A TaqMan minor groove binder probe-based quantitative reverse transcription polymerase chain reaction for detection and quantification of chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus with widespread distribution across the globe. Since 2016, CHIKV re-emerged in several countries including Indian subcontinent and Southeast Asia. A proper diagnostic tool for early diagnosis of CHIKV infection is crucial to facilitate patient management and control virus transmission at the earliest stage of outbreak. Therefore, a TaqMan minor groove binder (MGB) probe-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify the CHIKV. The primers and probe were designed based on a conserved genomic region of 730 global CHIKV sequences that is located between nsP1 and nsP2 genes. The nucleotide mismatches of primers and probe with 730 global CHIKV sequences and 13 alphaviruses were then analysed in silico. In this study, the last 5 nucleotides at 3' end of primers and 5' end of probe were considered to be the critical regions for priming. In silico analysis revealed that the critical regions of primers and probe were at least 99.6% matched with the 730 global CHIKV sequences. Besides, the primers and probe showed at least 5/20 (25.0%) and 4/17 (23.5%) nucleotide mismatches with 13 alphaviruses respectively. The amplification efficiency of qRT-PCR assay was 100.59% (95% CI= 93.06, 109.33) with a R2 score of 0.957. Its limit of detection (LOD) at 95% probability level was 16.6 CHIKV RNA copies (95% CI= 12.9, 28.9). The qRT-PCR assay was specific to CHIKV without cross-reacting with all dengue virus serotypes, Getah virus, Tembusu virus and Zika virus. The diagnostic results of qRT-PCR assay were perfectly agreed (k=1.000, p=0.003) with a commercial trioplex assay, with sensitivity of 100% (95% CI= 61, 100) and specificity of 100% (95% CI= 44, 100). Overall, the developed qRT-PCR assay is ideal for rapid, sensitive and specific detection as well as quantification of CHIKV.

Metformin acts in the gut and induces gut-liver crosstalk.

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Diagnosis and Severity Evaluation of Ulnar Neuropathy at the Elbow by Ultrasonography: A Case-Control Study.

Background: Traditional diagnostic techniques such as clinical examination and electrodiagnosis are less sensitive in diagnosing ulnar neuropathy at the elbow (UNE). Ultrasonography (USG) is increasingly being used to diagnose UNE. However, clinical applicability is limited by the lack of uniformity in the previous studies. Therefore, we aimed to study in the Indian patients the diagnostic utility of the ulnar nerve cross-sectional area (CSA) and a novel parameter-entrapment index (EI) in UNE measured by USG and to find if both these parameters correlate with the electrodiagnostic severity. Methods: This retrospective casecontrol study included 28 patients (36 nerves) of UNE and 12 (24 nerves) age- and gender-matched healthy controls. Electrodiagnostic severity was graded using the Padua classification. USG was performed in both groups, and CSA was measured at the medial epicondyle (ME) and 5 cm proximally and distally. EI was calculated by multiplying the ratio of CSA above ME over CSA at ME by 100. Best cutoffs were derived by the receiver operating characteristic curve analysis. Results: UNE group had significantly higher CSA at all three locations and lower EI than the control group. CSA at ME ≥9.7 mm2 and EI ≤61.5 has sensitivity and specificity of 88.9%/87.5% and 72.2%/79.2%, respectively. There was no significant difference in CSA and EI between nonsevere and severe UNE groups. Conclusion: CSA at ME and EI have good sensitivity and specificity in diagnosing UNE. However, they cannot differentiate nonsevere from severe UNE.

Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus.

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM).

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

@#Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Health impact of exposure to asbestos in polluted area of Southern Italy.

The three main sources of asbestos pollution in the city of Bari, Puglia, the former Fibronit asbestos factory, the Torre Quetta beach, the former Rossani barracks and the history of their reclamation are described. The results of cohort studies on factory workers and case-control studies on asbestos exposure to the resident population and the onset of mesothelioma are also reported. Finally, the data of the regional register of mesothelioma related to residents in the city of Bari and four new cases with environmental exposure due to the former Rossani barracks are presented.

Contemporary pollen and seed dispersal in natural populations of Bertholletia excelsa (Bonpl.).

Due to the nutritional content and commercial value of its seeds, Bertholletia excelsa is one of the most important species exploited in the Amazon region. The species is hermaphroditic, insect pollinated, and its seeds are dispersed by barochory and animals. Because the fruit set is dependent on natural pollinator activity, gene flow plays a key role in fruit production. However, to date, there have been no studies on pollen and seed flow in natural populations of B. excelsa. Herein, we used microsatellite loci and parentage analysis to investigate the spatial genetic structure (SGS), realized pollen and seed dispersal, and effective pollen dispersal for two B. excelsa populations in the Brazilian Amazon forest. Two plots were established in natural forests from which adults, juveniles, and seeds were sampled. Realized and effective pollen flow was greater than realized seed flow. The distance of realized pollen dispersal ranged from 36 to 2060 m, and the distance of realized seed dispersal ranged from 30 to 1742 m. Both pollen and seeds showed a dispersal pattern of isolation by distance, indicating a high frequency of mating among near-neighbor trees and seed dispersal near to mother trees. Both populations present SGS up to 175 m, which can be explained by isolation by distance pollen and seed dispersal patterns. Our results suggested that fragmentation of these forest populations may result in a significant decrease in gene flow, due to the isolation by distance pollen and seed dispersal patterns.

Morphological diversity of cassava accessions of the south-central mesoregion of the State of Mato Grosso, Brazil.

Genetic variability of cassava (Manihot esculenta Crantz) in Brazil is wide, being this the result of natural and cultural selection during pre- and post-domestication of the species in different environments. Given the number of species of the genus found in the region (38 of a total of 98 species), the central region of Brazil was defined as the primary center of cassava diversity. Therefore, genetic diversity characterization of cassava accessions is fundamental, both for farmers and for plant breeders, because it allows the organization of genetic resources and better utilization of available genetic diversity. This research aims to assess genetic divergence of cassava accessions from the south-central region of the State of Mato Grosso, based on multi-categorical morphological traits. For this purpose, 38 qualitative and quantitative morphological descriptors were used. Genetic diversity was expressed by the genetic similarity index, with subsequent clustering of accessions by the modified Tocher's procedure and UPGMA. Of 38 descriptors, only growth habit of stem showed no variability. Tocher and UPGMA methods were efficient and corroborated on group composition. Both methods were able to group accessions of different localities in distinct group consistency.

Genetic variability of garlic accessions as revealed by agro-morphological traits evaluated under different environments.

The cultivated garlic (Allium sativum L.) displays a wide phenotypic diversity, which is derived from natural mutations and phenotypic plasticity, due to dependence on soil type, moisture, latitude, altitude and cultural practices, leading to a large number of cultivars. This study aimed to evaluate the genetic variability shown by 63 garlic accessions belonging to Instituto Agronômico de Campinas and the Escola Superior de Agricultura "Luiz de Queiroz" germplasm collections. We evaluated ten quantitative characters in experimental trials conducted under two localities of the State of São Paulo: Monte Alegre do Sul and Piracicaba, during the agricultural year of 2007, in a randomized blocks design with five replications. The Mahalanobis distance was used to measure genetic dissimilarities. The UPGMA method and Tocher's method were used as clustering procedures. Results indicated significant variation among accessions (P < 0.01) for all evaluated characters, except for the percentage of secondary bulb growth in MAS, indicating the existence of genetic variation for bulb production, and germplasm evaluation considering different environments is more reliable for the characterization of the genotypic variability among garlic accessions, since it diminishes the environmental effects in the clustering of genotypes.

TGFß3 / SfaN1 gene variant and the risk factor of nonsyndromic cleft palate only among Indonesian patients.

Non-syndromic cleft palate only (NS CPO) is one of the most common congenital malformations that affect between 1 in 1000 - 2500 live births worldwide. The etiopathogenesis of clefts including NS CPO has been widely studied but is still poorly understood. NS CPO is considered to be a genetically complex, multifactorial disease. Based on several studies, mutations of TGFß3 gene emerged as the strong candidate gene associated with NS CPO. The purpose of this study was to analyze the relationship between the TGFß3 / SfaN1 gene variant and the risk of NS CPO in Indonesian patients. This study was case control design using samples from 31 NS CPO subjects and 35 control subjects. DNA was extracted from venous blood and the segment of TGFß3 gene/ SfaN1 were amplified by using polymerase chain reaction (PCR) technique, then digestion products by SfaN1 restriction enzyme which can detect locus of gene variant / polymorphism from restriction fragment length polymorphisms (RFLP) method were evaluated. The results indicated that the gene variant as substitution of base G into A was identified in TGFß3 gene and the frequency of heterozygous mutant GA genotype was 63,6% in NS CPO subjects and 36,4% in control subjects. The frequency of heterozygous mutant GA genotype was associated with increased risk of NS CPO (odds ratio (OR) = 2,260, 95% CI = 0,592 - 8,625). In conclusion, TGFß3 gene / SfaN1 polymorphism can be considered as the risk factor associated with NS CPO in Indonesian patients.

Genetic diversity in cassava landraces grown on farms in Alta Floresta-MT, Brazil.

Brazil is considered one of the domestication centers of cassava (Manihot esculenta), containing a large part of the biological diversity and traditional knowledge of the species. The aim of the present study was to evaluate the genetic diversity of cassava landraces grown by farmers in the north of Mato Grosso State, Brazil, using inter simple sequence repeat (ISSR) molecular markers. The study was carried out in the municipality of Alta Floresta, MT, on farms located in two rural areas. Seventeen cassava landraces were selected. The DNA was extracted and polymerase chain reaction amplifications were performed using 15 ISSR primers. Genetic similarity estimates were calculated using Jaccard's index and the generated matrix was used for clustering the genotypes by using UPGMA and Tocher's methods. The 15 ISSR primers amplified 120 fragments, revealing 61.67% polymorphism. The polymorphism information content ranged from 0.04 to 0.61, averaging 0.39. The most similar genotypes were AF5 and AF8, whereas the least similar were AF1 and AF16. The UPGMA clustering method formed five groups. Group I included twelve landraces, Group II contained two, and the other groups contained one landrace each. Tocher's method resulted in six groups: 12 landraces clustered in one group, and the other groups each contained one landrace. The ISSR markers proved efficient in revealing genetic diversity among the cassava landraces. The landraces grown by farmers in the two rural areas of Alta Floresta have a great variability and, thus, can be exploited in programs for breeding and preservation of the species.

Traceability from governmental producers of radiopharmaceuticals in measuring (18)F in Brazil.

Since the inception of its proficiency test program to evaluate radionuclide measurement in hospitals and clinics, the National Metrology Laboratory of Ionizing Radiation-LNMRI, that represents Brazilian National Metrology Institute (NMI) for ionizing radiation has expanded its measurement and calibration capability. Requirements from the National Health Surveillance Agency from Ministry of Health (ANVISA), to producers of radiopharmaceuticals provided an opportunity to improve the full traceability chain to the highest level. Fluorodeoxyglucose (FDG-(18)F) is the only radiopharmaceutical simultaneously produced by all Brazilian radiopharmaceutical production centers (RPCs). By running this proficiency test, LNMRI began to provide them with the required traceability. For evaluation, the ratio of RPC to reference value results and ISO/IEC17043:2010 criteria were used. The reference value established as calibration factor on the secondary standard ionization chamber was obtained from three absolute measurements systems, and routinely confirmed in each round of proficiency test by CIEMAT/NIST liquid scintillation counting. The γ-emitting impurities were checked using a High-Purity Germanium (HPGe) detector. The results show that Brazilian RPCs are in accordance with (accuracy within ±10%) the Brazilian standard for evaluation of measurements with radionuclide calibrators (CNEN NN 3.05., 2013). Nevertheless, the RPCs should improve the methodology of uncertainty estimates, essential when using the statistical criteria of ISO/IEC 17043 standard, in addition to improving accuracy to levels consistent with their position in the national traceability chain.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.