Aspergillus flavus spores simultaneously in the form of microspheres using calcium chloride as a cross-linking agent. The formulations were characterized by Fourier transform infrared spectroscopy (FTIR), a scanning electron micrograph (SEM), and thermogravimetry (TGA). Formulation characteristics, including the bead size, entrapment efficiency, swelling ratio of the beads, and rheological properties, were analyzed. The release behavior of beads with different formulations was evaluated. The addition of kaolin and rice husk powder in starch–alginate beads retarded the release profile of spores and metalaxyl. The release of the active ingredient from starch–alginate–kaolin beads and starch–alginate–rice husk powder beads occurred in both a controlled and sustained manner. Additionally, the release rate decreased with the increase of kaolin or rice husk powder content. The beads added with kaolin were slower than the release of rice husk powder. In comparison, spores released slower and lasted longer than metalaxyl. The starch–alginate–kaolin formulations could be used as controlled release material in the field of biocontrol and reduce the harm of fungicides to the environment.For the wise use of fungal biocontrol and metalaxyl fungicide, starch–alginate-based formulations have been developed by encapsulating metalaxyl and non-toxigenic  Aspergillus flavus or Aspergillus parasiticus. Additionally, aflatoxin contamination is a serious problem in many crops, such as corn, peanuts, and other agricultural products, in many countries. Biological control methods have proved to be effective in preventing aflatoxin contamination in several crops, including peanuts, cotton, and corn [A. flavus or A. parasiticus in soil at a particular time of developing crops. The amount of aflatoxin can be reduced due to the competitive inhibition of a non-aflatoxigenic strain. Abbas et al. proved that the aflatoxin in corn displayed a significant decline after applying the non-aflatoxin producing A. flavus strain NRRL30797 [Aflatoxin is a kind of mycotoxin mainly produced by and corn . BiocontRRL30797 .Sterilized barley, paddy, and rice usually act as the carrier of the strain, and the spores are attached to the surface of the grain. After application to the soil surface, these starch-rich grains can absorb moisture and provide an environment for fungal growth and sporulation. Other carriers for fungal biocontrol, including alginate pellets and pre-gelatinized starch–flour granules, have been applied to soil . RecentlPolysaccharides in nature have attracted considerable attention as controlled release materials . Their sAgrochemicals including pesticides, herbicides, fungicides, etc., have contributed greatly to crop protection in modern agriculture . In prevMetalaxyl is a widely used acylanilide fungicide with residual and systemic activity against plant diseases caused by the Oomycetes or water-mold fungi ,27,28. UAspergillus flavus. In the present work, we intend to develop starch–alginate-based formulations for the biological control and prevention of aflatoxin by encapsulating metalaxyl and non-toxigenic Aspergillus flavus spores simultaneously. Additionally, the feasibility of the formulation as a controlled release substance will be verified. The ingredients in the formulation are starch, alginate, kaolin, and rice husk. All of them are biodegradable, low cost, biocompatible, easily available, and environmentally friendly. Kaolin, as a common clay, has been utilized in controlled-release materials due to its larger surface area and noticeable ion-exchange capacity, and these advantages played a role in absorbing/desorbing the agrochemicals [Aspergillus flavus and metalaxyl based on starch–alginate.In terms of the existing research, most have focused on the separate encapsulation of the pesticide, bacteria, or fungus. Additionally, literature on the simultaneous and sustained release of fungi and agrochemicals is scanty. Since metalaxyl has often been used on crops such as peanuts and corn that were easily contaminated with aflatoxins , it woulhemicals . Rice huhemicals , which ahemicals . Therefo2, KBr, and phosphate buffer solution (PBS) were purchased from Sinopharm Chemical Reagent Co., Ltd. . DG18 agar was obtained from Beijing Aoboxing bio-tech Co., Ltd. Rice husk powder came from a local market in Changchun China and passed through a 100-mesh sieve. The non-toxic Aspergillus flavus strains (H4-5) were screened in our laboratory, which is located in Changchun China. The water used in the experiment was ultrapure water. All chemicals and reagents were used as received.Soluble starch (maize starch), sodium alginate, Tween 20, and metalaxyl were obtained from Aladin Ltd. . Kaolin was purchased from Xincheng Fine Chemical Co., Ltd. . CaClThe isolated non-toxic fungus was grown and maintained on dichloran 18% glycerol (DG18) agar. After incubation for two weeks at 30 °C, spores were removed by gently scraping DG18 plates and suspended in aqueous 0.2% Tween 20. The density of the spore suspension was measured using a hemocytometer and adjusted as described .2 solution under constant stirring. Thus, the beads were formed and cured for 30 min in the solution. After being taken out of the solution, the beads were washed three times with ultrapure water, and were allowed to dry for 12 hours at 40 °C in a circulating air oven [The quantitative starch, alginate, kaolin, or rice husk powder were dispersed in 50 mL of water with heating and magnetic stirring in a water bath. After a homogeneous solution was formed, 5 mg of metalaxyl and 5 mL of spore suspensions were then added to the glue solution and stirred for half an hour. The dispersion was added dropwise by a 50 mL syringe, from a 30 cm height, into 100 mL of 0.1 M CaClair oven . The comThirty thoroughly dried samples of each formulation were collected, and their outer diameters were measured using a Vernier caliper. The average bead diameter of each formulation is presented in 2 atmosphere and a flow rate of 50 mL/min, and the temperature ranged from 30 to 700 °C. Swelling properties of the beads were investigated in a buffer solution at 30 °C by monitoring the weight changes during incubation. A certain amount (Wd) of the completely dried beads was immersed in PBS (pH 6.0) and kept at 30 °C. The bloated beads were taken out and instantly weighed (Ws) after the excess of fluid lying on the surfaces was removed by filter paper. The swelling ratio (SR) was obtained by the equation: SR = (Ws − Wd)/Wd. Each group of measurements was repeated three times.The beads’ formulations were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron micrograph (SEM), thermogravimetry, and swelling tests. FTIR spectra of the beads were measured with KBr pellets by a Nexus670 FTIR  spectrometer. SEM was taken by HITACHI S-4800 . The samples were placed on a copper plate and sputtered with gold. Thermogravimetric analysis was achieved under atmospheric pressure using a SHIMADZU DTG-60H Thermal Analyzer , and the thermal properties of the samples were investigated. Quantitative samples were deposited in a Pt crucible. All experiments employed a linear heating rate of 10 °C/min under NThe rheological properties of the samples were measured with a small amplitude oscillatory test by an AR500 rheometer . After the metalaxyl and spore suspensions were mixed with different starch–alginate-based hydrogels, the samples were carefully transferred into the surface of the lower Peltier plate, and the upper sandblasted plate declined until it reached a 1000 μm gap distance. The storage modulus (G′) and loss modulus (G″) that differed with angular frequency were explored with the parallel plate geometry (diameter = 40 mm). The frequency ranged from 0.1 to 100 rad/s, the amplitude (γ) was 2%, and the temperature was 30 °C.The calibration curve was prepared with the absorbance of a series of standard solutions of metalaxyl spectrophotometrically by using a SHIMADZU UV-2550 UV-visible spectrophotometer . The experimental method proposed by Chen et al.  has beenAspergillus flavus within a certain concentration range. Encapsulation of non-toxic Aspergillus flavus spores and metalaxyl was implemented by the process of synthesizing bead formulations, which was stated in It was verified by a pre-experiment that metalaxyl had no effect on the sporulation of non-toxic Aspergillus flavus spores was measured by the method of a colony-forming unit (CFU/g). In order to determine the spore viability, 0.1 g of beads was dissolved in 10 mL of sodium citrate solution 2.0% (w/v) and then gradient dilutions of each sample in 0.85% w/v sodium chloride solution were prepared. Aliquots of 100 μL were inoculated on a DG18 medium and cultured for three to five days at 28 ± 2 °C. The results were obtained in terms of colony-forming units per gram of beads. The metalaxyl left in the CaCl2 solution from the beads was determined to get the entrapment efficiency of the fungicide. The method is depicted in The entrapment efficiency of non-toxic The controlled-release characteristics of starch–alginate-based beads were studied by immersing the dried and loaded samples of each formulation (1 g) in 50 mL of phosphate buffer solution (PBS) at 25 ± 1 °C. The pH of the PBS was adjusted to 6.0 in order to simulate soil acidity. The samples were placed in the dark to avoid the effects of light on matalaxyl.Aspergillus flavus colonies were recorded. After centrifugation at 10,000 rpm, the supernatant was determined by spectrophotometry at 220 nm after every 12 h to get the amount of metalaxyl released. After the sample was removed from the solution, an equivalent amount of fresh medium was poured in to maintain the osmotic pressure stability. All experiments were conducted in triplicate.The concentration of spores in the release medium was measured by the plate count method. After shaking until homogeneous, 200 μL of suspension was taken out and serially diluted in PBS prior to plating. Suspensions were diluted and 100 μL of aliquots were spread on DG18 medium. Plates were incubated at 28 ± 2 °C for three to five days and the results of To better understand the release process of spores and metalaxyl from the beads, mathematical modeling of the release was applied ,38,39. Ip < 0.05 was taken as the level of significance.Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Duncan’s multiple comparison tests using the software SPSS Statistics 20.0 . A value of p > 0.05). This may be due to the constant feed of conidial suspension and metalaxyl when synthesizing different formulations [The components of starch–alginate beads are shown in ulations −1. This is related to the –OH group together with some complex bands in the region 1200–1030 cm−1 due to C-O, C-H, and C-O-C stretching vibrations, which are the characteristic of natural polysaccharides. Furthermore, the absorption bands near 780 cm−1 are due to vibrational modes of pyranose rings of polysaccharides that have been espied [−1 was due to the stretching in aromatic rings [−1 was observed in all three figures due to the existence of carboxylate anions of calcium alginate [−1 in three cases was due to the presence of the C-N bond, which belonged to metalaxyl. This proved that metalaxyl was entrapped in the beads.FTIR spectra of the samples were measured with KBr pellets and are presented in n espied . The peaic rings . The stralginate . The absIt was observed that all beads were ellipsoidal shapes by SEM a–f. ObseThe shape of SA was more oval-shaped, while SAK and SAH were closer to a sphere. This was different from Baljit Singh’s results, which stated that the SA bead had a spherical shape and became elliptical with the incorporation of clay . This ma2, and H2O, which eventually formed polynuclear aromatic and graphitic carbon structures [The thermal stability of beads was investigated by thermogravimetry analysis (TGA) and derivative thermogravimetry (DTG), which is shown in ructures ,46. In tructures . The iniructures ,49. Simiructures . In eachructures .The influence of kaolin and rice husk powder on the swelling ratio of starch–alginate beads after incubation in PBS for 24 h is presented in The rheological properties of the composite gel were determined by measuring the storage modulus (G′) and loss modulus (G″) as a function of angular frequency at 30 °C . In nearThe release of the polymer-encapsulated substance undergoes two processes; first, the releasing medium penetrates the network of polymers and swells, followed by diffusion along the aqueous pathways to the surface of the granules. In the present study, the dried and loaded beads were immersed in PBS (pH 6.0) at 25 ± 1 °C in the light-proof condition, to simulate the soil environment. The release of spores and metalaxyl from the beads of different formulations has been investigated at the interval of 24 h and 12 h for 400 h and 300 h, respectively. The results are depicted in −1 after 336 h, whereas, with the addition of rice husk powder and kaolin, the amount of released spores decreased to 6.30 (SAH4) and 6.17 (SAK4) log CFU mL−1, respectively  versus ln t. The values of ‘n’ and ‘k’ and the diffusion mechanism for the release dynamics of spores and metalaxyl were evaluated and the results are presented in To investigate the mechanisms of spores and metalaxyl release from the starch–alginate beads, the diffusion characteristic constant ‘k’ and diffusion exponent ‘n’ were obtained by calculating the intercept and slope of the plot of ln (Starch–alginate-based beads were successfully developed by the ionic gelation method using calcium chloride as a crosslinking agent. Spores of a non-aflatoxigenic strain and metalaxyl were encapsulated in the beads confirmed by the measurement of entrapment efficiency and FTIR. It is concluded that the release of spores and metalaxyl from the beads was controllable and sustainable, which is important in the biocontrol of aflatoxin and management of pesticides’ harm to the environment and ecosystem.Aspergillus flavus, such as flowering or podding stages.The attendance of kaolin and rice husk powder in starch–alginate-based beads made the release rate slower, which prolonged the release of spores and metalaxyl and enhanced the bioavailability of the formulation. Moreover, the increase in kaolin or rice husk powder contents in these constitutions decreased the release of spores and metalaxyl. Further, the release rate of starch–alginate–kaolin beads was slower than that of rice husk powder-containing formulations. The spores’ release kinetics were consistent with the Normal Fickian diffusion mechanism, while the release of metalaxyl from most of the formulations occurred through the Case II diffusion mechanism. Thus, it can be concluded that the use of kaolin and rice husk powder as an addition to the starch–alginate-based encapsulation systems is very effective for the retarded release of bioactive substances. The starch–alginate–kaolin or starch–alginate–rice husk powder beads could be considered carriers of fungal biocontrol in the field of biological control and prevention, and also have the effect of a slow release of pesticides. In view of the slow release rate, starch–alginate–rice husk powder beads are suitable for use from the beginning of crop growth, while the starch–alginate–kaolin beads are suitable for later use, starting from the key stages of crops affected by


We report a disease outbreak caused by chikungunya virus in Zhejiang Province, China, in August 2017. Phylogenic analysis indicated that this virus belonged to the Indian Ocean clade of the East/Central/South African genotype and was imported by a traveler returning from Bangladesh. Aedes mosquitoes, specifically the species Ae. aegypti and Ae. albopictus  and has a humid subtropical monsoon climate. Its average annual temperature is 15°C–18°C, and it has abundant rainfall, an average of 1,100–2,200 mm annually. The mosquito vector Zhejiang Province does not belong to the endemic area of chikungunya; however, we report an outbreak of chikungunya caused by a traveler returning from Bangladesh in August 2017 in Quzhou, Zhejiang Province. The patient with the imported case was living in Huangdun village; another 3 CHIKV infections were confirmed in the same village  Figure. https://www.thermofisher.com) and performed phylogenetic analysis with 45 worldwide CHIKV strain sequences from GenBank. Results showed that the genome sequence of CHIKV ZJQZ3 was highly similar to the strain (GenBank accession no. MF773566) isolated from a patient in Australia who returned from Bangladesh in 2017 and belongs to the Indian Ocean clade of the East/Central/South African genotype  1 gene showed that 4 sequences were 100% identical with one another, which indicated that the infected traveler who returned from a disease-endemic area led to this local transmission and outbreak. We sequenced the complete genome sequence of CHIKV ZJQZ3  using Ion Torrent PGM  gene P3-D372E in CHIKV ZJQZ3 but found no Ae. albopictus mosquitoes in Zhejiang Province and may have the potential for further emergence and spread in northern China. Clinicians should be educated about the diagnosis of this disease, and public health organizations should work to overcome the diagnostic challenges of multiple arboviruses, carefully monitor imported cases, strengthen vector control, and conduct surveillance for CHIKV-infected vectors in high-risk areas to prevent local establishment of this new emerging virus.This outbreak indicates that CHIKV can be transmitted by Location of the sites of chikungunya virus infection in Quzhou, Zhejiang Province, China.


The binding of SYL1712 to the vial glycoprotein was computationally modeled and was predicted to interact with specific residues of GP. We used the time of the addition assay to show that compound SYL1712 blocks Ebola GP-mediated entry. Finally, consistent with being an Ebola virus entry inhibitor, compound SYL1712 inhibited infectious Ebola virus replication in tissue culture under biosafety level 4 containment, with an IC50 of 2 μM. In conclusion, we identified several related molecules with a diaryl-quinoline scaffold as potential anti-EBOV entry inhibitors, which can be further optimized for anti-Ebola drug development.Ebola virus is the causative agent of Ebola virus disease in humans. The lethality of Ebola virus infection is about 50%, supporting the urgent need to develop anti-Ebola drugs. Glycoprotein (GP) is the only surface protein of the Ebola virus, which is functionally critical for the virus to attach and enter the host cells, and is a promising target for anti-Ebola virus drug development. In this study, using the recombinant HIV-1/Ebola pseudovirus platform we previously established, we evaluated a small molecule library containing various quinoline compounds for anti-Ebola virus entry inhibitors. Some of the quinoline compounds specifically inhibited the entry of the Ebola virus. Among them, compound SYL1712 was the most potent Ebola virus entry inhibitor with an IC Filoviridae is considered a significant public health concern due its high fatality rate. In humans, the infection can cause Ebola virus disease (EVD), which is a lethal acute hemorrhagic disease . T. T35]. Te: 6F6S) . Overall50 of 1 μM using the pseudotyped virus, and an IC50 of 2 μM using infectious Ebola virus. The time-of-addition (TOA) experiment and computational docking study support the notion that this compound likely binds to the same cavity of Ebola GP, as do toremifene and other small molecule inhibitors [In this study, we used an HIV-1-based pseudotyped virus screening platform to evaluate a small library of diarylquinoline compounds, and we identified a number of Ebola virus-specific entry inhibitors. The most potent inhibitor of this series of compounds, SYL1712, has an IChibitors , and bloNumerous small molecule therapeutic candidates that directly target the EBOV GP or critical host factors for viral entry have been reported. Most of these inhibitors target events occurring in the endosome, such as GP proteolytic processing, endosomal trafficking, interactions with NPC1, and fusion, including: (1) CA-074, FY-DMK, CID23631927, K11777, and cathepsin B and L inhibitors ,39,40,41A number of SERMS, such as toremifene, and GPCR antagonists, such as benztropine, were shown to bind to a pocket at the GP1/GP2 subunit interface of the Ebola GP ,34,35,36


HIV self‐testing (HIVST) is recommended by the World Health Organization in addition to other testing modalities to increase uptake of HIV testing, particularly among harder‐to‐reach populations. This study provides the first empirical evidence of the costs of door‐to‐door community‐based HIVST distribution in Malawi, Zambia and Zimbabwe.HIVST kits were distributed door‐to‐door in 71 sites across Malawi, Zambia and Zimbabwe from June 2016 to May 2017. Programme expenditures, supplemented by on‐site observation and monitoring and evaluation data were used to estimate total economic and unit costs of HIVST distribution, by input and site. Inputs were categorized into start‐up, capital and recurrent costs. Sensitivity and scenario analyses were performed to assess the impact of key parameters on unit costs.In total, 152,671, 103,589 and 93,459 HIVST kits were distributed in Malawi, Zambia and Zimbabwe over 12, 11 and 10 months respectively. Across these countries, 43% to 51% of HIVST kits were distributed to men. The average cost per HIVST kit distributed was US$8.15, US$16.42 and US$13.84 in Malawi, Zambia and Zimbabwe, respectively, with pronounced intersite variation within countries driven largely by site‐level fixed costs. Site‐level recurrent costs were 70% to 92% of full costs and 20% to 62% higher than routine HIV testing services (HTS) costs. Personnel costs contributed from 26% to 52% of total costs across countries reflecting differences in remuneration approaches and country GDP.These early door‐to‐door community HIVST distribution programmes show large potential, both for reaching untested populations and for substantial economies of scale as HIVST programmes scale‐up and mature. From a societal perspective, the costs of HIVST appear similar to conventional HTS, with the higher providers’ costs substantially offsetting user costs. Future approaches to minimizing cost and/or maximize testing coverage could include unpaid door‐to‐door community‐led distribution to reach end‐users and integrating HIVST into routine clinical services via direct or secondary distribution strategies with lower fixed costs. Despite such gains, 24% of people living with HIV (PLWH) remain undiagnosed ® HIV self‐test now costing US$2 per kit in 50 low‐ and middle‐income countries HIVST is defined as a process where a person collects his/her own specimen  and then performs an HIV test and interprets the result, often in a private setting, either alone or with someone they trust. The World Health Organization recommends HIVST to reach the “at risk” and “untested” populations including men as a complement to current conventional testing approaches, including facility‐based and targeted community outreach‐based testing The HIV Self‐Testing AfRica (STAR) project has delivered over one million HIVST kits in Malawi, Zambia and Zimbabwe between 2016 and 2017 through a combination of distribution approaches, including facility‐based distribution at outpatient departments, within voluntary medical male circumcision (VMMC) services and in the community. This study presents the costs of the model that uses community‐based distribution agents (CBDAs) to deliver HIVST either at people's homes or within the community setting, hereafter “the CBDA model,” to generate evidence to inform the scale‐up of cost‐effective HIV testing services (HTS).22.1Table In the CBDA model, all individuals aged ≥16 years who were present in the homestead at the time of CBDAs’ home visit were eligible for self‐testing. Testing was done by the self‐tester themselves after kit use demonstration and information on test result interpretation and linkage to follow‐on care by the CBDAs. CBDAs provided a self‐referral card to all testers to facilitate linkage to the local health facility for confirmatory testing and care for individuals with reactive HIVST results. In some cases, CBDAs were present during the self‐test to provide reassurance and support if testers requested their presence or assistance. Table 2.2We estimated the full economic cost of delivering HIVST within the CBDA model from the providers perspective, following international costing guidelines This top‐down costing collated all financial expenditures and categorized each line item by input type and distribution model. Inputs were allocated to distribution sites following predefined allocation factors, based on project monitoring and evaluation (M&E) data, including the percentage of kits distributed, percentage of distributors based in each site, distance from central office and percentage of direct expenditures, which is a weighted average of the preceding allocation factors. Table To estimate economic costs, the expenditure analysis was complemented by a valuation of all other resources used in the CBDA model. Observations of distribution in each site strengthened the economists’ understanding of the intervention and allowed for collection of data on donated goods and services. As a vertical model, these were relatively limited, and include a value for district or health facility storage contributed by the public health system. During the life of the project, the price of HIVST kits dropped from nearly $4 per kit to $2 per kit. The latter was imputed in place of the higher observed prices as it was considered the relevant kit price for any decision‐making building upon this analysis. Total costs, total kits distributed and average cost per kit distributed were estimated at the country level, and for each country, at the site level. The latter provides a range of average costs by site and allows for identification of economies of scale.2.3We undertook a series of one‐way sensitivity analyses to assess the impact of key cost assumptions on the unit cost per HIVST kit distributed. We varied the discount rate used to annualize costs from the base case of 3% to 0% and 15% to capture the impact of not discounting or using a higher local central bank discount rate. Prevailing discount rates during the study period were 15% in Malawi, 12.5% in Zambia and 7% in Zimbabwe 2.4In anticipation of planned programme scale‐up by respective country ministries of health, we conducted scenario analysis varying salaries ±10% to assess the impact of integration into public health services, and variation in kit distribution by ±10%. We also modelled the impact of HIVST kit price between the observed average kit price (US$3.40), a recent Bill and Melinda Gates Foundation subsidized price (US$2) and a hypothetical price approximately equal to current rapid finger prick test price (US$1) 2.5The study did not involve patient‐level data collection; we did, however, obtain permission from ministries of health in the three countries to collate data from administrative, M&E records at facility level for cost allocation. Ethical approvals for the parent study were obtained from the Medical Research Council of Zimbabwe, Malawi College of Medicine Research Ethics Committee, University of Zambia Biomedical Research Ethics Committee, London School of Hygiene and Tropical Medicine Ethics Committee and University College London Ethics Committee. The trials are registered under the Clinical Trials Network  under registration numbers NCT02793804; NCT02718274; Pan African clinical trials registry PACTR201607001701788 for Malawi, Zambia and Zimbabwe.33.1During the costing period, 152,671, 103,589 and 93,459 HIVST kits were distributed in Malawi, Zambia and Zimbabwe against the approximate targets of 62,500, 416,294 and 224,116 through a total of 138, 139 and 1009 CBDAs respectively. The average number of HIVST kits distributed was 12,538  across 11 sites in Malawi, 7206  across 16 sites in Zambia and 2124 (range: 319 to 4201) across 44 sites in Zimbabwe, where distribution was intentionally restricted by campaign duration Table . Nearly 3.2Table 3.3The country‐level costs per HIVST kit distributed were US$8.15 for Malawi, US$16.42 for Zambia and US$13.84 in Zimbabwe. The cost per HIVST kit distributed across the sites ranged from US$7.20 to US$17.04 in Malawi, US$7.90 to U$50.00 in Zambia and from US$10.19 to US$54.44 in Zimbabwe. Figure 3.4Figures Varying HIVST kit price between US$1 and US$3.40 yielded costs of US$6.44, US$15.15 and US$12.25 versus US$8.87, US$17.60 and US$14.99 in Malawi, Zambia and Zimbabwe respectively. Varying salaries by ±10% yielded costs of US$7.94, US$15.57 and US$13.24 versus US$8.37, US$17.27 and US$14.43 respectively. Varying kit quantity by ±10% yielded costs of US$7.41, US$15.63 and US$12.83 versus US$9.06, US$17.60 and US$15.07 respectively. The best‐case scenario was US$6.14, US$13.99 and US$12.32 per kit distributed, whereas the worst‐case scenario was US$10.27, US$20.12 and US$21.85 per kit distributed.4This is the first published study to present costs of door‐to‐door CBDA delivery of HIVST kits in Malawi, Zambia and Zimbabwe. Costs ranged from as low as US$7.20 at a very large distribution site where CBDA distribution of HIVST kits was integrated with the delivery of other health products, to US$54.55 with campaign‐style delivery in a very small community in Zimbabwe that would otherwise not have access to testing. Staff costs contributed a substantial portion of the costs highlighting potential opportunities for lower cost models from reconfiguring distribution to rely on unpaid volunteers within door‐to‐door community‐led distribution models. Additionally, economies of scale can clearly be optimized. In this analysis, we showed how unit costs fall as the number of kits distributed increases. As all modes of testing are scaled up and testing coverage increases, it will be critical to target populations efficiently, with special focus on communities underserved by facility‐based HTS.Although costs are presented from a provider's perspective, door‐to‐door community HIVST distribution relieves users from substantial direct and indirect costs of attending health facilities. A study in these same communities in Malawi showed the mean costs of accessing HIV testing among women and men as US$1.83 and US$3.81, respectively, with men reporting significantly higher opportunity costs (i.e. lost income) HIVST costs reflected across all three countries are not dissimilar to those reported previously in Malawi $8.78 in 2016 US$) 16 US$ 20We anticipate potential for substantial economies of scale as HIVST programmes scale‐up and mature. The door‐to‐door community HIVST distribution model costed for this current study was implemented by a non‐governmental organization, under a research protocol, using paid and incentivized CBDAs and delivered to predominantly rural communities with no previous knowledge of, or experience with, HIVST. Interventions delivered in a research context tend to be associated with higher costs, as the primary objective is achieving effectiveness. Large‐scale implementation through door‐to‐door community‐led HIVST distribution with ordinarily paid government providers or community residents is likely to be significantly less costly. There are additional potential costs savings. First, we found costs were lower in high kit distribution sites suggesting economies of scale and ability to deliver at lower costs in more densely populated communities. Second, 10% to 20% of the costs were start‐up and initial capital costs, which would decrease as services mature. Third, as general populations and providers gain a better understanding of HIVST as a screening technology, we would expect less intense need for CBDAs  and community sensitization activities.Additionally, CBDAs could incorporate HIVST delivery into other health service activities thereby delivering cost savings to providers through economies of scope in services delivered by the CBDAs. Finally, as the HIVST market grows, technology advances and newer manufacturers enter, the price of HIVST kits will likely fall to prices comparable to blood‐based kits currently used in health facilities and in‐person support requirements could, in theory, could become cheaper than provider‐supervised testing. In this case, HIVST could save costs and allow providers to focus on confirmatory testing and strengthening linkage to ART From a research perspective, the wide cost variations highlight the importance of evaluating costs across a variety of settings in order to generate means and confidence intervals. Future analyses of these data may generate useful insights into efficiency and provide key inputs into modelled cost‐effectiveness analyses. It would also be important to expand conventional sensitivity analyses to assess unit costs when these observed ranges are included or when unit costs are incorporated as a function of scale. Furthermore, considering that our analysis only shows the costs of implementing CBDA model for a non‐governmental perspective and that these costs can vary if the kits were distributed differently, an important next research question will be to explore the costs of possible HIVST distribution modalities such as secondary distribution and social marketing models among others.4.1The findings of our cost analyses are limited to unit costs per kit distributed as the private nature of the HIVST did not allow us to estimate the costs of identifying new HIV‐positive individuals or those HIV‐positive individuals linked to treatment through HIVST. In addition, our results are borne out of a research trial setting and may not truly reflect a real‐world situation: for example, site fixed transport costs are likely higher due to the distances between the trial communities, while in routine scale‐up, all communities would receive HIVST kits and transport would be shared across far higher scale.Additionally, as HIVST was a new product, distribution was conservative, restricting the numbers of kits that each CBDA could distribute in Zimbabwe, and so constraining opportunities to operate at larger scale. Consequently, costs were likely higher than future routine implementation. The benefits of HIVST distribution may also be restricted by test performance characteristics such as sensitivity, specificity and ability of the user to read the test as well as rates of linkage to care. An important consideration would be the optimal, setting‐specific incentive structure for door‐to‐door community‐based distribution of the kits. It is important to highlight that for purposes of this analyses authors had not collated and analysed data on self‐test kit utilization. However, previous work has not only shown high uptake of HIVST but also high levels of kit utilization by recipients 4.2Countries keen to achieve impact and meet the global testing and treatment targets will likely need to invest in a mixture of HIV testing approaches, including door‐to‐door community delivered HIVST targeted at populations with financial or other barriers to obtaining HIV testing in health services, that is people living in settings with high undiagnosed HIV or remote communities, and groups such as men and adolescents. Reducing costs during short‐term scale‐up and implementation of this model should focus on economies of scope and scale and ensure efficiencies in personnel and transportation costs. Alternative cost‐minimization approaches also need to be explored for acceptability, impact and affordability, aiming to provide affordable access to HIVST nationally, for example integrating HIVST within the existing facility and community health services, secondary distribution from facilities including partner delivered and peer‐network approaches.5Staff costs were a substantial cost contributor highlighting the potential for lower cost models if distribution relied on unpaid volunteers within door‐to‐door community‐led distribution models.Economies of scale can also be optimized with our costs showing reductions when kits are distributed in higher numbers. Across all three countries, our HIVST cost estimates were not dissimilar to previous door‐to‐door community‐based HIVST and standard community‐based HIV testing models costed in sub‐Saharan Africa. Although the costs of CBDA delivered HIVST were higher than facility‐based HIV testing the evidence shows HIVST reaches many more individuals. A significant portion  of HIVST kits were distributed to men (key contributors to the HIV testing gap) compared to only 26% to 37% for facility HIV testing.The authors have no conflicts of interest to declare.CM, LM, LS, NA, MD, HM and FTP conceptualized and designed the study. CM, LM, LS, NA, PC, TC and SK collected and facilitated the collection of data. CM, LM, LS, NA, PC, TC, SK, MD, JJO, HM and FTP analysed and interpreted the data. CM, LM, LS, NA, MD, PC, TC, SK, JJO, MM, MN, RC, PI, ELS, MNE, GN, OM, KH, CJ, HA, ELC, FC, HM and FTP drafted the manuscript and revised it critically. MM, MN, RC, PI, ELS, MNE, GN, OM, KH, CJ, HA, ELC, FC, HM and FTP supervised the study and facilitated the acquisition of the cost data. All co‐authors approved the final version to be published.Table S1: Cost allocation factors across the interventions by cost input type.Table S2: Site‐level total and unit costs of HIVST and facility‐based testing.Data S1: Narrative description of the CBDA models across countries.Data S2: Definitions of cost category and cost inputs and allocation factors.Click here for additional data file.


Cryptococcus gattii represents an emerging fungal pathogen of immunocompromised and immunocompetent hosts in the United States. To our knowledge, this is the first case of posttransplant immune reconstitution syndrome due to C. gattii meningoencephalitis successfully treated with corticosteroids. We also report successful maintenance phase treatment with isavuconazole, a novel triazole, following fluconazole-induced prolonged QT syndrome. A 55-year-old male with history of renal transplantation 9 years earlier presented to the hospital with confusion, headaches, neck pain, cough, nausea, and vomiting. The patient, now disabled, previously was an airline mechanic who had travelled remotely to China, the Philippines, Hong Kong, and Surinam. No significant changes to his immunosuppressive regimen were noted in the months prior to admission .6/mL, creatinine of 1.3 mg/dL (baseline creatinine 1.2 mg/dL), and non-reactive fourth generation HIV antigen/antibody test. A lumbar puncture performed upon admission showed a cerebrospinal fluid (CSF) opening pressure (OP) of 36 cm H2O , WBC of 70 cells/μL (62% lymphocytes), glucose 25 mg/dL, and protein 115 mg/dL; CSF India ink stain was positive for yeast forms, meningoencephalitis CSF polymerase chain reaction  was positive for Cryptococcus neoformans/gattii, and culture yielded Cryptococcus gattii. A cryptococcal antigen was reactive at a titer of 1:2560 in serum and CSF. Dilated fundoscopic examination was negative for signs of cryptococcal intraocular invasion. Computed tomography (CT) scan of the chest demonstrated 2 solid nodules in the right lung (Cryptococcus (Admission laboratory tests showed a white blood cell count (WBC) of 7.2 x 10ght lung . Biopsy tococcus .He received liposomal amphotericin B and flucytosine as induction therapy. Tacrolimus was restarted after day 5 of his hospitalization . Given p2O in his CSF , and an OP of 15 cm H his CSF . Althougf values . Dexametf values . TransitC. gattii represents an emerging fungal pathogen that inhabits eucalyptus-type flora, soil, and bird droppings in tropical areas. A North American outbreak of C. gattii was observed in the Pacific Northwest in 2004 [C. gattii infection (versus C. neoformans) was distinguished by a higher MIC to fluconazole and its ability to promote aggressive CNS infection [C. gattii infection in immunocompetent hosts [C. gattii given the low fluconazole MIC and absence of travel to the Pacific Northwest. He likely acquired C. gattii either domestically or during international travel earlier in his life. Prior global surveys show VGI and VGIII subtypes predominate in Asia and North America (except the Pacific Northwest), respectively [ in 2004 . C. gattnfection . It has nt hosts . Multilont hosts . Althougectively .C. gattii infection caused his disseminated infection. C. gattii infection in solid organ transplant (SOT) recipients shows smaller nodular or interstitial pulmonary infiltrates, more frequent leptomeningeal enhancement, and higher rates of intensive care unit admission and mortality (70%) [We suspect reactivation of latent ty (70%) . Conversty (70%) , a resulty (70%) .C. neoformans and by limiting the interaction of calcineurin binding protein 1 (CBP1) and calcineurin [C. gattii infection include female sex, CNS disease, and higher median CD4 counts [C. gattii-induced posttransplant IRS.Immune reconstitution syndrome represents a paradoxical worsening of signs and symptoms after an initial response (despite effective therapy and microbiologic cure) and cannot be explained by a persistent primary infection, new secondary infection, or adverse drug events. Posttransplant IRS following cryptococcosis is seen in 5%–11% of patients approximately 4 weeks to 9 months after initiation of antifungal therapy . Manifescineurin , 18. Ris4 counts . In our C. gattii infection are currently limited to case series and reports [The immunologic mechanisms mediating posttransplant IRS are obscure. It is suggested that the reduction of immunosuppression and administration of antifungal therapy favor a shift from an anti-inflammatory Th2/Treg-mediated cytokine response to a proinflammatory Th1/Th17-mediated response. It is this overzealous reversion to a proinflammatory response that potentially promotes IRS in the wake of cryptococcal clearance. Prevalence estimates of IRS during  reports . Interes reports . WhetherC. neoformans meningoencephalitis [In patients diagnosed with IRS, corticosteroids are employed to tip the net state of immunosuppression towards an anti-inflammatory response. Precedence for the treatment of posttransplant IRS in cryptococcosis with corticosteroids is limited to case reports . Aside fphalitis . Corticophalitis . Infectiphalitis . In our C. gattii isolate was not obtained; although, to date, our patient has experienced a successful clinical response to this agent. The therapeutic efficacy of isavuconazole in treating CNS cryptococcosis is limited. The VITAL trial showed a successful response to isavuconazole in 6 out of 9 patients treated for cryptococcosis [C. gattii associated CNS infection (none with SOT), and all with successful responses. Prior in vitro susceptibility data indicates low epidemiological cutoff values to isavuconazole and effective brain tissue penetration of radiolabeled isavuconazole in healthy wistar rats [C. neoformans meningoencephalitis [C. gattii meningoencephalitis in humans is unknown.His course was complicated further by the development of prolonged increase in the QTc interval, which correlated with the start of fluconazole  and 1E. coccosis . Three otar rats , 32. Isaphalitis . WhetherOpen Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofz460_Suppl_Supplementary_FiguresClick here for additional data file.


Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus–neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%). Orthomyxoviridae, genus Thogotovirus, which is distributed worldwide and includes Araguari, Aransas Bay, Dhori, Jos, Thogoto, and Upolu viruses  is involved with BRBV transmission  was first isolated from a febrile patient with a history of tick bites in Bourbon County, Kansas, USA; the patient later died from severe illness in 2014 (Odocoileus virginianus), raccoon (Procyon lotor), Virginia opossum (Didelphis virginiana), and various other mammals and birds from northwest Missouri, USA, for neutralizing antibodies against BRBV to identify naturally exposed host species and to implicate potential zoonotic amplifiers.We evaluated banked animal serum and plasma for evidence of BRBV infection by using the plaque-reduction neutralization test (PRNT) to detect BRBV-reactive antibodies. We tested specimens of white-tailed deer  of 156 mammalian serum samples were positive at the 70% neutralization level . Mammals>5 mammalian species, 2 of which are domestic animals (dogs and horses). Of the wildlife species, the seropositivity rate for white-tailed deer was high (86%), whereas Virginia opossums, despite a moderate sample size (n = 28), showed no evidence of virus exposure. Deer and raccoons  could be useful wildlife sentinels for tracking the geographic distribution of BRBV. Dogs  and horses  merit further consideration among domestic animals for use as sentinels for either tracking virus activity or as an early warning system for mitigation of human risk. Because of limited sampling, we observed no statistically significant difference in seroprevalence between these 2 species.BRBV is probably transmitted to humans and other vertebrates by the lone star tick, an abundant arthropod in the south-central United States . However, a closely related congener could exist and generate cross-reactive antibodies to BRBV, causing false-positive results in our assay. Nevertheless, the PRNT is generally considered the standard for serologic assays.In conclusion, we have demonstrated that nonhuman vertebrates are exposed to BRBV. These findings are useful for future public health efforts and to better understand the ecology of BRBV. Specifically, we identified 2 candidate wildlife sentinels and potential domestic sentinels for tracking and possible early warning of BRBV transmission risk. However, whether any of these mammalian species are competent amplifier hosts for BRBV remains to be determined.Additional information on Bourbon virus in wild and domestic animals, Missouri, USA, 2012–2013.


Background: Several pathophysiological mechanisms are shared in both gallbladder stone disease (GSD) and migraines. We assessed the migraine risk for patients diagnosed with GSD. Methods: We identified 20,427 patients who were diagnosed with GSD between 2000 and 2011 from Taiwan’s National Health Insurance Research Database (NHIRD) as the study cohort. We randomly selected 81,706 controls from the non-GSD population with frequency matching by age and index year for the control cohort. All patient cases were followed until the end of 2011 to measure the incidence of migraines. Results: The cumulative incidence of migraines was greater in patients with GSD than in those without GSD (log-rank test: p < 0.001). The risk of migraine  = 1.56, 95% confidence interval (CI) = 1.41–1.73) was greater for the GSD cohort than that for the non-GSD cohort. The risk of migraine increased with the time of follow-up after a diagnosis of GSD. The risk of migraine contributed by GSD was greater for all age groups. The risk of migraine for GSD patients with depression , anxiety , and coronary artery disease (CAD)  tended to be greater than that for GSD patients without depression , anxiety , and CAD , respectively. Compared with the patients without GSD, the risk of developing migraines was greater in those GSD patients either with  or without  cholecystectomy. Compared with the GSD patients that have not had a cholecystectomy, the risk of developing migraines was lower in the GSD patients that had a cholecystectomy . Conclusions: GSD is associated with an increased risk of migraines in the Taiwanese population, but the risk diminishes after a cholecystectomy. Furthermore, in the development of migraines, GSD is synergic with some migraine-associated comorbidities, such as CAD, depression, and anxiety. Further study is necessary to clarify whether GSD is a causal risk factor for migraine. A migraine is characterized by repeated attacks of headaches, lasting 4–72 h, in patients who appear normal under physical examination and who exhibit no other obvious cause for the headache. Moreover, a migraine has at least two of unilateral pain, throbbing pain, and aggravation by movement, and is characterized by at least one of nausea or vomiting, and photophobia or phonophobia . A migraGallbladder stone disease (GSD) is a common gastrointestinal disorder worldwide, and its prevalence increases with the degree of socioeconomic development. It is present in 10% of the populations of Western countries and 3–10% of the populations of Asian countries; 5% of adults in Taiwan suffer from GSD . HoweverMigraines and GSD share many risk factors, particularly the metabolic disorders, and the association between migraines and upper abdominal symptoms implies a common pathophysiological mechanism between migraines and biliary tract disorders . FurtherIn this study, we hypothesized that a history of GSD might be associated with an increased risk of migraine development. We conducted a nationwide population-based cohort study by analyzing data from the National Health Insurance Research Database (NHIRD) of Taiwan to determine the relationship of GSD to the subsequent development of migraines and to assess the risk difference of migraines between GSD patients with and without having had a cholecystectomy.Since 1995, all residents of Taiwan have been required by law to be enrolled in the National Health Insurance (NHI) program. In 2014, the NHI program coverage rate was 99.9% [Patients newly diagnosed with gallbladder stone disease (GSD)  from 2000 to 2011 were identified from LHID2000. The index date was set as the date of GSD diagnosis. Moreover, we divided the patients with gallstones into two groups according to the history of having had a cholecystectomy or not. Patients with a history of migraines (ICD-9-CM code 346) diagnosed before the index date, aged younger than 20 years, or missing information for age or sex were excluded. For each GSD case, four non-GSD controls without a history of cholelithiasis (ICD-9-CM code 574) or migraines were identified from LHID2000, and were frequency-matched to the case with regard to age (every 5-year span), sex, and year of GSD diagnosis. All participants were followed from the index date until the occurrence of migraine, withdrawal from the NHI, or the end of 2011, whichever came first. The pre-existing comorbidities included diabetes (ICD-9-CM code 250), hypertension (ICD-9-CM codes 401–405), hyperlipidemia (ICD-9-CM code 272), stroke (ICD-9-CM codes 430–438), chronic obstructive pulmonary diseases (COPD) , coronary artery disease (CAD) (ICD-9-CM codes 410–414), depression , and anxiety (ICD-9-CM code 300.00).p < 0.05 was considered statistically significant. Distributions of demographic characteristics, including age, sex, and comorbidities, were compared between GSD patients and the non-GSD cohort by using chi-squared tests. Incidence densities of migraines for the two cohorts were calculated by age, sex, and comorbidity. To estimate the cumulative incidence rates of migraines in the GSD cohort and the non-GSD cohort, we performed a survival analysis using the Kaplan–Meier method, with significance based on the log-rank test. Both univariable and multivariable versions of Cox’s proportion hazard regression were used to examine the effect of GSD on the risk of migraine, as shown by hazard ratio (HR) with 95% confidence intervals (CI). The risk difference of migraine between GSD patients with and without having had a cholecystectomy was also assessed in our study. All analyses were performed with SAS statistical software . A two-tailed p < 0.001). The mean follow-up time was 6.74 (SD = 3.89) years in the GSD cohort and 6.83 (SD = 3.91) years in the non-GSD cohort. The log-rank test results for the cumulative incidence of migraines are shown in p < 0.001).Demographic characteristics and comorbidity values for both study cohorts are presented in The overall incidence density rate of migraine in the GSD cohort was 3.89 per 10,000 person-years and 1.69-fold higher than that in the non-GSD cohort , with an adjusted HR (aHR) of 1.56 (95% CI = 1.41–1.73) . In a stIn the multivariable model, GSD was associated with the development of migraines  . The risOur results support other findings stating that the prevalence of GSD increases with age and most (54.2%) of the GSD patients in our study were female. The aging process can increase biliary cholesterol secretion, decrease bile salts synthesis and secretion, impair gallbladder motility, and increase exposure time to the environmental factors to increase the relative risk of GSD . NonetheThe risk of migraine was associated with GSD, female sex, CAD, depression, and anxiety in our study. The female predominance of migraine was suggested to be related to estrogen, which can increase nitric oxide synthesis in endothelial cells to increase vascular dilatation capacity ,33. FurtAfter adjustments for age, sex, and comorbidities of hypertension, hyperlipidemia, coronary artery disease, depression, and anxiety, our results reveal that GSD is related to the subsequent development of migraines. Moreover, our results also suggest that the risk of migraine increases with an increase in the time without a follow-up, after a diagnosis of GSD . The cauThe exact mechanisms for the association between GSD, cholecystectomy, and migraines remain undetermined. The possible explanations for the association between GSD and migraines may be shared risk factors between GSD and migraines, the common peptides such as CCK and CGRP involved in both the pathogenesis of GSD and that of migraines, and multidirectional interaction between GSD and migraine through psychosocial stress ,16,17,18Our study has several strengths. First, our study is the first population-based study to examine the association between GSD and the subsequent development of migraines. Second, large data from a longitudinal database with a 12-year observation period for a representative cohort of 1,000,000 citizens strengthened the statistical power of the calculations. Third, the recruited patients were sampled from a stable population, namely, the approximately 99% of Taiwan residents who are covered by the Taiwan NHI program. Therefore, our findings can provide the generalizability to Taiwan. Finally, our longitudinal cohort study enabled assessing the temporal association between GSD and migraines; the association between GSD and migraines is supported in the present study. Our study has several limitations. First, we might have neglected potential confounding factors because the NHIRD cannot provide detailed information on migraine-related lifestyle, socioeconomic status, family history, and body mass index (BMI). However, we used the diagnosis of COPD to replace smoking habits for adjustment in this study, because COPD is well known to be related to smoking. In addition, BMI is closely related to diabetes, hypertension, hyperlipidemia, stroke, and CAD. The aforementioned factors were adjusted for the partial exclusion of the confounding effect of BMI. However, GSD was consistently related to migraine development in the multivariable Cox’s proportion hazard regression. Second, patients who did not seek medical consultation for GSD could not be recorded in our study. Because more than 99% of Taiwan’s population was covered by the NIH program and the well-established social benefit system, the accessibility and affordability of healthcare were both too high to compromise our findings. Moreover, we also compared the incidence of migraines between the clinically relevant gallstones patients with having had a cholecystectomy and without having had a cholecystectomy, rather than between the asymptomatic gallstones patients and general population, to lower the risk of surveillance bias, and our findings supported the ameliorating effect of having had a cholecystectomy against the development of migraines in patients with gallstones. However, migraines have been regarded as an under-identified and undertreated disease; therefore, the correlation between GSD and migraines might be underestimated in our study . FinallyIn conclusion, the results from our population-based cohort study indicate that GSD is associated with an increased risk of developing migraine in the Taiwanese population and the risk diminished after having had a cholecystectomy. Furthermore, GSD was synergic to some migraine-associated comorbidities, such as CAD, depression, and anxiety, in the subsequent development of migraines. However, further study is necessary to clarify whether GSD is an epiphenomenon or independent risk factor for migraines.


Pattern recognition receptors sense pathogen components and initiate the host antiviral innate immune response, such as inducing interferons (IFNs). Long non-coding RNAs (lncRNAs) are emerging regulators of multiple biological processes. However, their role in antiviral response, especially through regulating the human innate immune, is largely unexplored. Here we characterized that lncATV, a human specific lncRNA, was up-regulated upon type I/III IFN stimulations and virus infection. LncATV was cytoplasmic localized and relatively high expressed in human monocytes, erythroleukemia cells and hepatoma cells. Notably, lncATV knockdown significantly inhibited the replication of multiple RNA viruses, such as hepatitis C virus, Zika virus, Newcastle disease virus, and Sendai virus. Mechanistically, RIG-I antiviral signaling and IFN effective pathway were enhanced when lncATV expression was knocked down but inhibited by overexpressed lncATV. RNA immunoprecipitation results demonstrated an association between LncATV and RIG-I. Collectively, our findings reveal the functional role of a novel human specific lncATV as a regulatory lncRNA restricting virus associated innate immune response. Host response to viral infection consists of innate and adaptive defenses. The innate immune response is considered the first line of antiviral defense because it is active even at the same time of infection initiation , 2. ReceSo far, most studies on the host innate response to virus infection typically focus on protein-coding genes. However, non-coding RNAs (ncRNAs) are substantially transcribed from human genome, and the roles of many of these ncRNAs remain enigmas , 10. LonTo address this issue, we systematically characterized the IFN-regulated lncRNAs in type I or type III IFN stimulated human hepatoma cells. By screening antivirus activity, we focused on the functional characterization of a novel human specific IFN-induced lncRNA, lncATV, for its regulation of multiple RNA virus infections. Furthermore, we provide mechanistic data showing that lncATV participates in the RIG-I-mediated antiviral innate immune response.in situ hybridization, and statistical analysis is described in the The detailed information of cells, reagents, virus infection, plasmids, RNA interference, RNA pull-down assay, In-Cell Western, luciferase reporter assay, quantitative real-time reverse transcription-PCR, Western blotting, confocal microscopy, fluorescence in situ hybridization (FISH) in Huh7 cells. This analysis revealed that lncATV was predominantly enriched in the cytosol  in Huh7 cells and the specific repression of lncATV expression by siRNAs was confirmed . StrikinBecause the inhibition of HCV by lncATV knockdown could be attributed to either virus specific blocking or stimulation of host antiviral status. Therefore, we further selected several other RNA viruses, including Zika virus (ZIKV), Newcastle disease virus (NDV), and Sendai virus (SeV), for further assessment. ZIKV is a positive strand RNA flavivirus and has recently attracted global attention. As shown in The broad-spectrum antiviral effects demonstrated by lncATV disruption had drawn our attention whether innate immune pathways might involve in this regulation. Therefore, we assessed the related pathways after knocking down or over-expressing lncATV in Huh7 cells. As expected, silencing lncATV by RNAi strongly enhanced the NDV-mediated induction of both IFNβ and ISGs, including ISG15, MxA, PKR, IFIT1, and RSAD2 . IRF3 isRIG-I-like receptor (RLR) pathways are major defense cascades against RNA viruses. To further define the mechanism of lncATV affecting virus replication and innate immune response, we first investigated the effect of lncATV on RLR pathways. Knockdown of lncATV acted to significantly increase IFNβ promoter activity in Huh7 cells with the transfection of full-length RIG-I but not N-terminal RIG-I, MDA5, MAVS, or the constitutively activated IRF3(5D) . Highly Dynamic gene expression during host antiviral response is tightly coordinated by transcriptional and post-transcriptional mechanisms. An emerging theme is the increasing important role of lncRNAs in the regulation of innate immunity , 22. RIGIn this study, high-throughput transcriptome analysis has allowed us to identify differentially expressed human lncRNAs in response to type I and type III IFNs, which are major cellular defense effectors against virus infection. These IFN-regulated lncRNAs are valuable for future studies about infection, inflammation, and even cancer biology. With a functional screening of the top ranked differentially expressed lncRNAs, we focused on the IFN up-regulated lncATV. We provide experimental evidence showing that LncATV as a novel regulatory lncRNA to limit the innate immune response and promote viral infection. Crucially, our results show that lncATV acts through targeting RIG-I for the negative regulation of cell antiviral response.RIG-I is considered as an important PRR to percept 5′-triphosphate dsRNA produced by pathogens for a long time , 27. UntTaken together, we reveal a novel molecular mechanism, in which host self LncATV associates with RIG-I and inhibits RIG-I-mediated innate immunity activation. This unique regulation mode helps to understand the precise control of host immune homeostasis.All datasets generated for this study are included in the manuscript and/or the JF and MC: generation and analysis of data and writing of the manuscript. XC and XL: validation of data. WY: study supervision, design of the experiments, and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The global economic impact of canine rabies has been estimated by several studies. Asia bears a disproportionate burden of this zoonosis due to high levels of human deaths and rates of post-exposure prophylaxis (PEP), but low investment in preventative dog vaccination. The same factors that cause rabies to burden much of Asia are also present in Viet Nam. This study estimated the economic burden of canine rabies in a societal perspective including direct and indirect cost of rabies in dogs, livestock, and humans. Using data collected from personal interviews, published literature, published and supplementary reports, and primary data collection, we estimated the economic impact of canine rabies in Viet Nam over a ten year period (2005–2014). We incorporated the direct and indirect costs for PEP, dog vaccination efforts, livestock losses, and disability adjusted life years (DALYs) into the analysis. General findings from this analysis indicated that over the 10 year study period, the total economic impact of canine rabies was over $719 million USD. The largest portion of impacts (92%) were made up of PEP-related costs. Canine rabies created between 36,560 and 45,700 DALYs, measured in years of life lost (YLL). A total of 914 human deaths were reported over the study period. Deaths/100,000 people were 0.11, which is lower than the reported level for Asian countries. The cost per dog vaccinated was $1.75 USD. Our results indicate that canine rabies impacts in Viet Nam are consistent with the burden elsewhere reported in Asia, with large expenditures on PEP and very small investments in dog vaccination. With few exceptions, canine rabies is endemic in developing countries. The mortality rate of rabies approaches 100%. However, disease is preventable via the timely administration of post-exposure prophylaxis (PEP) following a rabid animal bite. Additionally, canine rabies can be controlled in dog populations through mass vaccination. Economics drive the majority of policy and decision making and as such, a robust understanding of the cost of prevention and control methods is useful for the development and implementation of rabies management strategies. The majority of afflicted individuals are poor and reside in rural regions, where knowledge of rabies is minimal and access and affordability of PEP are limited. A greater emphasis on mass dog vaccination is a cost effective method of eliminating canine rabies at the source, which decreases the need for human PEP and reduces the burden of human mortality within vulnerable populations. Canine rabies is an economically unique zoonosis, as most of its associated costs do not result from illness in the infected individual, but rather are the consequences of human deaths and efforts to prevent the disease in humans, livestock, and companion animals. This pattern of costs reflects two basic facts: the case fatality rate of rabies in humans is nearly 100%, and the disease is preventable through timely post-exposure prophylaxis (PEP) with rabies immune globulin (RIG) and multiple doses of rabies vaccine .Rabies exposures in humans or livestock result in economic impacts associated with vaccination or death. Because rabies patients die quickly, and there is no effective therapy, the cost of illness is relatively small, especially in the developing world. In contrast, the major direct costs arising from factors such as PEP and livestock deaths have been characterized in numerous studies –6. ResulOther impacts of rabies on the broader economy can be captured by examining changes in different sectors that result from the direct and indirect impacts of the disease . Knobel This study examined the economic impact of canine rabies in Viet Nam for a ten year period (2005–2014) using data collected from personal interviews, published literature, published and supplementary reports, and primary data collection (during a site visit in March 2016). Rabies is endemic in the Vietnamese dog population with about 100 human cases reported annually. During the study period, an average of over 400,000 PEPs were administered each year. Overall, canine rabies in Viet Nam appears consistent with the general rabies burden characterized by other Asian countries, in that a considerable amount of human deaths occur, a significant amount of PEP is administered, dog vaccination coverage is relatively low, and there is not a national comprehensive enforced dog vaccination campaign to eliminate canine rabies , 15–16. Common metrics to measure the impact of canine rabies were incorporated into this analysis, including the direct and indirect costs for PEP, dog vaccination efforts, livestock losses, and disability adjusted life years (DALYs). The results from this analysis detail the level of burden created by canine rabies in Viet Nam, examine whether canine rabies impacts in Viet Nam are consistent with other Asian countries, and determine if savings can be realized by reducing the impacts of PEP. Importantly, this study focuses exclusively on canine rabies. However, wildlife species, including bats, may be a reservoir for rabies in Viet Nam, although potential impacts associated with other animals are beyond the scope of this analysis.In Viet Nam, rabies in dogs is a notifiable disease to the Department of Animal Health, Ministry of Agriculture and Rural Development, which reports to the World Organisation for Animal Health (OIE). There is a general surveillance and monitoring program as well as a dog vaccination program. However, like many dog vaccination programs in Asian countries, rabies control through dog vaccination and management is underfunded and rabies remains a neglected disease. Identification of the current economic costs inflicted by rabies and the cost of control measures are imperative to building widespread investment and support for canine rabies elimination.Estimation of the economic impact of canine rabies to Viet Nam from 2005 to 2014 required coalescing information from several sources on the direct and indirect impacts of disease. Specifically, estimates of human mortality, direct and indirect medical costs, direct and indirect costs of PEP, dog vaccinations, and livestock mortality were required, along with the costs associated with each. Here we relied on estimates from numerous sources . The NatThe human population and quantity of PEP administration associated with rabies in Viet Nam during the study period is provided in The dog population in Viet Nam was estimated to range between 6 and 8 million during the study period. Reliable data regarding the dog population was only available from the DAH (S2) between 2011 and 2014 . To deriOne of the most critical animal species considered in this analysis is livestock. In Viet Nam, important livestock populations that are impacted by canine rabies include cattle, pigs, and buffalo. Reliable data on the prevalence of canine rabies in livestock could not be obtained through any Vietnamese source including the DAH, MARD, or NIHE. Therefore, estimates regarding the impact of canine rabies to livestock were derived from Hampson et al., 2015. Through primary data collection for the purpose of estimating the global burden of canine rabies, Hampson et al., 2015, inferred a relationship between rabies in livestock (IL) and dog vaccination coverage (VC), as presented in It follows from To estimate the total impacts associated with canine rabies in livestock the value of each species must be incorporated. The information regarding the average market prices associated with each livestock type was obtained from MARD. A national census is conducted every other year (even years) and the price per kilogram for meat from each species was derived from this survey. The market price for odd years was an average of the prior and subsequent even years where data were available.t was calculated by subtracting the average age of patients at death (31 years) from the life expectancy in Viet Nam (76 years) for each year of the study and multiplying by the number of reported deaths. The average age of patients at death was determined from three years of available data . Ages were reported categorically, so the mean age at death was calculated by multiplying the mean age of each age category by the average number of deaths in that category over three years, summing and dividing by the average number of deaths per year. The median age at death falls between two age categories (15–24 years and 25–34 years) so is likely around 25 years. The low median age reflects the reality that rabies disproportionately affects children. As a sensitivity analysis, patients’ age at death was allowed to vary by five years to account for the categorical nature of the data . Although the incubation period of rabies may be years, the time from onset of symptoms to death is just a few days. For this reason, YLD was assumed to be 0, meaning DALY = YLL.Disability-adjusted life years (DALYs) are the sum of years of life lost (YLL) and years lost due to disability (YLD) and were calculated for each study year (t). YLLRH) includes the variables that are listed in The cost of prevention of human rabies includes the direct cost of administering PEP (which includes RIG in 9% of cases), costs of the vaccines, and direct non-medical and indirect costs of PEP . Any pre-exposure prophylaxis costs are assumed to be negligible. Based on data derived from other similar countries, it was assumed that each patient receiving PEP was administered three doses of vaccine . The total cost of rabies in humans (TCRD) include the vaccine, salaries for the animal health workers, and the costs that fall to the dog owners  is the sum across livestock species at the applicable level of incidence for each livestock population priced at the average slaughter weight  associated with rabies is the sum of the human, dog, and livestock components . The valOver the ten-year period between 2005 and 2014, 25,539 pigs, 6,032 cattle, and 2,892 buffalo were estimated to have died from rabies. The total cost of lost livestock exceeded $10 million .Between 36,560 and 45,700 years of human life were lost due to rabies-caused deaths. A substantial number of lives were lost over the study period and it is likely that our estimate is an underrepresentation of the disease burden, primarily resultant from underreporting of rabies-related human deaths. Many reasons exist for underreporting, including an inability to diagnose rabies from the symptoms presented and a lack of verification of rabies positivity post-mortem. Examining the number of human deaths over the study period indicates that deaths tend to trend close to the average (91.4 per year). However, 2007 was the year with the most recorded deaths and 2014 tallied the fewest.Utilizing the data on the number of rabies deaths, it is possible to determine the costs associated with patients infected with rabies, including medical and non-medical costs . The aveFor this analysis, rabies prevention is composed of two parts: prevention of rabies in dogs through vaccination and prevention of rabies in humans through PEP. During the study period, over 25 million dogs were vaccinated against rabies. After examining the costs associated with the prevention of rabies in dogs, the vaccinator/animal health worker costs composed the largest portion of total costs. After examining the costs of rabies prevention in humans, it is evident that the majority of the costs are a result of direct non-medical and indirect costs of PEP. Components of direct non-medical costs include costs to the patient and family due to transportation, meals, accommodation; time lost as a result of PEP; time lost by caregivers; and indirect costs of PEP, including cost of work absence. For this analysis, it was provided by NIHE that RIG is only given to approximately 9% of individuals that received PEP. The total cost of rabies in Viet Nam from 2005 to 2014 was over $719 million in 2017 USD with 914 human lives lost and between 36,560 and 45,700 years of human life lost .The largest portion of rabies costs in Viet Nam are associated with PEP and only minimal expenditures are associated with dog vaccination . LivestoA substantial number of DALYs are lost, which is the result of a combination of the number of human deaths and the younger average age associated with individuals that succumb to canine rabies. Over the 10-year study period, 914 human deaths from rabies were reported. A myriad of reasons for this number of deaths have been implicated, including some individuals using traditional medicine and other homeopathic remedies as opposed to proper PEP, other individuals improperly assume that a bite from the household pet dog will not result in rabies, children often do not recognize the risk of rabies and fail to alert an adult to a dog bite, and individuals with very limited resources do not seek care as they cannot afford to do so.Average dog vaccination coverage is less than half of the dogs in Viet Nam, which is an insufficient level of coverage to achieve canine rabies elimination. Likely the dog vaccination coverage is similar to other canine rabies endemic countries in Asia, in that coverage levels are higher in urban and suburban areas and lower in rural areas. This only exacerbates canine rabies impacts as most of the human and livestock exposures happen in rural areas as a result of a lower vaccination rate among dogs in these areas.This study is potentially subject to limitations associated with the reliability of the data acquired. Some of the values used in this manuscript are from other publications or worldwide entities which maintain global records (ie: the FAO). However, other values are derived from supplementary reports or internal documents provided in-country. In each case, it is difficult to ascertain the accuracy of the values. Importantly, global human rabies incidence is believed to be grossly underreported with some data suggesting that the true rabies incidence in humans is upwards of 100 times greater than that reported . This stComparing annual disease burden against prevention and control measures reveals that Viet Nam is consistent with other rabies endemic countries in Asia as more money is spent on PEP than preventing human rabies through dog vaccination. This is very evident when examining costs associated with PEP and dog vaccination. PEP costs compose 92% of the burden of rabies in Viet Nam whereas dog vaccination costs are only 6% of the costs or burden associated with the disease. Examining the costs per dog vaccinated against the cost per PEP reveals the same trend. Prevention cost per PEP is 93 times greater than the costs per dog vaccinated. The results of this study indicate that canine rabies impacts in Viet Nam are consistent with how these impacts have been characterized in Asia, specifically that there are large expenditures on PEP and very small expenditures on dog vaccination. Canine rabies elimination requires a level of dog vaccination coverage that exceeds 70% . To achiThe Ministry of Agriculture and Rural Development and the Ministry of Health crafted a report outlining a national program toward rabies control and elimination between 2017 and 2021 . This doS1 TextThe conversion rate used from Vietnamese dong (VND) to USD was from 2009 ; the values were then grown to 2017 USD.(DOC)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Hybridization has been routinely practiced in agriculture to enhance the crop yield. Principally, it can cause hybrid vigor where hybrid plants display increased size, biomass, fertility, and resistance to diseases, when compared to their parents. During hybridization, hybrid offspring receive a genomic shock due to mixing of distant parental genomes, which triggers a myriad of genomic rearrangements, e.g., transpositions, genome size changes, chromosomal rearrangements, and other effects on the chromatin. Recently, it has been reported that, besides genomic rearrangements, hybridization can also alter the somatic mutation rates in plants. In this review, we provide in-depth insights about hybridization triggered genomic rearrangements and somatic mutations in plants. In 1876, Charles Darwin described heterosis or hybrid vigor as a phenomenon, which results in enhanced crop yields by hybridizing distantly related maize varieties. Since then, hybridization in plants has been routinely practiced in agriculture to increase the productivity of crop plants . This unHybridization is also inextricably linked with speciation and therefore has an important evolutionary significance. Speciation allows unification and functioning of diverged genomes from different species naturally . SpeciatArabidopsis unveiled a spontaneous point mutation rate of 7 × 10−9 base substitutions per site per generation . Ce. CeNicotl length . It willT. aestivum) is a product of natural hybridization and allopolyploidy. The most frequent chromosomal abnormality observed in wheat includes single translocation events, followed by multiple translocation or complex rearrangements, and also inversion events, in the descending degree of their frequencies of occurrence [It has been argued that chromosomal rearrangements can play an important role in the process of speciation as emergence of new species is often accompanied with structural changes in the chromosomes by means of translocations, deletions, and inversion events . Many aucurrence .Tragopogon miscellus have also revealed enormous chromosomal variations. Moreover, intergenomic translocations were seen in more than 75% of the population and close to 70% of the individuals displayed aneuploidy for one or more chromosomes [Arabidopsis thaliana × Arabidopsis lyrata and this deleted fragment constitutes the nucleolus organizing regions (NOR2) [A. suecica, which is a natural allopolyploid . Interestingly, transposition of NORs—from A. thaliana to A. arenosa chromosomes—has also been reported in these hybrids [Genomic and fluorescence in situ hybridization studies on the natural allopolyploid populations of omosomes . Partials (NOR2) . Similar hybrids . Such ch hybrids .Aegilops and Triticum [Work on interspecific hybridization from non-plant species has shown that centromeric regions remain prime targets for genomic instabilities. In marsupial hybrids, maternally derived centromeric regions remain hot sites for extensive chromosomal rearrangements, chromatin structural changes, satellite repeat, and transposable element amplification . VariabiTriticum . In wheaHelianthus, revealing genome size variations in different hybrids makes it intriguing to understand whether such changes can also happen in reciprocal hybrids. It should be possible to address this by using next generation sequencing to get in-depth insights, or with flow cytometric observations of the DAPI stained nuclei, to get some sneak peeks into the genome size variations.Hybridization has the potential to bring genetic diversity, increase the vigor in progeny, and it may also reduce the fitness of the individual. Genomic rearrangements seen in hybrids  by virtuDrosophila hybrids, hybrid plants also show altered mutation rates [Recent literature convincingly points out the fact that like on rates . In thison rates . Lastly,


In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α All messenger RNAs in eukaryotic cell are synthesized by RNA polymerase II (RNAPII), a complex enzyme, which consists of 12 subunits of the total weight over 0.8 MDa. During transcription, RNAPII is a target of transient assembly of numerous transcription factors, such as the general transcription factors, coactivators , elongation factors, proteins for RNA processing and transcription termination.Saccharomyces cerevisiae and huge breakthrough progress was made in the past two decades by resolving 3D structures of yeast RNAPII as well as of its complexes with several factors [Until recently, RNAPII has been mostly investigated in yeast  factors ,10,11,12 factors ,14,15. T factors . Despite2 homodimer, which is involved in RNA polymerase assembly and promoter recognition [2 assembly intermediate of prokaryotic RNA polymerase [All 12 subunits of RNAPII are closely homologous or identical to the subunits of nuclear RNA polymerase I and III, five of which are related to the core subunits of bacterial RNA polymerase . Rpb11 sognition . Togethelymerase ,17. Rpb3lymerase ,19. ConsPOLR2J1 (RPB11a) gene encodes hRPB11a subunit representing the major component of mammalian RNAPII complex. Other members of the family, the POLR2J2 (RPB11b) and POLR2J3 (RPB11c) genes, yield several minor mRNAs resulting from alternative splicing [POLR2J2 (RPB11b) gene are translated remain to be established. Remarkably, using complementation assay, it was demonstrated that only minor isoform hRPB11bα (the product of expression of the POLR2J2 gene) is functional in yeast whereas the major  hRPB11a is not [In humans, a gene family has been identified that potentially encodes several hRPB11 (POLR2J) proteins differing mainly in their C-terminal regions ,21,22,23splicing ,21. Wheta is not . Obviousa is not ,25,26. THomo sapiens: The strength of the hRPB11–ATF4 interaction in corresponding transcription complexes appeared to be isoform-specific.Here, we provide the first evidence about in vivo functional distinction between the previously discovered ,24,25,26Homo sapiens by four different genes located on chromosome 7 [POLR2J1 (~5.5 kb long), is orthologous to the corresponding rpb11 genes in other species and encodes the usual , main isoform of hRPB11 (POLR2J) subunit called hRPB11a (117 aa long) which, as judged by its evolutionary history, is the subject of strong purifying selection because is strictly conserved in all warm-blooded animals.Previously, we have shown that, in drastic difference with all other species, small indispensable subunit Rpb11 of RNA polymerase II is encoded in mosome 7 ,21. One POLR2J genes of Homo sapiens (POLR2J2–POLR2J4), which are much longer (approximately 34.5 kb in length each), two, POLR2J2 and POLR2J3, not only have preserved all the hRPB11 (POLR2J) subunit coding potential, but can even produce plural variants of this subunit, including the most interesting isoforms hRPB11bα  and hRPB11bβ  [Among other AC18330) ,21. The AC18330) ,21.To characterize the hRPB11bα isoform and to discover unique functional features, which can distinguish it from the major isoform hRPB11a, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library and identified transcription factor ATF4, a member of the ATF/CREB (activating transcription factor/cAMP response element binding protein) family of basic region-leucine zipper proteins ,28,29, ahRPB11bα (POLR2J2) mRNA was most abundant in the brain [GAL1 promoter to express library clones as fusions to a transcriptional activation domain B42. Twenty-five positive clones capable of simultaneously activating both reporters LEU2 and lacZ in a galactose-specific manner were obtained and further characterized. Five of them contained the plasmids encoding the C-terminal part of RNA polymerase II subunit hRPB3, thus confirming the specificity of the screening. Four positive clones encoded the C-terminal part of transcription factor ATF4 (aa 221–351)  . To defi221–351) B.E. coli. The recombinant vector carries an M15 replication origin, the kanamycin resistance gene, the LacI gene, and the cloning expression region of pET15b (construction details will be described elsewhere). This vector can be used for co-expression together with any available ColE1-containing vector with a resistance selection marker other than kanamycin.To confirm the in vivo interaction found by yeast two-hybrid system, we performed in vitro binding assay. We constructed a new vector pEXP2 to co-express interacting proteins in 2+-based TALON agarose, the beads were washed, and the absorbed proteins were eluted with imidazole. ATF4 as well as its C-terminal part were efficiently recovered with hRPB11bα, although they were not co-purified with hRPB11a  were cloned in pET21d and expressed as untagged proteins. We co-transformed plasmids encoding hRPB11a and hRPB11bα with the plasmids harboring cDNA of ATF4 and co-expressed the recombinant proteins in the same bacterial cell. The soluble supernatants were incubated with the Co hRPB11a . Due to hRPB11a differs from the minor isoform hRPB11bα in its C-terminal region, encoding by the fourth exon, and in the additional lysine residue (Lys-17) in the N-terminal part ,21. To dHomo sapiens RNA polymerase II interacts with transcription factor ATF4 through its C-terminal b-Zip domain and that the integrity of the hRPB11bα isoform protein structure is important for productive binding.In summary, as is schematically represented on The interaction of hRPB11bα and ATF4 can be directly involved in activation of RNA polymerase II. To test this hypothesis, ATF4 and hRPB11bα cloned in mammalian expression plasmids and a luciferase reporter containing tandem ATF4 binding sites were cotransfected into HeLa cells. As shown in Eukaryotic RNA polymerase II subunit Rpb11 is an essential component of the transcriptional machinery that forms a heterodimer with Rpb3 subunit. This heterodimer plays an important structural role in the formation of the RNA polymerase II complex . It was POLR2J and POLR2J2, were characterized in in vitro experiments, hRPB11a , hRPB11bα, and hRPB11bβ [Homo sapiens as part of human chromosome 7 [POLR2J1, POLR2J2, and POLR2J3, are located as a single cluster with a total length of 214,530 bp in the genetic region 7q22.1 on the long arm of the chromosome; another gene, POLR2J4 , is located in the cytogenetic locus 7p13 of the short arm of chromosome 7. It was also shown that the expression of four human POLR2J genes (POLR2J1–POLR2J4) could lead to the synthesis of at least 14 species of mature mRNAs encoding slightly different isoforms of the hRPB11 subunit, 12 of which were detected by us as full-sized copies or clearly correlated cDNAs fragments in the available EST (expressed sequence tags) and cDNA databases [In 2001, in collaboration with French scientists, we first established that one of the subunits of human nuclear RNA polymerase II is encoded by at least several genes: Three isoforms of human hRPB11 (POLR2J) subunit, synthesized by the expression of two different genes: mosome 7 . Three gHomo sapiens proteins interacting with these human-specific isoforms using genetic and biochemical approaches [In order to study the function of the most interesting isoforms hRPB11bα and hRPB11bβ, for the first time, we discovered proaches ,26. The proaches ,26. Indeproaches .Here, in this work, we analyzed in detail the specificity of interaction of major RNA polymerase II subunit hRPB11a  and human-specific isoform hRPB11bα  with the b-Zip transcription factor ATF4, which was identified as one of the most prominent interactor of these α-like subunits under study of the human RNA polymerase II.We have established that ATF4 through its C-terminal b-Zip domain interacts with one of the human specific isoforms of Rpb11, i.e., the minor subunit hRPB11bα, in the yeast two-hybrid system, and this interaction was also confirmed by coprecipitation of the proteins in vitro after their heterologous expression in a bacterial system. Overexpression of ATF4 and hRPB11bα in the human HeLa cell line increases transcription levels in 12.5 times (in luciferase test).We found that the intensity of interaction of ATF4 with hRPB11a was lower than that with hRPB11bα, indicating selectivity of ATF4 binding to the two different human isoforms. Previously, it was shown that another α-like subunit of RNA polymerase II, hRPB3, directly binds ATF4 in vivo and in vitro and enhances ATF4 transactivating activity, indicating that this interaction (hRPB3–ATF4) provides the main platform for ATF4 activation of the major form of human  RNA polymerase II enzyme . We demoATF4 is a transcription factor activated during stress conditions. Thus, the ATF4–hRPB11bα interaction probably is necessary for rapid gene activation in cellular response to different stress signals. Indeed, mRNA of hRPB11bα is found in polysomes after treatment of cells with 5′-fluorouracil and may be implicated in the resistance formation to this anticancer drug .EcoRI/BamHI insert. pMW103-hRPB11bα was introduced along with the LacZ reporter plasmid pDR8 (Invitrogen) into the S. cerevisiae strain SKY 191  by standard lithium acetate transformation [LEU2 gene are replaced by two copies of the LexA operator and the pDR8 contains the LacZ gene also under the control of 6 LexA operators. Absence of transcriptional activation capability of the LEU2 and LacZ reporters by the baits in the SKY 191 was verified prior to library screening. Human fetal brain cDNA library (Invitrogen) based on the expression vector pJG4-5, which utilizes the galactose-inducible GAL1 promoter to express library clones as fusions to a transcriptional activation domain , was directly transformed into yeast containing pMW103-hRPB11bα and pDR8. Cells were plated on each of 15 SD-Glu plates (240 × 240 mm) lacking triptophan, histidine and uracil (-WHU) and allowed to grow for 3 days at 30 °C. In the next step, a homogenized slurry was prepared from the pool of primary transformants (approximately 3 × 105 colonies), aliquoted, frozen, and stored at −70 °C. Then, an aliquot was thawed, diluted with SD-Gal/Raff/-WHU  liquid medium, and incubated with shaking at 30 °C for 5 h. About 106 cells were plated on each of 5 of 100 mm Gal/Raff/-WHUL plates (lacking leucine) and incubated for 5 days at 30 °C. Positive clones, which were able to grow on galactose/raffinose media lacking leucine, were tested for galactose-inducible transcriptional activation of lexAop-lacZ reporter by plating on 5-bromo-4-chloro-3-indoyl-d-galactoside  containing medium. Plasmids from positive yeast clones were isolated, electroporated into E. coli XL-blue cells, and plated on media containing 50 µg/mL ampicillin. The recovered library-derived plasmids were further analyzed as positive candidates. To investigate the specificity of interaction of the hRPB11bα with the prey, cDNA of hRPB11a was cloned into pMW103. Resulting plasmid pMW103-hRPB11a was transformed into SKY 191 as described above along with pDB20 and library-derived plasmid.For two-hybrid selection, the complete open reading frame of human isoform hRPB11bα was cloned into bait vector pMW103  in frameormation . Cells w2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0), and resuspended in 2 mL of Z-buffer. Then, 200 μL of cells suspension were mixed with 800 μL of Z-buffer (1:5 dilution) and the optical density at 600 nm (OD600) was measured. Then, 20 μL of 0.1% SDS, 40 μL of chloroform were added, and tubes were vortexed for 10 s. The permeabilized cells were equilibrated to 30 °C for 15 min, then 200 μL of 4 mg/mL ONPG  solution was added, and the reaction proceeded at 30 °C until a yellow color was observed. After the addition of Na2CO3 and centrifugation, the formation of o-nitrophenol was determined by measuring the optical density at 420 nm (OD420). β-galactosidase activity was calculated with the following equation: u = 1000 × OD420/(t × OD600 × 5).Exponentially growing cells in 2 mL SD Gal-Raff/−Ura –His −Trp media were collected by centrifugation, washed with Z-buffer , isolated by two-hybrid selection, was cloned into the BamHI-HindIII sites of pET21d. Full-length cDNA of ATF4 was amplified from the plasmid pCGN-ATF4 kindly provided by Dr. Tsonwin Hai  and cloned into pET21d as BamHI-XhoI insert. To construct deletion variant of hRPB11bα without the fourth exon, the corresponding part of cDNA was amplified with the pair of specific primers  and cloned into the NdeI-BamHI sites of pEXP2. Plasmids were transformed into E. coli BL21(DE3) strain. For co-expression, the two pairs of plasmids were transformed into the same strain. The cells were grown at 37 °C in LB media until the A600 0.5. Then, IPTG was added to final concentration of 1 mM, and bacterial culture was incubated at 30 °C or at room temperature for 4 h or overnight, respectively. The cells were centrifuged, resuspended in TG500  buffer, and lysed by sonication. The insoluble materials were removed by centrifugation. Next, TG500-equilibrated Talon Co2+ affinity bead suspension  was added to the lysate and the suspension was incubated for 1 h at 4 °C with slight agitation. Then, the beads were washed five times with TG500. Absorbed proteins were batch-eluted with TGI buffer  and analyzed by electrophoresis in 15% SDS polyacrylamide gels. The gels were visualized by Coomassie Blue R250 staining essentially as described in [Full-length cDNA of isoforms hRPB11a and hRPB11bα were cloned into pEXP2 as ribed in .Human HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. Transient transfections were performed using Lipofectamine 2000 reagent  according to the manufacturer’s instructions.The reporter pSp1ATF-Luc containing one SP1 and three ATF binding sites (3×ATF4) downstream from SP1 and pCGN-ATF4 plasmids were kindly provided by Dr. Tsonwin Hai (Ohio State University).Luciferase assay was performed using Luciferase Assay Kit  according to the manufacturer’s instructions.Homo sapiens has been shown for the first time. We have demonstrated that minor hRPB11bα isoform of human RNA polymerase II specifically interacts with C-terminal b-Zip domain of ATF4, providing novel platform for binding of this transcription factor to heterodimeric region of the α-like subunits (Rpb3–Rpb11) in RNAPII complexes containing hRPB11bα isoform of POLR2J subunit.The functional distinction between the previously discovered original isoforms of the hRPB11 (POLR2J) subunit of RNA polymerase II of