Marine macroalgae (Enteromorpha intestinalis) for improving the growth performance, meat quality traits, and serum biochemical parameters in broilers.

Objective: The purpose of the current study was to examine the effectiveness of green macroalgae as a novel, natural feed additive for broilers that have a greater concentration of active ingredients. Materials and Methods: Four experimental groups of 180-day-old male broiler chicks (Cobb-500) were randomly assigned, with three replicates in each group: the control group [T0, maintained only with basal diet] and three treatment groups supplemented with macroalgae for 35 days along with basal diet [T1 = 0.05% (w/w); T2 = 0.1% (w/w); T3 = 0.2% (w/w) macroalgae]. Live weight, carcass weight, and organs' weight were noted at the conclusion of the experiment. The meat quality was examined using the muscles of the thighs and breasts, and blood serum was obtained for biochemical assessment. Results: The results revealed that dietary supplementation of green macroalgae (0.1%) in broiler rations significantly (p < 0.05) improved the growth performance compared to other treated groups and controls. With increasing weight, it enhanced meat quality traits assessed by increased water holding capacity, ultimate pH, redness and yellowness, and decreased lightness of muscles in the thighs and breasts. Both the levels of serum cholesterol and abdominal fat decreased and showed no unwholesome effects on liver and kidney functions. Conclusion: For the production of safe and high-quality poultry meat, marine green macroalgae (Enteromorpha intestinalis) could be used as a potential feed additive. It enhanced the growth rate in broilers and improved meat quality and serum biochemical parameters for supplying healthy meat in the human food chain.

Role of Clove and Tulsi on broiler health and meat production.

The role of Clove (Cv) and Tulsi (Ts) supplementation on broiler growth performance and gut health as an alternative to antibiotic growth promoters has already been established. Therefore, the objectives of this study were to investigate the role of Cv and Ts on the serum biochemical profile and meat quality traits in broilers. A total of sixty (60) one-day-old commercial broiler chicks were randomly allotted into four homogenous groups (15 birds per group). They were then fed Cv powder and Ts extract from day (d) 8 to d 28 with drinking water i.e. zero level of Cv or Ts (T0), 0.5% Cv + 2% Ts (T1), 1.0% Cv + 3% Ts (T2), 1.5% Cv + 4% Ts (T3). Blood and meat samples were collected on d 14, 21, and 28 to analyze the serum-biochemical profile and meat quality. Supplementation of Cv and Ts improved serum-biochemical profile by reducing total cholesterol and low-density lipoprotein levels in broilers. However, they did not affect the serum protein levels while the 0.5-1% Cv and 2-3% Ts supplemented groups had higher glucose levels on d 21. Production of breast and thigh meat increased with increased size, and density of myofibers while fed 0.5-1% Cv and 2-3% Ts. On the contrary, 1.5% Cv powder and 4% Ts extract supplementation improved thigh meat color, breast and thigh meat pH as well as the water-holding capacity. The current study findings suggest that Cv and Ts can be used in combination to improve broiler health, production, and meat quality.

Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation.

In an epoch of the growing risk of antibiotic resistance, there is a dire need to establish an effective novel feeding practice for broiler nutrition as an alternative to antibiotics. Hence, the aim of the current study was to evaluate the impact of clove powder and tulsi extract on the growth performance, gut morphologic and morphometric indices, and cecal microbial status of broiler, as an alternative to antibiotic growth promoters (AGPs). Sixty day-old chicks of Cobb-500 strain were randomly divided into 4 groups, each having 15 birds. Chicks of the control group (T0) were fed commercial broiler feed with no additional supplementation. The treatment groups were offered commercial broiler feed and received clove powder and tulsi extract with drinking water at the rate of 0.5% + 2% (T1), 1% + 3% (T2), and 1.5% + 4% (T3), respectively. Results showed a nonlinear relationship with the dosage of clove and tulsi. All the growth parameters substantially (P < 0.05) improved in T2 while T1 and T3 showed no significant improvement compared to T0. The final body weight was significantly (P < 0.05) higher in T2. Giblet and offal weights showed no noticeable differences except in the intestine and heart where intestine weight markedly (P < 0.05) decreased in T3 and heart weight significantly (P < 0.05) increased in T1 and T2. Clove and tulsi supplementation substantially improved the villus height and villus surface area of the small intestine in T2 while the large intestine remained mostly unaffected by the treatment. Cecal microbial status significantly improved in all the treatment groups having increased (P < 0.05) Lactobacillus spp. count and decreased (P < 0.05) E. coli count compared to T0. Based on the aforementioned findings, it can be concluded that the combination of clove and tulsi can improve the growth performance and gut health of broilers which is largely dose-dependent and might be supplied as a potential alternative to AGPs.

C-to-U RNA Editing: A Site Directed RNA Editing Tool for Restoration of Genetic Code.

The restoration of genetic code by editing mutated genes is a potential method for the treatment of genetic diseases/disorders. Genetic disorders are caused by the point mutations of thymine (T) to cytidine (C) or guanosine (G) to adenine (A), for which gene editing (editing of mutated genes) is a promising therapeutic technique. In C-to-Uridine (U) RNA editing, it converts the base C-to-U in RNA molecules and leads to nonsynonymous changes when occurring in coding regions; however, for G-to-A mutations, A-to-I editing occurs. Editing of C-to-U is not as physiologically common as that of A-to-I editing. Although hundreds to thousands of coding sites have been found to be C-to-U edited or editable in humans, the biological significance of this phenomenon remains elusive. In this review, we have tried to provide detailed information on physiological and artificial approaches for C-to-U RNA editing.

The hazardous effects of formalin and alcoholic fixative in mice: A public health perspective study.

Formalin is used for different purposes due to its preservation capability. But continuous exposure to formalin may result various health related issues leading to cancer and death. A new alcohol-based fixative, EMA (ethanol, methanol and acetic acid = 3:1:1) could be a safer option in this regard. To compare the health hazards of formalin and EMA, a total 15 adult male mice were randomly distributed into three groups- exposure groups (formalin and EMA) and control group. The mice were subjected to natural inhalation exposure of the fixatives followed by behavioral depression test (forced swimming test), histopathology and serum biochemical tests. Our results showed that the hazardous effects of formalin were remarkably higher than that of EMA. Formalin exposed group showed severe depression (P < 0.001) in the forced swimming test compared to EMA and control groups. Histopathologically, diffuse lymphocytic infiltrations around the lung alveoli and bronchioles and severe inflammation with accumulation of reactive cells in the cerebral cortex were detected in the formalin exposed group, whereas little or no inflammation with fibrinous exudates in the bronchioles was reported in the EMA group and no inflammatory cells were detected in the cerebral tissues. The serum biochemical analysis of the inflammatory mediators (Interleukin-6 and C-reactive protein) revealed that both significantly (P < 0.001) increased in the formalin exposed group compared to EMA and control groups. These results confer that EMA could be a safer option to reduce health hazards of formalin in the workplace environment.

Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues.

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.

Biochemical and morphological attributes of broiler kidney in response to dietary glucocorticoid, dexamethasone.

Glucocorticoids (GCs) initiate oxidative stress and cause renal damage which lead to hypertension, heart failure and ultimately death. The current study aimed to investigate the alterations in serum biochemical parameters i.e. HDL and LDL; gross anatomy, histomorphology and histomorphometry of broiler kidney in response to dietary GC, dexamethasone (DEX). Day old chicks (DOCs) were randomly assigned into four groups: control and three treatment groups (T1, T2 and T3). The control group was fed commercial broiler type ration and the treated groups were fed commercial broiler type ration containing GC (Dexamethasone @ 3, 5 and 7 mg/kg in T1, T2 and T3 group respectively). To measure the biochemical parameters, blood samples were collected on days 7, 14, 21, and 28 of the experiment. For histological investigation, kidney (left) samples were collected from the individual birds after sacrificing on days 7, 14, 21, and 28 of the experiment. Histomorphological alterations of the kidney were assessed by routine hematoxylin and eosin (H&E) staining. Biochemical analysis showed significantly increased serum HDL and LDL level compared to the control. In gross study, dark congested kidney was found with significantly decreased weight, length and width. Treatment with DEX augmented congestion, inflammation and fibrosis in kidney, as evidence by histomorphometric study. Extensively degenerated and atrophied glomeruli, degenerated tubular epithelium with distorted tubules and inter tubular empty spaces were seen. Percentage of atrophied glomeruli increased significantly and maximum percentage of glomerular atrophy was seen at day 28. These changes were found more explicitly in the higher dose group. Histomorphometric study also revealed significant decrease in the diameter of glomerulus. The findings of this study suggest that DEX may alter the serum biochemical parameters as well as kidney gross and histomorphology.

Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon.

The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, specific adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bp were designed to be complementary to target mRNAs, with either 6X stem-loops downstream of the guide RNA and a CMV promoter, or a 1X MS2 stem-loop on either side of the guide RNA and a U6 promoter. The engineered ADAR1 deaminase domain could convert adenosine to inosine at the desired editing site in EGFP, which was edited to contain an amber (TAG), opal (TGA) or ochre (TAA) stop codon. The system could convert the stop codons to a read-through tryptophan codon (TGG) in a cellular system, leading to fluorescence emission, observed using JuLi microscopy. PCR-RFLP and Sanger sequencing of the target transcript were also conducted, revealing an editing efficiency of 20.97 % for the opal stop codon, and 26 % and 17 % for the 5' and 3' A residues, respectively, in the ochre stop codon, using the double MS2. This was a higher editing rate than that achieved using the MS2-6X guide RNA. Observation of restoration of the read-through codon from the three different stop codons over time demonstrated a relatively low percentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.

RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.

Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR-RFLP and Sanger's sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.

Artificial RNA Editing with ADAR for Gene Therapy.

Editing mutated genes is a potential way for the treatment of genetic diseases. G-to-A mutations are common in mammals and can be treated by adenosine-to-inosine (A-to-I) editing, a type of substitutional RNA editing. The molecular mechanism of A-to-I editing involves the hydrolytic deamination of adenosine to an inosine base; this reaction is mediated by RNA-specific deaminases, adenosine deaminases acting on RNA (ADARs), family protein. Here, we review recent findings regarding the application of ADARs to restoring the genetic code along with different approaches involved in the process of artificial RNA editing by ADAR. We have also addressed comparative studies of various isoforms of ADARs. Therefore, we will try to provide a detailed overview of the artificial RNA editing and the role of ADAR with a focus on the enzymatic site directed A-to-I editing.

Male and Female Mice Exhibit Divergent Responses of the Cortical Vasculature to Traumatic Brain Injury.

We previously reported that traumatic brain injuries (TBI) alter the cerebrovasculature near the injury site in rats, followed by revascularization over a 2-week period. Here, we tested our hypothesis that male and female adult mice have differential cerebrovascular responses following a moderate controlled cortical impact (CCI). Using in vivo magnetic resonance imaging (MRI), a new technique called vessel painting, and immunohistochemistry, we found no differences between males and females in lesion volume, neurodegeneration, blood-brain barrier (BBB) alteration, and microglia activation. However, females exhibited more astrocytic hypertrophy and heme-oxygenase-1 (HO-1) induction at 1 day post-injury (dpi), whereas males presented with increased endothelial activation and expression of ß-catenin, shown to be involved in angiogenesis. At 7 dpi, we observed an increase in the number of vessels and an enhancement in vessel complexity in the injured cortex of males compared with females. Cerebrovasculature recovers differently after CCI, suggesting biological sex should be considered when designing new therapeutic agents.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase.

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.