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Introduction. Hepatitis B virus (HBV) infection leads to a chronic liver disease that is distributed worldwide. The characterization of HBV into genotypes/subgenotypes is not only a mere procedure for distinguishing different HBV strains around the world because determining their geographic distribution is crucial to understanding their spread across the world.
Material and methods. We characterized different HBV genotypes and subgenotypes in five municipalities located in northeastern Maranhão, in the Brazilian north Atlantic coast. 92 HBsAg-positive individuals were submitted to PCR (polymerase chain reaction). Fifty samples were sequenced using automated Sanger sequencing and classified by phylogenetic methods.
Results. Subgenotypes D4 and A1 were found in 42 (84%) and eight (16%) samples, respectively. To our knowledge, this is the first study to describe a high frequency of subgenotype D4 in any population. Subgenotype A1 is frequently found across Brazil, but D4 has been rarely detected and only in a few Brazilian states. This study shows the characterization of HBV subgenotypes from a population based study in the state of Maranhão, particularly in populations that do not have frequent contact with populations from other regions of the world.
Conclusion. Our findings showed a HBV subgenotype profile that probably reflect the viruses that were brought with the slave trade from Africa to Maranhão. This study also reinforces the need to evaluate the status of HBV dispersion not only in large urban centers, but also in the hinterland, to enable the implementation of effective control and treatment measures.
Even though an efficient vaccine to prevent infection from the hepatitis B virus (HBV) is available since the 1980s more than 700,000 people have died worldwide from its complications in 2010.1,2 HBV is a highly prevalent and silent infection.3 HBV infection is a public health problem in regions where complete vaccination coverage has been difficult to achieve, particularly in infants, who are more prone to develop the chronic course of the disease.4,5
East Asia and Sub-Saharan Africa show high HBV prevalence, whereas Tropical Latin America, Central Latin America, Western Europe, and North America are considered low prevalence areas.6 According to a national survey, Brazil is considered a low prevalence area,7 although some areas with higher prevalence have been reported in the Amazon Basin and in the Southeast and South regions.8
HBV is a DNA virus classified into eight genotypes (AH) that differ by 7.5% or more in their genomic sequence and have distinct geographical distributions.9,10 A putative genotype I has also been described,11 although some authors have contested it, based on new recommendations for sorting a new genotype.12,13 Additionally, a genotype J has also been described in Japan.14 This tentative genotype shows similarities to gibbon and orangutan viruses and further studies are needed to ratify its classification.15 HBV genotypes may also be further divided into subgenotypes based on a nucleotide intragenotypic difference between 4 and 7.5%.9
The classification of HBV into genotypes/subgenotypes is not only a mere procedure for distinguishing different strains around the world because determining their geographic distribution is crucial to understanding their spread and analyzing evolutionary pathways.16,17
The distribution of genotypes around the world is a reflection of human population movements and is related to each ethnical background.15,18 In Brazil, genotype A is the most frequent genotype found followed by D, and F,19–26 whereas A1, D3, and F2a seem to be the most prevalent subgenotypes.19–22,24,27
The population of Brazil is composed of three main ancestral backgrounds: Europeans, Amerindians, and Africans.28 The latter group consisted of African slaves brought to work in many regions of the New World. Brazil received the largest numbers of slaves, followed by the Caribbean.29 Maranhão is a state in the north Atlantic Coast of Brazil that has particular features in the history of African slave trade: later establishment of slavery, isolation from other regions of the country due to the lack of river connection with the continent, and the similarity of slave trading routes with those in North Atlantic regions.30
The state of Maranhão is one of the Brazilian states with a higher percentage of Afro-descendants and remained isolated from the rest of the country until the mid-19th century.30 There are only two previous studies analyzing the distribution of HBV genotypes in the state of Maranhão: one that analyzed patients followed up in the city of São Luís, the state capital,19 and another that analyzed a quilombo community in northwestern Maranhão.31 In both studies, HBV genotypes also found in Africa were the most frequent, but genotype D4 found at the São Luís Referral Center for Liver Studies, São Luís, Maranhão, Brazil, has rarely been detected in other Brazilian regions.
This study characterized two different HBV subgenotypes found in five municipalities located in northeastern Maranhão.Material and MethodsSample selection and ethical approval
A total of 3,860 samples were screened for HBsAg serological marker using commercial kits according to the manufacturer's instructions (DiaSorin, Italy) in the five municipalities located in northeastern Maranhão along the Brazilian north Atlantic Coast (Figure 1) from 2012 to 2014. The representative sample number for the study region was calculated by considering an HBsAg prevalence of 0.5% in Brazil and the total number of individuals in each municipality. The collection procedure was performed by two-stage cluster sampling: first by choosing sectors according to population proportion and second by selecting the squares in these sectors by random sampling.
Geographic location of the five municipalities sampled in northeastern Maranhão, Brazil. Each star represents one municipality. The red circle indicates de state capital, São Luis. Source. Modified from: Wikimedia Commons -https://commons.wikimedia.org/wiki/ File:Maranhao_MesoMicroMunicip.svg#/media/File:Maranhao_MesoMicroMunicip.svg
The study was approved by the research ethics committee at University Hospital, Federal University of Maranhão (HUUFMA), São Luís, Maranhão, Brazil, and written consent was obtained from all individuals that agreed to participate in the study.HBV DNA amplification and sequencing
HBsAg positive samples were analyzed by nested polymerase chain reaction (nested PCR) to detect HBV DNA. HBV DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen®, Hilden, Germany) following the manufacturer's instructions. Fragments of 1,306 base pairs (bp) comprising the whole HBsAg and part of the DNA polymerase genes were amplified by nested PCR. Primers' sequences, PCR conditions, and sequencing procedures are described elsewhere.19,27 All procedures were strictly done following measures for avoiding contamination as well as negative and positive controls were used in PCR assays.32Viral load
PCR negative samples were analyzed for detection of viral load by real time PCR. We used the TaqMan® system (Life Technologies, Carlsbad, CA, USA) and probes, primers, and thermocycling conditions used are described elsewhere.33 The amplification was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, USA).HBV genotyping and subgenotyping
Chromatograms were evaluated using Phred-Phrap software34,35 and a quality score of 20 was used to select good quality readings. The consensus sequence of each sample was obtained using CAP3 software available at the Electropherogram quality analysis webpage (http:// asparagin.cenargen.embrapa.br/phph/). Sequences were aligned using BioEdit (v. 7.0.8) software and edited using CLUSTAL W software.36 HBV genotypes and subgeno-types were classified by phylogenetic reconstructions with published reference sequences from the GenBank database (http://www.ncbi.nlm.nih.gov/).
Phylogenetic trees were constructed using the Bayesian Markov Chain Monte Carlo (MCMC) method implemented in the BEAST package v.1.6.137 under a relaxed molecular clock using GTR + G + I as nucleotide substitution model; MCMC was run for 20 million generations and trees were sampled every 2000 generations. Maximum clade credibility tree was summarized using TreeAnnotator v.1.6.1 and the tree was visualized in FigTree v1.4.2. software (available at: http://tree.bio.ed.ac.uk/software/ figtree).
Serotypes were sorted according to the position of amino acids in the S gene by visual analysis.10
All sequences were submitted to GenBank under accessions numbers KX302085 - KX302134.Statistical analysis
The Student's t-test was used to determine the difference in mean age between subgenotypes; the Fisher's exact test was used to compare the other variables between sub-genotypes. Differences were considered significant at p < 0.05. All analyses were performed using STATA software version 11.0.ResultsDemographic characteristics of HBV-DNA positive individuals
Of the 3,860 samples, 92 were positive for HBsAg serological marker. Fifty-one (55.4%) of the 92 HBsAg-positive individuals had detectable HBV DNA by nested PCR. Most HBV-DNA positive individuals were men (56.9%; 29/51) and their mean age was 44.3 (SD = 21.1) years. In addition, most HBV-positive samples were from Humberto de Campos (43.1%; 22/51). We also found that most HBV-positive individuals were from the rural zone (70.6%; 36/51), had a family income of < 1 minimum wage (64.7%; 33/51), and declared themselves as being Afro-American or mestizo/mullato (76.5%; 39/51).Viral load
Forty-one of the 92 positive HBsAg samples were negative for HBV-DNA. These negative samples were submitted to viral load detection which was performed in 39 of the 41 samples because two serum samples were unusable. In total, 36 of the 39 samples analyzed had undetectable HBV viral load confirming our findings by nested PCR.Genotype/subgenotype and serotype distribution
All 51 positive samples had their partial HBV sequence determined. One sample was excluded from the final analysis because the sequence did not achieve a good quality index, but the remaining 50 samples had their HBV genotype and subgenotype determined (Table 1).
Genotypes A and D were only found in our sample and genotype D was the most frequent (84%; 42/50). The phylogeny showed that all genotype A sequences were subgenotype A1 (adw2) and all genotype D individuals were subgenotype D4 (ayw2). Additionally, subgenotype D4 clustered in a monophyletic group together with other sequences from Maranhão, whereas subgenotype A1 samples grouped with different HBV/A1 branches, except with the one containing sequences from Africa only (Figure 2).
Phylogenetic analysis of HBV subgenotype isolates from different regions of Maranhão state, Brazil, detailing subgenotypes A1 (A) and D4 (B). Analyses were performed by Bayesian Inference using the Bayesian Markov chain Monte Carlo (MCMC) method. Sequences identified in this study are highlighted (#) and reference sequences from GenBank are indicated by their corresponding genotype/subgenotype accession number and origin. The posterior probability values are shown for key nodes.
Phylogenetic analysis of HBV subgenotype isolates from different regions of Maranhão state, Brazil, detailing subgenotypes A1 (A) and D4 (B). Analyses were performed by Bayesian Inference using the Bayesian Markov chain Monte Cario (MCMC) method. Sequences identified in this study are highlighted (#) and reference sequences from GenBank are indicated by their corresponding genotype/subgenotype accession number and origin. The posterior probability values are shown for key nodes.
We found no significant difference in most sociodemographic characteristics between subgenotypes A1 and D4, except for municipality and family income (Table 1). There were more subgenotype D4 individuals in Morros, Humberto de Campos, and Urbano Santos than in Axixá and Icatu (P = 0.015). Additionally, the frequency of subgenotype D4 was higher in families with income < 1 minimum wage (P = 0.019).
Demographic characteristics of HBV-carriers and subgenotypes found in this study.
|Subgenotype (%) D4
|8 (16)
|42 (84)
|5 (17.86)
|23 (82.14)
|28 (56)
|3 (13.64)
|19 (86.36)
|22 (44)
|Mean age (± SD)
|41.5 (±18.7)
|45.3 (±21.8)
|44.7 (±21.2)
|1 (50)
|1 (50)
|2 (4)
|2 (16.7)
|10 (83.3)
|12 (24)
|4 (50)
|4 (50)
|8 (16)
|Humberto de Campos
|1 (4.55)
|21 (95.4)
|22 (44)
|Urbano Santos
|6 (100)
|6 (12)
|3 (21.4)
|12 (78.6)
|15 (30)
|5 (13.9)
|30 (86.1)
|35 (70)
|Ethnic group*
|3 (25)
|9 (75)
|12 (24)
|5 (100)
|5 (10)
|Native American
|2 (40)
|3 (60)
|5 (10)
|3 (11.5)
|23 (88.5)
|26 (52)
|2 (100)
|2 (4)
|Education level
|No education
|12 (100)
|12 (24)
|Incomplete Elementary education
|5 (23.81)
|16 (76.2)
|21 (42)
|Complete Elementary education
|4 (100)
|4 (8)
|Incomplete High school education
|1 (20)
|4 (80)
|5 (10)
|Complete High school education
|2 (28.6)
|5 (71.4)
|7 (14)
|Higher education
|Not applied
|1 (100)
|1 (2)
|Family Income
|< 1 mw**
|2 (6.3)
|30 (93.7)
|32 (64)
|1-3 mw
|6 (33.3)
|12 (66.7)
|18 (36)
Up to the present time, this is the third study to determine HBV genotype/subgenotype distribution in the state of Maranhão, in the north Atlantic coast of Brazil19,31 but it is the first to obtain HBsAg-positive samples from representative number of individuals of a regional population-based survey. The study region is located in northeastern Maranhão and consists of five municipalities with low socio-economical index.38
Most infected individuals lived with less than one minimum wage for the whole family and the level of education was often low. Thus, because of poverty and low educational status, these individuals are often unaware about means of transmission and prevention of diseases such as hepatitis, and thus may be more susceptible to infection. The delayed improvements in living and hygiene conditions and the difficulties in launching prevention, control, and treatment strategies that are important to control viral hepatitis and other diseases are known and have been described elsewhere.8
Two different HBV genotypes were found in our sample. Genotype D was the most prevalent followed by few genotype A cases. This finding is contrary to the HBV genotype prevalence described for Maranhão and Brazil as a whole, where genotype A is the most prevalente.19,21–26,31,39 An exception to that pattern is found in southern Brazil, where genotype D is the most frequent genotype,20,40,41 which is related to the immigration of European people from highly HBV/D prevalent regions.42
Only two subgenotypes were identified in our study, A1 and D4 being the latter the most frequent. Subgenotype A1 is the most frequent subgenotype found in cases of genotype A across Brazil27 and it was also the most frequent subgenotype found in the previous study conducted in São Luís, in Maranhão.19 In addition to subgenotype A1 that study also identified other four subgenotypes (D2, D3, F2a, and D4). Unlike the current study, subgenotype A1 was the most prevalent in that study, even though subgenotype D4 also had a relatively high frequency (24%; 29/ 119).19 The difference between our results and those of Barros, et al. (2014) is noteworthy and may be related to the samples analyzed: the population from the latter study consisted of chronic HBV carrier patients treated at a referral center for treatment of liver disease, and even though it covers the entire state of Maranhão, more than 75% of participants were from the metropolitan area of São Luís, the state capital. Thus, subgenotype A1 seems to be more prevalent in the metropolitan region, whereas subgenotype D4 is more frequent in other regions of the state such as the northeast.
Alvarado-Mora, et al. (2011) only found subgenotype A1 in four sequenced samples from quilombo Frechal, located in the municipality of Mirinzal, northwestern Maranhão.31 More studies in other regions of Maranhão should be conducted to analyze a larger number of HBV-positive samples for a better understanding of subgenotype prevalence in this region.
Subgenotype D4 was previously found in the aforementioned study conducted in Maranhão,19 but it is not frequently found in Brazil. This genotype was initially described in two different studies conducted in the Brazilian Amazon, in the states of Amazonas21 and Rondônia.24 Genotype D4 was also found in 5.1% (36/702) of samples collected across Brazil,27 mostly in Maranhão, but also in other states in the southeast (São Paulo and Minas Gerais) and north (Pará) of Brazil.43 We hypothesize that internal migrations among different Brazilian states may explain the presence of subgenotype D4 in these regions, particularly in Pará, which has a large border with the state of Maranhão.
The high prevalence of subgenotype D4 in Maranhão supports the hypothesis that this strain was not introduced in Brazil through European immigration because this sub-genotype is not frequent in Europe.42 Zehender, et al. (2012) performed for the first time a HBV-D epidemiological reconstruction by phylodynamics and phylogeographic analysis in Europe.44 Although they did not included D4 sequences in their analysis, a preliminary review of them showed that D4 and D7 subgenotypes are close related. Furthermore, other study also found this relation between these two subgenotypes.45 HBV-D7 is more often restricted to the Northern Africa46,47 and may give support to the hypothesis of HBV-D4 distribution from this continente.19
In fact, subgenotype D4 has been previously described in many countries worldwide.10,48–55 HBV D4 has also been found in Caribbean countries of significant African descent such as Haiti, Martinique, and Cuba.56–58 Interestingly, in Kenya, only subgenotypes A1 (85.7%) and D4 (14.3 %) have been reported,49 but in proportions that are the opposite of those found in Maranhão. As suggested by Barros, et al. (2014), subgenotype D4 may have infected individuals frequently in the past, and during the period of slavery these strains may have reached other continents where African slaves were also traded.19 For instance, Haiti and Cuba are regions outside Africa with a high frequency of subgenotype D4 cases.56,58 The Caribbean had intense trading of slaves from Africa, lower only than that in Brazil.29 Thus, the history of population formation, combined with molecular evidence of virus traits, may support a better understanding of the spread of infections such as HBV.
Subgenotype D4 strains found in Brazil clustered in a separate branch in the phylogenetic tree with other samples described elsewhere (except from three from India), suggesting the introduction of a unique strain in the country. It is noteworthy that only these India sequences appeared to be related to our sequences apart from any other described in the world. These Indian strains are from Tripura State which is the first region in India where subgenotype D4 was found.53 Although the authors did not found a high prevalence of the HBV-D4 (13/76; 17.1), Portuguese vessels might have brought from India some strains of this infection to Maranhão, mainly when British begun to suppress the Atlantic slave trade and forced the Northern Atlantic traders to change their routes to SouthEast Africa.30,53,59,60 Subgenotype A1 strains also clustered in a branch different than the one where most African sequences were grouped and differently than subgenotype D4 strains, A1 strains did not aggregate in a few branches but rather in many branches containing samples from across the world, with only few African samples from Somalia and South Africa. Thus A1 strains cannot be considered an African clade. In a study that analyzed A1 strains from Brazil,60 the authors concluded that Brazilian A1 genotypes are not of Central or Western Africa origin, but originated from slaves brought from southeastern Africa during the mid-19th century, because the study's samples clustered into an "Asian-American" clade similarly to the samples in our study. However, there are few available sequences with these genotypes from East African countries, despite the reports of slaves coming to Maranhão from these countries.61
The Brazilian population was formed by three ancestral backgrounds in decreasing order of contribution: Europeans, Africans, and Amerindians. Nevertheless, the state of Maranhão is one of the regions in Brazil with the greatest contribution of African ancestry.28 Our results show that most individuals who were assigned to subgenotypes A1 and D4 declared themselves as being Afro-American (24%) or mestizo/mulatto (52%), reinforcing the association between these two subgenotypes and African ancestry. Moreover, the pattern of slavery establishment in Maranhão was peculiar: due to geographic features (isolation from other regions in the country because of a lack of river connection to the continent) and navigation issues (wind and ocean currents), slave trade routes in Maranhão were more similar to those in Caribbean and North Atlantic countries than those in the rest of Brazil.30 Thus, we expected to find the same subgenotype in Maranhão and in Caribbean countries, which is not the same one found in Bahia, another Brazilian state with a large number of Afro-descendants.27 Finally, the recent finding of HDV-8 in Maranhão co-infecting individuals that carried HBV/ D4 corroborates the African origin of the latter subgenotype.62,63
This study emphasized the presence of HBV genotypes that might be originated from Africa in the state of Maranhão, particularly in populations that do not have frequent contact with populations from other regions of the world. This pattern probably reflects the viruses that were brought with the slave trade from Africa to Maranhão that was interrupted in the middle of the 19th century. Maranhão has a great contribution of African ancestors originated from a particular slave trade route that is similar to the one followed in the Caribbean islands and both areas share a similar pattern of HBV subgenotype distribution.
This study has unexpectedly shown a low prevalence of HBV DNA among the HBsAg individuals. This may be explained by the nature of the sample. The individuals who participated of this study were from an epidemiologic survey rather than individuals searching for medical care. However, the high prevalence of HBV D4 strain may scatter light on the interaction of this virus and the human host, that is, genotype D4 may influence the immunologic state of HBsAg marker.64 Only with more studies related to this issue with follow up of patients from this population would explain this finding.
This study also reinforces the need to evaluate the status of HBV dispersion not only in large urban centers, but also in the hinterland, to enable the implementation of effective control and treatment measures. Studies involving population samples from wide geographic areas and hard-to-access regions are difficult to perform, mainly due to the high costs and lack of specialized human recourses. Nevertheless, further investigations in isolated areas are needed because a better knowledge of HBV infection across the world is essential for the effective control of this infectious disease.Acknowledgments
The authors thank the State Health Department of Maranhão -Epidemiological Surveillance Secretariat; Central Laboratory of Public Health- Maranhão (LACEN-MA) as well as the municipal government of all participating Municipalities.
This work was supported by Fundação de Amparo à Pesquisa do Estado do Maranhão - (FAPEMA) [grant: PPSUS-03348/13 and PPP-01263/12]; São Paulo Research Foundation (FAPESP) - [grant: 2009/53946-3]; Brazilian Ministry of Health [grant: 1/2013]. Max Diego Cruz Santos received a Ph.D. fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant: 141138/2012-2]. João Renato Rebello Pinho receives fellowship from CNPq (Bolsista de Produtividade em Pesquisa do CNPq - Nível 2).


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Monthly Archives: May 2019
CSA 2019: ITDS Sessions and Meeting Next Week
The annual meeting of the Canadian Sociological Association is upcoming next week at the University of British Columbia (June 3-6). The Internet, Technology, & Digital Sociology (ITDS) research cluster is affiliated with five sessions at the conference. Check out the … Continue reading
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CSA 2019: Workshops, Events, and Tours
We would like to publicize a few opportunities for attendees to participate in during the conference. For each of the following, registration or ticket purchases are required in advance. Musqueam Nation Community and Cultural Centre Tour Tuesday, June 4 from … Continue reading
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I am final, I am sorry, but, in my opinion, there is other way of the decision of a question.
- 04.06.2019
- by Shaktirn
- 1 comments
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no future reckoning. Oh, sir ! there is a hell, dreadful, inconceivable, eternal!" Here, through the excess of anguish, the poor fellow fainted away. Mr. Fantom, who did not at all relish this scene, said to his friend, "Well, sir, we will go, if you please, for you see there is nothing to be done."
"Sir," replied Mr. Trueman, mournfully, "you may go if you please, but I shall stay, for I see there is a great deal to be done." "What !" rejoined the other, " do you think it possible his life can be saved ?"—"No, indeed," said Trueman; " but I hope it is possible his soul may be saved."— "I do not understand these things," said Fantom, making toward the door.—"Nor I neither," said Trueman; "but, as a fellow-sinner, I am bound to do what I can for this poor man. Do you go home, Mr. Fantom, and finish your Treatise on Universal Benevolence, and the Blessed Effects of Philosophy; and, hark ye, be sure you let the frontispiece of your book represent William on the gibbet : that will be what our minister calls a practical illustration. You know I hate theories : this is realizing; this is philosophy made easy to the meanest capacity. This is the precious fruit which grows on that darling tree; so many slips of which have been transplanted from that land of liberty, of which it is the native, but which, with all your digging, planting, watering, dunging, and dressing, will, I trust, never thrive in this blessed land of ours."
Mr. Fantom sneaked off, to finish his work at home; and. Mr. Trueman staid to finish his in the prison. He passed the night with the wretched convict; he prayed with him and for him, and read to him the penitential psalms, and some portions of the Gospel. But he was too humble and too prudent a man to venture out of his depth by arguments and consolations which he was not warranted to use: this he left for the clergyman; but he pressed on William the great duty of making the only amends now in his power, to those whom he had led astray. They then drew up the following paper, which Mr. Trueman got printed, and gave away, at the place of execution :
The last Words, Confession, and Dying Speech of WILLIAM Wilson, who was executed at Chelmsford, for Murder.
I was bred up in the fear of God, and lived with credit in many sober families, in which I was a faithful servant ; but, being tempted by a little higher wages, I left a good
place to go and live with Mr. Fantom, who, however, made good none of his fine promises, but proved a hard masterfull of fine words and charitable speeches in favor of the poor; but apt to oppress, overwork, and underpay them. In his service I was not allowed time to go to church. This troubled me at first, till I overheard my master say, that going to church was a superstitious prejudice, and only meant for the vulgar. Upon this I resolved to go no more; for I thought there could not be two religions, one for the master, and one for the servant. Finding my master never prayed, I, too, left off praying: this gave Satan great power over me, so that I from that time fell into almost every sin. I was very uneasy at first, and my conscience gave me no rest; but I was soon reconciled by overhearing my master and another gentleman say, that death was only an eternal sleep, and hell and judgment were but an invention of priests, to keep the poor in order. I mention this as a warning to all masters and mistresses, to take care what they converse about while servants are waiting at table. They cannot tell how many souls they have sent to perdition by such loose talk. The crime for which I die is the natural consequence of the principles I learnt of my master. A rich man, indeed, who throws off religion, may escape the gallows, because want does not drive him to commit those crimes which lead to it; but what shall restrain a needy man, who has been taught that there is no dreadful reckoning? Honesty is but a dream, without the awful sanctions of heaven and hell. Virtue is but a shadow, if it be stripped of the terrors and the promises of the gospel. Morality is but an empty name, if it be destitute of the principle and power of Christianity. Oh, my dear fellow-servants ! take warning by my sad fate : never be tempted away from a sober service for the sake of a little more wages; never venture your immortal souls in houses where God is not feared. And now hear me, O my God, though I have blasphemed thee! forgive me, O my Savior, though I have denied thee! O Lord most holy, O God most mighty, deliver me from the bitter pains of eternal death, and receive my soul, for His sake who died for sinners.
Mr. Trueman would never leave this poor penitent till he was launched into eternity, but attended him with the minister in the cart. This pious clergyman never cared to say what he thought of William's state. When Mr. Trueman ventured to mention his hope, that though his penitence was
late, yet it was sincere, and spoke of the dying thief on the cross as a ground of encouragement, the minister, with a very serious look, made this answer:-"Sir, that instance is too often brought forward on occasions to which it does not apply: I do not choose to say any thing to your application of it in the present case, but I will answer you in the words of a good man speaking of the penitent thief: "There is one such instance given, that nobody might despair ; and there is but one, that nobody might presume.'".
Poor William was turned off just a quarter before eleven ; and may the Lord have had mercy on his soul !
MR. BRAGWELL and Mr. Worthy happened to meet last year at Weyhill-fair. They were glad to see each other, as they had but seldom met of late; Mr. Bragwell having removed some years before from Mr. Worthy's neighborhood, to a distant village, where he had bought an estate.
Mr. Bragwell was a substantial farmer and grazier. He had risen in the world by what worldly men call a run of good fortune. He had also been a man of great industry; that is, he had paid a diligent and constant attention to his own interest. He understood business, and had a knack of turning almost every thing to his own advantage. He had that sort of sense which good men call cunning, and knaves call wisdom. He was too prudent ever to do any thing so wrong that the law could take hold of him; yet he was not, over scrupulous about the morality of an action, when the prospect of enriching himself by it was very great, and the chance of hurting his character was small. The corn he sent home to his customers was not always quite so good as the samples he had produced at market; and he now and then forgot to name some capital blemish in the horses he sold at the fair. He scorned to be guilty of the petty frauds of cheating in weights and measures, for he thought that was a beggarly sin; but he valued himself on his skill in making a bargain, and fancied it showed his superior knowledge of the world, to take advantage of the ignorance of a dealer.
It was his constant rule to undervalue every thing he was about to buy, and to overvalue every thing he was about to sell; but as he seldom lost sight of his discretion, he avoided every thing that was very shameful ; so that he was considered merely as a hard dealer, and a keen hand at a bargain. Now and then, when he had been caught in pushing his own advantage too far, he contrived to get out of the scrape by turning the whole into a jest, saying it was a good take in, a rare joke, and that he had only a mind to divert himself with the folly of his neighbor, who could be so easily imposed on.
Mr. Bragwell, however, in his way, set a high value on character; not, indeed, that he had a right sense of its worth; he did not consider reputation as desirable because it increases influence, and for that reason strengthens the hands of a good man, and enlarges his sphere of usefulness; but he made the advantage of reputation, as well as of every other good, centre in himself. Had he observed a strict attention to principle, he feared he should not have got on so fast in the world as those do who consult expediency rather than probity; while, without a certain degree of character, he knew also, that he should forfeit that confidence which put other men in his power, and would set them as much on their guard against him, as he, who thought all mankind pretty much alike, was on his guard against them.
Mr. Bragwell had one favorite maxim; namely, that a man's success in life was a sure proof of his wisdom; and that all failure and misfortune was the consequence of a man's own folly. As this opinion was first taken up by him fom vanity and ignorance, so it was more and more confirmed by his own prosperity. He saw that he himself had suceeded greatly without either money or education to begii with; and he therefore now despised every man, however excellent his character or talents might be, who had not the same success in life. . His natural disposition was not particularly bad, but prosperity had hardened his heart. He made his own progress in life the rule by which the conduc of all other men was to be judged, without any allowance for their peculiar disadvantages, or the visitations of providence. He thought, for his part, that every man of sense could command success on his undertakings, and contrd and dispose the events of his own life.
But though he considered those who nad had less success than himself as no better than fools, yet he did not extend this opinion to Mr. Worthy, whom he looked upon not only as a good but a wise man. They had beer bred up when children in the same house ; but with ths difference, that


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Iranian Army is ready to help in all crisis: Top official
Tehran (IP) - Coordinating Deputy of the Islamic Republic of Iran Army Rear Admiral Habibollah Sayyari said that besides the main responsibility of the Army which is protecting borders, the Army helps the nation in all crises.
Iran Press/Iran News: On the sideline of the inauguration ceremony of the 2000-bed hospital in an exclusive interview with Iran Press, the top military official noted that "The main responsibility of the army is protecting borders and no crisis can affect this responsibility."
Army has been active in all conditions such as floods and earthquakes, he added.
"We could have an active role in facing Coronavirus crisis which spreads all over the country and help the medical organization in all provinces," Rear Admiral Sayyari said.
He went on to say that we have supported human resources for the hospitals which were in need too, in addition to it, over 32 hospitals, all medical centers and more than 4,600 beds were allocated to the fight against Covid-19.
"We control the transportation in more than 80 cities for disinfecting and checking peopleو" the top official highlighted.
The present field hospital shows that the Army is able to give the best services in the least time, he concluded.
Read More:
Daily production of 100,000 masks in Shiraz
Rouhani: We have no shortage of hospital beds, nurses
Help that does not meet current needs will not be operational; Health Ministry spox


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So I rarely log into AR15.com, because I'm not one to post much on weapons boards, and their search capability sucks.
- Public search is limited to ONE SEARCH PER 60 SECONDS.
- Public search is limited to SUBJECTS only.
But they sent me a bit of
SPAM email today informing me that they have updated their SPAM newsletter capability. Or something. So I was forced to go look at the options, and then to log in and at least change my PW, which was hopelessly out of date. (Gotta love password managers!)
Back to the subject of search: Don't they know that their entire site is indexed by Google? Could it be that in 2019, people still don't know how to limit a Google search to a particular site? (No, I'm not going to explain it to you. That would make the folks who run ar15.com mad at me.)


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The Sudbury Annual Camp Fair is coming back! Join us on Sunday January 26th from 11-2 at Lincoln-Sudbury Regional High. This event is free for all. There are over 80 camps attending. You will be able to get information about day, overnight and travel camps. For more information visit sudburycampfair.org
390 Lincoln Road
Tanvi Devimane
Event Organizer/Sponsor: Loring PTO
When: January 26, 2019 from 11:00 am
Where: Lincoln Sudbury Regional High School
390 Lincoln Road
Sudbury, Massachusetts 01776
Cost: free
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by Conscious Reminder
During our whole lifetimes, there are a lot of lessons which we may learn, and which can also help us to overcome the problems we have and live happily and in contentment.
Here, we are going to share the best such lessons which we all have learned.
Here is a list comprised of 15 spiritual lessons which we find really important. We should all try to comprehend them and remember them too:
1. We should never let the behavior of others tell us what we should do. We are free beings, and we have the responsibility for our own actions.
2. Every one of us is connected. This means that if we help someone, we are helping ourselves. If we hurt someone, we will hurt ourselves too.
3. We shouldn't fear of some painful experiences. In fact, pain can be bad or good, but we are able to deal with such pain in bad or good ways too.
4. When we drop all expectations and desires about the way people have to be, we are never going to feel hurt or deceived again.
5. Every fear comes down to the fear of death. Until we become quite familiar with what death is, we are never going to have the ability to live without being afraid.
6. We can't know anything more about others than we can know only about ourselves. Getting to know ourselves better will help us understand other people better too.
7. Real love will never hurt as it actually gives without even asking for something in return. This means that even when love is rejected or unseen, it will always be what it truly is.
8. Pain and pleasure are two different aspects of one same coin. The first one cannot be possessed without the second one.
9. When we treat the symptom, we cannot cure a particular disease. Rather than that, we should try to remove the disease's root-cause.
10. We should remember that there isn't God that is higher than the Truth.
11. We should never focus our attention on the things other people do or say. Instead, we should try to find the motive for their speaking or acting.
12. We should always tell the truth, regardless of the consequences. In fact, being honest will be the single way in which we can feel at peace with other people and ourselves too.
13. Possessions may also possess us. We should let go of our attachments to our belongings.
14. Every one of us comes to the world alone so that we also leave the world alone. We should not be afraid to experience loneliness as only in that way we are going to have the ability to confront ourselves as we truly are.
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text: Yasticaki 89 Report post Posted May 8 (edited) Hi there guys! So some of you may or may not know I am currently rebuilding my bank. Currently my bank value is showing at around 327M on Grand Exchange Tab. It's probably inaccurate however, so I'm guessing it's about 270M. I will check what this value will be at the end of my Vorkath Session and seeing the difference will be interesting! How I woox-walk:https://imgur.com/a/cHPjZ3x So starting off, I set off to buy the supplies for 300 Vorkath Trips. My inventory and setup is as follows: Thus, the required supplies would be: 300 x Range potion(4) 300 x Anti-venom(3) 300 x Extended super antifire(2) 300 x 3 Prayer potions(4) = 900 Prayer potions(4) 300 x 18 Sharks = 5400 Sharks Bringing Total Supplies at 37M. Will hope for some visages maybe!! Perhaps an improvement in my pb? Current PB is 0:51!! Will be interesting to see the final loot tab!!! UPDATE: This Vorkath Grind has now ended! So my final loot tab for those that are interested: Final Loot tab of 227m. I have inserted 6K plat tokens to represent 6m of loot that I sold off for something I had to buy. Overall, I had approx 2.4m of bolts left, so the total profit was 227-38+2.4 = 191.4M Pure Profit. Key Info: Starting KC: 4470 Ending KC: 6208 PB: 0:51 End PB: 0:51 Uniques: 1 x Jar of Decay, 2 x Dragonbone Necklace Edited June 10 by Yasticaki 2 1 batfire, Corbula and azaethor reacted to this Quote Share this post Link to post Share on other sites


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Other bug subscribers Subscribe someone else. They hope these examples will help you to get a better understanding of the Linux system and that you feel encouraged to try out things on your own. With TurboPrint FreeEdition you can test your printer's features: All times are GMT Linux – General This Linux forum is for general Linux questions and discussion. The Product is warranted against defects in materials and workmanship under normal use and service a for a period of one 1 year from the date of original purchase when delivered to you in new condition in its original container or b for 1 year from the date of original purchase for refurbished Products.
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These drivers comes with gutenprint 5. Quality and dpi can't be set.
Canon i Free Driver Download | Driver Supports | Pinterest | Canon and Free
Paying and heavily for a printer driver seems to be quite contradictory of all things Linux Uninstall Procedures If needed, you can uninstall this program using the Control Panel. Beautiful luster finish provides an elegant surface that resists fingerprints and virtually eliminates glare. Canon i and Linux support?
Package includes 15 sheets. Double click on the program that you want to uninstall. It is a Canon i USB printer. The replacement Product you receive may be a refurbished or reconditioned unit and will be lniux for the balance of the period remaining on your original limited warranty.
Printer not found
Canon offers a wide range of compatible supplies and accessories that can enhance your user experience with you i that you can purchase direct. HR High Resolution Paper – 8. Unfortunately, there doesn't seem to be a good solution, the drivers are nearly the same price as the printer was and you may as well buy a printer that is supported by CUPS because you will certainly get better support if you run into trouble as it is much more popular AND you are supporting a higher purpose.
Find More Posts by frostschutz. Package includes 20 sheets. All times are GMT There is no utility for the OS Version you selected. Glossy, top-quality paper for vibrant, lifelike color images you won't be able to tell from the original. Canon USA shall have no responsibility for such items except for compatible Canon brand peripheral equipment covered by a separate warranty "Separate Warranty".
You can always use the freeware variant of the driver, if high quality printing is not that important to you To post a comment you must log in.
Plain Paper – sheets paper weight 17 lb. At bugreport sf on Gutenprint developer gernot wrote: If you need to reset your password, click here. The steps below are for Windows 7. Repairs of such Canon brand peripheral ljnux shall canno governed by the terms of the Separate Warranty. Last edited by osio; at In Demo mode, the only one allowed in the Free 'Trial', the logo prints all over the place.
Learn about the technology behind the paper. Please visit this page to clear all LQ-related cookies. Probably 10 Euros would get floods of customers in for any company releasing the driver.
This paper is great when you want high-quality color output to produce winning reports and eye-catching graphics. Well here is the updated quote from the Turboprint web site: In the event service is required for the Product during the limited warranty period, Canon USA offers two hardware support options: You need to log in to change this bug's status.
Till Kamppeter till-kamppeter on But in Ubuntu


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As part of its ongoing initiative to connect residents and visitors with high quality healthcare in Southwest Florida, the Collier County Medical Society is hosting their 12th Annual Women's Health Forum.
With the theme "Hot Topics in Wellness: Mind & Body." the forum will feature breakout sessions on a spectrum of women's health topics from 24 local physicians. Breakout session topics include holistic living, breast health, cardiology, healthy vision, patient advocacy, mental health, hearing impairment, gynecology, vein health and more.
Drop-ins are welcome during the no-cost event at Naples United Church of Christ, McSpadden Hall from 8:30 a.m.-1 p.m.
– David Wilkinson, M.D., Collier County Medical Society


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David Beckham hailed the collective spirit of the Major League Soccer All-Stars after they beat West Ham 3-2 at BMO Field last night.
The MLS side extended their unbeaten All-Star Game record against overseas opposition to 5-0 in front of a stadium-record sell-out crowd of 20,844, with impressive first-half goals from Christian Gomez and Mexico's Cuauhtemoc Blanco and a second-half penalty by Canada's Dwayne De Rosario.
Striker Dean Ashton netted both goals for West Ham as the Londoners took the lead in the 26th minute and then levelled 2-2 in the 67th in a competitive match.
'Any good player that's passionate about soccer is committed in every game that we play,' Beckham said.
'And every player out there was committed to winning the game.
'We said we were going to take it seriously and we wanted to win the game and we did that.
'We also wanted to put in a good team performance because we are a bunch of players that play against each other every week and haven't played with each other.
'But we proved tonight that we can come together and we played as a team, we worked as a team and we won as a team.
'So, overall, it was a very good performance.'
MLS coach and former Liverpool player Steve Nicol was delighted with the victory, hailing it as a great boost for the MLS' standing in world football.
'It's always a pleasure to be involved with good players and I think we showed tonight that not only do we have good players in this league but great players,' Nicol said.
'It was a fantastic performance, we thoroughly deserved to win and it was a pleasure for me.'


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In the summer, people health education instructors and the hands-on focus on problems that are critical
Health instruction is crucial within our own children's lives. Summer vacations at which it isn't unusual for the children to own a deficiency of self-esteem , stress, and depression are usually carried over into by A carefree perspective.
A wellbeing program requires that students be exposed towards the benefits of physical exercise. It can help them develop their bodies along with their heads. Children are fast since it may be really fun, to take part. Rather than focusing on the effects of non-physical activities, children will learn continue being lively and to rip out it.
Physical activities include running, swimming, and other types of exercise. If kids participate in a health education program at school or at summer camp, they may learn how to swim, http://nemgiogiatruyen.com/csun-biology-advisement-secrets-that-no-one-else-knows-about/ how to run, and how to work out safely. They will also learn how to protect themselves from being injured or sick while swimming.
Health teachers do not limit the quantity of activities that children do during the summer. They train children how to restrain their bodies also breathe properly while doing physical activities.
Mental health education, including group discussions, face-to-face therapy, and working with children with autism, allows children to address their fears and learn how to manage their own bodies. They will be encouraged to talk about different fears or problem areas in their bodies that they have been avoiding.
Swimming is a great activity for children to engage in as part of their summer health education. http://www.domyigaraze.pl/forum/viewtopic.php?f=68&t=8249 Parents should not be afraid to ask their children what they do or how they feel about the water, especially if they were not allowed to swim as a child.
Children and adults will want to get a swimming pool near to where they live because it is so convenient. Most states have these pools available for rent during the summer months. Some pools even have the activity units attached to them so that children can participate in swimming and physical fitness activities while not having to worry about them being dirty.
The youngsters will need to find a healthful dose of daily training. They can do it throughout walking, riding bicycles, visiting the gym, or performing lawn work. Adults may add such a day-to-day exercise in their daily schedule.
Summer mental health education programs often include games that give children a sense of accomplishment and have them laughing. These games are simple ones such as dress up, coloring, or do-it-yourself. One game that is particularly effective is a scavenger hunt.
Adults and children can put together clues and puzzles to find out about a child's anxiety and how it came about. Many physical activities also teach children how to be independent and help them develop a sense of independence.
Because summer is not necessarily the time for our children to stay indoors, teachers can implement a physical activity program during one of our children's summer health education sessions. We do not have to ignore our children's natural needs or suppress them. The sun is a terrific source of Vitamin D and an hour of physical activity in the sun can be more than enough for children to build muscle and bone.
Health education teachers who are devoted to improving our children's health should incorporate summer mental health education in one of their students' summer health education programs. When children are encouraged to participate in these sessions, they will feel healthier, happier, and connected to others. Having a regular physical activity in the summer will help strengthen both minds and bodies.


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Price: $99.95
Quantity: 1 available
Book Condition: Good
240 pages. Index. "In all those qualities which mark the born leader of men, James Outram had very few rivals among the best and greatest of the soldier statesmen who rose to fame in the service of the old East India Company." - from Preface. Average wear to unmarked book. Binding sound. Moderage age-toning to contents. Above-average wear to dust jacket which is now preserved in mylar. A sound copy.
Title: The Bayard of India: A Life of General Sir James Outram - Everyman's Library 396
Categories: History, Biography, Asia,
Edition: Reprint
Publisher: London, J.M. Dent & Sons Ltd: 1925
Binding: Hardcover
Book Condition: Good
Jacket Condition: Fair
Item: 1.00 Item
Seller ID: 322G2273
Keywords: The Bayard of India: A Life of General Sir James Outram - Everyman's Library 396 Mutiny India Indian East India Company Generals British Military,


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