B1 Gene Research Articles

Molecular and Seroprevalence of Toxoplasmosis in Goats' Blood and Milk in Iraq.

Toxoplasmosis is one of the most acute parasitic and zoonotic infections, which causes severe economic losses in animals due to abortion and reproductive problems worldwide. Therefore, this study was conducted in Baghdad province to detect the prevalence of Toxoplasmosis in blood and milk samples of 384 adult female goats using the serological indirect-enzyme-linked immunosorbent assay (iELISA) and the molecular polymerase chain reaction (PCR) test. The positive iELISA results were 20.57% in sera and 5.99% in milk samples. Regarding cross-classification results, the iELISA results revealed that 5.73% of goats were positive by testing both sera and milk samples, 14.32% and 0.26% were positive for testing sera and milk only, respectively, and 79.69% were negative by testing sera and milk. Targeting the B1 gene, the total positive results of the PCR assay showed that 13.92% and 30.43% of blood and milk samples, respectively, were positive at 546bp. Concerning cross-classification results, the total positive goats by testing of both sera and milk was 8.86%, while 5.06% of goats were positive only for testing of blood, and 86.08% were negative for testing of both samples. At the same time, no positive PCR results were detected in milk samples. In conclusion, there is a wide prevalence and incidence of Toxoplasmosis among goats in study areas. Furthermore, studies are essential to detect the parasite in different ages and sexes of goats and other domestic and wild animals using ELISA as a reliable, automated, and rapid test and PCR as a highly confirmative test.

Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

Molecular detection and dense granule antigen 6 genotyping of feline Toxoplasma gondii in Phayao, Thailand.

Globally, toxoplasmosis is an important zoonotic parasite infection of many warm-blooded animals (including humans). Toxoplasma gondii oocysts are widespread, and their contamination can be primarily attributed to the members of the Felidae family. This study aimed to estimate the prevalence and determine the dense granule antigen 6 (GRA6) genotype of T. gondii among domestic cats in the Phayao Province, Thailand. A total of 124 fecal samples were collected from owned cats in the Muang district, Phayao, Thailand, from January to December 2020. Fecal samples were tested for the presence of T. gondii DNA using targeted B1 gene polymerase chain reaction (PCR) amplification, and positive samples were subsequently analyzed for their T. gondii genotype through PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the GRA6 gene. Among the 124 samples, 46 (37.1%) were tested positive for T. gondii. Only 10 positive DNA samples were successfully amplified for the GRA6 marker. Subsequent PCR-RFLP and sequence analyses indicated that all T. gondii isolates from cats in Phayao belonged to GRA6 genotype I. Data revealed that toxoplasmosis is remarkably distributed among (studied) domestic cats in Phayao, Thailand. Moreover, the virulent GRA6 allele was found to be circulated among domestic cats in this area. However, no significant correlation was observed between infection rates and different risk factors, which indicated that pet cats of any age, gender, or breed have similar risks of being infected with T. gondii. Our results further suggested that infective oocysts of T. gondii are widely distributed and that environmental contamination with these oocysts will introduce more risks of disease transmission to humans and other animals.

Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimer's dementias.

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences – Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.

Investigation of Toxoplasma infection in zoo animals using multispecies ELISA and GRA7 nested PCR

BackgroundToxoplasma is an obligate intracellular protozoan that causes an important zoonotic disease with a worldwide distribution. Felids are the definitive hosts of this parasite, while virtually all warm-blooded animals, including birds, serve as intermediate hosts. Four ring-tailed lemurs (Lemur catta) in the Taipei Zoo died of acute Toxoplasma infection in June 2019. Since then, Toxoplasma has occasionally been identified in this Zoo during necropsy of dead animals and PCR of animal blood samples. Therefore, a general survey of Toxoplasma infection in animals in the Zoo seems to be needed.Methods and resultsAn indirect multispecies ELISA was used for the first time to screen for Toxoplasma infection in 326 serum samples collected from 75 species of animals. The infection rate of Toxoplasma was 27% (88/326). A commercial latex agglutination (LAT) assay was used to re-examine the samples with doubtful and uncertain ELISA results (151 samples from 42 species). The infection rate increased to 36.2% (118/326), and the indirect multispecies ELISA appeared to be applicable to 31 of 75 species animals included in this study. Nested PCR assays targeting the dense granule protein 7 (GRA7) gene and B1 gene were also used to detect Toxoplasma in DNA samples extracted from 10 liver or blood specimens from 8 animals. GRA7 gene fragments were amplified from 8 samples from 7 animals, while B1 gene fragments were amplified from only 4 samples from 4 animals. From the B1 nested PCR and the sequence data of GRA7 fragments amplified from infectious specimens, the animals in the Zoo were speculated to have been infected by at least three different Toxoplasma variants.ConclusionsAccording to the serological investigation, we speculated that over one-third (36.2%) of animals in Taipei Zoo presented the infection of Toxoplasma, and the indirect multispecies ELISA we used can be applied to detect Toxoplasma infection in 31 animal species included in this study. Sequence analysis revealed that at least three Toxoplasma variants were infecting the animals of Taipei Zoo.

Using Elisa and nested PCR for detection of the toxoplasmosis in milk and the influence of infection and some factors on milk composition in the Iraqi local and Shami goats

This study concluded detection of Toxoplasma gondii in milk, immunologically by using Elisa and nested PCR)nPCR (based on B1 gene, also to investigate the effect of toxoplasmosis, parity, breed and flock on some milk composition in the Iraqi local and Shami goats in the middle of Iraq. A total of 80 milk samples of the lactating goats were collected. Results of this study showed the prevalence of Toxoplasmosis was 21.25% and 28.75% by Elisa and nPCR respectively without significant differences. The sensitivity of Elisa was a low (30.43%) whereas the specificity was a high (82.45%). The degree of agreement estimated by Kappa coefficient revealed a slight agreement (0.14) between two methods. The results indicated that goats infected with toxoplasmosis have significantly (P&lt;0.05) higher in protein and lactose percentage whereas the effect was not significant for each of fat, SNF and pH. On the other hand, the effect of parity was significant (P&lt;0.05) on fat, protein, lactose, solid nonfat and non-effect on PH. As well as effect of breed and flock was significant (P&lt;0.05) on protein, lactose and solid nonfat without significant effect on fat and PH.

Identification of vacuolar phosphate influx transporters in Brassica napus.

Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homoeostasis in Arabidopsis thaliana and Oryza sativa under fluctuating external Pi supply, but the identity and involvement of VPTs in cellular Pi homoeostasis in Brassica napus is poorly understood. Here, we identified two vacuolar Pi influx transporters B. napus, BnA09PHT5;1b and BnCnPHT5;1b, and uncovered their necessity for cellular Pi homoeostasis through functional analysis. Both Brassica proteins are homologs of Arabidopsis AtPHT5;1 with a similar sequence, structure, tonoplast localization, and VPT activity. Brassica pht5;1b double mutants had smaller shoots and larger shoot cellular Pi concentrations than wild-type B. napus, which contrasts with a previous study of the Arabidopsis pht5;1 mutant, suggesting that PHT5;1-VPTs play different roles in cellular Pi homoeostasis in seedlings of B. napus and A. thaliana. Disruption of BnPHT5;1b genes also caused Pi toxicity in floral organs, reduced seed yield and impacted seed traits, consistent with the proposed role of AtPHT5;1 in floral Pi homoeostasis in Arabidopsis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved roles of BnPHT5;1bs in cellular Pi homoeostasis in this plant species.

Molecular identification and genotyping of Toxoplasma gondii isolated from sheep and cattle in northern Iran.

Toxoplasmosis, a foodborne disease, in human occurs commonly after the ingestion of tissue cysts via the raw and/or undercooked meat of different infected intermediate hosts such as sheep and cattle. The current study aimed to detect the genetic structure of Toxoplasma gondii isolated from various organs of sheep and cattle in the north of Iran. Conventional PCR was carried out by B1 and REP-529 genes of T. gondii. Nested and RFLP-PCR were performed for all positive samples using SAG2 and GRA6 genetic markers. Amplicons from second round of nested-PCR were sequenced and analyzed with NCBI database. Among of 179 examined samples, 38(21.20%) were positive. The highest of positive cases were found in kidney (28.60%). PCR-RFLP of SAG2 and GRA6 genes demonstrated the alleles of clonal type III in the all of isolates. Sequence analysis of the amplicons revealed the alleles of clonal type III and atypical isolates (Tg-67, Tg-100 and Tg-106). Phylogenetic analyses showed separate clade for the atypical isolates from others in the present study and the reference strains clades. In conclusion, the genetic characterization of T. gondii isolates from sheep and cattle showed high genetic diversity compared with standard type I, II and III genotypes. These results support the hypothesis of the existence of polymorphic and overlapping strains within livestock in Iran. It also suggested the necessity of increased genotyping and sampling efforts to accurately estimate T. gondii intra specific genetic diversity.

Neuroprotective effects of ergosterol against TNF-α-induced HT-22 hippocampal cell injury

Neuroinflammation is a brain pathology that involves the expression of high levels of pro-inflammatory mediators, including tumor necrosis factor-alpha (TNF-α). An excessive TNF-α expression could result in neuronal cell death and subsequently lead to neurodegeneration. Auricularia polytricha (AP; an edible mushroom) has been reported as a rich source of ergosterol with several medicinal benefits. The current study reports on the neuroprotective effects of AP extracts and ergosterol against the TNF-α-induced HT-22 hippocampal cell injury. The hexane extract of AP (APH) demonstrated a neuroprotective effect against the TNF-α-induced HT-22 cell toxicity, taking place through the activation of the antioxidant pathway. Ergosterol, a major component of APH, could attenuate the toxicity of TNF-α on HT-22 cells, by increasing the expression of a major antioxidant enzyme (superoxide dismutase-1) and by facilitating the scavenging of reactive oxygen species through antioxidant signaling. Moreover, an antibody array was performed to screen the possible molecular targets of ergosterol in HT-22 cells exposed to TNF-α. Based on the antibody array, the phospho-Akt was activated in the presence of ergosterol, and this finding was also supported by Western blotting analysis. Furthermore, ergosterol inhibited the transcriptional expressions of the glutamate ionotropic receptor N-methyl-D-aspartate (NMDA) type subunit 2B gene (Grin2b) through an early growth response-1 (EGR-1) overexpression in TNF-α-treated HT-22 cells. Our findings suggest that a novel therapeutic effect of AP and ergosterol against neuroinflammation, that it is mediated by an NMDA gene modulation occurring through the overexpression of the EGR-1 transcription factor.

High prevalence of Toxoplasma gondii in Nigerian wild rats by molecular detection

Toxoplasmosis has been reported in Nigeria using several diagnostic tools with high prevalence in humans and some food animals. Rodents have been recognised as vital intermediate hosts of Toxoplasma gondii. However, there is paucity of information on the occurrence of T. gondii in wild rats found in Nigeria. This study aimed at molecular detection of T. gondii in Zyzomys pedunculatus and to evaluate its involvement in the epidemiology of toxoplasmosis in Nigeria. A total of 84 rats were sampled across three states of the North Central Nigeria, and DNA was extracted from the brain, lungs, kidney and intestine of the rats for the detection of T. gondii DNA by nested PCR to amplify the multicopy B1 gene. Sixty-four of the 84 samples (76.2%) were positive for T. gondii out of which 5 samples were sequenced and had an identity score of between 97.73% and 99.35% with the reference B1 gene of T. gondii in GenBank. This study suggests Nigerian wild rats may be an important intermediate hosts of T. gondii and may play a role in the epidemiology and maintenance of T. gondii circulation in Nigeria.

Terrestrial runoff influences the transport and contamination levels of Toxoplasma gondii in marine organisms

There is a growing concern regarding the potential adverse impact of Toxoplasma gondii contamination of the marine environment on marine wildlife and public health. Terrestrial runoff is a significant route for dissemination of T. gondii oocysts from land to sea. Yet, the influence of terrestrial runoff on T. gondii prevalence in marine animals in China is largely unknown. To address this concern, we examined the presence of T. gondii in marine oysters Crassostrea spp., rockfish Sebastes schlegelii (S. schlegelii), fat greenling fish Hexagrammos otakii (H. otakii), and Asian paddle crab Charybdis japonica (C. japonica) using a PCR assay targeting T. gondii B1 gene. A total of 1920 samples were randomly collected, in Jan-Dec 2020, from terrestrial runoff areas (TRA, TRB, and TRC) and non-terrestrial runoff area (Grape bay) in Weihai, China. T. gondii prevalence in TRB and TRC was 6.04 % and 5.83 %, respectively, which was higher than 2.29 % detected in the non-terrestrial runoff area. The highest prevalence was detected in Crassostrea spp., and a correlation was observed between T. gondii prevalence and weight of Crassostrea spp. The temperature, but not precipitation, significantly correlated with T. gondii prevalence. Understanding the fate of T. gondii delivered to oceans by terrestrial runoff is critical for predicting future disease risks for marine wildlife and humans.

Detection of Giardia duodenalis and Toxoplasma gondii in soil and water samples in the Quindío River basin, Colombia

Two zoonotic protozoan pathogens, Giardia duodenalis and Toxoplasma gondii, are important causes of waterborne infections in the Quindío region in Colombia. No previous data exist on how contamination occurs at the source for drinking water consumed by the human population in this region. Our aim was to describe the frequency of G. duodenalis and T. gondii DNA in 11 sampling points during a five-month period in water and adjacent soil at the Quindío River basin (Andean region in the central western part of Colombia). The study employed nested PCR for T. gondii, using the B1 gene as the amplification target, and single-round PCR for G. duodenalis assemblage A and assemblage B, amplifying the gdh gene, followed by DNA sequencing. In 50 soil samples, 28% (14/50) were positive for T. gondii. For G. duodenalis, distribution was in equal parts for assemblage A (8%; 4/50) and assemblage B (8%, 4/50). Genotyping of T. gondii sequences showed two soil samples with type I strain, another two samples of soil with type III strain, but most samples were of unidentified strains. In water samples, T. gondii was detected in 9.1% (5/55), G. duodenalis assemblage A in 34.5% (19/55), and G. duodenalis assemblage B in 12.7% (7/55). T. gondii DNA positivity was associated with lower soil temperature (p = 0.0239). Presence of G. duodenalis and T. gondii was evidenced in soil and water samples in the Quindío River basin, indicating soil as the potential source of contamination for the river that it is destined for human consumption. Monitoring these protozoa in drinking water is necessary to prevent public health risks in human populations.

Transgenic human HOXB1-9 directs anterior-posterior axial skeleton pattern in Hoxb1-9 deficient mice

The cervical and anterior thoracic regions of mammals generally exhibit similar vertebral numbers and identities along the anterior-posterior axis. The position of the forelimbs along the axial skeleton is also generally conserved. In contrast, the number of lumbar and sacral vertebrae and pelvic position exhibit more variation, correlating with posture and locomotion. The molecular mechanisms that lead to these conserved and variable axial skeletal patterns between species are not fully understood. Here we use a human HOXB1-9 transgene to complement a HoxB1-9 deficiency in the mouse. In TgHOXB1-9 mice, human HOXB1, B2, B3, and B4 (HOXB1-4) genes were expressed in mouse embryos in patterns similar to mouse Hoxb1-4 genes. Human transgene expression rescued the cervical and anterior thoracic vertebral patterning defects of HoxB1-9Δ/Δ mice. In addition, the posterior shift in forelimb position of HoxB1-9Δ/Δ mice was rescued by the transgene. Interestingly, the position of the lumbar-sacral transition in both TgHOXB1-9; HoxB1-9Δ/Δ and TgHOXB1-9; HoxB1-9+/+ mice was altered from six lumbar and four sacral vertebrae found in wild-type controls to five lumbar and five sacral vertebrae. The change in the position of the lumbar-sacral transition consequently altered the position of the pelvis. In contrast to the conserved expression of human HOXB1-4 genes in TgHOXB1-9 mouse embryos, the anterior border of human HOXB9 expression in the neural tube and paraxial mesoderm was shifted posteriorly by 2–3 somites compared to the anterior boundary of endogenous Hoxb9 expression. These findings suggest that conservation and variation in Hoxb/HOXB expression contributes to conserved and species-specific vertebral pattern and limb position.

Molecular Incidence of Toxoplasma gondii, Neospora caninum, and Cryptosporidium parvum in Dissimilar Kinds of Raw and Pasteurized Milk Samples of Naturally Infected Animal Species

Milk of naturally infected animal species is considered an essential reservoir of Toxoplasma gondii, Neospora caninum, and Cryptosporidium parvum. An extant survey was performed to measure the T. gondii, N. caninum, and C. parvum incidence and periodic frequency amongst milk samples. One-thousand and one-hundred and sixty raw and 400 pasteurized milk samples were collected. Milk samples were used for DNA extraction. The nested-polymerase chain reaction was applied to diagnose B1, NP, and hsp70 genes of the T. gondii, N. caninum, and C. parvum, respectively. The total incidence of T. gondii, N. caninum, and C. parvum in examined milk samples were 2.69%, 10.51%, and 2.94%, respectively. Co-contamination of examined milk samples with all three protozoa was 0.38%. T. gondii had the highest distribution amongst raw sheep (5.00%) milk samples, while N. caninum, and C. parvum had the highest distribution in raw buffalo (23.68% and 6.31%, respectively) milk samples. Marked periodicity with the higher distribution of all protozoa among raw milk samples collected during autumn and summer seasons were found (P &lt;0.05). Raw milk of animal species and pasteurized cow milk may be reservoirs of N. caninum, T. gondii, and C. parvum. Accurate monitoring of raw and pasteurized milk, particularly in autumn and summer seasons, can diminish the risk of human and animal toxoplasmosis, cryptosporidiosis, and neosporosis. However, further investigations are essential to understand the exact epidemiological aspects and other risk factors associated with the N. caninum, C. parvum, and T. gondii presence in raw and pasteurized milk.

Molecular occurrence and risk factors for Toxoplasma gondii infection in equids in Jilin, China

Toxoplasma gondii, one of the important zoonotic parasites, has been detected in lots of hosts including humans, with a widespread prevalence. The products of equids, such as meat and milk, have been closely related to humans’ life. As the intermediate hosts, little is known about equids toxoplasmosis in Jilin province. Therefore, the present study aimed to evaluate the occurrence and risk factors of Toxoplasma gondii infections in equids from Jilin, northeastern China. In this study, a total of 245 blood samples of equids (192 horses, 25 donkeys and 28 mules) were collected from six localities in Jilin Province from March 2018 to August 2020 and detected by PCR. The occurrence rate of T. gondii B1 gene was analyzed using multivariable logistic regression to evaluate risk factors associated with the positive rates in equids. Among 245equids, T. gondii molecular occurrence was 9.0% (22/245). The highest positive rate was observed in equids from Dongfeng (16.3%) followed by Taonan (10.0%), Wangqing (8.3%), Antu (8.0%), Tonghua (8.0%) and Shulan (2.3%). Statistical analysis revealed that farming model and region may be two main risk factors. Data analysis indicated that the positive rate in captive farm (3.2%, 95% CI: 0.0–6.7%) was significantly lower than those in cage-free farm (P < 0.05), and the region of Shulan was protective factor (OR: 0.063, 95% CI: 0.007–0.559).The results of our study alert people to be aware that the present of equids T. gondii infection in this region, and contribute to a prevention and treatment program for toxoplasmosis in Jilin, China.

Molecular detection of toxoplasma gondii DNA in goats (Capra hircus), sheep (Ovis aries), and donkey (Equus asinus) milk using PCR in East Azerbaijan province, Iran

Toxoplasmosis, a zoonotic infection that is significant for public health (immunocompromised patients, pregnant women) and veterinary medicine (economic losses in the herd), is caused by an intracellular protozoan parasite belonging to the phylum Apicomplexa called Toxoplasma gondii. Consumption of unpasteurized milk and contaminated undercooked meat is a significant source for humans. The present study aimed to determine Toxoplasma gondii DNA in sheep, goats and donkeys Milk kept in East Azerbaijan province using the PCR method based on the B1 gene. For this purpose, 100 milk samples, including 45 sheep, 45 goats and 10 donkeys, were collected from different regions of northwestern Iran using direct milking and then transferred to the Food and Aquatic Health Laboratory under refrigerated conditions. The results showed that out of 100 milk samples examined, 16 samples (16%) were contaminated, and Toxoplasma gondii DNA was detected in 5 (11.11%) sheep, 9 (20%) goats and 2 (20%) donkeys milk specimen, respectively. These findings indicated that Toxoplasma gondii contaminated the raw milk, a human infection source.

Bioinformatics Analysis of Hub Genes Involved in Alcohol-Related Hemifacial Microsomia Pathogenesis.

Alcohol is a recognized teratogen, and alcohol exposure increases the risk for hemifacial microsomia (HFM) of the fetus during maternal pregnancy. The present study aimed to explore potential mechanisms and verify hub genes of HFM associated with alcohol by bioinformatics methods. First, HFM and alcohol pathogenic genes were obtained. Thereafter, a protein-protein interactional (PPI) network was constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and molecular complex detection were performed by Metascape. Finally, we used the cytoHubba plugin to screen the hub genes. A total of 43 HFM genes and 50 optimal alcohol candidate genes were selected. The PPI networks for pathogenic genes contained 93 nodes and 503 edges. Functional enrichment analysis largely focused on tissue formation and development. Two modules were identified from the PPI network, and 10 hub genes were screened out. The genes most relevant to alcohol-induced HFM pathogenesis included CTNNB1, TP53, MYC, HDAC1, and SOX2. This study identified some significant hub genes, pathways, and modules of HFM related to alcohol by bioinformatics analyses. Our results suggest that the CTNNB1, TP53, MYC, HDAC1, and SOX B1 gene subfamilies may have played a major role in alcohol-induced HFM.

Snail acetylation by autophagy-derived acetyl-coenzyme A promotes invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells.

BackgroundAutophagy is elevated in metastatic tumors and is often associated with active epithelial‐to‐mesenchymal transition (EMT). However, the extent to which EMT is dependent on autophagy is largely unknown. This study aimed to identify the mechanisms by which autophagy facilitates EMT.MethodsWe employed a liquid chromatography‐based metabolomic approach with kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1) gene co‐mutated (KL) cells that represent an autophagy/EMT‐coactivated invasive lung cancer subtype for the identification of metabolites linked to autophagy‐driven EMT activation. Molecular mechanisms of autophagy‐driven EMT activation were further investigated by quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blotting analysis, immunoprecipitation, immunofluorescence staining, and metabolite assays. The effects of chemical and genetic perturbations on autophagic flux were assessed by two orthogonal approaches: microtubule‐associated protein 1A/1B‐light chain 3 (LC3) turnover analysis by Western blotting and monomeric red fluorescent protein‐green fluorescent protein (mRFP‐GFP)‐LC3 tandem fluorescent protein quenching assay. Transcription factor EB (TFEB) activity was measured by coordinated lysosomal expression and regulation (CLEAR) motif‐driven luciferase reporter assay. Experimental metastasis (tail vein injection) mouse models were used to evaluate the impact of calcium/calmodulin‐dependent protein kinase kinase 2 (CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVIS luciferase imaging system.ResultsWe found that autophagy in KL cancer cells increased acetyl‐coenzyme A (acetyl‐CoA), which facilitated the acetylation and stabilization of the EMT‐inducing transcription factor Snail. The autophagy/acetyl‐CoA/acetyl‐Snail axis was further validated in tumor tissues and in autophagy‐activated pancreatic cancer cells. TFEB acetylation in KL cancer cells sustained pro‐metastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)‐independent manner. Pharmacological inhibition of this axis via CAMKK2 inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KL cancer cells in vivo.ConclusionsThis study demonstrates that autophagy‐derived acetyl‐CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRAS‐LKB1 co‐mutated lung cancer cells and that inhibition of the autophagy/acetyl‐CoA/acetyl‐Snail axis using CAMKK2 or ACLY inhibitors could be a potential therapeutic strategy to suppress metastasis of KL lung cancer.

Assessment of SARS-CoV-2-Infected Patients and Their Clinical Outcomes During the Third Wave in India: A Single-Center Observational Study.

BackgroundThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that emerged from the Chinese mainland has spread throughout the world affecting the normal lives of the people. Both developed and developing nations have been equally affected and coronavirus disease-19 (COVID-19) resulted in the death of millions of people worldwide. The virus is undergoing mutations and is evolving into variants that are responsible for wave after wave. This study was carried out to assess the clinical outcomes of people infected with the novel virus during the third wave of the COVID-19 pandemic in India.MethodsThe study was carried out between November 2021 and January 2022 and included 100 consecutive patients attending the hospital attached to the BLDE (Deemed to be University) Shri B.M. Patil Medical College, Bijapur, Karnataka, South India. All patients included in the study returned a positive report in a real-time polymerase chain reaction (RT-PCR). The patient details collected included age, sex, cycle threshold (Ct) values for envelope (E)/nucleocapsid (N), and Orf1b (open reading frame 1b) genes, hospitalization status, vaccine status, C-reactive protein (CRP), D-dimer, interleukin-6 (IL-6), and final clinical outcome. The data were entered into Microsoft Office Excel sheets, and statistical inferences were drawn using SPSS 24 (IBM Corp., Armonk, NY).ResultsOf the 100 patients included in the study, only 14 (14%) patients were vaccinated. The patient's mean age was 34.22±17.50. Among the vaccinated patients, the majority had taken COVISHIELD™ (85.71%) compared to COVAXIN® (14.29%). Only 14% of patients were symptomatic, and the mean Ct values among all the patients were 29.92±3.74 (E gene/N gene) and 27.6±4.78 (Orf1B gene). Eight (8%) patients were hospitalized, and all the patients recovered from the infection. Among the hospitalized patients, six (75%) were vaccinated. The mean age of the hospitalized patients was 43.8±14.25 years. The mean CRP, D-dimer, and IL-6 concentrations among the hospitalized patients were noted to be 22.375±16.58 mg/L, 654.325±577.24 ng/mL, and 5.075±2.15 ng/mL, respectively.ConclusionThe study results demonstrate that despite unvaccinated status, most patients in the third wave had only suffered from asymptomatic infection. Moreover, people who developed a clinical infection and those who required hospitalization had an uneventful recovery irrespective of their vaccination status.