This study assessed the neurotoxicity of cobalt (Co) on SH-SY5Y human neuroblastoma cells in vitro. The results showed that CoCl2 reduced cell viability in a dose-dependent manner when measured by MTT and NR assays at 24, 48, and 72 hours. CoCl2 treatment caused cell shrinkage and apoptosis. Antioxidants provided some protection against Co toxicity at lower concentrations but not at higher concentrations, suggesting oxidative stress may not be the main mechanism of toxicity. Overall, the results indicate that Co is toxic to neurons and this toxicity could relate to neurological symptoms reported in patients with cobalt-chromium hip implants who have elevated blood Co levels.
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
CC3TM:A Stable,Sterile Analog of Poly-D-Lysine that is Optimal for the Cultur...Daniel Schroen, PhD
Poly-D-Lysine (PDL) improves attachment and growth of certain fastidious cells. PDL surfaces cannot be considered formally sterile, because current sterilization methods would compromise cell adhesion and often require controlled storage conditions over the usual 1-2 year shelf life. To address these issues, a non-biological analog of PDL was investigated for function and stability in cell culture. This synthetic organic polymer, CC3TM, displays a high amine group density and positive charge in neutral media.
HepG2 cell model for genotoxicity and steatosis assessmentHCS Pharma
Early detection of toxic events induced by drug cantidats is mandatory in order to avoid late attrition in the process of R&D. Here we present two assays that can be done with the HepG2 human hepatoma cell line: genotoxicity assay (DNA double strand break) and steatosis.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
The roles of group C and F streptococci in causing endemic pharyngitis are still controversial, although group C streptococci are implicated in the outbreaks of pharyngitis and associated disorders.
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
CC3TM:A Stable,Sterile Analog of Poly-D-Lysine that is Optimal for the Cultur...Daniel Schroen, PhD
Poly-D-Lysine (PDL) improves attachment and growth of certain fastidious cells. PDL surfaces cannot be considered formally sterile, because current sterilization methods would compromise cell adhesion and often require controlled storage conditions over the usual 1-2 year shelf life. To address these issues, a non-biological analog of PDL was investigated for function and stability in cell culture. This synthetic organic polymer, CC3TM, displays a high amine group density and positive charge in neutral media.
HepG2 cell model for genotoxicity and steatosis assessmentHCS Pharma
Early detection of toxic events induced by drug cantidats is mandatory in order to avoid late attrition in the process of R&D. Here we present two assays that can be done with the HepG2 human hepatoma cell line: genotoxicity assay (DNA double strand break) and steatosis.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
The roles of group C and F streptococci in causing endemic pharyngitis are still controversial, although group C streptococci are implicated in the outbreaks of pharyngitis and associated disorders.
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
Anticancer Activity of New Di-Nuclear Copper (I) ComplexTaghreed Al-Noor
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
Results: Compared to the control group, MDA levels were high while GSH and CAT levels were low in the deltamethrin group. Histopathological analysis showed spaces between the pigment epithelium, irregularity in the delimiting membrane, degenerated ganglion, cone and bacillus cell, pyknotic nuclei, thinned inner limitation membrane, and thickened vascular wall. The control group showed FAS expression in the pigment layer limiting membranes, in the nuclei of many cone and bacillus cells, and ganglion cells in the control group sections. In the deltamethrin group, FAS expression was observed in the inner and outer limiting membranes of the pigment epithelium, cone and bacillus cells, and ganglion cell nuclei. In the control group, negative NOS expression in the pigment epithelium and outer limiting membranes, internal limitation membrane, and ganglion cells in the cone and bacillus cell nuclei were observed. In the deltamethrin group, NOS expression was positive in the pigment epithelium, cone and bacillus, and ganglion cell nuclei.
Conclusion: We suggest that deltamethrin toxicity induced apoptotic process due to increased inflammation in the retina and may cause visual impairment as a result of neural damage.
Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
High content screening in MCF7 and MDA-MB231 cells show differential response...Thermo Fisher Scientific
Oxygen levels in typical cell culture conditions do not accurately reflect the oxygen levels cells are exposed to within the body. Furthermore, oxygen levels can vary within the tumor microenvironment. These variances can affect how cells respond to a variety of drugs and small molecules. To further understand how oxygen levels affect drug sensitivity, the response of hormone-dependent MCF7 cells were compared to hormone-independent MDA-MB231 cells, cultured under low and high oxygen.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
1. The Neurotoxicity of Cobalt on SH-SY5Y Cells in vitro
Holly Cribbes, Prof. Mary Helen Grant
*Dept of Biomedical Engineering
University of Strathclyde, Glasgow G4 0RE, Scotland, United Kingdom
Introduction
Cobalt (Co) wear particles released from cobalt-chromium (CoCr) alloy metal-on-metal
(MoM) hip implants have been shown to cause symptoms of toxicity. Neurological sympto
ms such as memory loss, cognitive decline, blindness and loss of hearing, have been repo
rted in patients with these implants, all of whom have elevated blood Co levels beyond the
toxic threshold of 7µg/L. Normal Co serum levels in humans without implants is <0.19µg/L
(Bocca et al., 2006) and in those with MoM implants this is significantly elevated
( >400µg/L). The scientific literature provides evidence that Co is cytotoxic to many cell
types in vitro however there is a lack of toxicological data on neurons. It is unknown how
Co exerts its neurotoxic effects and what the cellular mechanism(s) of action could be. Co
is thought to generate reactive oxygen species (ROS) within the cells inducing oxidative
stress and this may contribute to its neurotoxic effects. Here we assessed the toxicity of
Co on SH-SY5Y cells. Once this was established, the effect of added antioxidants n-acetyl
cysteine (NAC), Vitamin B12 and the hydroxyl scavenger dimethylthiourea (DMTU) were
assessed to determine whether the Co effects were counteracted or modulated following
co-treatment. This was done to provide further insight into the mechanisms of Co-
mediated neurotoxicity and whether oxidative processes were involved.
Conclusions
The results suggest that CoCl2 is toxic to SH-SY5Y cells in vitro and this could
relate to the toxicity of CoCl2 in vivo. This could therefore have consequences
for patients with MoM hips. CoCl2 reduced cell viability measured by MTT
assays in a dose-dependant manner at high concentrations. Clear
morphological differences can be seen comparing controls to 500µM CoCl2
treated cells. Cells containing CoCl2 appear shrunken with their processes
completely retracted and appear to have formed apoptotic bodies. This
suggests that high concentrations of CoCl2 induces apoptosis in SH-SY5Y
cells. From 48h, longer incubation times with CoCl2 caused significantly
greater effects on cell viability and this is reflected in their LD50 values. The
MTT results at 24 hours suggest that CoCl2 may be more toxic at this point as
the LD50 value is lower than that at 48 hours and 72 hours. These data
suggest that CoCl2 may rapidly interfere with the reductive metabolism of the
cell before a neuronal defence mechanism is activated. Consequently, the
mitochondria could be a target in CoCl2 toxicity.
Antioxidants added to CoCl2 treated cells increase cell viability at lower
concentrations but only to some extent. The antioxidants had no significant
effect on cell viability at higher CoCl2 concentrations suggesting that CoCl2 at
this point is too toxic. Since the antioxidants did not greatly modulate the
effects of CoCl2 toxicity, oxidative stress may not be the main mechanism of
CoCl2 toxicity. Future research could focus on identifying the underlying
cellular mechanisms of cobalt toxicity for therapeutic benefits in those
exposed to high amounts from their MoM implant.
Figure 1: CoCl2 treatment on SH-SY5Y cells measured by MTT and NR assays. (A) At 24h.
(B) At 48h. (C) At 72h. Results are % of control values (Mean+ range, n=12). Control contains no
CoCl2. Microscopy cell pictures showing control with no CoCl2 and cells treated with 500µM at
24h (D), at 48h (E) and at 72h (F).Scale bar = 50µm. (G) shows LD50 values for MTT and NR
assays at 24, 48, 72h
Figure 2: CoCl2 treatment with and without antioxidants, 1mM NAC, 1mM DMTU
+ 500µM Vit B12. (A) Cell viability at 48h with/without antioxidant treatment. Results are % of
control containing no CoCl2. *sig dif to control (Mean + range, n=12 (B) Cell pictures showing
controls without CoCl2 treatment, with 300µM CoCl2 only, and 300µM CoCl2 with antioxidant
treatment at 48h. Scale bar = 50µm (C) Table showing MTT significant differences compared to
CoCl2 only treated cells. results for 24h (blue), 48h (red) and 72h (green). +sig diff compared to
CoCl2 only control. – not sig diff from CoCl2 only control.
Methods
Effect of Cobalt SH-SY5Y Cells
SH-SY5Y neuroblastoma cells were cultured in supplemented DMEM/ F-12 nutrient
mixture (Gibco, Life Technologies). Solid cobalt (II) hexahydrate (CoCl2) was
dissolved in sterile d-H2O to obtain a stock solution which was used to prepare
concentrations of CoCl2 diluted in DMEM/F-12 medium (25-500µM).
Cell Viability measured by MTT and NR assays
Cells were seeded in a 96-well plate at a density of 5 x 104
cells/cm2.
.Cells were
treated with various concentrations of CoCl2 and incubated for 24, 48 and 72 hours.
After incubation MTT and NR assays were performed and cell viability was measured
at 540nm where absorbance is proportional to the number of viable cells. MTT and
NR assays were also carried out in antioxidant/scavenger CoCl2-treated cell
experiments.
Effect of antioxidants/scavenger on Co-treated cells
Stock solutions of antioxidants were made and
concentrations of 1mM DMTU, 500µM Vitamin B12 and
1mM NAC were added separately with CoCl2 at toxic
concentrations (300-600µM). Viability was assessed
by performing MTT and NR assays after incubation
for 24, 48 and 72 hours.
Cell Morphology
Cell morphology was assessed in CoCl2 treated cells and CoCl2 with
antioxidant/scavenger treated cells using microscopy (Nikon) and cell photographs
were taken using Moticam 10.0MP microscopy camera.
Figure 1. 96 well plate arrangement
X – Cells + Medium; X - CoCl2 controls (300-
600µM); Y – Antioxidant/scavenger controls; Y
– Antioxidant + CoCl2 at 300-600µM
A
B
CResults
D E F
G
Cell Viability after 48h Cobalt
treatment with/ without AntioxidantsA B
C