Genes Hsp70 Research Articles

Dynamic expression of heat-shock and acid-tolerance related genes of Lactobacillus delbrueckii ssp. bulgaricus LBB.B5 in milk

Lactobacillus delbrueckii ssp. bulgaricus is a central species in the production of fermented dairy food. However, from the point of view of starter culture production this species is extremely sensitive to freeze-drying. Therefore, it is essential to understand the physiological processes and adaptation mechanisms of the bacterial cell, especially its response to factors such as heat-, cold- and acid stress. Here we report the results of the dynamic expression monitoring of heat/cold-shock related genes (hsp60 and cspA) and genes putatively contributing to acid-tolerance (ornB, thrB and thrC) in L. delbr. ssp. bulgaricus LBB.B5 grown in milk. These genes were selected as hsp60 and cspA encode a heat-shock and a cold-shock protein, respectively, while ornB encodes an ornithine decarboxylase, related to acid tolerance in this species. Genes thrB and thrC are involved in threonine synthesis and are of particular interest as unlike the majority of amino-acids, the threonine synthesis pathway is conserved in all L. delbr. ssp. bulgaricus strains. Expression levels were monitored by RT-qPCR for 7 hours of fermentation at 42oC and then until the 24th hour under cold storage at 4oC. Samples at 3h were used as control. Two distinct patterns in the expression dynamics of the analysed genes were observed. Genes cspA, ornB, thrB and thrC, followed a pattern with maximal expression at 5h of the fermentation with levels of 11.6, 6.8, 3.9 and 2.4 times the control, respectively. Maximal expression levels coincided with the transition of the culture from exponential to stationary phase at a pH threshold of 5.0. Gene hsp60 showed a different pattern with gradually increasing expression throughout the hole fermentation process, including after cold storage when expression level reached 6.4 times the control. The upregulation of threonine biosynthesis and of a cold-shock protein synthesis with the onset of the stationary phase may suggest that together with the activity of ornithine decarboxylase, they go beyond their function of serving the amino-acid anabolism or managing cold stress, but rather facilitate the transition of the cells to stationary phase and/or their adaptation to acidic conditions. The gradual upregulation of hsp60 on the other hand may reflect the adaptation of the cells to growth at higher temperature (42oC) and subsequent transfer to cold storage.

Cloning and functional analysis of heat shock protein Hsp70 from Sclerotinia sclerotiorum

To clarify the roles of the heat shock protein gene Hsp70 in the sclerotial formation and pathogenicity of Sclerotinia sclerotiorum, we employed reverse transcription PCR (RT-PCR) to clone Hsp70 from S. sclerotiorum and performed sequence analysis. Quantitative real-time PCR (qRT-PCR) was employed to determine the relative expression levels of Hsp70 at different growth stages and under the stress induced by cyclic adenosine monophosphate (cAMP) and low and high temperatures. The thermal stability of Hsp70 was measured. The Agrobacterium-mediated method was employed to construct the Hsp70-silenced strain. The pathogenicity and fungicide resistance of strains were tested by inoculation in detached rapeseed leaves and cultivation in the media containing procymidone and thiophanate-methyl, respectively. The results showed that the cloned Hsp70 had a total length of 1 890 bp and close relationship with the Hsp70 gene of Ciborinia. Hsp70 showcased the highest expression level in sclerotia, which was more than 30 times higher than that in hyphae. The cAMP stress significantly induced the expression of Hsp70. The expression level of Hsp70 showed an increasing-decreasing-increasing trend at 40 ℃ and no significant change at 4 ℃. Recombinant strain with high expression of Hsp70 showed good thermal stability. The Hsp70-silenced transformant did not form sclerotia, with decreased pathogenicity and fungicide resistance. This study reveals that Hsp70 plays an important role in the sclerotial formation and stress resistance of S. sclerotium, providing reference for further in-depth research on the biological roles of Hsp70 in S. sclerotium.

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation. Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced. Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

Genetics and ontogeny are key factors influencing thermal resilience in a culturally and economically important bivalve

Increasing seawater temperatures coupled with more intense and frequent heatwaves pose an increasing threat to marine species. In this study, the New Zealand green-lipped mussel, Perna canaliculus, was used to investigate the effect of genetics and ontogeny on thermal resilience. The culturally and economically significant mussel P. canaliculus (Gmelin, 1971) has been selectively-bred in New Zealand for two decades, making it a unique biological resource to investigate genetic interactions in a temperate bivalve species. Six selectively-bred full sibling families and four different ages, from early juveniles (6, 8, 10 weeks post-fertilisation) to sub-adults (52 weeks post-fertilisation), were used for experimentation. At each age, each family was exposed to a three-hour heat challenge, followed by recovery, and survival assessments. The shell lengths of live and dead juvenile mussels were also measured. Gill tissue samples from sub-adults were collected after the thermal challenge to quantify the 70 kDa heat shock protein gene (hsp70). Results showed that genetics, ontogeny and size influence thermal resilience in P. canaliculus, with LT50 values ranging between 31.3 and 34.4 °C for all studied families and ages. Juveniles showed greater thermotolerance compared to sub-adults, while the largest individuals within each family/age class tended to be more heat sensitive than their siblings. Sub-adults differentially upregulated hsp70 in a pattern that correlated with net family survival following heat challenge, reinforcing the perceived role of inducible HSP70 protein in molluscs. This study provides insights into the complex interactions of age and genotype in determining heat tolerance of a key mussel species. As marine temperatures increase, equally complex selection pressure responses may therefore occur. Future research should focus on transcriptomic and genomic approaches for key species such as P. canaliculus to further understand and predict the effect of genetic variation and ontogeny on their survival in the context of climate change.

Fermentation and fortification of Sargassum linearifolium with multi-strain probiotics improves mucosal barrier status, inflammatory response and resistance to Vibrio harveyi infection in barramundi Lates calcarifer

While aquaculture intensification increases productivity and profitability, it can lead to increased susceptibility of fish to disease outbreaks. As such, the production of aquafeeds with immunomodulatory properties is currently of significant interest to the aquaculture sector with a view of improving on-farm welfare. This study aimed to assess the effectiveness of the brown seaweed Sargassum linearifolium fortified with the probiotics Lactobacillus casei and Saccharomyces cerevisiae through submerged fermentation in improving growth, overall health and the ability to resist Vibrio harveyi infection in barramundi, Lates calcarifer. Two seaweed-based diets, probiotic-fortified S. linearifolium (referred to as PF-SL) and S. linearifolium without probiotic fortification (referred to as SL), were compared with a standard barramundi diet (control). Barramundi juveniles weighing 4.25 ± 0.09 g were fed the test diets three times daily to satiety for 56 days. The results demonstrated a significantly higher growth performance in fish fed the PF-SL diet when compared to the control. However, there were no notable differences in the apparent digestibility coefficient (ADC) of dry matter, protein, and lipid between PF-SL and control. Fish fed the PF-SL diet showed improved mucosal barrier function in both the skin and intestine, as indicated by the increased density of mucous cells. Similarly, fish fed the PF-SL diet exhibited higher glutathione peroxidase and total antioxidant activity than the control. The qRT-PCR revealed significant upregulation in the expression of anti-inflammatory genes (occludin, claudin1 and nrf2) and downregulation in the expression of proinflammatory genes (hsp70 and tnf-α) in fish fed the PF-SL diet. Furthermore, the PF-SL diet resulted in a 27% mortality in a 14-day challenge trial with V. harveyi, which was lower than the mortality rates of the SL (46%) and control (67%). Overall, this study demonstrates that feeding juvenile barramundi with probiotic-fortified brown seaweed S. linearifolium was able to improve growth, immunity, and resistance against Vibrio harveyi infection, suggesting significant potential for application across the aquaculture industry for improved fish welfare.

Occurrence, Sources and Virulence Potential of Arcobacter butzleri in Urban Municipal Stormwater Systems.

A. butzleri is an underappreciated emerging global pathogen, despite growing evidence that it is a major contributor of diarrheal illness. Few studies have investigated the occurrence and public health risks that this organism possesses from waterborne exposure routes including through stormwater use. In this study, we assessed the prevalence, virulence potential, and primary sources of stormwater-isolated A. butzleri in fecally contaminated urban stormwater systems. Based on qPCR, A. butzleri was the most common enteric bacterial pathogen [25%] found in stormwater among a panel of pathogens surveyed, including Shiga-toxin producing Escherichia coli (STEC) [6%], Campylobacter spp. [4%], and Salmonella spp. [<1%]. Concentrations of the bacteria, based on qPCR amplification of the single copy gene hsp60, were as high as 6.2 log10 copies/100 mL, suggesting significant loading of this pathogen in some stormwater systems. Importantly, out of 73 unique stormwater culture isolates, 90% were positive for the putative virulence genes cadF, ciaB, tlyA, cjl349, pldA, and mviN, while 50-75% of isolates also possessed the virulence genes irgA, hecA, and hecB. Occurrence of A. butzleri was most often associated with the human fecal pollution marker HF183 in stormwater samples. These results suggest that A. butzleri may be an important bacterial pathogen in stormwater, warranting further study on the risks it represents to public health during stormwater use.

Combined toxicity of abamectin and carbendazim on enzymatic and transcriptional levels in the soil-earthworm microcosm.

To reveal the toxicological mechanisms of pesticide mixtures on soil organisms, this study concentrated on evaluating enzymatic activity and gene expression changes in the earthworm Eisenia fetida (Savigny 1826). Despite being frequently exposed to multiple pesticides, including the common combination of abamectin (ABA) and carbendazim (CAR), environmental organisms have primarily been studied for the effects of individual pesticides. Acute toxicity results exhibited that the combination of ABA and CAR caused a synergistic impact on E. fetida. The levels of MDA, ROS, T-SOD, and caspase3 demonstrated a significant increase across most individual and combined groups, indicating the induction of oxidative stress and cell death. Additionally, the expression of three genes (hsp70, gst, and crt) exhibited a significant decrease following exposure to individual pesticides and their combinations, pointing toward cellular damage and impaired detoxification function. In contrast, a noteworthy increase in ann expression was observed after exposure to both individual pesticides and their mixtures, suggesting the stimulation of reproductive capacity in E. fetida. The present findings contributed to a more comprehensive understanding of the potential toxicity mechanisms of the ABA and CAR mixture, specifically on oxidative stress, cell death, detoxification dysfunction, and reproductive capacity in earthworms. Collectively, these data offered valuable toxicological insights into the combined effects of pesticides on soil organisms, enhancing our understanding of the underlying risks associated with the coexistence of different pesticides in natural soil environments.

Central inflammatory mechanism of celastrol in intervention of obesity-depression comorbidity in mice from amygdala-dorsal raphe nucleus

This study aims to further elucidate the efficacy targets of celastrol(CEL) intervention in central inflammation in mice with obesity-depression comorbiditiy, based on the differential mRNA expression in the amygdala(AMY) and dorsal raphe nucleus(DRN) after CEL intervention. C57BL/6J mice were randomly divided into a normal diet group(Chow), a obesity-depression comorbidity(COM) group, and low-, medium-, and high-dose CEL groups(CEL-L, CEL-M, CEL-H, 0.5, 1.0, 2.0 mg·kg~(-1)). The Chow group received a normal diet, while the COM group and CEL-L, CEL-M, CEL-H groups received a high-fat diet combined with chronic stress from wet bedding. After 10 weeks of feeding, the mice were orally administered CEL for three weeks. Subsequently, the AMY and DRN of mice in the Chow, COM, and CEL-H groups were subjected to transcriptome analysis, and the intersection of target differentially expressed genes in both nuclei was visualized using a Venn diagram. The intersected genes were then imported into STRING for protein-protein interaction(PPI) analysis, and Gene Ontology(GO) analysis was performed using DAVID to identify the core targets regulated by CEL in the AMY and DRN. Independent samples were subjected to quantitative real-time PCR(qPCR) to validate the intersection genes. The results revealed that the common genes regulated by CEL in the AMY and DRN included chemokine family genes Ccl2, Ccl5, Ccl7, Cxcl10, Cxcr6, and Hsp70 family genes Hspa1a, Hspa1b, as well as Myd88, Il2ra, Irf7, Slc17a8, Drd2, Parp9, and Nampt. GO analysis showed that the top 5 nodes Ccl2, Cxcl10, Myd88, Ccl5, and Irf7 were all involved in immune-inflammation regulation(P&lt;0.01). The qPCR results from independent samples showed that in the AMY, compared with the results in the Chow group, chemokine family genes, Hsp70, Myd88, Il2ra, Irf7, Slc17a8, Parp9, and Nampt were significantly up-regulated in the COM group, with Drd2 showing a decreasing trend; these pathological changes were significantly improved in the CEL-H group compared to the COM group. In the DRN, compared with the results in the Chow group, chemokine family genes, Hsp70, Myd88, Il2ra, Irf7, Parp9, and Nampt were significantly down-regulated, while Slc17a8 was significantly up-regulated in the COM group; compared with those in the COM group, Cxcr6, Irf7, and Drd2 were significantly up-regulated, while Slc17a8 was significantly down-regulated in the CEL-H group. In both the AMY and DRN, the expression of Irf7 by CEL showed both inhibition and activation in a dose-dependent manner(R~2 were 0.709 8 and 0.917 2, respectively). These findings suggest that CEL can effectively improve neuroinflammation by regulating bidirectional expression of the same target proteins, thereby intervening in the immune activation of the AMY and immune suppression of the DRN in COM mice.

Toxicity of Ammonia Stress on the Physiological Homeostasis in the Gills of Litopenaeus vannamei under Seawater and Low-Salinity Conditions.

Ammonia is a major water quality factor influencing the survival and health of shrimp, among which the gill is the main effector organ for ammonia toxicity. In this study, we chose two types of Litopenaeus vannamei that were cultured in 30‱ seawater and domesticated in 3‱ low salinity, respectively, and then separately subjected to ammonia stress for 14 days under seawater and low-salinity conditions, of which the 3‱ low salinity-cultured shrimp were domesticated from the shrimp cultured in 30‱ seawater after 27 days of gradual salinity desalination. In detail, this study included four groups, namely the SC group (ammonia-N 0 mg/L, salinity 30‱), SAN group (ammonia-N 10 mg/L, salinity 30‱), LC group (ammonia-N 0 mg/L, salinity 3‱), and LAN group (ammonia-N 10 mg/L, salinity 3‱). The ammonia stress lasted for 14 days, and then the changes in the morphological structure and physiological function of the gills were explored. The results show that ammonia stress caused the severe contraction of gill filaments and the deformation or even rupture of gill vessels. Biochemical indicators of oxidative stress, including LPO and MDA contents, as well as T-AOC and GST activities, were increased in the SAN and LAN groups, while the activities of CAT and POD and the mRNA expression levels of antioxidant-related genes (nrf2, cat, gpx, hsp70, and trx) were decreased. In addition, the mRNA expression levels of the genes involved in ER stress (ire1 and xbp1), apoptosis (casp-3, casp-9, and jnk), detoxification (gst, ugt, and sult), glucose metabolism (pdh, hk, pk, and ldh), and the tricarboxylic acid cycle (mdh, cs, idh, and odh) were decreased in the SAN and LAN groups; the levels of electron-transport chain-related genes (ndh, cco, and coi), and the bip and sdh genes were decreased in the SAN group but increased in the LAN group; and the level of the ATPase gene was decreased but the cytc gene was increased in the SAN and LAN groups. The mRNA expression levels of osmotic regulation-related genes (nka-β, ca, aqp and clc) were decreased in the SAN group, while the level of the ca gene was increased in the LAN group; the nka-α gene was decreased in both two groups. The results demonstrate that ammonia stress could influence the physiological homeostasis of the shrimp gills, possibly by damaging the tissue morphology, and affecting the redox, ER function, apoptosis, detoxification, energy metabolism, and osmoregulation.

Piroplasmid Infections Among Domestic Dogs in the Mountain City of Rio de Janeiro, Brazil.

The aim of the present study was to analyze the frequency of thepiroplasmids inblood from dogs and ticks recovered from these animals in Teresópolis city, located in the mountain region of Rio de Janeiro state, Brazil. In addition to the clinical and hematological profile. A total of 400 dogs attended in a veterinary clinic in this city between 2020 and 2021 were included. The blood was collected from the dogs, along with ticks and information on these dogs was obtained through a questionnaire applied to the owners. Thin-smear analyses and complete blood counts were performed. All forms characteristic of piroplasmids were measured and classified morphologically. The blood was also subjected to PCR assays based on the genes 18S rRNA and hsp70. In addition, the ixodid ticks were classified morphologically and subjected to PCR for piroplasmids research. The amplified products were sent for gene sequencing. Piroplasmids were detected in 2.3% of the dogs. The variables statistically associated with infections in these animals were hemorrhage/bleeding, jaundice, anisocytosis, activated monocytes and macroplatelets (p ≤ 0.05). Piriform, ring-shaped, oval and aberrant structures were viewed in erythrocytes, neutrophils and monocytes, with lengths greater than and less than 2.5µm. The nine positive samples from these dogs were characterized as due to Rangelia vitalii. However, one sequence from B. vogeli was detected in a single adult specimen of R. sanguineus. Although circulation of two species of piroplasmids potentially infective for domestic dogs has been observed in the mountain city of Rio de Janeiro, infection due to R. vitalii was mostly seen in the dogs of the present study.

Co-exposure to deltamethrin and cyazofamid: variations in enzyme activity and gene transcription in the earthworm (Eisenia fetida).

Pesticide formulations are typically applied as mixtures, and their synergistic effects can increase toxicity to the organisms in the environment. Despite pesticide mixtures being the leading cause of pesticide exposure incidents, little attention has been given to assessing their combined toxicity and interactions. This survey purposed to reveal the cumulative toxic effects of deltamethrin (DEL) and cyazofamid (CYA) on earthworms (Eisenia fetida) by examining multiple endpoints. Our findings revealed that the LC50 values of DEL for E. fetida, following 7- and 14-day exposures, ranged from 887.7 (728-1095) to 1552 (1226-2298) mg kg-1, while those of CYA ranged from 316.8 (246.2-489.4) to 483.2 (326.1-1202) mg kg-1. The combinations of DEL and CYA induced synergistic influences on the organisms. The contents of Cu/Zn-SOD and CarE showed significant variations when exposed to DEL, CYA, and their combinations compared to the untreated group. Furthermore, the mixture administration resulted in more pronounced alterations in the expression of five genes (hsp70, tctp, gst, mt, and crt) associated with cellular stress, carcinogenesis, detoxification, and endoplasmic reticulum compared to single exposures. In conclusion, our comprehensive findings provided detailed insights into the cumulative toxic effects of chemical mixtures across miscellaneous endpoints and concentration ranges. These results underscored the importance of considering mixture administration during ecological risk evaluations of chemicals.

Geographic origin shapes the adaptive divergences of Rotaria rotatoria (Rotifera, Bdelloidea) to thermal stress: Insights from ecology and transcriptomics.

Global warming has raised concerns regarding the potential impact on aquatic biosafety and health. To illuminate the adaptive mechanisms of bdelloid rotifers in response to global warming, the ecological and transcriptomic characteristics of two strains (HX and ZJ) of Rotaria rotatoria were investigated at 25°C and 35°C. Our results showed an obvious genetic divergence between the two geographic populations. Thermal stress significantly reduced the average lifespan of R. rotatoria in both strains, but increased the offspring production in the ZJ strain. Furthermore, the expression levels of genes Hsp70 were significantly upregulated in the HX strain, while GSTo1 and Cu/Zn-SOD were on the contrary. In the ZJ strain, the expression levels of genes Hsp70, CAT2, and GSTo1 were upregulated under thermal stress. Conversely, a significant decrease in the expression level of the Mn-SOD gene was observed in the ZJ strain under thermal stress. Transcriptomic profiling analysis revealed a total of 105 and 5288 differentially expressed genes (DEGs) in the HX and ZJ strains under thermal stress, respectively. The PCA results showed clear differences in gene expression pattern between HX and ZJ strains under thermal stress. Interestingly, compared to the HX strain, numerous downregulated DEGs in the ZJ strain were enriched into pathways related to metabolism under thermal stress, suggesting that rotifers from the ZJ strain prioritize resource allocation to reproduction by suppressing costly metabolic processes. This finding is consistent with the life table results. This study provides new insights into the adaptive evolution of aquatic animals in the context of global climate change.

Comparative Toxicity of TiO2 and Sn-Doped TiO2 Nanoparticles in Zebrafish After Acute and Chronic Exposure.

This study was conducted to assess the toxicological potential of synthesized pure and Sn-doped TiO2 NPs (Sn-TiO2 NPs) in zebrafish after acute and chronic exposure. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were synthesized and characterized using X-ray diffraction, Scanning Electron Microscope, diffuse reflectance spectra, dynamic light scattering, and zeta potential analyses. The pure TiO2 NPs, 4%, and 8% Sn-TiO2 NPs were spherical with average sizes of about 40, 28, and 21 nm, respectively, indicating significant size reduction of TiO2 NPs following Sn doping. According to our results, the LC50-96h increased in the order of 8% Sn-TiO2 NPs (45 mg L-1) < 4% Sn-TiO2 NPs (80.14 mg L-1) < pure TiO2 NPs (105.47 mg L-1), respectively. Exposure of fish to Sn-TiO2 NPs after 30 days resulted in more severe histopathological alterations in gills, liver, intestine, and kidneys than pure TiO2 NPs. Furthermore, Sn-doping significantly elevated malondialdehyde levels and micronuclei frequency, indicating increased oxidative stress and genotoxicity. Expression analysis revealed altered expression of various genes, including upregulation of pro-apoptotic Bax gene and downregulation of anti-apoptotic Bcl-2 gene, suggesting potential induction of apoptosis in response to Sn-doped NPs. Additionally, antioxidant genes (Gpx, Sod, Cat, and Ucp-2) and stress response gene (Hsp70) showed altered expression, suggesting complex cellular responses to mitigate the toxic effects. Overall, this study highlights the concerning impact of Sn-doping on the toxicity of TiO2 NPs in zebrafish and emphasizes the need for further research to elucidate the exact mechanisms underlying this enhanced toxicity.

Analysis of stress-related gene expression in lobster seeds (Panulirus sp.) post formalin stress test

Indonesia is the potential international-scale lobster economic activity due to the abundant supply of tropical lobster seeds Panulirus sp., however the lobster cultivation sector have still resulted in problems of low survival during the rearing period. This study aimed to analyze the expression levels of the HSP70 gene related to stress response in lobster seeds exposed to formalin dose of LC70 for 1 h. Seeds were obtained from catching lobsters at Ujung Genteng Beach, Sukabumi City, West Java Province, with an average weight of 0.0254±0.004 g and a length of 2.3314±0.1549 cm. The observation parameters tested were the gene expression and survival rates of the post-soaking lobsters. The results showed that formalin immersion at a dose of 220 ppm on juvenile-stage lobster seeds resulted in the death of 70% of the population and the ability to express the stress gene HSP70 at different fitness levels were different with the highest expression level found in fit condition lobster seeds was 2.949±0.420.

Molecular Typing of Leishmania spp. Causing Tegumentary Leishmaniasis in Northeastern Italy, 2014-2020.

Tegumentary leishmaniasis (TL) is endemic but neglected in southern Europe. Therefore, this study aimed to analyze the Leishmania strains causing TL cases in northeastern Italy, where an upsurge of TL cases has been observed in the last decade. Sections from 109 formalin-fixed and paraffin-embedded (FFPE) biopsies of skin and mucosal tissues were collected from TL cases in the selected area. Two DNA targets were amplified and sequenced: the ribosomal internal transcribed spacer 1 (ITS1) and the heat-shock protein 70 gene (hsp70). An in silico analysis was also performed on 149 genomes belonging to the Leishmania donovani complex. A total of 88 out of 109 (80.7%) samples from 83 TL cases were successfully typed by ITS1 and/or hsp70. ITS1 analysis identified L. infantum in 67 cases (91.8%), while L. major (n = 4, 5.5%) and L. tropica (n = 2, 2.7%) were detected in the remaining cases that were categorized as imported. Further, the hsp70 typing of 75 autochthonous cases showed the presence of eight distinct sequence variants belonging to the Leishmania donovani complex, with high genetic variability when compared to known L. infantum populations. In conclusion, our findings show that peculiar L. infantum variants are emerging in the novel focus on TL in northeastern Italy.

Validation of Oxford nanopore sequencing for improved New World Leishmania species identification via analysis of 70-kDA heat shock protein

BackgroundLeishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp.MethodsA dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform.ResultsThe results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection.ConclusionsThis study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases.Graphical abstract

Huoxin pill protects verapamil-induced zebrafish heart failure through inhibition of oxidative stress-triggered inflammation and apoptosis

Heart failure (HF) is a major and growing public health concern. Although advances in medical and surgical therapies have been achieved over the last decades, there is still no firmly evidence-based treatment with many traditional Chinese medicines (TCMs) for HF. Huoxin Pill (HXP), a TCM, has been widely used to treat patients with coronary heart disease and angina pectoris. However, the underlying molecular mechanism is poorly understood. In this study, using a verapamil-induced zebrafish HF model, we validated the efficacy and revealed the underlying mechanism of HXP in the treatment of HF. Zebrafish embryos were pretreated with different concentrations of HXP followed by verapamil administration, and we found that HXP significantly improved cardiac function in HF zebrafish, such as by effectively alleviating venous congestion and increasing heart rates. Mechanistically, HXP evidently inhibited verapamil-induced ROS and H2O2 production and upregulated CAT activity in HF zebrafish. Moreover, transgenic lines Tg(mpx:EGFP) and Tg(nfkb:EGFP) were administered for inflammation evaluation, and we found that neutrophil infiltration in HF zebrafish hearts and the activated NF-kB level could be reduced by HXP. Furthermore, HXP significantly downregulated the level of cell apoptosis in HF zebrafish hearts, as assessed by AO staining. Molecularly, RT‒qPCR results showed that pretreatment with HXP upregulated antioxidant-related genes such as gpx-1a and gss and downregulated the expression of the stress-related gene hsp70, proinflammatory genes such as tnf-α, il-6 and lck, and apoptosis-related indicators such as apaf1, puma and caspase9. In conclusion, HXP exerts a protective effect on verapamil-induced zebrafish HF through inhibition of oxidative stress-triggered inflammation and apoptosis.

Gene Expression of HSP70 Gene when Ascorbic Acid is Added to Broiler Diets Meat and Its Relationship to Meat Quality

This experiment was conducted in the animal production field of the Agricultural Research Station/College of Agriculture and Marshlands/Dhi Qar University for the period from (30/11/2022 to 4/1/2023) for the purpose of studying the gene expression of the HSP 70 heat shock gene when ascorbic acid was added to the diets provided to birds. And the extent of its effect on the quality of meat. In this study, 150 non-sexed Ross 308 birds, one day old, were used in this study. The study included four treatments. Incubation was randomly distributed among the experimental treatments, with three replicates of 12 replicated birds. The treatments were divided on the basis of adding ascorbic acid to the diet without adding it, as well as shedding heat by 2-3 m for a period of 8 hours / day at the normal temperature rate and without heat shedding. The division of treatments began immediately after the second week of the age of the birds. The gene expression level of the HSP70 gene was measured at the age of 21 and 35days, and the sensory evaluation test of the thigh and chest segments was measured at the age of 21. and 35 days The results of the study showed the: 1-The high rate of gene expression of the HSP70 gene in the liver tissue in the T2 treatment exposed to a high temperature negatively affected the quality of meat through conducting a sensory evaluation of the thigh and breast segments, where the evaluation rate decreased in this treatment, which indicates that the relationship is inverse between the rate of the gene HSP70 and average sensory evaluation of the thigh and thoracicseg ments. 2-Adding ascorbic acid at a rate of 1 gm / kg feed to the T4 treatment that was also exposed to high temperature erecorded an increase in the sensory evaluation rate of the thigh and breast segments, which led to an improvement in flavor, taste and tenderness in poultry meat, which indicates that the addition of ascorbic acid has a role in relieving stress Thus decreased, the rate of gene expression of the HSP70 gene.

Effects of High-Lipid Dietary Protein Ratio on Growth, Antioxidant Parameters, Histological Structure, and Expression of Antioxidant- and Immune-Related Genes of Hybrid Grouper

The hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus) is a new species of grouper crossed from giant grouper (E. lanceolatus) as the male parent and brown-marbled grouper (E. fuscoguttatus) as the female parent. We hypothesized that optimal levels of dietary protein may benefit liver function. High-lipid diets are energetic feeds that conserve protein and reduce costs, and are a hot topic in aquaculture today. Therefore, the objective of the research is to investigated the effects of dietary protein level in high-lipid diets on serum and liver biochemistry, liver histology, and liver immune and antioxidant indexes and gene mRNA expression of the juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus). Six iso-lipidic (161 g/kg) diets were formulated containing graded levels of protein (510 as control, 480,450, 420, 390 and 360 g/kg). Each treatment consisted of three replicates and 30 fish (6.70 ± 0.02 g) in one replicate. After an 8-week feeding experiment, the results indicated the following: (1) With the decreasing of dietary protein level, the specific growth rate (SGR) of groupers increased gradually and then decreased; SGRs of the 390 and 360 g/kg groups were significantly lower than other groups (p &lt; 0.05). (2) In terms of serum and liver, the activity of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD), and the total antioxidant capacity (T-AOC) content, and the activity of immune enzymes such as lysozyme (LYS) and immunoglobulin (IgM) was significantly increased under the appropriate protein level. (3) Based on liver histology, we know that high or low dietary protein levels cause liver damage. (4) Dietary protein levels can significantly affect the mRNA expression levels of an anti-inflammatory factor gene (tgfβ), pro-inflammatory factor genes (il6, il8), heat shock proteins, and antioxidant and immune genes (hsp70 and hsp90, gpx, nrf2, keap1). It is concluded that the appropriate protein level can promote the growth performance of groupers, improve antioxidant activity and immune enzyme activity in serum and liver, and enhance the expression of immune genes.

Parental exposure to environmental cadmium causes developmental defects in the rare minnow (Gobiocypris rarus) embryos: The association between ROS-mediated oxidative stress and developmental dysfunction

Cadmium is widely distributed in aquaculture systems. The exposure of parental fish species to cadmium is known to retard of growth and cause death in offspring. However, the potential mechanisms responsible for how parental cadmium exposure effects embryonic development yet to be elucidated. Herein, we investigated the effects of parental cadmium exposure on embryonic developmental abnormalities in rare minnow. Our results demonstrated that significantly increased rates of malformation were recorded in the parental cadmium exposure groups. The most significant impairments and morphological changes were egg condensation and spine curvature malformations. Parental cadmium exposure significantly increased the cadmium content of rare minnow embryos, thus increasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Further investigation found that parental cadmium exposure reduced superoxide dismutase (SOD) activity and significantly up-regulated the expression of antioxidative genes (gpx-1, cat and mn-sod) and detoxifying metallothionein (MT) in rare minnow embryos. In addition, stress-related genes (hsp70, ccyp1α and jnk) were significantly upregulated while developmental-related genes (wnt8α, vezf1 and mstn) were significantly downregulated in rare minnow embryos. Our analysis revealed that parental cadmium exposure in rare minnow caused developmental abnormalities in embryos via the accumulation of cadmium content which induced oxidative stress and developmental dysfunction. Collectively, our analysis suggested that environmental cadmium may pose a severe threat to the continuation of certain fish species.