The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with ...conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.
Abstract
Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been ...extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells
via
new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from
Nelumbo nucifera
, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells,
via
calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.
Cadmium is an important widely distributed heavy metal in the environment due to its several industrial uses, while milk thistle is an important herb and is a source of several antioxidant ...particularly silymarin which is a pharmacological active substance present in seeds of milk thistle plant (
Silybum marianum
). The current study investigated pathological effects of cadmium (Cd) and their amelioration with silymarin (SL) and milk thistle (MT) quails. A total of 144 quails were equally divided into 9 groups and given different combinations of cadmium chloride (150 and 300 mg/kg feed), SL (250 mg/kg), and MT (10 g/kg) feed. Parameters studied were clinical signs, mortality, organ weights, testes weight and volume, and gross and microscopic pathology. Results of this study indicated an increased mortality and reduced body weight in cadmium-treated quails. Quails were dull, depressed compared with control. Swollen hemorrhagic liver along with atrophied testes were also observed in these groups. No active spermatozoa were observed in lumen of seminiferous tubules of Cd-treated birds presenting arrest of spermatogenesis. Supplementing MT and SL ameliorated mortality, organ weights, spermatogenesis, and histopathological lesions. It may be concluded that MT and SL proved beneficial in cadmium-induced toxicities in Japanese quails.
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•Coxiella burnetii is prevalent in the environment (soil) and potential reservoirs (small ruminants) in Punjab province of Pakistan.•The prevalent strains are genetically distinct and ...their occurrence in soil have predisposition to some soil characteristics and risk factors.•Future studies are much essential to reveal further disease epidemiology and potential risk to direct-contact population, the farmers and veterinarians.
Coxiella burnetii causes query (Q) fever, an important zoonotic disease with worldwide significance. The role of environment in the ecology of C. burnetti, and its influence on seroconversion in animals has not been elucidated in Pakistan. We carried out a cross-sectional study in Punjab province to (1) determine the prevalence and distribution of C. burnetii in soil using an ISIIII gene-based real time-polymerase chain reaction (RT-PCR) assay, (2) analyze association between the occurrence of C. burnetii in soil and its predictors i.e. soil characteristics (macro- and micro-nutrients) and several likely risk factors including the seroconversion in small ruminants at places where its genome had or had not been detected, and (3) predict homology and genetic diversity of the identified strains using sequences originated from different hosts worldwide. A total of 2425 soil samples from nine districts of Punjab province were processed. C. burnetii DNA was detected in 47 samples (1.94%, 95% CI: ±0.55) originating from 35 villages of studied districts (7.22%, 95% CI: ±2.30). The highest prevalence was found in Attock (7.11%, 95% CI: ±3.36), followed by Lahore (4.83%, 95% CI: ±3.49), Sahiwal (4.70%, 95% CI: ±2.6), Dera Ghazi Khan (2.33%, 95% CI: ±2.02), Faisalabad (1.35%, 95% CI: ±1.18) and Sheikhupura (0.68%, 95% CI: ±0.94). The odds of detecting bacterial DNA in soil was increased with a unit increase in organic matter 2.511 (95% CI: 1.453–4.340), p=0.001 and sodium 1.013 (95% CI: 1.005–1.022), p=0.001, whereas, calcium 0.984 (95% CI: 0.975–0.994), p=0.002 and potassium 0.994 (95% CI: 0.990–0.999), p=0.011 had protective effect where a unit increase in each analyte decreased odds for its occurrence by 1.0% approximately. Likewise, for categorical variables (risk factors), the odds of detecting C. burnetii were higher at locations >500m away from a main road 1.95 (95% CI: 1.06–3.78), p=0.04. The enzyme-linked immunosorbent assay (ELISA) revealed an increased prevalence of antibodies in sheep (17.9%, 95% CI: ±5.54) compared with goats (16.4%, 95% CI: ±4.34). When determining the association between soil DNA and C. burnetii antibodies in small ruminants, the odds of detecting these antibodies were significant in sheep at the livestock barns 2.81 (95% CI: 1.20–7.37), p=0.02. The IS1111 gene-based sequence analysis revealed a clustering of the DNA into two distinct groups with much genetic divergence (0.76–68.70%): the first group that contained sequences from Lahore district clustered with human and buffalo origin isolates, whereas the second group that contained the sequences from the remaining study districts clustered with goat-, rodent- and human-origin isolates. This study provides the first evidence of the presence of C. burnetii in the environment in Punjab province, Pakistan. Future studies are needed to ascertain the bacteria’s molecular epidemiology over a wide geographical area, type the isolates, and evaluates the potential risks to human populations, particularly farmers and veterinarians.
Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of ...cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified
N
-desmethyldauricine (LP-4), isolated from
rhizoma of Menispermum dauricum DC
as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca
2+
/Calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.
A kinetic assay for total calcium in serum was developed which is based on the activation of Ca(++)-ATPase by free Ca(++) Ca(++)(f) maintained by EGTA in the reaction mixture. The concentration of ...Ca(++)(f) was dependent on total reference calcium added or serum calcium. Ca(++)-ATPase activity was coupled to the reduction of NADH by pyruvate kinase (PK) and lactate dehydrogenase (LDH) and monitored by change in absorbance at 340 nm. The calcium in normal serum was 10.08 +/- 0.24 mg/dl (n = 35) by our method while with o-cresolphthalein complexone (CPC) method, the total calcium in the same 35 serum samples was 10.14 +/- 0.54 mg/dl. The range of within-run coefficient of variations (CVs) by this method was 0.9-2.87% at 8-12 mg/dl and day-to-day CVs were 0.72-3.17%. The presence of other ions and standard clinical interfering agents did not affect this assay system. The correlation between values obtained with our method (y) and CPC method (x) for normal serum was: y = 1.064x-0.580 mg/dl (r = 0.912, n = 59).
Throughout the world, millions of Muslims fast daily during the month of Ramadan from sunrise (Sahar) until sunset (Iftar). Considering the impacts of environment on physiological functions, we ...performed this study in order to examine the effects of Ramadan fasting on some blood parameters of healthy volunteers and hypertensive patients. According to the results, there were no significant changes in the weight, heart rate (HR), blood pressure (BP), serum total cholesterol and packed cell volume (PCV) of volunteers before and after fasting (P<0.05). However, considering hypertensive patients, blood urea significantly increased, while blood pressure (both systolic and diastolic pressures) significantly decreased during the fasting period (P>0.05). Therefore, it can be concluded that hypertensive patients, while continuing their previous medications, can safely fast during the month of Ramadan.
Throughout the world, millions of Muslims fast daily during the month of Ramadan from sunrise (Sahar) until sunset (Iftar). Considering the impacts of environment on physiological functions, we ...performed this study in order to examine the effects of Ramadan fasting on some blood parameters of healthy volunteers and hypertensive patients. According to the results, there were no significant changes in the weight, heart rate (HR), blood pressure (BP), serum total cholesterol and packed cell volume (PCV) of volunteers before and after fasting (P0.05). Therefore, it can be concluded that hypertensive patients, while continuing their previous medications, can safely fast during the month of Ramadan.
Throughout the world, millions of Muslims fast daily during the month of Ramadan from sunrise (Sahar) until sunset (Iftar). Considering the impacts of environment on physiological functions, we ...performed this study in order to examine the effects of Ramadan fasting on some blood parameters of healthy volunteers and hypertensive patients. According to the results, there were no significant changes in the weight, heart rate (HR), blood pressure (BP), serum total cholesterol and packed cell volume (PCV) of volunteers before and after fasting (P<0.05). However, considering hypertensive patients, blood urea significantly increased, while blood pressure (both systolic and diastolic pressures) significantly decreased during the fasting period (P>0.05). Therefore, it can be concluded that hypertensive patients, while continuing their previous medications, can safely fast during the month of Ramadan.
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