6-Methoxyflavone antagonizes chronic constriction injury and diabetes associated neuropathic nociception expression.

Neuropathy is a disturbance of function or a pathological change in nerves causing poor health and quality of life. A proportion of chronic pain patients in the community suffer persistent neuropathic pain symptoms because current drug therapies may be suboptimal so there is a need for new therapeutic modalities. This study investigated the neuroprotective flavonoid, 6-methoxyflavone (6MF), as a potential therapeutic agent and gabapentin as the standard comparator, against neuropathic models. Thus, neuropathic-like states were induced in Sprague-Dawley rats using sciatic nerve chronic constriction injury (CCI) mononeuropathy and systemic administration of streptozotocin (STZ) to induce polyneuropathy. Subsequent behaviors reflecting allodynia, hyperalgesia, and vulvodynia were assessed and any possible motoric side-effects were evaluated including locomotor activity, as well as rotarod discoordination and gait disruption. 6MF (25-75 mg/kg) antagonized neuropathic-like nociceptive behaviors including static- (pressure) and dynamic- (light brushing) hindpaw allodynia plus heat/cold and pressure hyperalgesia in the CCI and STZ models. 6MF also reduced static and dynamic components of vulvodynia in the STZ induced polyneuropathy model. Additionally, 6MF reversed CCI and STZ suppression of locomotor activity and rotarod discoordination, suggesting a beneficial activity on motor side effects, in contrast to gabapentin. Hence, 6MF possesses anti-neuropathic-like activity not only against different nociceptive modalities but also impairment of motoric side effects.

Hydrazyl hydroxycoumarins as new potential conquerors towards Pseudomonas aeruginosa.

A class of unique hydrazyl hydroxycoumarins (HHs) as novel structural scaffold was developed to combat dreadful bacterial infections. Some HHs could effectively suppress bacterial growth at low concentrations, especially, pyridyl HH 7 exhibited a good inhibition against Pseudomonas aeruginosa 27853 with a low MIC value of 0.5 µg/mL, which was 8-fold more active than norfloxacin. Furthermore, pyridyl HH 7 with low hemolytic activity and low cytotoxicity towards NCM460 cells showed much lower trend to induce the drug-resistant development than norfloxacin. Preliminarily mechanism exploration indicated that pyridyl HH 7 could eradicate the integrity of bacterial membrane, result in the leakage of intracellular proteins, and interact with bacterial DNA gyrase via non-covalent binding, and ADME analysis manifested that compound 7 gave good pharmacokinetic properties. These results suggested that the newly developed hydrazyl hydroxycoumarins as potential multitargeting antibacterial agents should be worthy of further investigation for combating bacterial infection.

Toxicological assessment of dietary exposure of polyethylene microplastics on growth, nutrient digestibility, carcass and gut histology of Nile Tilapia (Oreochromis niloticus) fingerlings.

This study was conducted to ascertain the negative effects of dietary low-density polyethylene microplastics (LDPE-MPs) exposure on growth, nutrient digestibility, body composition and gut histology of Nile tilapia (Oreochromis niloticus). Six sunflower meal-based diets (protein 30.95%; fat 8.04%) were prepared; one was the control (0%) and five were incorporated with LDPE-MPs at levels of 2, 4, 6, 8 and 10% in sunflower meal-based diets. A total of eighteen experimental tanks, each with 15 fingerlings, were used in triplicates. Fish were fed at the rate of 5% biomass twice a day for 60 days. Results revealed that best values of growth, nutrient digestibility, body composition and gut histology were observed by control diet, while 10% exposure to LDPE-MPs significantly (P < 0.05) reduced weight gain (WG%, 85.04%), specific growth rate (SGR%, 0.68%), and increased FCR (3.92%). The findings showed that higher level of LDPE-MPs (10%) exposure in the diet of O. niloticus negatively affects nutrient digestibility. Furthermore, the results revealed that the higher concentration of LDPE-MPs (10%) had a detrimental impact on crude protein (11.92%) and crude fat (8.04%). A high number of histological lesions were seen in gut of fingerlings exposed to LDPE-MPs. Hence, LDPE-MPs potentially harm the aquatic health.

Evaluating the impact of sprinkler cycle and flow rate on dairy buffalo performance during heat stress.

The aim of this study was to evaluate the effect of the sprinkler cycle and flow rate on physiological, behavioural, and productive responses in dairy buffaloes. Nine Nili Ravi lactating buffaloes were subjected to three sprinkler cycles and two flow rates using a double replicated 3 × 3 Latin square design. The flow rates were 1.25 and 2 l/min, and the sprinkler cycles (minutes water on/off, number of cycles/h) were: 3/3, 10 cycles; 3/6, 7 cycles and 3/9, 5 cycles. The showering was applied from 0800 till 1630 h daily. In the first square of 21 d, each of the three sprinkler cycles was applied using a 1.25 l/min flow rate for 7 d per cycle. In the later square, the same treatments (sprinkler cycles) were applied using the 2 l/min flow rate. The average temperature humidity index during the study period was 85.7 ± 3.8 (Mean ± sd). The result showed that the 3/3 treatment group had lower body temperature and respiration rate than the other groups. The buffaloes in the 3/3 group produced 0.5 and 0.7 kg more milk with 1.4 and 2.4% more fat than the 3/6 and the 3/9 treatment groups, respectively. Similarly, the 2 l/min flow rate had a lower core body temperate and respiration rate and higher milk yield than the 1.25 l/min group. The 3/3 showering cycle with a 2 l/min flow rate appeared effective in improving physiological responses and milk yield in dairy buffaloes.

Resveratrol Enhances Temozolomide Efficacy in Glioblastoma Cells through Downregulated MGMT and Negative Regulators-Related STAT3 Inactivation.

Chemoresistance blunts the efficacy of temozolomide (TMZ) in the treatment of glioblastoma (GBM). Elevated levels of O6-methylguanine-DNA methyltransferase (MGMT) and activation of signal transducer and of transcription 3 (STAT3) have been reported to correlate with GBM resistance to alkylator chemotherapy. Resveratrol (Res) inhibits tumor growth and improves drug chemosensitivity by targeting STAT3 signaling. Whether the combined therapy of TMZ and Res could enhance chemosensitivity against GBM cells and the underlying molecular mechanism remains to be determined. In this study, Res was found to effectively improve chemosensitivities of different GBM cells to TMZ, which was evaluated by CCK-8, flow cytometry, and cell migration assay. The combined use of Res and TMZ downregulated STAT3 activity and STAT3-regulated gene products, thus inhibited cell proliferation and migration, as well as induced apoptosis, accompanied by increased levels of its negative regulators: PIAS3, SHP1, SHP2, and SOCS3. More importantly, a combination therapy of Res and TMZ reversed TMZ resistance of LN428 cells, which could be related to decreased MGMT and STAT3 levels. Furthermore, the JAK2-specific inhibitor AG490 was used to demonstrate that a reduced MGMT level was mediated by STAT3 inactivation. Taken together, Res inhibited STAT3 signaling through modulation of PIAS3, SHP1, SHP2, and SOCS3, thereby attenuating tumor growth and increasing sensitivity to TMZ. Therefore, Res is an ideal candidate to be used in TMZ combined chemotherapy for GBM.

Effect of Wide-Spectrum Monochromatic Lights on Growth, Phytochemistry, Nutraceuticals, and Antioxidant Potential of In Vitro Callus Cultures of Moringa oleifera.

Moringa oleifera, also called miracle tree, is a pharmaceutically important plant with a multitude of nutritional, medicinal, and therapeutic attributes. In the current study, an in-vitro-based elicitation approach was used to enhance the commercially viable bioactive compounds in an in vitro callus culture of M. oleifera. The callus culture was established and exposed to different monochromatic lights to assess the potentially interactive effects on biomass productions, biosynthesis of pharmaceutically valuable secondary metabolites, and antioxidant activity. Optimum biomass production (16.7 g/L dry weight), total phenolic contents (TPC: 18.03 mg/g), and flavonoid contents (TFC: 15.02 mg/g) were recorded in callus cultures placed under continuous white light (24 h), and of other light treatments. The highest antioxidant activity, i.e., ABTS (550.69 TEAC µM) and FRAP (365.37 TEAC µM), were also noted under white light (24 h). The analysis of phytochemicals confirmed the significant impact of white light exposures on the enhanced biosynthesis of plant secondary metabolites. The enhanced levels of secondary metabolites, i.e., kaempferol (1016.04 µg/g DW), neochlorogenic acid (998.38 µg/g DW), quercetin (959.92 µg/g DW), and minor compounds including luteolin, apigenin, and p-coumaric acid were observed as being highest in continuous white light (24 h with respect to the control (photoperiod). Similarly, blue light enhanced the chlorogenic acid accumulation. This study shows that differential spectral lights demonstrate a good approach for the enhancement of nutraceuticals along with novel pharmacologically important metabolites and antioxidants in the in vitro callus culture of M. oleifera.

Effects of Saccharomyces cerevisiae live cells and culture on growth and productive performance in lactating Nili-Ravi buffaloes.

An experimental work was conducted to evaluate the effects of Saccharomyces cerevisiae live cells and its culture on dry matter intake (DMI), milk yield, milk composition, body condition score, selected blood metabolites, feed conversion efficiency (FCE), nutrient digestibility, body weight gain, and economics of milk production in lactating multiparous Nili-Ravi buffaloes. In total, 20 buffaloes of age 5 years ± 6 months and weighing 550 ± 20 kg were selected and assigned to four dietary treatments (n=5 buffalo/treatment) under completely randomized design. The dietary treatments include treatment 1 (T1) control, treatment 2 (T2) 5g/head live yeast, treatment 3 (T3) 5g/head yeast culture, and treatment 4 (T4) 10 g/head yeast culture per day for 60 days excluding 14 days as an adjustment period. The results indicated that T4 showed significant (p<0.05) improvement in DMI, milk yield and components, blood glucose level, digestibility of nutrients, and body weight gain while significant decrease in blood urea nitrogen as compared to other treatment groups. Body condition score was not affected among treatments. In conclusion, yeast culture supplementation significantly improved (p <0.05) milk yield, milk composition, DMI, body weight gain, blood glucose level, and digestibility while significantly decreased blood urea level as compare to control. Economic return was also improved. BCS was not improved. Comparatively, yeast culture showed significant improvement in growth and productive performance as compare to live yeast. Meanwhile, 10-g yeast culture showed better results as compare to 5-g yeast culture.

Validation of a body condition scoring system in Nili Ravi dairy buffaloes (Bubalus bubalis): inter- and intra-assessor variability.

The aim of the research reported in this Research Communication was to apply the 5-point body condition scoring (BCS) system to dairy buffaloes and subjectively validate it by assessing the intra- and inter-assessor agreement. For this purpose, the BCS system developed for dairy cows was applied to buffaloes. A total of 230 Nili Ravi buffaloes of varying parity, lactation stages and pregnancy status were enrolled from the Buffalo Research Institute, Pattoki, Pakistan. Four observers independently assigned BCS values to each enrolled buffalo in two phases, as follows: (1) during phase I, the assessors were trained for BCS assessment using a BCS chart developed by Elanco Animal Health Ltd.; and (2) during phase II, the assessors were trained using live buffaloes for BCS assessment. Kappa statistics (kw) were used to determine the intra- and inter-assessor agreement. The results revealed that the exact overall inter- and intra-assessor agreement was moderate (kw = 0.48-0.55) and increased to substantial levels after training on live animals (kw = 0.63-0.87). Furthermore, the intra- and inter-assessor exact agreement was higher (kw = 0.57-0.58) for buffaloes tied to the mangers compared to the buffaloes standing in the loafing area (kw = 0.50). The inter-assessor agreements within 0.25 and 0.5 points were almost perfect (kw = 0.97-1.0). The current results suggested that the 5-point BCS system (using a scale from 1 to 5 with 0.25 increments) had substantial agreement for assessment and repeatability when applied to buffaloes.

Characterization and Evaluation of Antimicrobial Potential of Trigonella incise (Linn) Mediated Biosynthesized Silver Nanoparticles.

The goal of the research was to explore a new green method used to synthesize silver nanoparticles (Ag NPs) from an aqueous extract of Trigonella incise, which serves as a reducing and stabilizing agent. The obtained results showed an 85% yield of nanoparticles by using 2:5 (v/v) of 5% plant extract with a 0.5 M solution of AgNO3. Different techniques were used to characterize the synthesized Ag NPs, including X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and UV-visible spectroscopy. The UV-visible spectra of green synthesized silver nanoparticles showed maximum absorption at a wavelength of 440 nm. The FT-IR studies revealed the stretching oscillation frequency of synthesized silver nanoparticles in the absorption band near 860 cm-1. Similarly, the bending and stretching oscillation frequencies of the NH function group were assigned to the band in the 3226 cm-1 and 1647 cm-1 regions. The bending vibration of C-O at 1159 cm-1 confirmed the carbonyl functional group that was also assigned to the small intensity band in the range of 2361 cm-1. The X-ray diffraction analysis of Ag NPs revealed four distinct diffraction peaks at 2θ of 38°, 45°, 65° and 78°, corresponds to (111), (200), (220) and (311) of the face-centered cubic shape. The round shape morphology of Ag NPs with a mean diameter in the range 20-80 nm was analyzed via SEM images. Furthermore, the nanoparticles showed more significant antimicrobial activity against Salmonella typhi (S. typhi) and Staphylococcus aureus (S. aureus) with an inhibition zone of 21.5 mm and 20.5 mm at 6 µg/mL concentrations, respectively, once compared to the standard reference. At concentrations of 2 µg/mL and 4 µg/mL, all of the bacterial strains showed moderate activity, with inhibition zones ranging from 11 mm to 18.5 mm. Even at high concentrations of AgNPs, S. typhi showed maximum resistance. The best antifungal activity was observed by synthesized Ag NPs against Candida albicans (C. albicans) with 21 mm zone of inhibition, as compared to a standard drug which gives 22 mm of inhibition. Therefore, we conclude that the antibacterial and antifungal activities showed satisfactory results from the synthesized Ag NPs.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

Detection of toxoplasmosis in pets and stray cats through molecular and serological techniques in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Toxoplasma gondii is an important parasite that belongs to the phylum Apicomplexa, distributed globally, causing major health issues for a wide range of hosts, including humans, native and wild animals. METHODS: In the present study, we detected IgG and IgM antibodies through an ELISA kit and DNA of T. gondii through PCR in 197 pets and stray cats in Peshawar, Charsadda, Mardan, and Kohat districts of Khyber Pakhtunkhwa (Pakistan) to estimate the existence of feline toxoplasmosis. RESULTS: The current study revealed that stray cats have a significant infection rate of T. gondii (74.6%) as compared to pet cats (25.4%). In all the four districts, the prevalence of T. gondii was pointedly higher in district Kohat (95.5%) in the feline population. In comparison to the female (75.18%) and male (both pets and stray) cats have a maximum infection of (81.66%) non-significantly. The prevalence of T. gondii was observed to be significantly higher (91.66%) in the older and greater than 4 year old population of cats as compared to the younger ones. In poor health condition, the cat populations has a higher risk of infection of 92.3% as compared to healthy and poor body condition (73.91%) and (82.6%) respectively. The chronic and reactivated chronic conditions of toxoplasmosis were higher (58.37%) as compared to the acute condition. CONCLUSION: It has been concluded that toxoplasmosis is widely spread in the studied population.The outcomes of the present study show that T. gondii infection has a significant impact on the type of cat, age, and area, which implies a serious threat to human beings. Therefore, genotyping of T. gondii strains from different hosts is needed to forecast the current approach for prevention and control of this zoonotic parasite.

Elicitation of Submerged Adventitious Root Cultures of Stevia rebaudiana with Cuscuta reflexa for Production of Biomass and Secondary Metabolites.

Stevia rebaudiana is an important medicinal plant that belongs to the Asteraceae family. The leaves of Stevia rebaudiana are a rich source of many health-promoting agents such as polyphenols, flavonoids, and steviol glycoside, which play a key role in controlling obesity and diabetes. New strategies such as the elicitation of culture media are needed to enhance the productivity of active components. Herein, the Cuscuta reflexa extracts were exploited as elicitors to enhance the productivity of active components. Cuscuta reflexa is one of the parasitic plants that has the ability to elongate very fast and cover the host plant. Consequently, it may be possible that the addition of Cuscuta reflexa extracts to adventitious root cultures (ADR) of Stevia rebaudiana may elongate the root more than control cultures to produce higher quantities of the desired secondary metabolites. Therefore, the main objective of the current study was to investigate the effect of Cuscuta reflexa extract as a biotic elicitor on the biomass accumulation and production of antioxidant secondary metabolite in submerged adventitious root cultures of Stevia rebaudiana. Ten different concentrations of Cuscuta reflexa were added to liquid media containing 0.5 mg/L naphthalene acetic acid (NAA). The growth kinetics of adventitious roots was investigated for a period of 49 days with an interval of 7 days. The maximum biomass accumulation (7.83 g/3 flasks) was observed on medium containing 10 mg/L extract of Cuscuta reflexa on day 49. As the concentration of extract increases in the culture media, the biomass gradually decreases after 49 days of inoculation. In this study, the higher total phenolics content (0.31 mg GAE/g-DW), total flavonoids content (0.22 mg QE/g-DW), and antioxidant activity (85.54%) were observed in 100 mg/L treated cultures. The higher concentration (100 mg/L) of Cuscuta reflexa extract considerably increased the total phenolics content (TPC), total phenolics production (TPP), total flavonoids content (TFC), total flavonoids production (TFP), total polyphenolics content (TPPC), and total polyphenolics production (TPPP). It was concluded that the extract of Cuscuta reflexa moderately improved biomass accumulation but enhanced the synthesis of phenolics, flavonoids, and antioxidant activities. Here, biomass's independent production of secondary metabolites was observed with the addition of extract. The present study will be helpful to scale up adventitious roots culture into a bioreactor for the production of secondary metabolites rather than biomass accumulation in medicinally important Stevia rebaudiana.

Chemical Elicitors-Induced Variation in Cellular Biomass, Biosynthesis of Secondary Cell Products, and Antioxidant System in Callus Cultures of Fagonia indica.

Fagonia indica is a rich source of pharmacologically active compounds. The variation in the metabolites of interest is one of the major issues in wild plants due to different environmental factors. The addition of chemical elicitors is one of the effective strategies to trigger the biosynthetic pathways for the release of a higher quantity of bioactive compounds. Therefore, this study was designed to investigate the effects of chemical elicitors, aluminum chloride (AlCl3) and cadmium chloride (CdCl2), on the biosynthesis of secondary metabolites, biomass, and the antioxidant system in callus cultures of F. indica. Among various treatments applied, AlCl3 (0.1 mM concentration) improved the highest in biomass accumulation (fresh weight (FW): 404.72 g/L) as compared to the control (FW: 269.85 g/L). The exposure of cultures to AlCl3 (0.01 mM) enhanced the accumulation of secondary metabolites, and the total phenolic contents (TPCs: 7.74 mg/g DW) and total flavonoid contents (TFCs: 1.07 mg/g DW) were higher than those of cultures exposed to CdCl2 (0.01 mM) with content levels (TPC: 5.60 and TFC: 0.97 mg/g) as compared to the control (TPC: 4.16 and TFC: 0.42 mg/g DW). Likewise, AlCl3 and CdCl2 also promoted the free radical scavenging activity (FRSA; 89.4% and 90%, respectively) at a concentration of 0.01 mM, as compared to the control (65.48%). For instance, the quantification of metabolites via high-performance liquid chromatography (HPLC) revealed an optimum production of myricetin (1.20 mg/g), apigenin (0.83 mg/g), isorhamnetin (0.70 mg/g), and kaempferol (0.64 mg/g). Cultures grown in the presence of AlCl3 triggered higher quantities of secondary metabolites than those grown in the presence of CdCl2 (0.79, 0.74, 0.57, and 0.67 mg/g). Moreover, AlCl3 at 0.1 mM enhanced the biosynthesis of superoxide dismutase (SOD: 0.08 nM/min/mg-FW) and peroxidase enzymes (POD: 2.37 nM/min/mg-FW), while CdCl2 resulted in an SOD activity up to 0.06 nM/min/mg-FW and POD: 2.72 nM/min/mg-FW. From these results, it is clear that AlCl3 is a better elicitor in terms of a higher and uniform productivity of biomass, secondary cell products, and antioxidant enzymes compared to CdCl2 and the control. It is possible to scale the current strategy to a bioreactor for a higher productivity of metabolites of interest for various pharmaceutical industries.

Molecular epidemiology of Coxiella Brunetii in small ruminants in Punjab, Pakistan: a novel reporting analytical cross sectional study.

Coxiella burnetii, an intracellular zoonotic bacterium, causes query (Q) fever in ruminants. Its role has never been elucidated in small ruminants from Pakistan. The current study is designed to (a) determine the prevalence of coxiellosis in small ruminants, (b) evaluate the association of various potential risk factors and biomarkers in the occurrence of Coxiella burnetii, (c) and determine phylogeny and genetic variability of its various isolates identified during the study. For this purpose, 320 blood samples from sheep (n = 160) and goats (n = 160) were collected from 9 Union Councils of district Kasur, Punjab, and processed for DNA extraction. C. burnetii was confirmed by amplification of IS1111 transposase gene with an amplicon size of 294 bp. The results showed that the overall positive percentage of C. burnetii is 36.87% (sheep: 46.9% and goats: 30%). The phylogenetic tree was also constructed which described the possible origin of this pathogen from environment. Besides, after translation into amino acid, the resultant alignment showed several unique changes at position numbers 18 and 27 in the isolates from goats and at 27 and 66 from those of sheep. These mutations can have major impact on the infectious characteristics of this pathogen. Furthermore, different potential risk factors and clinical biomarkers like age, tick infestation, abortion, mastitis, and infertility were also studied and found that these are significantly (p < 0.05) associated with the occurrence of coxiellosis. It is concluded from the study that C. burnetii is endemic in small ruminants in Punjab, Pakistan. The outcomes of this study are alarming for scientific community as well as for policy makers because coxiellosis is an emerging threat to both humans and animals in this region due to its interspecies transmission ability.

SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response.

Plants are major sulfur reducers in the global sulfur cycle. Sulfate, the major natural sulfur source in soil, is absorbed by plant roots and transported into plastids, where it is reduced and assimilated into Cys for further metabolic processes. Despite its importance, how sulfate is transported into plastids is poorly understood. We previously demonstrated using single Arabidopsis (Arabidopsis thaliana) genetic mutants that each member of the sulfate transporter (SULTR) subfamily 3 was able to transport sulfate across the chloroplast envelope membrane. To resolve the function of SULTR3s, we constructed a sultr3 quintuple mutant completely knocking out all five members of the subfamily. Here we report that all members of the SULTR3 subfamily show chloroplast membrane localization. Sulfate uptake by chloroplasts of the quintuple mutant is reduced by more than 50% compared with the wild type. Consequently, Cys and abscisic acid (ABA) content are reduced to ∼67 and ∼20% of the wild-type level, respectively, and strong positive correlations are found among sulfate, Cys, and ABA content. The sultr3 quintuple mutant shows obvious growth retardation with smaller rosettes and shorter roots. Seed germination of the sultr3 quintuple mutant is hypersensitive to exogenous ABA and salt stress, but is rescued by sulfide supplementation. Furthermore, sulfate-induced stomatal closure is abolished in the quintuple mutant, strongly suggesting that chloroplast sulfate is required for stomatal closure. Our genetic analyses unequivocally demonstrate that sulfate transporter subfamily 3 is responsible for more than half of the chloroplast sulfate uptake and influences downstream sulfate assimilation and ABA biosynthesis.

miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis.

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3'UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

Health Risks in Different Age Group of Nitrate in Spring Water Used for Drinking in Harnai, Balochistan, Pakistan.

This study aims to determine the nitrate exposure and their health assessment in spring water used for drinking in Harnai. Total 24 water samples were collected from four springs used for drinking. Three samples from starting point and three from the end point of each spring were collected. DR/890 multi-parameter portable calorimeter was used for the measurement of nitrate. The concentration of nitrate was ranged from 0.1 to 1.1 mg/l with an average of 0.389 mg/l. The results show that the concentration of nitrate in spring water is 93%, and 99% less than permissible limits recommended by Pakistan Standards & Quality Control Authority (PSQCA) and World Health Organization (WHO), respectively. The values of physiochemical parameters like potential of Hydrogen (pH), Electrical Conductivity (EC) and Total Dissolved Solid (TDS) were ranged from 7.8 to 8.3, 564 to 749 µS/cm, and 36 to 479 mg/l with average values 8.025, 630.5 µS/cm, and 403.5 mg/l, respectively. The calculated mean chronic daily intake (CDI) in three age groups was found 0.01, 0.02, and 0.03 in adults, children, and infants, respectively. Hence, it is concluded that spring water used for drinking in Harnai was considered safe and do not pose any health hazards associated with nitrate.

Initial assessment of a child with suspected disorder of sex development.

Disorders of sex development (DSD) are defined as discrepancy between chromosomal, gonadal and anatomic sex. The basic principles for the management of DSD include a multidisciplinary approach for gender assignment. Clinical assessment includes a detailed history and examination of external genitalia. Most of the disorders with symmetrical gonades indicate hormonal cause while asymmetrical gonades are found in chromosomal DSDs. Karyotyping will indicate a 46XX DSD, 46 XY DSD or mosicism. Internal anatomy is defined by ultrasonography, genitoscopy and laparoscopy. Human chorionic gonadotrophins (hCG) stimulation test is carried out in under-virilised males to see the function of Leydig cells in testes. The Most common cause of 46XX DSD is congenital adrenal hyperplasia (CAH). The decision of gender assignment surgery is to be taken in a multidisciplinary environment and with informed consent of the parents. Most of 46 XX CAH patients, even if markedly virilised, and 46 XY complete androgen insensitivity syndrome are raised as females. Similarly, most of 5-α reductase deficiency and 17-ß hydroxysteroid dehydrogenase deficiency patients are assigned to the male gender. The decision in cases of mixed gondal dysgenesis and ovotesticular DSD is based on the development of external and internal genitalia. Patients with androgen biosynthetic defects, partial androgen insensitivity syndrome are usually assigned to the male gender.

Dual Inhibition of Rip2 and IRAK1/4 Regulates IL-1ß and IL-6 in Sarcoidosis Alveolar Macrophages and Peripheral Blood Mononuclear Cells.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1ß mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1ß and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4(+)CD25(+) cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.

Prevalence of Toxoplasma gondii oocysts through Copro-PCR in cats at Pet Center (UVAS), Lahore, Pakistan.

Toxoplasmosis is a major zoonotic disease of warm-blooded animals caused by Toxoplasma gondii. Cats are the only definitive host and they excrete environmentally resistant T. gondii oocysts in their faeces. Coproscopy was used to detect oocysts of enteric coccidians and then Copro-PCR was employed to test specifically for T. gondii in 470 cat samples. The prevalence of T. gondii oocysts was 2.3% (11/470) based on PCR. We observed 15 (3.2%) of 470 samples positive for coccidian oocysts by microscopy. The presence of Copro-DNA of T. gondii was found significantly higher (p<0.05) in males than females. We tested 11 samples of T. gondii oocysts in which 9 samples were from coccidian oocysts positive samples and 2 samples from negative faecal samples. Our results showed that PCR is the reliable method for the detection of faecal oocysts of T. gondii in cats as compared to microscopy. As per our knowledge, ours is first study for Copro-PCR prevalence of cats' T. gondii oocysts excretion in Pakistan.