Vanillin serves as a potential substitute for chemical chelator desferal in iron-overloaded mice.

PURPOSE: Iron toxicity occurs under iron-overloaded settings, such as a high iron diet and blood transfusion, and damages important organs. Vanillin has been proven to have potential iron chelation capability. Given the negative effects of commonly used iron chelators like deferoxamine (DFO), we sought to examine the iron chelation potency of vanillin and evaluate its potential effect in the treatment of iron overload-related disorders. METHODS: 42 male NMRI mice were prepared for this purpose, and except for the negative control group, iron overload conditions were generated in them by injecting iron. Then normal saline (as a control), vanillin, and DFO (n = 7) were subsequently given to iron-overloaded mice. In the following, the activity of antioxidant enzymes catalase and superoxide dismutase were measured in the blood serum, brain, kidney, spleen, lung, and liver tissues of mice. Furthermore, the level of lipid peroxidation was determined by measuring the amount of malondialdehyde. Also, Perl's and H&E staining were used to examine the physiopathology changes of tissues. FINDINGS: Vanillin, a natural antioxidant compound, outperformed deferoxamine, a chemical iron chelator. Along with a decrease in iron content, the activity of catalase and superoxide dismutase enhanced in the iron-overloaded groups that were treated with vanillin. The level of lipid peroxidation was also declined in the iron-overloaded mice receiving vanillin. CONCLUSION: Vanillin can be used as a suitable substitute for chemical chelators with fewer side effects and equivalent efficiency. We encourage the use of this compound as a natural iron chelator following performing additional safety and efficacy studies.

The natural iron chelators' ferulic acid and caffeic acid rescue mice's brains from side effects of iron overload.

Studies have shown that iron accumulation in the brain leads to neurogenic disorders. Novel iron chelating agents such as natural remedies are useful to decrease the side effects of iron in the brain. In addition, flavones and polyphenols are capable of chelating metals. In the current study, we evaluated the iron chelating capacity of ferulic acid and caffeic acid in the brain tissues of iron-overloaded mice. The mice received iron dextran intraperitoneally four times a week for 6 weeks. Next, blood samples were taken from the mice. In addition, brain tissues were excised for tissue staining as well as total iron and catalase (CAT) activity assessment. Ferulic acid and caffeic acid significantly decreased iron content in both brain and serum samples. Ferulic acid decreased iron by 50 and 51% more than the iron dextran-treated mice and by 43 and 2% more than desferal (DFO)-treated mice in serum and brain, respectively. In addition, caffeic acid reduced iron 57% more than the iron-treated group and 49 and 2% more than the desferal-treated group in the serum and brain, respectively. The catalase activity decreased with the increase in iron. By administering natural compounds, the catalase activity was increased equal to that of the control group. Thus, ferulic acid and caffeic acid might be possible natural iron chelators for brain iron overload therapy.

Reduction of iron toxicity in the heart of iron-overloaded mice with natural compounds.

BACKGROUND: Iron-overload is one of the risk factors in susceptible individuals. Iron-overload causes complications such as diastolic dysfunction, arrhythmias, ventricular dilation, and systolic dysfunction in the heart. Therefore, particular care is needed for those who need blood transfusions or patients with underlying heart diseases. PURPOSE: In this study, we examined the ability of five compounds, hesperidin, coumarin, caffeic acid, ferulic acid, and vanillin, to reduce the effects of iron-overdose in the heart of iron-overloaded mice. METHODS: For this purpose, 84 mice were prepared and except for the control group, iron-overload conditions were created in them by injecting iron. The hearts of mice were then harvested and the activities of the antioxidant enzymes catalase and superoxide dismutase were evaluated. Additionally, the amount of lipid peroxidation was measured by assessing the quantity of malondialdehyde. The physiopathology of cardiac tissue was considered by Perl's and H&E staining. RESULTS: According to the results, almost all natural compounds showed better performance than desferal, as an iron chelator chemical. Meanwhile, hesperidin, vanillin, and ferulic acid were the best antioxidant compounds and were able to improve the activity of antioxidant enzymes by reducing the amount of deposited iron. CONCLUSION: We recommend the use of the above compounds as natural iron chelators after completing additional studies.

Naringin is a promising natural compound for therapy of iron-overload disorders

Abstract Naringin has been shown to exhibit satisfying iron chelation capacity. Considering the side effects of routinely-used iron chelator (desferrioxamine, DFO), we decided to evaluate the iron chelation potency of naringin to discover whether or not it can be a promising natural substitute for treatment of excessive iron-related diseases. 35 mice were classified into five groups of 7 and subjected to iron dextran administration to induce the iron-overload condition. Iron-overloaded mice were then treated with normal saline (as control), naringin or DFO Morphology changes, and iron deposition in liver tissues were studied using H&E and Perl's staining. The results revealed that naringin is more potent than DFO in removing excessive iron ions deposited in liver tissues, indicating that naringin is a promising natural compound for therapy of iron overload disorders

Naringin Chelates Excessive Iron and Prevents the Formation of Amyloid-Beta Plaques in the Hippocampus of Iron-Overloaded Mice.

Metal chelating agents are antioxidant agents, which decrease the reductive potential and stabilize the oxidized metal ion form. In this study, we evaluated the naringin capacity in chelating iron and preventing amyloid-beta plaque formation in the hippocampus of iron-overloaded mice. Thirty-five NMRI male mice (8-10 weeks old) were provided. The mice were classified into five groups. Iron dextran was administered as i.p. injection (100 mg/kg/day) four times a week for four subsequent weeks. The treated groups received 30 and 60 mg/kg/day naringin for a month. After histological processing, the brain sections were stained with Perls' stain kit for iron spots, and Congo red was used to stain the brain and hippocampus for amyloid-beta plaques. 30 mg/kg/day of naringin was shown to decrease nonheme iron in an efficient manner; iron content in this group decreased to 16.83 ± 0.57 µg/g wet weight, a quantity as low as that observed in the normal saline-receiving group. The nonheme iron content in the mice receiving 60 mg/kg/day of naringin was 20.73 ± 0.65 µg/g wet weight. In addition, Aß plaque numbers in CA1, CA3, and DG areas of the hippocampus decreased significantly following treatment with 30 or 60 mg/kg/day naringin. Naringin has a strong iron chelation capacity and is able to reduce the formation of amyloid plaques. So it can be useful for neuroprotection and prevention of Alzheimer's disease.

Comparison Between Hesperidin, Coumarin, and Deferoxamine Iron Chelation and Antioxidant Activity Against Excessive Iron in the Iron Overloaded Mice.

OBJECTIVE: Iron accumulation in the brain leads to the development of Alzheimer's and Parkinson's diseases. Nowadays, iron chelation therapy is the best way to decrease the side effects of iron and amyloid plaques accumulation. Iron chelators are commonly used for the treatment of Alzheimer's disease. Previous studies have shown that natural products such as phenol and flavonoid compounds could chelate heavy metals. In the current study, we examined the iron chelation activity of hesperidin and coumarin on the brain tissue of iron-overloaded mice. METHODS: 48 NMRI male mice were divided into eight groups (n = 6). Six groups were treated with iron dextran (100 mg/kg/day) four times a week for 6 weeks. After stopping the injections for a month, five groups of iron-overloaded mice were treated with hesperidin, coumarin, and desferal four times a week subsequent for four subsequent weeks. Finally, the mice were anesthetized, and blood samples were collected from the ventricle of the heart for subsequent examination. The brain tissues were isolated and fixed in the 4% paraformaldehyde solution for Perl's staining. RESULTS: The results show that hesperidin and coumarin could strongly chelate excessive iron from the serum and deposit iron from the brain tissue compared to desferal group. Catalase and super oxidase activity were decreased in the iron-overloaded group, but in the treated group by hesperidin and coumarin, the enzyme's activity was increased significantly. CONCLUSION: Hesperidin and coumarin, as natural products, are powerful options to chelate iron ions and increase the activity of antioxidant enzymes.

6-OHDA mediated neurotoxicity in SH-SY5Y cellular model of Parkinson disease suppressed by pretreatment with hesperidin through activating L-type calcium channels.

OBJECTIVES: Parkinson's disease (PD) is a neurological condition with selective progressive degeneration of dopaminergic neurons. Routine therapies are symptomatic and palliative. Although, hesperidin (Hsd) is known for its neuroprotective effects, its exact cellular mechanism is still a mystery. Considering the important role of calcium (Ca2+) in cellular mechanisms of neurodegenerative diseases, the present study aimed to investigate the possible effects of Hsd on Ca2+ channels in cellular model of PD and the possible association between the selective vulnerability of neurons in cellular models of PD and expression of the physiological phenotype that changes Ca2+ homeostasis. METHODS: SH-SY5Y cell line was used in this study; cell damage was induced by 150 µM 6-OHDA and the cells' viability was examined using MTT assay. Intracellular calcium, reactive oxygen species (ROS) and mitochondrial membrane potential were determined by the fluorescence spectrophotometry method. The expressions of calcium channel receptors were determined by gel electrophoresis and immunoblotting. RESULTS: Loss of cell viability and mitochondrial membrane potential were confirmed in 6-OHDA treated cells. In addition, intracellular ROS and calcium levels, calcium channel receptors significantly increased in 6-OHDA-treated cells. Incubation of SH-SY5Y cells with hesperidin showed a protective effect, reduced the biochemical markers of cell damage/death, and balanced calcium hemostasis. CONCLUSIONS: Based on our findings, it seems that hesperidin could suppress the progression of the cellular model of PD via acting on intracellular calcium homeostasis. Further studies are needed to confirm the potential benefits of preventive and therapeutic effects of stabilizing cellular calcium homeostasis in neurodegenerative disease.

Aqueous Cichorium intybus L. seed extract may protect against acute palmitate-induced impairment in cultured human umbilical vein endothelial cells by adjusting the Akt/eNOS pathway, ROS: NO ratio and ET-1 concentration.

BACKGROUND: Endothelial dysfunction, which is a vascular response to oxidative stress and inflammation, involves a cascade of downstream events that lead to decreased synthesis of insulin-mediated vasodilator nitric oxide (NO) and increased production of vasoconstrictor protein endothelin-1 (ET-1). NO, and ET-1 production by endothelial cells is regulated by phosphatidylinositol 3-kinase (PI3K)-Akt-eNOS axis and mitogen-activated protein kinase (MAPK) axis of the insulin signaling pathway, respectively. METHODS: After treating the human umbilical vein endothelial cells (HUVECs) with either palmitate complexed with bovine serum albumin (BSA) (abbreviated as PA) or the aqueous Cichorium intybus L. (chicory) seed extract (chicory seed extract, abbreviated as CSE) alone, and simultaneously together (PA + CSE), for 3, 12, and 24 h, we evaluated the capacity of CSE to reestablish the PA-induced imbalance between PI3K/Akt/eNOS and MAPK signaling pathways. The level of oxidative stress was determined by fluorimeter. Insulin-induced levels of NO and ET-1 were measured by Griess and ELISA methods, respectively. Western blotting was used to determine the extent of Akt and eNOS phosphorylation. RESULTS: Contrary to PA that caused an increase in the reactive oxygen species (ROS) levels and attenuated NO production, CSE readjusted the NO/ROS ratio within 12 h. CSE improved the metabolic arm of the insulin signaling pathway by up-regulating the insulin-stimulated phospho-eNOS Ser1177/total eNOS and phospho-Akt Thr308/total Akt ratios and decreased ET-1 levels. CONCLUSIONS: CSE ameliorated the PA-induced endothelial dysfunction not only by its anti-ROS property but also by selectively enhancing the protective arm and diminishing the injurious arm of insulin signaling pathways.

Effects of BDNF receptor antagonist on the severity of physical and psychological dependence, morphine-induced locomotor sensitization and the ventral tegmental area-nucleus accumbens BDNF levels in morphine- dependent and withdrawn rats.

This study examined the effects of systemic administration of the TrkB receptor antagonist (ANA-12) on the severity of physical and psychological dependence and morphine-induced locomotor sensitization, the ventral tegmental area (VTA)-nucleus accumbens (NAc) BDNF levels in morphine-dependent and withdrawn rats. Rats were injected with bi-daily doses (10 mg/kg, at 12 h intervals) of morphine for 10 days. Then, rats were tested for naloxone-precipitated morphine withdrawal signs, the anxiety (the elevated plus maze-EPM) after the last morphine injection and injection of ANA12 (ip). Also, morphine-induced locomotor sensitization was evaluated after morphine challenge followed by an injection of ANA-12 in morphine-withdrawn rats. The VTA-NAc BDNF levels were assessed in morphine-dependent and withdrawn rats. The overall Gellert-Holtzman score was significantly higher in morphine-dependent rats receiving ANA-12 than in those receiving saline. Also, the percentage of time spent in the open arms in control and morphine-dependent rats receiving ANA-12 were higher compared to the Cont/Sal and D/Sal rats, respectively. There was no significant difference in the locomotor activity and the VTA-NAc BDNF levels between D/Sal/morphine and D/ANA-12/morphine groups after morphine withdrawal. We conclude that the systemic administration of ANA-12 exacerbates the severity of physical dependence on morphine and partially attenuates the anxiety-like behavior in morphine-dependent rats. However, ANA-12 did not affect morphine-induced locomotor sensitization and the VTA-NAc BDNF levels in morphine-dependent and withdrawn rats.

Induction of transient cell cycle arrest by H2 O2 via modulation of ultradian oscillations of Hes1, Socs3, and p-Stat3 in fibroblast cells.

Biological clocks, time-keeping systems, enable the living organisms to synchronize their biochemical processes with their environment. Among these molecular oscillators, ultradian oscillators have been identified with volatility less than 24 h. Transcription factor Hes1, a member of the basic Helix-loop-Helix (bHLH) protein family, has an oscillation duration of 2 h in vertebrates. Due to the pivotal role of oxidative stress in many human diseases, we evaluated the effect(s) of oxidative stress on Hes1 oscillator, its upstream regulators, and its downstream cell cycle regulators. NIH/3T3 mouse fibroblast cells were treated with sublethal (250 µM) and lethal (1000 µM) doses of H2 O2 for 30 min. H2 O2 generated a delay in p-Stat3 and Socs3 mRNAs followed by suppression of Hes1 protein. These events were accompanied by simultaneous upregulation of p21 and downregulation of cyclinD1, resulting in a temporary arrest of the cell cycle. In conclusion, the elimination of Hes1 protein oscillation by H2 O2 may represent a defense mechanism against oxidative stress in fibroblast cells.

Antioxidant activity of ethyl acetate and methanolic extracts of two marine algae, Nannochloropsis oculata and Gracilaria gracilis - an in vitro assay

ABSTRACT The aim of this study was to evaluate the antioxidant activity of ethyl acetate and methanolic extracts of two marine algae, Nannochloropsis oculata and Gracilaria gracilis. The extracts were assayed for total phenol and flavonoid content, DPPH free radical scavenging capacity, nitric oxide activity, iron chelation activity, and reducing power activity. Total phenol and flavonoid content were found to be high in both algae. Ethyl acetate extracts of both algae were found to exhibit significant antioxidant activity. Ethyl acetate extract of N. oculata exhibited a good capacity for iron chelation, nitrate oxide, and scavenging DPPH free radicals (72.95±2.30, 73.73±1.76, and 39.03±0.97% inhibition at 400 µg mL-1 respectively).

Antihypoxic activities of Crataegus pentaegyn and Crataegus microphylla fruits-an in vivo assay

ABSTRACT The aim of this study was to evaluate anti-hypoxia activity of polyphenolic extracts of Crataegus microphylla and Crataegus pentaegyn on mice. Three experimental models of hypoxia were considered, including asphyctic hypoxia, haemic hypoxia, and circulatory hypoxia. Polyphenolic extract of both plants exhibited significant anti-hypoxic activity and prolonged animal survival time. Anti-hypoia activity of C. pentaegyn was found to be superior to that of C. microphylla in circulatory and asphyctic hypoxia. Antihypoxic activity of these extracts may be attributed to their phenolic compounds.

Use of single chain antibody derivatives for targeted drug delivery.

Single chain antibodies (scFvs), which contain only the variable domains of full-length antibodies, are relatively small molecules that can be used for selective drug delivery. In this review, we display how scFv antibodies help improve the specificity and efficiency of drugs. Small interfering RNA (siRNA) delivery using scFv-drug fusion peptides, siRNA delivery using scFv-conjugated nanoparticles, targeted delivery using scFv-viral peptide- fusion proteins, use of scFv in fusion with cell penetrating peptides for effective targeted drug delivery, scFv-mediated targeted delivery of inorganic nanoparticles, scFv-mediated increase of tumor killing activity of granulocytes, use of scFv for tumor imaging, site-directed conjugation of scFv molecules to drug carrier systems, use of scFv to relieve pain, use of scFv for increasing drug loading efficiency are among the topics that are discussed here.

Antioxidant Activity of Different Fractions of Methanolic Extract of the Golden Chanterelle Mushroom Cantharellus cibarius (Higher Basidiomycetes) from Iran.

Natural compounds with antioxidant activity can be useful for treatment of reactive oxygen species-related diseases, comprising atherosclerosis, inflammatory injury, cardiovascular disease, cancer, diabetes, Alzheimer's disease, cataracts, autism, and aging. The current study was perform to assay the antioxidant activity different fractions of methanolic extract of golden chanterelle mushroom Cantharellus cibarius, a mushroom found in the north of Iran. Different fractions of methanolic extract of this mushroom, including n-hexane, chloroform, ethyl acetate, n-butanol, and water, were evaluated for antioxidant activity using six in vitro assay systems. Mushroom fruit was obtained from the local market, Sari (northern Iran). The n-hexane fraction had higher amounts of flavonoids contents (40.01 ± 1.30 mg quercetin equivalent g-1 of extract) and the highest exhibition of nitric oxide scavenging activity (77.21 ± 1.48%). The highest content of phenol was observed in the n-butanol fraction, which was 40.97 ± 0.99 mg gallic acid equivalent g-1 of extract. Among all the fractions, the ethyl acetate fraction was found to show higher DPPH scavenging activity (33.43 ± 1.30%) and the aqueous fraction to display the most reducing power. The highest Fe2+ chelating activity was observed in the chloroform fraction and then in the n-hexane fraction (86.13 ± 1.61 and 80.68 ± 2.07, respectively). The results all together signify C. cibarius as a valuable source of natural bioactive compounds with antioxidant activity.

Natural inhibitors of PI3K/AKT signaling in breast cancer: emphasis on newly-discovered molecular mechanisms of action.

Epidermal growth factor receptor (EGFR) plays a critical role in the initiation and progression of a variety of human cancers, including breast cancer. An important signaling pathway downstream of EGFR is the PI3K/AKt pathway, which regulates cellular processes as diverse as cell growth, survival, proliferation and migration. Deregulated activity of this pathway may lead to uncontrolled cell growth, survival, migration and invasion, contributing to tumor formation. In this review, we evaluate natural compounds that, in vitro (breast cancer cell lines) and/or in vivo (animal model, clinical) studies, suppress breast cancer cells or tumors mainly by suppressing the PI3K/AKT signaling pathway. The effect of these compounds on cell cycle arrest, inhibition of cell migration and invasion, tumor angiogenesis and metastasis in breast cancer are discussed.

In Vivo Iron-Chelating Activity and Phenolic Profiles of the Angel's Wings Mushroom, Pleurotus porrigens (Higher Basidiomycetes).

Pleurotus porrigens is an culinary-medicinal mushroom. It is locally called sadafi and is found in the northern regions of Iran, especially in Mazandaran. This mushroom is used to prepare a variety of local and specialty foods. Because of the phenol and flavonoid contents and the strong iron-chelating activity of this mushroom, it was selected for an assay of in vivo iron-chelating activity. Methanolic extract was administered intraperitoneally to iron-overloaded mice at two dosages (200 and 400 mg/kg/24 hours) for a total of 20 days, with a frequency of 5 times a week for 4 successive weeks. The total iron content was determined by atomic absorption spectroscopy. Plasma Fe3+ content was determined using a kit. Liver sections were stained by hematoxylin and eosin and Perls stain. A significant decrease in the plasma concentration of iron was observed in mice treated with extracts (P < 0.001). The animals showed a dramatic decrease in plasma Fe3+ content when compared with the control group (P < 0.001). Also, Perls stain improved the smaller amount of deposited iron in the liver of iron-overloaded mice treated with the extract. Liver sections revealed a marked reduction in the extent of necrotic hepatocytes, fibrous tissues, and pseudo-lobules. A high-performance liquid chromatography method was developed to simultaneously separate 7 phenolic acids in extract. Rutin (1.784 ± 0.052 mg g(-1) of extract) and p-coumaric acid (1.026 ± 0.043 mg g(-1) of extract) were detected as the main flavonoid and phenolic acids in extract, respectively. The extract exhibited satisfactory potency to chelate excessive iron in mice, potentially offering new natural alternatives to treat patients with iron overload. More studies are needed to determine which compounds are responsible for these biological activities.

Correlation between Sun Protection Factor and Antioxidant Activity, Phenol and Flavonoid Contents of some Medicinal Plants.

Long exposure of UV radiation increases risk of skin diseases such as cancer and photoallergic reactions. UV-B (280-320 nm) radiation is mainly responsible for inducing the skin problems. Skin protection is a suitable method against ultraviolet radiation-induced damage. Various synthetic agents have been used as photo protective but because of their potential toxicity in humans, they have limited usage. Natural substances have been recently considered as potential sunscreen resources due to their absorption in the UV region and their antioxidant activity. In the present study, the UV protective effects of 20 extracts from four common medicinal plants were evaluated. Their phenol and flavonoid contents and antioxidant activities were determined and correlation between SPF and these contents were evaluated. SPFs were between 0.102 and 24.470. The highest value was reached with ultrasonic extract of Crataegus pentagyna (SPF = 24.47) followed by methanolic extract of Feijoa sellowiana (SPF = 1.30). Good correlation was found between SPF and phenolic contents (Correlation Coefficient = 0.55 and p = 0.01) but no correlations were found between SPF and flavonoid contents or antioxidant activity. These extracts can be used alone or as additives in other sun screen formulations to enhance their SPF.

Antihypoxic activities of the golden Chanterelle Mushroom, Cantharellus cibarius (higher Basidiomycetes).

Cantharellus cibarius is an edible mushroom with worldwide distribution. Because of its good radical scavenging and strong iron-chelating activity, this mushroom was nominated for assay of antihypoxic activity. Protective effects of Chanterelle extract against hypoxia-induced lethality in mice were evaluated by 3 experimental models of hypoxia: asphyctic, hemic, and circulatory. Antihypoxic activity was especially pronounced in the hemic model. The effect was dose dependent. C. cibarius extract (600 mg kg-1) kept the mice alive for 10.07 ± 1.18 min. It significantly (P < 0.0001) and dose dependently prolonged survival time compared to control group (7.00 ± 0.63 min). Extract at 300 mg kg-1 prolonged survival time to 9.94 ± 0.87 min, which was statistically significant (P < 0.0001) compared to control group. In circulatory model, C. cibarius extract (600 mg kg-1) was effective. It prolonged latency for death significantly with regard to the control group (15.18 ± 4.21 vs. 9.84 ± 0.75 min; P < 0.001). At 300 mg kg-1, the extract also prolonged survival time (13.57 ± 0.87 min), and this effect was also statistically significant compared to the control group (P < 0.01). Extract showed no activity in the asphyctic model. Mice in the control group died of hypoxia in 28.20 ± 3.27 min. Extract (600 mg kg-1) prolonged latency for death, but this activity was not statistically significant (P < 0.05). Phenytoin prolonged latency for death to 55.00 ± 6.05 min (P < 0.0001).

Recent advances in head and neck squamous cell carcinoma--a review.

The current review presents the results of the most recent studies performed on different aspects of human head and neck squamous cell carcinoma, including radiosensitivity induction, efficiency improvement of monoclonal antibodies using low-intensity ultrasound, chemical compounds such as toll-like receptor (TLC) agonists, dasatinib, resveratrol and niclosamide, nuclear inhibition of cancer using STAT3 decoy oligonucleotide, efficiency of anti-EGFR monoclonal antibodies in detection of head and neck cancers and other related issues.

Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities.