A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal.

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans.

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the ß and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors.

Piwi-interacting (pi) RNAs are germline-expressed small RNAs linked to epigenetic programming. C. elegans piRNAs are thought to be transcribed as independent gene-like loci. To test this idea and to identify potential transcription start (TS) sites for piRNA precursors, we developed CapSeq, an efficient enzymatic method for 5' anchored RNA profiling. Using CapSeq, we identify candidate TS sites, defined by 70-90 nt sequence tags, for >50% of annotated Pol II loci. Surprisingly, however, these CapSeq tags failed to identify the overwhelming majority of piRNA loci. Instead, we show that the likely piRNA precursors are ∼26 nt capped small (cs) RNAs that initiate precisely 2 nt upstream of mature piRNAs and that piRNA processing or stability requires a U at the csRNA +3 position. Finally, we identify a heretofore unrecognized class of piRNAs processed from csRNAs that are expressed at promoters genome wide, nearly doubling the number of piRNAs available for genome surveillance.

The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

Enhancing the Efficiency of Resilient Multipath-Routed Elastic Optical Networks: A Novel Approach for Coexisting Protected and Unprotected Services with Idle Slot Reuse.

In this paper, we investigate a scenario in which protected and unprotected services coexist in an elastic optical network under dynamic traffic. In the investigated scenario, unprotected services can reuse the reserved idle bandwidth to provide protection to the protected services. Under this scenario, we propose a new heuristic algorithm that enables such reuse as well as define and introduce a new assignment problem in elastic optical networks, named a Transmission Spectrum Assignment (T-SA) problem. In this paper, we consider a scenario in which services may be routed using the multipath routing approach. Additionally, protection using bandwidth squeezing is also considered. We assess our proposal through simulations on three different network topologies and compare our proposal against the classical protection approach, in which bandwidth reuse is not allowed. For the simulated range of network loads, the maximum (minimum) blocking probability reduction obtained by our proposal is approximately 48% (10%) in the European topology, 46% (7%) in the NSFNET topology, and 32% (6%) in the German topology.

A Fragile Image Watermarking Scheme in DWT Domain Using Chaotic Sequences and Error-Correcting Codes.

With the rapid development of digital signal processing tools, image contents can be easily manipulated or maliciously tampered with. Fragile watermarking has been largely used for content authentication purposes. This article presents a new proposal for image fragile watermarking algorithms for tamper detection and image recovery. The watermarked bits are obtained from the parity bits of an error-correcting code whose message is formed from a binary chaotic sequence (generated from a secret key known to all legitimate users) and from bits of the original image. Part of the codeword (the chaotic bits) is perfectly known to these users during the extraction phase, adding security and robustness to the watermarking method. The watermarked bits are inserted at specific sub-bands of the discrete wavelet transform of the original image and are used as authentication bits for the tamper detection process. The imperceptibility, detection, and recovery of this algorithm are tested for various common attacks over digital images. The proposed algorithm is analyzed for both grayscale and colored images. Comparison results reveal that the proposed technique performs better than some existing methods.

SARS-CoV-2 IgG Seroprevalence among Blood Donors as a Monitor of the COVID-19 Epidemic, Brazil.

During epidemics, data from different sources can provide information on varying aspects of the epidemic process. Serology-based epidemiologic surveys could be used to compose a consistent epidemic scenario. We assessed the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG in serum samples collected from 7,837 blood donors in 7 cities of Brazil during March-December 2020. Based on our results, we propose a modification in a compartmental model that uses reported number of SARS-CoV-2 cases and serology results from blood donors as inputs and delivers estimates of hidden variables, such as daily values of SARS-CoV-2 transmission rates and cumulative incidence rate of reported and unreported SARS-CoV-2 cases. We concluded that the information about cumulative incidence of a disease in a city's population can be obtained by testing serum samples collected from blood donors. Our proposed method also can be extended to surveillance of other infectious diseases.

HCV infection in hemophilia A patients is associated with altered cytokines and chemokines profile and might modulate the levels of FVIII inhibitor.

Prevalence of hepatitis C virus (HCV) is high in hemophilia A patients and the development of FVIII inhibitor is another challenge in the management of these individuals. The influence of HCV infection in the occurrence of inhibitors was investigated by the comparison of clinical and laboratory data from noninfected (NI, n = 96) and chronically HCV-infected (HCV, n = 58) hemophilia A patients. Concentrations of plasmatic cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A) and chemokines (CCL2, CCL5, CXCL8, CXCL9, and CXCL10) were quantified from patients' samples. The results showed that older age, use of cryoprecipitate and fresh frozen plasma, and severe hemophilia were associated with HCV infection, whereas exclusive use of virus inactivated clotting factors was a protector factor to acquiring HCV infection. HCV infection was strongly associated with low levels of inhibitor (OR = 20.53, p < 0.001). Patients with a history of inhibitor (INB+) presented a mixed immune profile characterized by higher levels of pro-and anti-inflammatory cytokines than those without a history of inhibitor (INB-). The highest levels of CCL2 and CXCL8 were seen in HCVINB- , whereas CXCL9 and CXCL10 in HCVINB+ . Heatmap analysis of the set of cytokines and chemokines concentration distributed HCV patients into two distinct clusters, HCVINB+ and HCVINB- , both characterized by low concentrations of IL-4, while noninfected patients were grouped in a single block regardless of inhibitor development history (NIINB-/INB+ ). This finding suggests that the strong association between HCV infection and low levels of factor VIII inhibitors might be due to the modulation of the cytokine and chemokine network established by the antiviral response.

Pro-inflammatory immune profile mediated by TNF and IFN-γ and regulated by IL-10 is associated to IgG anti-SARS-CoV-2 in asymptomatic blood donors.

The SARS-CoV-2 virus has infected and killed millions of people, but little is known about the risk factors that lead to the development of severe, mild or asymptomatic conditions after infection. The individual immune response and the balance of cytokines and chemokines have been shown to be important for the prognosis of patients. Additionally, it is essential to understand how the production of specific antibodies with viral neutralizing capacity is established. In this context, this study aimed to identify positive individuals for IgG anti-SARS-CoV-2 in a large population of blood donors (n = 7837) to establish their immune response profile and to evaluate its viral neutralization capacity. The prevalence found for IgG anti-SARS-CoV-2 was 5.6% (n = 441), with male blood donors (61.9%) being more prevalent among the positive ones. The results showed that positive individuals for IgG anti-SARS-CoV-2 have high serum concentrations of chemokines, TNF, IFN-γ and IL-10. The analyses showed that the positivity index for IgG anti-SARS-CoV-2 is associated with the neutralizing capacity of the antibodies, which, in turn, is significantly related to lower serum concentrations of CCL5 and CXCL10. The results allow us to hypothesize that the development and maintenance of IgG anti-SARS-CoV-2 antibodies in infected individuals occurs in a pro-inflammatory microenvironment well regulated by IL-10 with great capacity for recruiting cells from the innate and adaptive immune systems.

Impact of COVID-19 on the efficacy of meeting the transfusion demand by a Brazilian blood banks network.

One of the effects of the pandemic in the hemotherapy services was the reduction in the attendance of blood donors and production of blood components. It is relevant to investigate how the capacity to meet the demand for blood components was affected, especially in blood centers located in the regions most affected by the pandemic, such as Brazil. This study aimed to describe the impact of the pandemic on the capacity to meet the demand for different types of blood components by a Brazilian blood center in 2020, compared to the historical series of 2016-2019 and to discuss the measures adopted to mitigate the effects of the pandemic. Retrospective cross-sectional study was carried out with comparative analysis of the blood components requested and attended in the period from 2016 to 2020. Data analysis was performed by Graphpad Prism 5. The spread of COVID-19 cases since March 2020 had impact on the blood components production and transfusions. The reduction in the production of blood components was observed prior to the restriction measures, in March 2020. In comparison to 2016-2019, there was a reduction in the number of transfusions performed in all months of 2020. The results suggest that the measures adopted in a Brazilian blood center to face the COVID-19 pandemic resulted in reasonable regularity in the supply of blood components. The sharing of experiences between blood banks in different regions, social and epidemiological contexts can contribute to the improvement of strategies to reduce the impact of COVID-19 in transfusion medicine.

Analysis of current SARS-CoV-2 infection in a large population of blood donors evidenced that RNAemia is rare in plasma.

BACKGROUND: Transmission of SARS-CoV-2 by asymptomatic individuals and by blood transfusion are important issues to understand to control the viral spread. In this work, we estimated the current SARS-CoV-2 infection rate in blood donors from Belo Horizonte, Brazil. STUDY DESIGN AND METHODS: Saliva and blood samples were collected from 4103 blood donors from June 15 to September 30, 2020. Saliva samples were tested by real-time RT-PCR for SARS-CoV-2 in mini-pools of four samples. Individual samples were tested for positive or inconclusive pools, and positive donors had their plasma tested. RESULTS: Twenty-seven (0.66%) blood donors were positive for SARS-CoV-2 in their saliva, but their plasma was negative, except for one, who presented a high viral load in saliva and nasopharyngeal samples and RNAemia in the plasma close to the limit of detection. Fourteen (56%) positive blood donors reported mild symptoms related to COVID-19 after donation, but the viral load levels were not statistically different between symptomatic and asymptomatic individuals. DISCUSSION: Despite the measures taken by Blood Centers to avoid blood donors with SARS-CoV-2 infection, asymptomatic or presymptomatic carriers are able to donate. The risk of the virus transmission by transfusion seems to be negligible since plasma RNAemia was seen at a very low level in only one (3.7%) of the positive donors, but other studies must be performed to confirm this finding.

ADAMTS-13-VWF axis in sickle cell disease patients.

Sickle cell disease (SCD) comprises a group of genetic disorders characterized by the presence of the hemoglobin (Hb) S in homozygosis or in heterozygosis with some other Hb variant or in interaction with thalassemia. SCD is characterized by a very complex pathophysiology, which determines a wide variability of clinical manifestations, including a chronic state of hypercoagulability responsible for the increased risk of thromboembolic events. ADAMTS13 and von Willebrand factor (VWF) play an important role in arterial and venous thrombosis. Thus, the aim of this study was to understand how the ADAMTS13-VWF axis behaves in sickle cell disease, as well as whether there is an association of these markers with the use of hydroxyurea (HU). This is a cross-sectional study conducted with 40 patients diagnosed with SCD and 40 healthy individuals. The analysis of the ADAMTS13-VWF axis was comparatively performed between groups of patients and controls and, afterwards, between patients with SCD who were users and non-users of HU. ADAMTS13 activity, ADAMTS13 activity/VWF:Ag, and ADAMTS13:Ag/VWF:Ag ratios were significantly lower and VWF:Ag levels significantly higher in SCD patients when compared to the controls. There was no statistically significant difference in ADAMTS13:Ag and VWF collagen binding (VWF:CB) levels between the groups evaluated. Among the categories of HU use, there was no statistically significant difference in any of the evaluated markers. As a conclusion, we could observe that the ADAMTS13-VWF axis is altered in SCD when compared to healthy individuals and that there is no association between these markers and the use of HU.

Impact of COVID-19 in the attendance of blood donors and production on a Brazilian Blood Centres.

BACKGROUND: One of the effects of the coronavirus disease 2019 (COVID-19) pandemic is the risk of shortages in Blood Centres. OBJECTIVES: To verify the impact of the COVID-19 pandemic on the blood donor's attendance and production of blood components in Fundação Hemominas, a Brazilian public institution was formed by several Blood Centres. METHODS: A cross-sectional study was carried out from January to June 2020. Data collected were compared to a historical series from 2016 to 2019. RESULTS: The study showed a reduction in the attendance of blood donors, whole blood collections and blood component production from March 2020, when the first case of COVID-19 was notified in Minas Gerais, Brazil. The results evidenced that Hemominas Blood Centres were affected in a very distinct way by the pandemic with a general mean reduction around 17% in attendance of blood donors and in production of blood components in the period of March to June. On the other hand, the return of blood donors rate increased. CONCLUSION: The reduction in blood donation during the pandemic period was significant, despite the measures adopted. Still, the recruitment of return donors appears to be an important measure to be considered to decrease the pandemic's effect on blood stocks.

Prevalence and risk factors associated with hepatitis C among Brazilian male patients with haemophilia: A long-term follow-up.

People with haemophilia represent a population with a high prevalence of HCV infection due to the use of blood components and plasma-derived clotting factor concentrates before the introduction of viral-inactivating procedures (in the 1980s) and screening for HCV (in the 1990s). About 80% of HCV-infected patients have chronic HCV infection, and at least 20% develop end-stage liver disease. The aim of the study was to assess current anti-HCV positivity in a large cohort of Brazilian haemophilia patients and to determine associated factors with HCV exposure. The study retrospectively analysed medical records of all male haemophilia patients attended the main public referral blood centre in Belo Horizonte, Brazil, from January 1985 to January 2015. Sociodemographic, epidemiological and serological characteristics were collected of all participants tested for anti-HCV. Among 724 patients enrolled in the study, anti-HCV was positive in 259 resulting in a seroprevalence of 35.8% (95% CI: 32.3%-39.3%). Factors independently associated with previous exposure to HCV were as follows: age older than 30 years, moderate to severe haemophilia, detection of inhibitor at least once in lifetime and previous exposure to hepatitis B virus (HBV) infection or HIV infection. Otherwise, exclusive previous use of inactivated clotting factors resulted in a significant decrease in the chance of positivity for anti-HCV. At the end of cohort period, patients with positive anti-HCV had a 3-fold higher risk of death. This study showed that hepatitis C infection remains a critical problem for Brazilian haemophilia patients and reinforced the need to unify efforts to eradicate it.

Genetic variants of VWF gene in type 2 von Willebrand disease.

INTRODUCTION: von Willebrand disease (VWD) is the most common inherited bleeding disorder. Few studies have explored the molecular basis of type 2 VWD. AIM: This study aimed to identify variants associated with type 2 VWD. METHODS: We collected clinical and laboratory data, as well as response to desmopressin and bleeding assessment tool (BAT) score in patients diagnosed with type 2 VWD. We sequenced exons 17, 18, 20 and 28 of the VWF gene. RESULTS: We identified 19 different variants in 40 unrelated patients (47.5%). Most of the variants (84.2%) were found in exon 28. A total of 10/19 variants (52.6%) were identified as "likely causative" in 17/40 patients (42.5%), according to the ISTH-SSC and EAHAD VWF gene mutations databases. Nine variants were initially identified as potentially benign. However, through analyses in silico, four of these variants were reclassified as "likely pathogenic" (Ile1380Val, Asn1435Ser, Ser1486Leu and Tyr1584Cys). Response to desmopressin was associated with three variants: Met740Ile, Arg1597Gln and Tyr1584Cys. Major bleeding was associated with variants related to VWD subtypes 2B and 2M. CONCLUSION: In conclusion, we identified 19 variants, of which 14 are "likely pathogenic" and therefore associated with VWD. We suggest a possible association of pathogenic variants with major bleeding, response to desmopressin and BAT score ≥10, although this requires further confirmation.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Quality control of B-lines analysis in stress Echo 2020.

BACKGROUND: The effectiveness trial "Stress echo (SE) 2020" evaluates novel applications of SE in and beyond coronary artery disease. The core protocol also includes 4-site simplified scan of B-lines by lung ultrasound, useful to assess pulmonary congestion. PURPOSE: To provide web-based upstream quality control and harmonization of B-lines reading criteria. METHODS: 60 readers (all previously accredited for regional wall motion, 53 B-lines naive) from 52 centers of 16 countries of SE 2020 network read a set of 20 lung ultrasound video-clips selected by the Pisa lab serving as reference standard, after taking an obligatory web-based learning 2-h module ( http://se2020.altervista.org ). Each test clip was scored for B-lines from 0 (black lung, A-lines, no B-lines) to 10 (white lung, coalescing B-lines). The diagnostic gold standard was the concordant assessment of two experienced readers of the Pisa lab. The answer of the reader was considered correct if concordant with reference standard reading ±1 (for instance, reference standard reading of 5 B-lines; correct answer 4, 5, or 6). The a priori determined pass threshold was 18/20 (≥ 90%) with R value (intra-class correlation coefficient) between reference standard and recruiting center) > 0.90. Inter-observer agreement was assessed with intra-class correlation coefficient statistics. RESULTS: All 60 readers were successfully accredited: 26 (43%) on first, 24 (40%) on second, and 10 (17%) on third attempt. The average diagnostic accuracy of the 60 accredited readers was 95%, with R value of 0.95 compared to reference standard reading. The 53 B-lines naive scored similarly to the 7 B-lines expert on first attempt (90 versus 95%, p = NS). Compared to the step-1 of quality control for regional wall motion abnormalities, the mean reading time per attempt was shorter (17 ± 3 vs 29 ± 12 min, p < .01), the first attempt success rate was higher (43 vs 28%, p < 0.01), and the drop-out of readers smaller (0 vs 28%, p < .01). CONCLUSIONS: Web-based learning is highly effective for teaching and harmonizing B-lines reading. Echocardiographers without previous experience with B-lines learn quickly.

Validation of a Spanish Version of the Information Needs in Cardiac Rehabilitation Scale to Assess Information Needs and Preferences in Cardiac Rehabilitation.

BACKGROUND: The identification of information needs is considered the first step to increase knowledge that ultimately could improve health outcomes in cardiac rehabilitation (CR). OBJECTIVE: The aim of this study was to psychometrically validate the Spanish Information Needs in Cardiac Rehabilitation (INCR). METHODS: The Spanish INCR was psychometrically tested in 184 patients undergoing CR. The internal consistency was assessed using Cronbach α, factor structure was assessed using exploratory factor analysis, and criterion validity regarding educational level, occupation, and duration in CR was assessed. RESULTS: Cronbach α was .97. Factor analysis revealed 10 factors, all internally consistent. Criterion validity was supported by significant differences in total INCR scores by educational level (P < .01), occupation (P < .01), and duration in CR (P < .05). Emergency/safety was the greatest information need perceived by patients. CONCLUSIONS: The Spanish INCR was demonstrated to have good reliability and validity. This tool can be applicable in clinical and research settings, assessing patients' information needs during CR and as part of education programming.