Transcription pattern of key molecular chaperones in heat shocked caprine cardiac fibroblasts.

The present study was attempted to unveil the impact of heat stress on transcription pattern of major heat shock response genes in caprine cardiac fibroblasts. Cardiac tissues (n = 6) were collected and primary cardiac cell culture was done. Cultured cardiac fibroblasts were kept in an atmosphere of 5% CO2 and 95% air at 38.5 °C. Cardiac cells achieved 70-75% confluence after 72 hours of incubation. Heat stress was induced on confluent cardiac fibroblasts at 42 °C for 0 (control), 20, 60, 100 and 200 min. Quantitative RT-PCR for ß2m (internal control), HSP60, HSP70, HSP90, and HSP110 was done and their transcription pattern was assessed by Pfaffl method. HSP60, HSP90, and HSP110 transcription did not differ at 20 min, up-regulated (p < 0.05) from 60 to 200 min and registered highest at 200 min of heat exposure. HSP70 transcription was gradually escalated (p < 0.05) time dependently from 20 to 200 min and reached zenith at 200 min of heat exposure. Differential induction in transcription of key molecular chaperones at various durations of heat exposure might reduce cardiac fibroblasts apoptosis and thus could maintain cardiac tissue function during heat stress.

Impact of heat stress on transcriptional abundance of HSP70 in cardiac cells of goat.

The present study was aimed to document the effect of heat stress on the transcriptional abundance of heat shock protein 70 (HSP70) mRNA in cultured cardiac cells of goat. The heart tissues (n = 6) from different goats were used for the culture study. The cardiac cells obtained from different heart tissues were cultured in 24 well cell culture plates and incubated in a humidified CO2 (5%) incubator at 37 °C. The cardiac cells were allowed to become 75-80% confluent after 72 h of incubation. Thereafter, the cardiac cells were subjected to heat exposure at 42 °C (heat exposed) for 0, 20, 60 and 100 min. The cardiac cells exposed to heat stress at 42 °C for 0 min was taken as control. The relative abundance of HSP70 mRNA was gradually up-regulated (p < .05) from 20 to 100 min of heat exposure and reached the zenith (p < .05) at 100 min of heat challenge. The present finding highlights that, HSP70 could possibly act as a cytoprotective factor and may promote cardiac cell survival against the detrimental effect of heat stress. Moreover, this study may serve as the harbinger to conduct further research work on expression kinetics of HSP70 in cardiac cells of goat including other livestock species.

First report of the isolation and phylogenetic characterization of equine Setaria digitata from India based on mitochondrial COI, 12S rDNA, and nuclear ITS2 sequence data.

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.

Histological anomalies and alterations in enzyme activities of the earthworm Glyphidrillus tuberosus exposed to high concentrations of phosphogypsum.

Phosphogypsum (PG) is the major solid waste generated by phosphate fertilizer plants and is used worldwide as sulfur and calcium supplement in agricultural soil. Considering the probability of elevated doses of PG during agricultural applications, this study was carried out to assess its impact on the connective tissue, tissue cholinesterase (ChE) activity, lactate dehydrogenase (LDH) activity, and lipid peroxidation (LPX) level of the tropical earthworm Glyphidrillus tuberosus (Stephenson) found in abundance in the rice fields in India. Consistent loss of connective tissue and sloughing of the intestinal epithelium were observed in worms exposed to 10%, 15%, and 20% concentrations of PG in soil over an incubation period of 30 days. ChE, LDH activities, and the level of LPX indicated highly significant variation (p < 0.01) between pre and postclitellar regions of the worm and concentrations of treatment. ChE activity was higher in postclitellar with respect to preclitellar region; however, the values for LDH activity and LPX level were higher in preclitellar region in comparison to postclitellar region in both PG treated and control worms. It was concluded that PG concentration at and beyond 10% could cause damage to muscle fibers and bring about significant alterations in these enzyme activities in G.tuberosus thus affecting the physiology and ecological functions of these worms.

Quantum Fluctuations in Quasi-One-Dimensional Dipolar Bose-Einstein Condensates.

Recent experiments have revealed that beyond-mean-field corrections are much more relevant in weakly interacting dipolar condensates than in their nondipolar counterparts. We show that in quasi-one-dimensional geometries quantum corrections in dipolar and nondipolar condensates are strikingly different due to the peculiar momentum dependence of the dipolar interactions. The energy correction of the condensate presents not only a modified density dependence, but it may even change from attractive to repulsive at a critical density due to the surprising role played by the transversal directions. The anomalous quantum correction translates into a strongly modified physics for quantum-stabilized droplets and dipolar solitons. Moreover, and for similar reasons, quantum corrections of three-body correlations, and hence of three-body losses, are strongly modified by the dipolar interactions. This intriguing physics can be readily probed in current experiments with magnetic atoms.

Malnutrition-free India: Dream or reality.

In spite of great efforts done by the Government of India to tackle the problem of malnutrition, proportion of population normal on nutrition scale has been stationary; nearly half of them have been a victim of under- and over-nutrition. Over a period, undernutrition has declined but this has been compensated by overnutrition. Hidden hunger is a still an unfinished agendum. The achievement of food grain security at the national level did not percolate down, and households' food insecurity and level of chronic food insecurity are still high. High economic growth rates have failed to improve food security in India. There is a need and scope for organizational, financial, and managerial reforms in this direction. The repercussion of malnutrition is not limited to physical deterioration, but it also affects the psychological level of victims. The legislative, service, and educational approaches should be optimized to fulfill the dream of malnutrition-free India.

Mapping of the QTLs governing grain micronutrients and thousand kernel weight in wheat (Triticum aestivum L.) using high density SNP markers.

Biofortification is gaining importance globally to improve human nutrition through enhancing the micronutrient content, such as vitamin A, iron, and zinc, in staple food crops. The present study aims to identify the chromosomal regions governing the grain iron concentration (GFeC), grain zinc concentration (GZnC), and thousand kernel weight (TKW) using recombinant inbred lines (RILs) in wheat, developed from a cross between HD3086 and HI1500. The experiment was conducted in four different production conditions at Delhi viz., control, drought, heat, and combined heat and drought stress and at Indore under drought stress. Grain iron and zinc content increased under heat and combined stress conditions, while thousand kernel weight decreased. Medium to high heritability with a moderate correlation between grain iron and zinc was observed. Out of 4,106 polymorphic markers between the parents, 3,407 SNP markers were used for linkage map construction which spanned over a length of 14791.18 cm. QTL analysis identified a total of 32 chromosomal regions governing the traits under study, which includes 9, 11, and 12 QTLs for GFeC, GZnC, and TKW, respectively. A QTL hotspot was identified on chromosome 4B which is associated with grain iron, grain zinc, and thousand kernel weight explaining the phenotypic variance of 29.28, 10.98, and 17.53%, respectively. Similarly, common loci were identified on chromosomes 4B and 4D for grain iron, zinc, and thousand kernel weight. In silico analysis of these chromosomal regions identified putative candidate genes that code for proteins such as Inositol 1,3,4-trisphosphate 5/6-kinase, P-loop containing nucleoside triphosphate hydrolase, Pleckstrin homology (PH) domains, Serine-threonine/tyrosine-protein kinase and F-box-like domain superfamily proteins which play role in many important biochemical or physiological process. The identified markers linked to QTLs can be used in MAS once successfully validated.

Virulence and genetic analysis of Puccinia graminis tritici in the Indian sub-continent from 2016 to 2022 and evaluation of wheat varieties for stem rust resistance.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), has re-emerged as one of the major concerns for global wheat production since the evolution of Ug99 and other virulent pathotypes of Pgt from East Africa, Europe, Central Asia, and other regions. Host resistance is the most effective, economic, and eco-friendly approach for managing stem rust. Understanding the virulence nature, genetic diversity, origin, distribution, and evolutionary pattern of Pgt pathotypes over time and space is a prerequisite for effectively managing newly emerging Pgt isolates through host resistance. In the present study, we monitored the occurrence of stem rust of wheat in India and neighboring countries from 2016 to 2022, collected 620 single-pustule isolates of Pgt from six states of India and Nepal, analyzed them on Indian stem rust differentials, and determined their virulence phenotypes and molecular genotypes. The Ug99 type of pathotypes did not occur in India. Pathotypes 11 and 40A were most predominant during these years. Virulence phenotyping of these isolates identified 14 Pgt pathotypes, which were genotyped using 37 Puccinia spp.-specific polymorphic microsatellites, followed by additional phylogenetic analyses using DARwin. These analyses identified three major molecular groups, demonstrating fewer lineages, clonality, and long-distance migration of Pgt isolates in India. Fourteen of the 40 recently released Indian wheat varieties exhibited complete resistance to all 23 Pgt pathotypes at the seedling stage. Twelve Sr genes were postulated in 39 varieties based on their seedling response to Pgt pathotypes. The values of slow rusting parameters i.e. coefficient of infection, area under disease progress curve, and infection rates, assessed at adult plant stage at five geographically different locations during two crop seasons, indicated the slow rusting behavior of several varieties. Six Sr genes (Sr2, Sr57, Sr58, Sr24, Sr31, and Sr38) were identified in 24 wheat varieties using molecular markers closely linked to these genes. These findings will guide future breeding programs toward more effective management of wheat stem rust.

Marfan syndrome: report of two cases with review of literature.

Marfan syndrome is a variable, autosomal dominant disorder of connective tissue whose cardinal features affect the cardiovascular system, eyes and skeleton. The minimal birth incidence is around 1 in 9800. About three quarters of patients have an affected parent; new mutations account for the remainder. The patient's prognosis depends on the severity of cardiovascular complications and is mainly determined by progressive dilation of the aorta. If signs of Marfan syndrome are recognized, it is important to refer to the correct health care professional for further testing to prevent associated complications. If not properly treated, premature death may be caused by the severe cardiovascular and pulmonary complications associated with Marfan syndrome. Therefore, it is important to identify this potentially life-threatening condition in general practice. This article reports two cases with a very typical features of Marfan syndrome.

Bacteriophages Can Make a Difference in Water Quality: Evidence From a Community-Based Study From North India.

Background and objectives Diarrhoea is a preventable and treatable faecal-oral disease. Despite significant inputs from the health and non-health sector in the treatment and prevention of diarrhoea, it remains a significant contributor to under-five-years children mortality and exerts profound effects on their growth and development. Bacteriophage has the potential to prevent diarrhoea. Bacteriophage status may influence the extent of diarrhoea. The objectives of the study were a) to assess the bacteriophage status and quality of water based on the Most Probable Number (MPN) count in the drinking water of under-five years children, and b) to find the association of the extent of diarrhoea with the bacteriophage status and quality of drinking water of under-five-years children in rural areas of Varanasi. Methods This is a community-based cross-sectional study done in the Chiraigaon community development block in a rural area of Varanasi. Water samples were collected and analysed in the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi. Results The result of the study was that bacteriophage is present in 118 samples of water, whereas bacteriophage for Escherichia coli (E. coli), Klebsiella, and Vibrio were present in the drinking water of 81.1%, 53.8%, and 25.8% of under-five-years children, respectively. The water quality was highly satisfactory in 41.7% of samples and unsatisfactory in 15.2% of samples (p<0.004). All samples with highly satisfactory water quality had bacteriophages. Diarrhoea was present in 57.14% of samples without bacteriophage and 24.5% in samples with bacteriophage (p<0.01). The unadjusted odds ratio is 4.09. Interpretation and conclusions The odds of diarrhoea are four times higher in the water sample without bacteriophage than in the water sample in which bacteriophage is present. Bacteriophage study in preventing diarrhoea in children under five and health risk assessment call for focus.

Exploring the missing link between soil total antioxidant capacity and herbicide-induced stress on the earthworm Eudrilus eugeniae (Kinberg).

Herbicide application and residue accumulation in farm soils have deleterious effects on non-target fauna such as earthworms. Although previous studies have documented both positive and deleterious effects of herbicides on soil biota, reports are rare on possible toxicity reduction by raising soil total antioxidant capacity (TAC). Here we review the impact of pretilachlor, a herbicide on the morpho-histology and physiology of the earthworm Eudrilus eugeniae in soil amended with farmyard manure (FYM), poultry manure (PM) and vermimanure (VM), sources of antioxidants over a period of 168 h. The results indicated a significant spike in the TAC of amended soils relative to control. Dermal undulation, setal aberrations, muscular anomaly, protein and lipid peroxidation variations in the activities of lactate dehydrogenase (LDH) and catalase (CAT) were significantly less in animals from amended soils. The maximum percent increase in protein (314%) and reductions in LPX (87%), LDH (87.9%) and CAT (87.3%) were observed in the earthworm from VM-amended soil. The increase in TAC was also maximum (109.9%) in soil amended with VM. A significant negative correlation between soil TAC with the biochemical parameters was observed and confirmed through receiver operator characteristics (ROC) and principal component analysis (PCA). The novelty of the present study includes exploring the missing link between the antioxidant level of organically amended soil and the herbicide-induced oxidative stress in the earthworm E. eugeniae. We concluded that soils with high levels of antioxidants could reduce oxidative damage in E eugeniae due to herbicide toxicity.

Identification of genomic regions of wheat associated with grain Fe and Zn content under drought and heat stress using genome-wide association study.

Wheat is the staple food crop of global importance for its grain nutrient quality. Grain iron and zinc content of the wheat grain is an important quantitatively inherited trait that is influenced by the environmental factors such as drought and heat stress. Phenotypic evaluation of 295 advanced breeding lines from the wheat stress breeding program of IARI was carried out under timely sown irrigated (IR), restricted irrigated, and late-sown conditions at New Delhi during the cropping season of 2020-21, and grain iron (GFeC) and zinc (GZnC) contents were estimated from both control and treatments. A statistically significant increase in GFeC and GZnC was observed under stress conditions compared to that of the control. Genotyping was carried out with the SNPs from the 35K Axiom Breeder's array, and marker-trait association was identified by GWAS analysis. Of the 23 MTAs identified, seven were linked with GFeC and sixteen were linked with GZnC. In silico analysis revealed a few important transcripts involved in various plant metabolism, growth, and development activities such as auxin response factor, root UVB sensitive proteins, potassium transporter, glycosyl transferase, COBRA, and F-box-like domain. The identified MTAs can be used for molecular breeding after validation and also for rapid development of micronutrient-rich varieties of wheat to mitigate hidden hunger.

Polyvinylchloride and polypropylene as adsorbents of the pesticide monocrotophos enhance oxidative stress in Eudrillus eugeniae (Kinberg).

The use of plastics has increased significantly with consequent rise in the generation of wastes. Microplastics (MPs) with particle size <5 mm are produced in natural terrestrial habitats by weathering of the discarded plastic debris and therefore are likely to impact soil biota. Earthworms are the dominant soil fauna which play vital role in soil formation and decomposition of organics. Since these animals are soil feeders, MP particles contaminating soil are likely to enter in to the gut of these animals affecting their physiology. MPs have been shown to be potent adsorbents of various other pollutants such as heavy metals and agrochemicals. This study reports the effects of two MPs, polyvinyl chloride (PVC) and polypropylene (PP) alone and in combination with the pesticide monocrotophos in soil on tissue protein, lipid peroxidation (LPX), activities of lactate dehydrogenase (LDH) and catalase (CAT) of an epigeic earthworm Eudrillus eugeniae over an exposure period of 48h. Results from molecular docking and laboratory experiment confirmed that both the MPs are potent adsorbents of the pesticide and enhanced oxidative stress on the animal with significant reduction in protein, increased LPX level and enzyme activities. PP indicated significantly higher pesticide adsorption relative to PVC.

Treatment seeking in reproductive age women with RTIs/STIs: a community based cross-sectional study.

The socio-cultural and economic contexts in developing countries influence the epidemiology or STIs and helps in making them an important public health priority. This study was carried out to explore the health care seeking pathway of the women suffering from RTls/STls. influence or major socio-demographic variables on treatment seeking pattern. This cross sectional study was carried out comprising or eight hundred reproductive age (15-49 years) women selected following a multistage sampling procedure. The information pertaining to health seeking behaviour were collected from them using a pre-designed pretested interview schedule. Quantum of RTIs/STIs in the study group was estimated and symptomatics were asked specially about time lag between appearance of symptoms and seeking acre; reason for not seeking care; outcome of treatment they have taken if any. Out of total 359 subjects with symptoms of RTls/STls only about one-third (37.3%) had sought treatment. Only a few (4.5%) had sought treatment within 1 month of appearance of symptoms. Considering it to be physiological about half of the subjects (45.3%) did not seek treatment. The treatment seeking pattern was highly associated with the level of education (p = 0.000). Reluctance in seeking treatment, delay in its initiation, prelerence for unqualified practitioners in first consultation and significant association between education and treatment seeking pattern emphasize BCC and service provision for control and prevention of RTls/STls.

Effect of phosphogypsum amendment on soil physico-chemical properties, microbial load and enzyme activities.

Phosphogypsum (PG) is produced as a solid waste from phosphatic fertilizer plants. The waste slurry is disposed off in settling ponds or in heaps. This solid waste is now increasingly being used as a calcium supplement in agriculture. This study reports the effectof PG amendmenton soil physico chemical properties, bacterial and fungal count and activities of soil enzymes such as invertase, cellulase and amylase over an incubation period of 28 days. The highest mean percent carbon loss (55.98%) was recorded in 15% PG amended soil followed by (55.28%) in 10% PG amended soil and the minimum (1.68%) in control soil. The highest number of bacterial colonies (47.4 CFU g(-1) soil), fungal count (17.8 CFU g(-1) soil), highest amylase activity (38.4 microg g(-1) soil hr(-1)) and cellulase activity (38.37 microg g(-1) soil hr(-1)) were recorded in 10% amended soil. Statistically significant difference (p<0.05) has been recorded in the activities of amylase and cellulase over the period of incubation irrespective of amendments. Considering the bacterial and fungal growth and the activities of the three soil enzymes in the control and amended sets, it appears that 10% PG amendment is optimal for microbial growth and soil enzyme activities.

Everything is not on track in management of diarrhoea in under-five children: Evidence from rural area of India.

CONTEXT: Diarrhea is one of the leading causes of death and morbidity in under-five children. Effective management can significantly influence survival of children with diarrhea. OBJECTIVES: This study was carried out to assess extent of diarrhea, its source of treatment, and type of therapy. SETTINGS AND DESIGN: This snapshot study was conducted in rural setting of India. METHODS AND MATERIAL: This study was conducted on 217 under-five children selected through multistage sampling procedure. STATISTICAL ANALYSIS USED: Statistical association of diarrhea with age and gender of children was established by computing Chi-square. RESULTS: Period prevalence (Recall period 2 weeks of visit) of diarrhea in rural under-five children was 35.9% (95%C.I. 29.3-42.2%). Highest prevalence in age group 0-12 months (47.8%) and lowest in 49-59 months (18.2%). Number of episode per child is 1.08. Diarrhea was watery in 69.4%. Quacks were treatment provider for 36.4% cases. In 17.6% episodes, no treatment was taken. Only 9.4% episodes were treated in government facilities. For the treatment of diarrhea, antibiotic was used in 71.8% cases, ORS use was 33.3% cases, and zinc was used 21.8% of diarrhea episodes. CONCLUSIONS: Diarrhea is a significant problem in under-five children from a rural area of India. In spite of existence of National Program for Control of Diarrheal Diseases (NPCDD) since four decades, management of diarrhea is not on track. Focusing on primary care provision to under-five children can help in bringing the management of diarrhea on track.

Ocular cysticercosis at a teaching hospital in Northern India.

BACKGROUND: Ocular cysticercosis (OC) is common in tropical countries. This study aimed to analyze the clinical presentation patterns, management and treatment outcomes of OC cases seen at a teaching hospital in North India. METHODS: This study took place between March 2014 and February 2019. A total of 36 patients with OC were analyzed to determine clinical presentation and outcomes. RESULTS: Of the 36 patients, 13 (36.11%) were male and 23 (63.89%) were female. The most frequently affected age group was 10-29 years (n = 22; 61.11%). All of the patients had unilateral lesions, with involvement of the left eye in 22 (61.11%) and the right in 14 (38.89%). The majority of cases were isolated to the ocular region; however, five (13.89%) demonstrated neural involvement as well. In terms of cyst location, 15 (41.67%) were orbital, 13 (36.11%) were subconjunctival and four each (11.11%) were intraocular or on the eyelid. The most common clinical presentations were subconjunctival masses or proptosis in 13 each (36.11%) and periorbital swelling in 12 (33.33%). Most patients received medical treatment (n - 23; 63.89%), while the others required surgical excision. Recurrence was noted in seven patients (19.44%), of which three underwent surgery while the rest were treated medically. Two patients (5.56%) developed phthisis. CONCLUSIONS: In this study, OC cysts were more often orbital or subconjunctival compared to findings reported from Western countries. In addition, a female preponderance was noted in contrast to previously reported findings. Advanced radioimaging is crucial to ensure early diagnosis and treatment.

Molecular epidemiology, risk factors and hematological evaluation of asymptomatic Theileria annulata infected cattle in Odisha, India.

BACKGROUND: Theileria annulata is a tick-borne apicomplexan parasite that affects bovine and causes severe economic losses. Aims: Our study aimed to determine the molecular prevalence of T. annulata infection in asymptomatic carrier cattle in Odisha, India, to study the association of potential risk factors with theileriosis, and to investigate the effect of the parasite infection on hematological parameters in naturally affected animals. METHODS: A total of 226 cattle blood samples were collected from seven districts of Odisha, India. Molecular diagnoses of tropical theileriosis by polymerase chain reaction (PCR), cloning, sequencing, and phylogenetic analysis of isolated parasites were performed. Potential risk factors were investigated by univariable and multivariable logistic regression statistical analysis. Hematological parameters were compared between positive and negative animals. RESULTS: All animals included in our study were clinically normal, however, 54.86% (124/226) of examined animals were positive by PCR for T. annulata. The multivariable logistic regression showed that contact with other cattle from different herds during grazing (P<0.0001; OR: 12.75; 95% CI: 5.21-31.21), previous history of clinical signs (P=0.002; OR: 3.31; 95% CI: 1.53-6.31), and frequency of a ectoparasiticides application pre year (P<0.0001; OR: 9.22; 95% CI: 3.03-28.09) were the potential risk factors for the occurrence of tropical theileriosis. Nucleotide sequence identity data demonstrated that T. annulata strain (MN818858) Odisha shared homology of 99.6%, 99.49%, and 99.36% with Uttar Pradesh, India (MF346035), Bahrain (AF214797), and Hyderabad, India (MK034702), respectively. CONCLUSION: This is the first study to gain insight into the molecular epidemiology, risk factors, phylogeny, and hematological analysis of asymptomatic T. annulata infected cattle from India.

Setal-epidermal, muscular and enzymatic anomalies induced by certain agrochemicals in the earthworm Eudrilus eugeniae (Kinberg).

Eudrilus eugeniae, the vermicomposing worm, is found in considerable numbers in agricultural fields in India due to their eventual transfer through vermimanure. These worms are very often exposed to pesticides, herbicides, chemical fertilisers and other soil amendments. This paper reports the effects of variable concentrations of urea, phosphogypsum (PG), paper mill sludge (PMS) and two organophosphorus agrochemicals, monocrotophos and glyphosate, on certain morphological, histological and biochemical parameters of E. eugeniae. Results indicated setal anomalies, epidermal lesions, clitellar swelling and constriction of the body. Disintegration of connective tissue, vacuolation of dermis and significant alterations in protein, lipid peroxidation levels and activities of lactate dehydrogenase, acetylcholinesterase and catalase have also been observed in the treated worms. It is proposed that setae, connective tissue, protein and enzymes in E. eugeniae could be useful markers to evaluate toxicity due to the test chemicals.