advanced to allow for multi-scale visualization of brain plasticity and pathology, but computational analysis of the big data sets involved forms the bottleneck toward the creation of multiscale models of brain structure and function. While a full accounting of techniques and progress toward a comprehensive model of brain anatomy and function is beyond the scope of this or any other single paper, this review serves to highlight the opportunities for analysis of neuronal spine anatomy and function provided by new imaging technologies and the high-throughput application of older technologies while surveying the strengths and weaknesses of currently available computational analytical tools and room for future improvement.
dendritic spines; microscopy; image analysis; systems biology; modeling and simulations; big data
To characterize the spectrum of CYP4V2 gene mutations in 92 unrelated Chinese probands with Bietti’s crystalline dystrophy (BCD) and to describe the molecular and clinical characteristics of four novel CYP4V2 mutations associated with BCD.
All study participants underwent a complete ophthalmological examination. Mutational screening of CYP4V2 coding regions and flanking intron sequences was examined via directional Sanger sequencing, with allele separation confirmed by screening other family members. Subsequent in silico analysis of the mutational consequence on protein function was undertaken, with the impact of the novel mutation on pre-mRNA splicing examined via RT–PCR.
Fifteen disease-causing variants were identified in 92 probands with BCD, including four novel mutations and eleven previously reported mutations. The most prevalent mutation was c.802_810del17insGC, which was detected in 69 unrelated families, with an allele frequency of 52.7% (97/184). Homozygosity was revealed in 35 unrelated families, and compound heterozygosity was observed in 43 subjects. Four patients harbored four novel variants, with these mutations cosegregated within all affected individuals and were not found in unaffected family members and 100 unrelated controls. Transcriptional analysis of a novel splice mutation revealed altered RNA splicing. In silico analysis predicted that the missense variant, p.Tyr343Asp, disrupted the CYP4V2 surface electrostatic potential distribution and spatial conformation. Among the patients with four novel mutations, genotype did not always correlate with age at onset, disease course, or electroretinogram (ERG) changes, with phenotypic variations even noted within the same genotype.
The c.802_810del17insCG mutation was the most common mutation in the 92 Chinese probands with BCD examined. Four novel mutations were identified, contributing to the spectrum of CYP4V2 mutations associated with BCD, with no clear link established between disease phenotype and genotype.
The western borderland between Yunnan Province, China, and Myanmar is characterized by a climate that facilitates year-round production of mosquitoes. Numerous mosquito-transmitted viruses, including Japanese encephalitis virus circulate in this area. This project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations.
Mosquitoes were collected in Mangshi and Ruili cities of Dehong Prefecture near the border of China and Burma in Yunnan Province, the Peoples Republic of China in 2010. We monitored mosquito species abundance for a 12-month period using ultraviolet light, carbon dioxide baited CDC light and gravid traps; and tested the captured mosquitoes for the presence of virus to evaluate mosquito-virus associations in rural/agricultural settings in the area.
A total of 43 species of mosquitoes from seven genera were collected, including 15 Culex species, 15 Anopheles spp., four Aedes spp., three Armigeres spp., one Mimomyia spp., two Uranotaenia spp. and three Mansonia spp.. Species richness and diversity varied between Mangshi and Ruili. Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Anopheles peditaeniatus were the most abundant species in both sampling sites. Ultraviolet light traps collected more specimens than CDC light traps baited with dry ice, though both collected the same variety of mosquito species. The CDC gravid trap was the most effective trap for capture of Culex quinquefasciatus, a species underrepresented in light trap collections. A total of 26 virus strains were isolated, which included 13 strains of Japanese encephalitis virus, four strains of Getah virus, one strain of Oya virus, one strain from the orbivirus genus, and seven strains of Culex pipien pallens densovirus.
The present study illustrates the value of monitoring mosquito populations and mosquito-transmitted viruses year-round in areas where the climate supports year-round adult mosquito activity.
Background: the number and proportion of adults diagnosed with HIV infection aged 50 years and older has risen. This study compares the effect of CD4 counts and anti-retroviral therapy (ART) on mortality rates among adults diagnosed aged ≥50 with those diagnosed at a younger age.
Methods: retrospective cohort analysis of national surveillance reports of HIV-diagnosed adults (15 years and older) in England, Wales and Northern Ireland. The relative impacts of age, CD4 count at diagnosis and ART on mortality were determined in Cox proportional hazards models.
Results: among 63,805 adults diagnosed with HIV between 2000 and 2009, 9% (5,683) were aged ≥50 years; older persons were more likely to be white, heterosexual and present with a CD4 count <200 cells/mm3 (48 versus 32% P < 0.01) and AIDS at diagnosis (19 versus 9%, P < 0.01). One-year mortality was higher in older adults (10 versus 3%, P < 0.01) and especially in those diagnosed with a CD4 <200 cells/mm3 left untreated (46 versus 15%, P < 0.01). While the relative mortality risk reduction from ART initiation at CD <200 cells/mm3 was similar in both age groups, the absolute risk difference was higher among older adults (40 versus 12% fewer deaths) such that the number needed to treat older adults to prevent one death was two compared with eight among younger adults.
Conclusions: the magnitude of benefit from ART is greater in older adults than younger adults. Older persons should be considered as a target for HIV testing. Coupled with prompt treatment, earlier diagnosis is likely to reduce substantially deaths in this group.
HIV; AIDS; antiretroviral therapy; epidemiology; surveillance; older people
Recent advances in automated high-resolution fluorescence microscopy and robotic handling have made the systematic and cost effective study of diverse morphological changes within a large population of cells possible under a variety of perturbations, e.g., drugs, compounds, metal catalysts, RNA interference (RNAi). Cell population-based studies deviate from conventional microscopy studies on a few cells, and could provide stronger statistical power for drawing experimental observations and conclusions. However, it is challenging to manually extract and quantify phenotypic changes from the large amounts of complex image data generated. Thus, bioimage informatics approaches are needed to rapidly and objectively quantify and analyze the image data. This paper provides an overview of the bioimage informatics challenges and approaches in image-based studies for drug and target discovery. The concepts and capabilities of image-based screening are first illustrated by a few practical examples investigating different kinds of phenotypic changes caEditorsused by drugs, compounds, or RNAi. The bioimage analysis approaches, including object detection, segmentation, and tracking, are then described. Subsequently, the quantitative features, phenotype identification, and multidimensional profile analysis for profiling the effects of drugs and targets are summarized. Moreover, a number of publicly available software packages for bioimage informatics are listed for further reference. It is expected that this review will help readers, including those without bioimage informatics expertise, understand the capabilities, approaches, and tools of bioimage informatics and apply them to advance their own studies.
To investigate inhibitory γ-aminobutyric acid (GABA) ergic postsynaptic currents (IPSCs) and postsynaptic currents (PSCs) in layer IV of the rat visual cortex during the critical period and when plasticity was extended through dissolution of the perineuronal nets (PNNs).
We employed 24 normal Long-Evans rats to study GABAA-PSC characteristics of neurons within layer IV of the visual cortex during development. The animals were divided into six groups of four rats according to ages at recording: PW3 (P21-23d), PW4 (P28-30d), PW5 (P35-37d), PW6 (P42-44d), PW7 (P49-51d), and PW8 (56-58d). An additional 24 chondroitin sulfate proteoglycan (CSPG) degradation rats (also Long-Evans) were generated by making a pattern of injections of chondroitinase ABC (chABC) into the visual cortex 1 week prior to recording at PW3, PW4, PW5, PW6, PW7, and PW8. Immunohistochemistry was used to identify the effect of chABC injection on CSPGs. PSCs were detected with whole-cell patch recordings, and GABAA receptor-mediated IPSCs were pharmacologically isolated.
IPSC peak current showed a strong rise in the age-matched control group, peaked at PW5 and were maintained at a roughly constant value thereafter. Although there was a small increase in peak current for the chABC group with age, the peak currents continued to decrease with the delayed highest value at PW6, resulting in significantly different week-by-week comparison with normal development. IPSC decay time continued to increase until PW7 in the control group, while those in the chABC group were maintained at a stable level after an initial increase at PW4. Compared with normal rats, the decay times recorded in the chABC rats were always shorter, which differed significantly at each age. We did not observe any differences in IPSC properties between the age-matched control and penicillinase (P-ase) group. However, the change in IPSCs after chABC treatment was not reflected in the total PSCs or in basic membrane properties in layer IV of the rat visual cortex.
Our results demonstrate that rather than rapidly increasing during the critical period for neuronal plasticity, IPSCs in layer IV of rat visual cortex are maintained at an immature level when PNNs are removed by chABC. This suggests that GABA receptor maturation involves the conformation of the CSPGs in PNNs.
gamma-aminobutyric acid receptor; plasticity; visual cortex; development; postsynaptic currents; chondroitinase ABC; chondroitin sulfate proteoglycans; whole-cell patch recording
To determine if transplantation of olfactory ensheathing cells (OECs) can reduce loss of optic nerve axons after raised intraocular pressure (IOP) in the rat.
OECs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. The IOP was raised by injection of magnetic microspheres into the anterior chamber.
At 4 weeks after raising the IOP, the transplanted OECs had migrated into the dorsal area of the optic nerve head (ONH) where they surrounded the optic nerve fibers with a non-myelinated ensheathment. The mean amount of damage to the ONH astrocytic area in rats was 51.0% compared with 85.8% in those without OEC transplants (P < 0.02) and the mean loss of axons in the optic nerve was 51.0% compared with 80.3% in the absence of OECs (P < 0.01).
OECs transplanted into the region of the ONH of the rat can reduce the loss of axons and the damage to ONH astrocytes caused by raised IOP.
Confirmation of these preliminary experimental data, further understanding of possible mechanisms of axonal protection by OECs, and the longer-term time course of protection could provide a basis for future human clinical trials of autografted OECs, which would be available from autologous nasal epithelial biopsies.
olfactory ensheathing cells; lamina cribrosa; transplantation; optic nerve head
Optogenetics is a new and rapidly evolving gene and neuroengineering technology that allows optical control of specific populations of neurons without affecting other neurons in the brain at high temporal and spatial resolution. By heterologous expression of the light-sensitive membrane proteins, cell type-specific depolarization or hyperpolarization can be optically induced on a millisecond time scale. Optogenetics has the higher selectivity and specificity compared to traditional electrophysiological techniques and pharmaceutical methods. It has been a novel promising tool for medical research. Because of easy handling, high temporal and spatial precision, optogenetics has been applied to many aspects of nervous system research, such as tactual neural circuit, visual neural circuit, auditory neural circuit and olfactory neural circuit, as well as research of some neurological diseases. The review highlights the recent advances of optogenetics in medical study.
light-sensitive proteins; optogenetics; optical manipulation; medical
Murine typhus; Rickettsia typhi; outbreak; China–Myanmar border; People’s Republic of China; typhus; Rickettsia; vector-borne infections
The present study investigated the prevalence of refractive error, visual impairment, and eye diseases in school-aged children in western China.
The survey was done in a representative county (Yongchuan District, Chongqing Municipality) of western China. Cluster random sampling was used to select children aged 6 to 15 years. We conducted door-to-door surveys and eye examinations including optometry, stereoscopic vision test, eye position and eye movement, slit lamp examination of the anterior segment, retinoscopy, and fundus examination after cycloplegia with 1% cyclopentolate.
Among 3469 children, data were available for 3079 (88.76%). The prevalences of eye diseases were, in descending order, refractive error (20.69%; 637/3079), conjunctivitis (11.76%; 362/3079), amblyopia (1.88%; 58/3079), color vision defect (0.52%; 16/3079), keratitis (0.36%; 11/3079), strabismus (0.29%; 9/3079), cataract (0.23%; 7/3079), pathologic myopia (0.19%; 6/3079), and ocular trauma (0.13%; 4/3079). The prevalence of corneal leucoma, corneal staphyloma, optic neuropathy, macular degeneration, and myelinated nerve fibers was 0.03% (1/3079) for each. The prevalence of visual impairment was 7.70% (237/3079), and the major causes of visual impairment were uncorrected refractive error (86.08%; 204/237), amblyopia (9.70%; 23/237), pathologic myopia (1.27%; 3/237), congenital cataract (0.42%; 1/237), and others (2.11%; 5/237).
Among school-aged children in a less developed area of western China, refractive error was the most prevalent eye disorder, and uncorrected refractive error was the main cause of visual impairment.
school-aged child; refractive error; visual impairment
The United Kingdom’s CD4 surveillance scheme monitors CD4 cell counts among HIV patients and is a national resource for HIV surveillance. It has driven public health policy and allowed auditing of national HIV testing, treatment and care guidelines.
We demonstrate its utility through four example outputs: median CD4 count at HIV diagnosis; late HIV diagnosis and short-term mortality; the timing of first CD4 count to indicate entry into HIV care; and the proportion of patients with CD4 counts <350 cells/mm3 receiving anti-retroviral therapy (ARV).
In 2009, 95% (61,502/64,420) of adults living with diagnosed HIV infection had CD4 counts available. The median CD4 count at diagnosis increased from 276 to 335 cells/mm3 between 2000 and 2009, indicating modest improvements in HIV testing. In 2009, 52% of patients were diagnosed at a late stage of HIV infection (CD4 <350 cells/mm3); these individuals had a ten-fold risk of dying within a year of their diagnosis compared to those diagnosed promptly. In 2008, the national target of performing a CD4 count within 14 days of diagnosis was met for 61% of patients. National treatment guidelines have largely been met with 83% patients with CD4 <350 cells/mm3 receiving ARV.
The monitoring of CD4 counts is critical to HIV surveillance in the United Kingdom enabling the close monitoring of efforts to reduce morbidity and mortality associated with late diagnosis and underpins the auditing of policies and guidelines. These routine surveillance outputs can be generated at national and local levels to drive and monitor public health policy and prevention efforts.
Surveillance; CD4 counts; patient outcomes; United Kingdom.
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2′-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3′- and 5′-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3′,5′-O-diisobutyryl-2′-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.
We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial “hit” improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.
Genetic interaction profiles are highly informative and helpful for understanding the functional linkages between genes, and therefore have been extensively exploited for annotating gene functions and dissecting specific pathway structures. However, our understanding is rather limited to the relationship between double concurrent perturbation and various higher level phenotypic changes, e.g. those in cells, tissues or organs. Modifier screens, such as synthetic genetic arrays (SGA) can help us to understand the phenotype caused by combined gene mutations. Unfortunately, exhaustive tests on all possible combined mutations in any genome are vulnerable to combinatorial explosion and are infeasible either technically or financially. Therefore, an accurate computational approach to predict genetic interaction is highly desirable, and such methods have the potential of alleviating the bottleneck on experiment design.
In this work, we introduce a computational systems biology approach for the accurate prediction of pairwise synthetic genetic interactions (SGI). First, a high-coverage and high-precision functional gene network (FGN) is constructed by integrating protein-protein interaction (PPI), protein complex and gene expression data; then, a graph-based semi-supervised learning (SSL) classifier is utilized to identify SGI, where the topological properties of protein pairs in weighted FGN is used as input features of the classifier. We compare the proposed SSL method with the state-of-the-art supervised classifier, the support vector machines (SVM), on a benchmark dataset in S. cerevisiae to validate our method's ability to distinguish synthetic genetic interactions from non-interaction gene pairs. Experimental results show that the proposed method can accurately predict genetic interactions in S. cerevisiae (with a sensitivity of 92% and specificity of 91%). Noticeably, the SSL method is more efficient than SVM, especially for very small training sets and large test sets.
We developed a graph-based SSL classifier for predicting the SGI. The classifier employs topological properties of weighted FGN as input features and simultaneously employs information induced from labelled and unlabelled data. Our analysis indicates that the topological properties of weighted FGN can be employed to accurately predict SGI. Also, the graph-based SSL method outperforms the traditional standard supervised approach, especially when used with small training sets. The proposed method can alleviate experimental burden of exhaustive test and provide a useful guide for the biologist in narrowing down the candidate gene pairs with SGI. The data and source code implementing the method are available from the website: http://home.ustc.edu.cn/~yzh33108/GeneticInterPred.htm