Asphodelus tenuifolius extracts arrested inflammation and arthritis through modulation of TNF-α, NF-κB, ILs, and COX-2 activities in in vivo models.

Asphodelus tenuifolius is traditionally used in the management of rheumatic pain and inflamed body parts. The current study validated its traditional use as an anti-arthritic and anti-inflammatory agent using a series of in vivo models. Carrageenan and histamine-induced acute oedema models were employed to study the effects of n-hexane (n-HeAT) and ethanolic (EeAT) extracts on acute inflammatory mediators and were found to inhibit oedema formation in a dose-dependent manner. Formalin and complete Freund's adjuvant (CFA) were injected into the hind paw of rats for the induction of arthritis. In the formalin model both n-HeAT and EeAT showed significantly better (p < 0.05) anti-oedema effects from day 6 onward. In CFA model rats were treated on 8th day of induction with extracts at the doses of 250, 500, and 750 mg/kg respectively. Piroxicam (10 mg/kg) and normal saline (10 mL/kg) were used as positive and negative controls respectively. Both n-HeAT and EeAT significantly (p < 0.05) decreased arthritis development in a time-dependent manner and at 28th day extent of inflammation was even less than that observed at day 8. The arthritic score was measured at day 12, 16, 20, 24, and 28 and was observed to be significantly less (p < 0.05) in animals treated with 750 mg/kg of n-HeAT and EeAT, respectively. Joint inflammation (p < 0.01), bone erosion (p < 0.001) and, pannus formation (p < 0.01) were significantly declined in A. tenuifolius treatment groups. Radiographic evaluations (X-ray) were conducted to check bone integrity and extent of inflammation and were observed to be diminished at day 28 in A. tenuifolius extracts treated groups. HPLC was performed to screen the phytochemical profile of n-HeAT and EeAT and were found to contain flavonoids and phenolic compounds. Quantitative real-time polymerase chain reaction (qPCR) was performed to detect effects of n-HeAT and EeAT treatments on inflammatory markers i.e., IL-4, IL-6, IL-10, COX-2, NF-κB, and I-κB using blood samples. ELISA assays were performed for the detection of levels of C-reactive proteins, respectively. Significant downregulation of TNF-α, IL-4, IL-6, IL-1ß, COX-2, NF-κB with simultaneous upregulation of IL-10 and I-κB was observed in n-HeAT and EeAT treatment groups. ELISA assays also showed significant (p < 0.05) down-modulation in the serum levels of CRP and TNF-α. Both extracts showed relatively weak antioxidant activities as compared with ascorbic acid in in vitro assay. Based on findings of the current study it is concluded that A. tenuifolius has anti-inflammatory and anti-arthritic effects and thus has potential to be used as an adjunct to standard NSAIDs therapy.Graphic abstract.

Mutation in pvcABCD operon of Pseudomonas aeruginosa modulates MexEF-OprN efflux system and hence resistance to chloramphenicol and ciprofloxacin.

Pseudomonas aeruginosa harbors pvcABCD operon that is responsible for the synthesis of paerucumarin. Here we report the involvement of pvcABCD operon in chloramphenicol and ciprofloxacin resistance. P. aeruginosa mutant defective in pvcB (PW4832) was more sensitive to chloramphenicol and ciprofloxacin in comparison with its parent strain (MPAO1). A mutation in pvcA gene in MPAO1 (PW4830) did not alter the sensitivity to either antibiotic. As chloramphenicol and ciprofloxacin are substrates of MexEF-OprN efflux pump, so we decided to investigate the modulation of MexEF-OprN and its transcriptional regulator MexT in PW4832, PW4830 and MPAO1 strains. We isolated and sequenced mexT gene from MPAO1, PW4830 and PW4832. The nucleotide sequence of mexT gene in all three strains was identical. Expression levels of mexEF-oprN, mexT and mexS genes were checked via quantitative real-time RT-PCR. All these genes showed significant repression in mRNA levels in PW4832 as compared to MPAO1. These results indicate that chloramphenicol and ciprofloxacin sensitivity in PW4832 is due to transcriptional repression of mexT and mexEF-oprN genes. Exogenous addition of paerucumarin resumed the expression of mexT and mexEF-oprN genes as well as resistance against chloramphenicol and ciprofloxacin in PW4832 strain. This is a novel finding linking pvcB gene of P. aeruginosa with chloramphenicol and ciprofloxacin resistance and MexEF-OprN pump modulation which needs to be further explored.

Prevalence and Spatial Distribution of Animal Brucellosis in Central Punjab, Pakistan.

Brucellosis is an important zoonotic disease of animals and humans caused by bacteria of the genus Brucella. Brucellae are Gram-negative intracellular bacteria which infect a wide variety of animals including goats, sheep, buffaloes, cows, pigs, and wildlife. The objectives of this study were to determine the seroprevalence and spatial distribution of brucellosis in Central Punjab, Pakistan. A total of 1083 blood samples of goats, sheep, buffaloes, and cows were collected from 38 villages of four districts (Kasur, Faisalabad, Lahore, and Okara) of Punjab, Pakistan, and screened for brucellosis by Rose Bengal Plate test (RBPT) and PCR confirmed. Epidemiological, demographic data and GPS coordinates for every sample were collected. By using interpolation of the Aeronautical Reconnaissance Coverage Geographic Information System (Arc GIS), a surface plot was generated applying inverse distance weight (IDW). It was found that 35 (3.23%) serum samples were positive for brucellosis. In eight (61.5%), six (75%), seven (87.5%), and eight (89%) villages, positive goats, sheep, buffaloes, and cattle were detected, respectively. In general, older animals are more often positive for brucellosis. In goats bucks were more often RBPT positive than females while in sheep, buffaloes, and cattle more females were positive. The spatial distribution of brucellosis shows that it is widely distributed in the western region of the study area in goats and in the South-West region in sheep. Similarly, for buffaloes it is restricted to the south-east and north-west regions, and in cattle brucellosis is present in western region of study area only. Reflected by this study, brucellosis poses a risk for livestock in developing countries due to lack of awareness by officials, owners, and consumers, and control measures are missing. A risk map of brucellosis was generated to develop effective strategies for awareness rising and to improve the quality of control programs in Pakistan.

Occurrence of Toxoplasma gondii antibodies and associated risk factors in women in selected districts of Punjab province, Pakistan.

Toxoplasmosis is a parasitic zoonotic disease caused by Toxoplasma (T.) gondii. Limited data are available on the occurrence of T. gondii in women especially pregnant women in Pakistan. The present study aimed to determine the occurrence and risk factors associated with T. gondii in pregnant and non-pregnant women in Punjab Province, Pakistan. A cross-sectional study was conducted and 593 samples were collected from pregnant (n = 293) and non-pregnant (n = 300) women of District Headquarter Hospitals of Chiniot, Faisalabad, Jhang and Okara, Pakistan. Data related to demographic parameters and risk factors were collected using a pretested questionnaire on blood sampling day. Serum samples were screened for antibodies (IgG) against T. gondii using ELISA. A univariant and binomial logistic regression was applied to estimate the association between seropositive and explanatory variables considering the 95% confidence interval. P value ⩽0.05 was considered statistically significant for all analysis. Out of 593, 44 (7.42%) women were seropositive for T. gondii IgG antibodies. Occupation, age, sampling location, socioeconomic status, contact with cat, pregnancy status and trimester of pregnancy were significantly associated with seropositivity for T. gondii antibodies. Location and trimester of pregnancy were identified as potential risk factors for T. gondii seropositivity based on binomial logistic regression. Toxoplasma gondii is prevalent in pregnant and non-pregnant women. Therefore, now a necessitated awareness is required to instruct the individuals about these infectious diseases (toxoplasmosis) and their control strategies to maintain the health of human population. Moreover, health awareness among public can help the minimization of T. gondii infection during pregnancy and subsequent risk of congenital toxoplasmosis.

Seroepidemiology and the Molecular Detection of Animal Brucellosis in Punjab, Pakistan.

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (B.), affecting both animals and humans, causing severe economic loses and severe illness, respectively. The objective of the present study was to determine the seroprevalence and the risk factors associated with caprine, ovine, and bovine brucellosis in selected districts of Punjab, Pakistan. A total of 1083 blood samples were randomly collected from animals (goats = 440, sheep = 203, cows = 206, and buffaloes = 234). Questionnaires were used to collect data on risk factors associated with brucellosis on the sampling day. All samples were initially screened for anti-Brucella antibodies using the rose bengal plate test (RBPT). The seropositive serum samples were confirmed by a quantitative real-time polymerase chain reaction (PCR) assay for the detection of the Brucella genus- and Brucella species-specific DNA (B. abortus and B. melitensis). Univariant and binary logistic regression were used to identify important risk factors of brucellosis. Anti-Brucella antibodies and DNA were detected in 35 (3.23%) serum samples. Thirty-four (97.1%) DNA samples were confirmed as B. melitensis by qRT-PCR. Abortion history and natural mating were found to be potential risk factors. Brucella melitensis was identified as the causative agent of caprine, ovine, and bovine brucellosis in the selected districts of Punjab, Pakistan. Diseased animals may act as a source of infection for other animals. The elimination of positive seroreactors, development of control strategies for brucellosis, and education programs regarding the control of zoonotic disease are highly needed in developing countries like Pakistan.

Isonitrile-functionalized tyrosine modulates swarming motility and quorum sensing in Pseudomonas aeruginosa.

Paerucumarin synthesized by pvc operon pvcABCD is an iron binding molecule which modulates biofilm formation in Pseudomonas aeruginosa but its direct function in bacterial pathogenesis needs further investigation. pvcA synthesizes isonitrile functionalized tyrosine (IFT) which is converted to mature paerucumarin by the proteins encoded by pvcB, pvcC and pvcD genes. Interruption of pvcB in MPAO1 resulted in accumulation of IFT as it cannot be converted to mature molecule. The MPAO1 pvcB mutant (PW4832) showed enhanced swarming motility, while complementation with plasmid pLL2 carrying pvcB reduced swarming motility. Enhanced levels of rhlA expression and rhamnolipid production were observed in PW4832 compared to the parent strain. Overexpression of ptxR, the positive regulator of pvcABCD, in PW4832 caused accumulation of more IFT and further elevated the level of rhlA expression. Expression of the quorum sensing system transcriptional activators lasR and rhlR, as well as the synthase genes lasI and rhlI, was enhanced in PW4832 compared to MPAO1, as was PQS accumulation. Exogenously added IFT, but not paerucumarin, enhanced the production of rhamnolipids in P. aeruginosa. These results suggest that IFT enhances swarming motility in P. aeruginosa either directly by enhancing rhamnolipid production or indirectly through modulation of the quorum sensing systems. This is the first report assigning an independent function to IFT in P. aeruginosa.

High frequency and molecular epidemiology of metallo-ß-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan.

Background: Metallo-ß-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the ß-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem). Methods: A total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of ß-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection. Results: The rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of bla TEM, bla SHV, bla OXA, bla IMP-1, and bla VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla MBL (bla IMP and bla VIM) and bla ESBL (bla TEM, bla SHV, bla OXA) was also detected through multiplex and singleplex PCR. bla OXA, bla TEM and bla SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion: To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla OXA, bla TEM and bla SHV with bla IMP-1 and bla VIM in MBL-producing gram-negative bacilli.

ETHNOBOTANICAL AND BIOLOGICAL ACTIVITIES OF Leptadenia pyrotechnica (Forssk.) Decne.: A REVIEW.

BACKGROUND: Leptadenia pyrotechnica is traditionally used for treating various diseases. This species holds variety of bioactive constituents that trigger healing properties. The present review was aimed to analyze nutritional, phytochemical and pharmacological activities of L. pyrotechnica. MATERIALS AND METHODS: The present review regarding Leptadenia pyrotechnica (Forssk.) Decne. Is the compilation of data from the previous research works conducted by various scientists across the world. Various published papers, medicinal plant databases, etc were utilized to compile the information. RESULTS: L. pyrotechnica is a wonderful desert plant belongs to the family Asclepiadaceae This plant possesses antifungal, antibacterial, anticancer, antioxidant, wound healing, anthelmintic, antiatheroscloretic, hypolipidemic, antdiabetic and hepatoprotective activities coupled with other multifarious uses. Almost all plant parts are used in the traditional medicinal system to treat various disorders. CONCLUSION: This review includes the substance of different ethnobotanical uses, phytochemistry and exclusive capability of this plant in the field of anti-microbial and human disease activities.