A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Effect of PEAR1, PTGS1 gene polymorphisms on the recurrence of aspirin-treated patients with ischemic stroke in the Han population of China: A 4-year follow-up study.

Platelet endothelial aggregation receptor 1 (PEAR1) and prostaglandin endoperoxide synthase 1 (PTGS1) polymorphisms can affect laboratory aspirin resistance. However, the impact of genetic polymorphisms on the recurrence of ischemic stroke (IS) patients treated with aspirin is not fully understood. This study aimed to examine the relationship between gene polymorphisms of PEAR1 and PTGS1 and IS recurrence in patients treated with aspirin. Peripheral blood samples were collected from 174 patients with nonrecurrent IS and 34 with recurrent IS after aspirin treatment. Follow-up was performed on all patients. PEAR1 rs12041331 and PTGS1 rs10306114 polymorphisms were determined using the PCR fluorescence probe method. And the correlations of them with the clinical characteristics were examined by multivariable logistic regression analysis. The distribution frequencies of PEAR1 rs12041331 and PTGS1 rs10306114 genotypes were in Hardy-Weinberg equilibrium, and there was no significant difference in the distribution of PEAR1 rs12041331 polymorphism. Compared to the nonrecurrent group, the AA genotype of the PTGS1 polymorphism was more frequent in the recurrent group (59.77% vs 35.29%, P = .003), and the A allele also showed a higher frequency than the G allele in the recurrent group (P = .001). Multivariable logistic regression analysis showed that smoking (OR = 5.228, 95% CI: 1.938-14.102, P = .001), coronary heart disease (OR = 4.754, 95% CI: 1.498-15.089, P = .008), and the polymorphism at PTGS1(A>G) AA/AG + GG (OR = 2.955, 95% CI: 1.320-6.616, P = .008) were independently associated with IS recurrence in Chinese patients. Our findings suggested that PTGS rs10306114 polymorphisms should receive more attention in the use of aspirin in patients with IS.

Exposure to nicotine regulates prostaglandin E2 secretion and autophagy of granulosa cells to retard follicular maturation in mammals.

Exposure to nicotine by cigarette smoking have shown strongly defectives on the physiological function of ovaries, which in turn leads to disorders of fertility in women. However, the potential molecular mechanisms remain to be elucidated. In this study, we notably found that nicotine was likely to specifically raise the expression of histone deacetylase 3 (HDAC3) to promote the apoptosis and autophagy of granulosa cells (GCs) and block follicular maturation. Moreover, prostaglandin E2 (PGE2) inhibited the apoptosis of GCs and facilitated follicular maturation, and nicotine appeared to inhibit PGE2 secretion by freezing the expression of cyclooxygenase 1 (COX1), which was the rate-limiting and essential enzyme for PGE2 synthesis. Epigenetically, the nicotine was observed to diminish the histone H3 lysine 9 acetylation (H3K9ac) level and compact the chromatin accessibility in -1776/-1499 bp region of COX1 by evoking the expression of HDAC3, with the deactivated Cas9-HDAC3/sgRNA system. Mechanistically, the COX1 protein was found to pick up and degrade the autophagy related protein beclin 1 (BECN1) to control the autophagy of GCs. These results provided a potential new molecular therapy to recover the damage of female fertility induced by nicotine from cigarette smoking.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Application of Nested-qPCR-High Resolution Melting (HRM) Technology on Strongyloides stercoralis Isolates from Iran.

PURPOSE: Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S. stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S. stercoralis from this country by focusing on evolutionary genetics analysis. METHODS: Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. RESULTS: The melting temperature (Tm) values of the isolates were 77.9 °C-78.3 °C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2 °C to 78.3 °C, while the isolates from Hormozgan Province showed Tm values of 77.9 °C, 78.0 °C, and 78.1 °C. The phylogenetic tree illustrated that the sequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = - 0.27). CONCLUSION: The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree.

Integrating network pharmacology and renal metabonomics to reveal the protective mechanism of resveratrol on gouty nephropathy.

Resveratrol (Res) has been demonstrated to have beneficial effects on gouty nephropathy (GN). However, the mechanisms of Res on GN remain unclear. This study aimed to investigate the mechanisms of Res on GN. In this study, network pharmacology technology was used to predict the Res targets in the prevention and treatment of GN. Renal metabonomics was used to identify differential metabolites in kidney tissue of GN model rats. Finally, molecular docking technology was used to verify the binding ability of Res to key targets. Metabonomics analysis showed that 24 potentially important metabolites were involved in the prevention and treatment of GN with Res. After exposure to Res, metabolite levels normalized. The network pharmacology analysis showed that 24 key targets were involved in the prevention and treatment of GN disease. According to the metabolite-gene network diagram, we identified two core genes, PTGS1 and PTGS2, and found that both were involved in the arachidonic acid metabolism pathway. Molecular docking further verified the affinity of Res binding to PTGS1 and PTGS2. In conclusion, the mechanism of Res against GN may be the regulation of arachidonic acid metabolism through the regulation of PTGS 1 and PTGS 2.

Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

BACKGROUND: Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation. OBJECTIVES: The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors. METHODS: The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies. RESULTS: Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen. CONCLUSION: We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants.

[Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province].

OBJECTIVE: To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. METHODS: Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. RESULTS: The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). CONCLUSIONS: Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.

15-lipoxygenase and cyclooxygenase expression profile and their related modulators in COVID-19 infection.

BACKGROUND: The role of the lipoxygenase (LOX) and cyclooxygenase (COX) enzymes in maintaining cellular homeostasis and regulating immune responses promoted us in this study to analyze the pattern of changes in 15-lipoxygenase and cyclooxygenase isoforms and their related cytokines in SARS-CoV-2 infection. METHODS: 15-LOX-1, 15-LOX-2, COX-1 and COX-2 gene expression levels were determined using qRT-PCR in nasopharynx specimens from patients with severe [N = 40] and non-severe [N = 40] confirmed SARS-CoV-2 infections and healthy controls. Circulating levels of lL-6, lL-10, PGE2, and IFN-γ were measured in patients and healthy controls using ELISA assay. The associations between the measured variables and the patient's clinic-pathological characteristics were assessed for all groups. RESULTS: The expression level of 15-LOX-1 was elevated significantly in male patients with severe infection; although female patients showed a different expression profile. 15-LOX-2 expression level was considerably increased in male patients with severe infection; while changes in its expression remained inconclusive in female patients. The relationship between 15-LOX expression and the male gender was prominent. Both COX isoforms expression showed elevation in male and female patients that were correlated with disease severity. The simultaneous increase in lL-6, PGE2 and IFN-γ levels also decrease in lL-10 in patients with severe infection indicating the possible regulatory network related to the COX and 15-LOX enzymes in the output of the SARS-CoV-2 infection. CONCLUSION: The results of this study determined the pattern of possible changes in key enzymes of prostaglandin and eicosanoids synthesis pathway and their mediators, which can be helpful in mapping the SARS-CoV-2 pathogenicity and pharmaceutical approaches.

The PTGS1 (rs1330344) CC Genotype Contributes to Susceptibility to Kawasaki Disease in Southern Chinese Children.

Kawasaki disease (KD) is an acute systemic vascular disease complicated by coronary artery injury. Although polymorphisms in prostaglandin-endoperoxide synthase 1 (PTGS1) are being increasingly explored in cardiovascular diseases, little is known regarding the connection between PTGS1 polymorphisms and KD risk. We evaluated 834 KD patients and 1474 healthy controls to explore the relationship between PTGS1 polymorphisms (rs1330344 and rs5788) and KD risk. Our results showed that the rs1330344 CC genotype was significantly associated with KD risk and coronary artery injury in children with KD. In combined analysis, individuals with 1-2 unfavorable genotypes had an increased risk of KD, compared with those with no risk genotype. Stratified analysis indicated that the rs1330344 CC genotype is strongly associated with increased risk of KD in children aged ≤60 months and females. Moreover, carrying 1-2 of these SNP genotypes had a higher risk of KD than those who harbored none of them in children ≤60 months of age and females; the risk of coronary artery dilatations/small aneurysms and medium/giant aneurysms was also significantly increased in KD patients. In summary, the PTGS1 rs1330344 CC genotype is associated with increased susceptibility to KD, which may contribute to KD pathogenesis and serve as a genetic biomarker.

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control.

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

The specific deletion of cyclooxygenase-1 in megakaryocytes/platelets reduces intestinal polyposis in ApcMin/+ mice.

Clinical and experimental evidence sustain the role of cyclooxygenase (COX)-1 in intestinal tumorigenesis. However, the cell type expressing the enzyme involved and molecular mechanism(s) have not been clarified yet. We aimed to elucidate the role of platelet COX-1 (the target of low-dose aspirin in humans) in intestinal tumorigenesis of ApcMin/+ mice, considered a clinically relevant model. To realize this objective, we generated an ApcMin/+ mouse with a specific deletion of Ptgs1(COX-1 gene name) in megakaryocytes/platelets (ApcMin/+;pPtgs1-/-mice) characterized by profound inhibition of thromboxane(TX)A2 biosynthesis ex vivo (serum TXB2; by 99%) and in vivo [urinary 2,3-dinor-TXB2(TXM), by 79%]. ApcMin/+ mice with the deletion of platelet COX-1 showed a significantly reduced number (67%) and size (32%) of tumors in the small intestine. The intestinal adenomas of these mice had decreased proliferative index associated with reduced COX-2 expression and systemic prostaglandin(PG)E2 biosynthesis (urinary PGEM) vs. ApcMin/+mice. Extravasated platelets were detected in the intestine of ApcMin/+mice. Thus, we explored their contribution to COX-2 induction in fibroblasts, considered the primary polyp cell type expressing the protein. In the coculture of human platelets and myofibroblasts, platelet-derived TXA2 was involved in the induction of COX-2-dependent PGE2 in myofibroblasts since it was prevented by the selective inhibition of platelet COX-1 by aspirin or by a specific antagonist of TXA2 receptors. In conclusion, our results support the platelet hypothesis of intestinal tumorigenesis and provide experimental evidence that selective inhibition of platelet COX-1 can mitigate early events of intestinal tumorigenesis by restraining COX-2 induction.

Morphological Identification and Phylogenetic Analysis of Laelapin Mite Species (Acari: Mesostigmata: Laelapidae) from China.

Laelapinae mites are involved in transmission of microbial diseases between wildlife and humans, with an impact on public health. In this study, 5 mite members in the subfamily Laelapinae (laelapin mites; LM) were morphologically identified by light microscopy, and the phylogenetic relationship of LM was analyzed in combination with the sequence information of part of the LM cytochrome oxidase subunit I (cox1) gene. The morphological identification revealed that 5 mites belonged to the genera Laelaps and Haemolaelaps, respectively. Sequence analysis showed that the ratio of non-synonymous mutation rate to synonymous mutation rate of LM was less than 1, indicating that the LM cox1 gene had undergone purifying selection. Phylogenetic analysis showed that the Laelapinae is a monophyletic group. The genera Haemolaelaps and Hyperlaelaps did not separated into distinct clades but clustered together with species of the genus Laelaps. Our morphological and molecular analyses to describe the phylogenetic relationships among different genera and species of Laelapinae provide a reference for the improvement and revision of the LM taxonomy system.

The influence of a nanoparticle gel loaded with siRNA-cyclooxygenase on flexor tendon healing: an in vivo animal study.

We investigated the influence of cyclooxygenase (COX)-1 and COX-2 siRNAs delivered through a nanoparticle-gel system on the strength of flexor tendon repairs. Sixteen flexor digitorum profundus (FDP) tendons of chicken toes were transected, repaired and wrapped with gels to evaluate gel adherence. We found that the gel adhered to the tendon surface firmly. Next, 56 tendons were used in a first set of in vivo experiments to compare the therapeutic effects of different doses of COX siRNAs. Another 15 tendons were added in a second set to further assess the effects of a dosage of 12 µg. After 4 weeks, the mean strength of the repaired tendons increased most notably in the toes treated with 12 µg COX siRNAs, and the number of samples with low strength (<35 N) was significantly smaller than in the group without molecular treatment. We conclude that COX-1 and COX-2 siRNAs delivered through a nanoparticle-gel system increased the healing strength of the repaired tendons.

Association between Vitamin Intake and Chronic Kidney Disease According to a Variant Located Upstream of the PTGS1 Gene: A Cross-Sectional Analysis of Shika Study.

Chronic kidney disease (CKD) patients have been advised to take vitamins; however, the effects have been controversial. The individual differences in developing CKD might involve genetic variants of inflammation, including variant rs883484 located upstream of the prostaglandin-endoperoxide synthase 1 (PTGS1) gene. We aimed to identify whether the 12 dietary vitamin intake interacts with genotypes of the rs883484 on developing CKD. The population-based, cross-sectional study had 684 Japanese participants (≥40 years old). The study used a validated, brief, self-administered diet history questionnaire to estimate the intake of the dietary vitamins. CKD was defined as estimated glomerular filtration < 60 mL/min/1.73 m2. The study participants had an average age of 62.1 ± 10.8 years with 15.4% minor homozygotes of rs883484, and 114 subjects had CKD. In the fully adjusted model, the higher intake of vitamins, namely niacin (odds ratio (OR) = 0.74, 95% confidence interval (CI): 0.57−0.96, p = 0.024), α-tocopherol (OR = 0.49, 95% CI: 0.26−0.95, p = 0.034), and vitamin C (OR = 0.97, 95% CI: 0.95−1.00, p = 0.037), was independently associated with lower CKD tendency in the minor homozygotes of rs883484. The results suggested the importance of dietary vitamin intake in the prevention of CKD in middle-aged to older-aged Japanese with minor homozygous of rs883484 gene variant.

Infection of the Asian gray shrew Crocidura attenuata (Insectivora: Soricidae) with Sarcocystis attenuati n. sp. (Apicomplexa: Sarcocystidae) in China.

BACKGROUND: Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host. METHODS: Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed. RESULTS: Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3-4.5 µm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9-16.7 × 9.2-10.6 µm with a prepatent period of 10-11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaia tana (i.e., 97.6-98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati. CONCLUSIONS: Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystis attenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystis scandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future.

COX-1, COX-2 and CYP2C19 variations may be related to cardiovascular events due to acetylsalicylic acid resistance.

BACKGROUND: In some stent implanted patients, cardiovascular events (CE) may occur. Acetylsalicylic acid (ASA) is routinely administered to these patients in order to prevent the occurrence of CE. CE may be related to gene variations which cause ASA resistance (AR). Therefore, it was aimed to investigate the relationship between COX-1, COX-2, CYP2C9 and CYP2C19 variations with CE due to AR. MATERIALS AND RESULTS: Seventy-four stent implanted patients, using 100 mg of ASA per day during five years were enrolled into the study. Following stent implantation, thirty-eight patients who had a CE within five years due to AR and 36 patients without CE were enrolled in patient and control group, respectively. AR was confirmed by platelet aggregation testing. After DNA isolation from blood; COX-1, COX-2, CYP2C19 and CYP2C9 variations were investigated with real-time polymerase chain reaction. At the end of this study, heterozygous genotype of COX-1 was found statistically high in patients whereas heterozygous genotype of CYP2C19*17 was found statistically high in controls. The presence of C and G allele in COX-1 and COX-2 were found statistically high in patients, respectively. The presence of T allele in CYP2C19*17 was found statistically high in controls. Heterozygous genotype of COX-1 variation was found statistically high in patients who have AR. Additionally heterozygous genotype of CYP2C19*17 was found statistically high in patients who have low thrombosis risk. CONCLUSIONS: COX-1 and COX-2 gene mutations may increase the risk of CE due to AR whereas CYP2C19*17 may have a protective effect in this process.

Musa sp. Leaves Extract Ameliorates the Hepato-Renal Toxicities Induced by Cadmium in Mice.

Heavy metals intoxication causes several health problems that necessitate finding new protective and therapeutic approaches. This study aimed to evaluate the impact of Musa sp. leaves extract (MLE) on hepato-renal toxicities induced by cadmium (Cd) in male mice. The phytochemical screening, metal chelating activity (MCA), and the median lethal dose (LD50) of MLE were determined. Fifty CD-1 male mice were used and intraperitoneally (i.p.) injected with MLE (1000 to 5000 mg/kg b.wt) for MLE LD50 determination. Another 50 mice were used for evaluating the effect of MLE on Cd toxicity. Blood samples were collected for hematological, liver, and kidney functions assessments. Liver tissue homogenates were used for determination of oxidant/antioxidant parameters. Liver and kidney tissues were harvested for histopathological and molecular investigations. MLE showed potent in vitro antioxidant activities. The MCA and LD50 of the MLE were 75 µg/mL and 3000 mg/kg b.wt, respectively. MLE showed beneficial therapeutic activity against hepato-renal toxicities in Cd-intoxicated mice, evidenced by improving the hematological, biochemical, histopathological, and molecular alterations.

Comparative subcellular localization of NRF2 and KEAP1 during the hepatocellular carcinoma development in vivo.

The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.