Chemical composition, antioxidant and antibacterial activity of Piper chaba stem extracts with preservative effects on storage of raw beef patties.

Piper chaba, a traditional South-east Asian medicinal herb and well-known curry spice, was studied to evaluate its suitability as a source of natural preservatives for beef products. Plant extracts that are high in phenolics and have high antimicrobial and antioxidant activities are likely to be useful as a natural preservative. Therefore, the phytochemical composition and the bioactivities of both ethanolic and methanolic extracts of P. chaba stem were examined first. The study revealed a significant antioxidant activities and potential antibacterial activity of P. chaba extracts. Next we investigated the preservation characteristics of P. chaba by using beef patties as a model system. Beef patties were produced and treated with 0.2 % ethanolic extract (mentioned as PEE) of P. chaba and 0.1 % commercial preservative (mentioned as PCP). They were then assessed for various storage quality parameters under refrigerated (4° C ± 1° C) conditions, including free fatty acid, antioxidant contents, and oxidative stability at 0, 6th, 16th, and 33rd days. No significant variations were observed across the products with regard to proximate composition study such as protein, ash and fat contents. In comparison to both PEE and PCP, the control product had higher free fatty acid values throughout the storage period. This indicates that the fat content of the PEE and PCP degraded at a slower rate than the control over the 33-day storage period. Our study also showed that both PCP and PEE had increased antioxidant capacity, implying that lipid oxidation is minimized. In contrast to the control, the oxidative stability of the P. chaba treated products was also higher. Altogether this study revealed that P. chaba could be utilized commercially, particularly in the food industry to preserve muscle foods. Practical Applications: Natural preservatives are becoming more popular as a result of the different carcinogenic and toxic side effects of conventional preservatives. P. chaba, an exquisite culinary herb in Bangladesh, has long been used as a traditional medicine, because of its antimicrobial and antioxidant properties. This study revealed that P. chaba can be utilized as a food preservative, which opens up new possibilities for its development and use in functional foods.

A Method to Quantify Drosophila Behavioral Activities Induced by GABAA Agonist Muscimol.

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria. As a potent GABAA receptor agonist, muscimol suppresses the activity of the central nervous system, reduces anxiety and induces sleep. We investigated the effects of muscimol on Drosophila behavior. Drosophila behavioral assays are powerful tools that are used to assess neural functions by focusing on specific changes in selected behavior, with the hypothesis that this behavioral change is due to alteration of the underlying neural function of interest. In this study, we developed a comparatively simple and cost-effective method for feeding adult flies muscimol, a pharmacologically active compound, and for quantifying the phenotypes of "resting" and "grooming+walking". This protocol may provide researchers with a convenient method to characterize small molecule-induced behavioral output in flies.

Cervical cancer in low-income countries: A Bangladeshi perspective.

Cervical cancer is the fourth most common cancer among women worldwide, with approximately 70% of cases involving infection with human papillomavirus (HPV) genotypes 16 and 18. According to International Agency for Research on Cancer, more than 50 million Bangladeshi women are at risk of developing cervical cancer, and 17 686 new cases and 10 362 deaths occur annually. If diagnosed at the precursor stage, however, cervical cancer is a condition that can be successfully treated. As a result, screening programs are necessary to identify the disease before it progresses to invasive cancer. In the present review, we discuss the overall situation of cervical cancer in Bangladesh, summarizing the sociodemographic status of affected women, associated risk factors, screening approaches, and treatment options. We emphasize the potential of visual inspection with acetic acid (VIA) as a cost-effective screening approach for detecting cervical lesions among poor women in the community. In a resource-limited country such as Bangladesh, VIA may represent an ideal model to build an effective awareness campaign through urban and rural hospitals, community-based clinics, and other health facilities available in industry.

Molecular Mechanisms Underlying Hepatocellular Carcinoma Induction by Aberrant NRF2 Activation-Mediated Transcription Networks: Interaction of NRF2-KEAP1 Controls the Fate of Hepatocarcinogenesis.

NF-E2-related factor 2 (NRF2) is a basic leucine zipper transcription factor, a master regulator of redox homeostasis regulating a variety of genes for antioxidant and detoxification enzymes. NRF2 was, therefore, initially thought to protect the liver from oxidative stress. Recent studies, however, have revealed that mutations in NRF2 cause aberrant accumulation of NRF2 in the nucleus and exert the upregulation of NRF2 target genes. Moreover, among all molecular changes in hepatocellular carcinoma (HCC), NRF2 activation has been revealed as a more prominent pathway contributing to the progression of precancerous lesions to malignancy. Nevertheless, how its activation leads to poor prognosis in HCC patients remains unclear. In this review, we provide an overview of how aberrant activation of NRF2 triggers HCC development. We also summarize the emerging roles of other NRF family members in liver cancer development.

Epidemiology, clinical features, and impact of food habits on the risk of hepatocellular carcinoma: A case-control study in Bangladesh.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer mortality worldwide. Infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) is the most predominant cause of HCC. Concerns arise for the presence of additional risk factors, as there is still a large proportion of patients without HBV or HCV infection. Previous studies have reported that higher intake of fruits and vegetables and reduced consumption of red/processed meat might play a protective role in HCC etiology, though the nationwide proof is limited. Hence, we studied multiple risk factors including food habit, lifestyle, and clinical implications of HCC patients in Bangladeshi. Demographic, clinical, and biochemical data, as well as data on food habits, were collected in this study. Our results indicated that a high intake of rice (AOR 4.28, 95% CI 1.48 to 14.07, p = 0.011), low intake of fruits (AOR = 4.41 95% CI 1.48-15.46; p = 0.012), leafy vegetables (AOR = 2.80, 95% CI 1.32-6.08; p = 0.008), and fish (AOR = 4.64 95% CI 2.18-10.23; p<0.001) increased the HCC risk. Moreover, a high intake of eggs (AOR = 2.07 95% CI 0.98-4.43; p = 0.058) also showed an increased risk. Roti, non-leafy vegetables, red meat, and tea were found to have no association with HCC risk. This study revealed that food habit patterns and lifestyle may have a profound effect on HCC development among Bangladeshi patients in addition to well established risk factors.

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges.

The world population is expected to increase from 7.3 to 9.7 billion by 2050. Pest outbreak and increased abiotic stresses due to climate change pose a high risk to tropical crop production. Although conventional breeding techniques have significantly increased crop production and yield, new approaches are required to further improve crop production in order to meet the global growing demand for food. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein9) genome editing technology has shown great promise for quickly addressing emerging challenges in agriculture. It can be used to precisely modify genome sequence of any organism including plants to achieve the desired trait. Compared to other genome editing tools such as zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), CRISPR/Cas9 is faster, cheaper, precise and highly efficient in editing genomes even at the multiplex level. Application of CRISPR/Cas9 technology in editing the plant genome is emerging rapidly. The CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crop plants to counteract harmful effects from climate change and ensure future food security of increasing population in tropical countries. This review updates current knowledge and potentials of CRISPR/Cas9 for improvement of crops cultivated in tropical climates to gain resiliency against emerging pests and abiotic stresses.

Soy undecapeptide induces Drosophila hind leg grooming via dopamine receptor.

ß-Conglycinin α subunit (323-333) [ßCGα(323-333)] is an exogenous neuromodulating undecapeptide found from enzymatic digest of ß-conglycinin, a soy major storage protein by mice behavior tests. We investigated effect of ßCGα(323-333) on Drosophila behavior. Oral administration of ßCGα(323-333) in Drosophila increased hind leg grooming, which may act through specific sets of neurons. It was reported that dopamine receptor (DopR) meditates hind leg grooming, and we tested involvement of DopR in ßCGα(323-333)-induced hind leg grooming by using DopR knockout flies. In the wild type but not in the DopR-knockout flies, ßCGα(323-333) increased hind leg grooming. These results suggest that ßCGα(323-333) induces hind leg grooming via activating the DopR. This is the first report showing that exogenously administered peptide changes fly behaviors.

Antioxidant, cytotoxic and antineoplastic effects of Carissa carandas Linn. leaves.

For scientific clarification of some traditional uses, this study was designed to explore the antioxidant, cytotoxic and antineoplastic properties of leaf extract of Carissa carandas Linn., a traditional medicinal plant of Bangladesh. The methanol extract of Carissa carandas leaves (MELC) was applied on DPPH and ABTS experiments to determine its antioxidant activity. In vitro the cytotoxic effect of MELC was evaluated against colonic adenocarcinoma cell lines (SW-480 and SW-48) whereas in vivo its antineoplastic property was tested against Ehrlich ascites carcinoma (EAC). The DPPH and ABTS assays revealed the antioxidant activity of MELC with IC50 10.5±1.2 and 1.75±0.3µg/ml that was comparable to L-ascorbic acid. In vitro cytotoxic study, MELC reduced the viability of adenocarcinoma cells in dose dependent manner and in vivo, administration of MELC (25mg/kg) resulted in a significant (p<0.05) decrease in viable EAC cell count thereby increasing the life span of the EAC cell bearing mice. Restoration of hematological parameters such as red blood cells (RBC), hemoglobin and white blood cells (WBC) to normal levels in MELC-treated mice was also observed. Moreover, treatment with MELC induced apoptosis of EAC cells as observed in fluorescence microscopic view of DAPI (4,6-diamidino-2-phenylindole) stained cells and also increased p53 gene expression MELC-treated cells in respect to untreated EAC control. In addition, the MELC was rich in polyphenol content and its GC-MS chromatogram confirmed the presence of some compounds all of which showed anticancer and cytotoxic activities in previous studies. In a word, this study supports the use of Carissa carandas in traditional medicine as well as highlights the need to further explore the potentials of MELC as an antineoplastic agent.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies.

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Constitutive activation of Drosophila CncC transcription factor reduces lipid formation in the fat body.

Accumulating evidence indicates that the vertebrate stress-response transcription factors Nrf1 and Nrf2 are involved in hepatic lipid metabolism. However, the underlying molecular mechanisms of Nrf1-and Nrf2-mediated lipid metabolism remain unclear. To elucidate the precise roles of Nrfs in this process, we analyzed the physiological role of CncC in lipid metabolism as a Drosophila model for vertebrate Nrf1 and Nrf2. We first examined whether CncC activity is repressed under physiological conditions through a species-conserved NHB1 (N-terminal homology box 1) domain, similar to that observed for Nrf1. Deletion of the NHB1 domain (CncCΔN) led to CncC-mediated rough-eye phenotypes and the induced expression of the CncC target gene gstD1 both in vivo and in vitro. Thus, we decided to explore how CncCΔN overexpression affects the formation of the fat body, which is the major lipid storage organ. Intriguingly, CncCΔN caused a significant reduction in lipid droplet size and triglyceride (TG) levels in the fat body compared to wild type. We found that CncCΔN induced a number of genes related to innate immunity that might have an effect on the regulation of cellular lipid storage. Our study provides new insights into the regulatory mechanism of CncC and its role in lipid homeostasis.

Whole mount immunolabeling of olfactory receptor neurons in the Drosophila antenna.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

The casein kinase 2-nrf1 axis controls the clearance of ubiquitinated proteins by regulating proteasome gene expression.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of human diseases, including neurodegenerative disorders. Thus, stimulating proteasome activity is a promising strategy to ameliorate these age-related diseases. Here we show that the protein kinase casein kinase 2 (CK2) regulates the transcriptional activity of Nrf1 to control the expression of the proteasome genes and thus the clearance of ubiquitinated proteins. We identify CK2 as an Nrf1-binding protein and find that the knockdown of CK2 enhances the Nrf1-dependent expression of the proteasome subunit genes. Real-time monitoring of proteasome activity reveals that CK2 knockdown alleviates the accumulation of ubiquitinated proteins upon proteasome inhibition. Furthermore, we identify Ser 497 of Nrf1 as the CK2 phosphorylation site and demonstrate that its alanine substitution (S497A) augments the transcriptional activity of Nrf1 and mitigates proteasome dysfunction and the formation of p62-positive juxtanuclear inclusion bodies upon proteasome inhibition. These results indicate that the CK2-mediated phosphorylation of Nrf1 suppresses the proteasome gene expression and activity and thus suggest that the CK2-Nrf1 axis is a potential therapeutic target for diseases associated with UPS impairment.

Chromatin modification of Notch targets in olfactory receptor neuron diversification.

Neuronal-class diversification is central during neurogenesis. This requirement is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. We discovered an epigenetic mechanism in which neuron diversity is maximized via locus-specific chromatin modifications that generate context-dependent responses from a single, generally used intracellular signal. Each ORN in Drosophila acquires one of three basic identities defined by the compound outcome of three iterated Notch signaling events during neurogenesis. Hamlet, the Drosophila Evi1 and Prdm16 proto-oncogene homolog, modifies cellular responses to these iteratively used Notch signals in a context-dependent manner, and controls odorant receptor gene choice and ORN axon targeting specificity. In nascent ORNs, Hamlet erases the Notch state inherited from the parental cell, enabling a modified response in a subsequent round of Notch signaling. Hamlet directs locus-specific modifications of histone methylation and histone density and controls accessibility of the DNA-binding protein Suppressor of Hairless at the Notch target promoter.

Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation. There are four main DA neuron classes (I-IV)(1). They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation(2-10). The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology(11-13) because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses. DA neuron activity modifies the output of a larval locomotion central pattern generator(14-16). The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses(14,16-20). Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)(21). These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field(7,22,23). Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping(7,22,23), as well as the wiring of a simple circuit modulating larval locomotion(14-17). We present here a practical guide to generate and analyze genetic mosaics(24) marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)(1,10,25) and Flp-out(22,26,27) techniques (summarized in Fig. 1).

Convergent local identity and topographic projection of sensory neurons.

Development of sensory neural circuits requires concurrent specification of neuron modality, position, and topographic projections. However, little is understood about how controls over these distinct parameters can unify in a single developmental sequence. To address this question, we have used the nociceptive class IV dendritic arborization neurons in the Drosophila larval body wall as an excellent model that allows precise spatiotemporal dissection of developmental-genetic control over sensory neuron positioning and wiring, and subsequent analysis of its functional significance for sensorimotor behavior. The class IV neurogenetic program is intrinsic to the anterior domain of the embryonic parasegment epithelium. Along the ventrolateral axis of this domain, nociceptive neuron induction requirements depend upon location. Near the ventral midline, both Hedgehog and Epithelial growth factor receptor signaling are required for class IV neurogenesis. In addition, close to the ventral midline, class IV neurogenesis is preceded by expression of the Iroquois factor Mirror that promotes local nociceptive neuron differentiation. Remarkably, Mirror is also required for the proper routing of class IV topographic axonal projections across the midline of the CNS. Manipulation of Mirror activity in class IV neurons retargeted axonal projections and caused concordant changes in larval nociceptive escape behavior. These findings indicate that convergent sensory neuron specification, local differentiation, and topographic wiring are mediated by Mirror, and they suggest an integrated paradigm for position-sensitive neural development.

Immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the Drosophila larva body wall.

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis(1), and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation(2-5) and degeneration(6). The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron(7,8). Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape(4,9-11). We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring (12,13). In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Evaluation of antitumour activity of Calotropis gigantea L. root bark against Ehrlich ascites carcinoma in Swiss albino mice.

OBJECTIVE: To investigate experimentally the possible antitumor effect of methanol extract (ME) of Calotropis gigantea L. (C. gigantean) root bark and its petroleum ether (PEF) and chloroform (CF) soluble fractions against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. METHODS: The effects of ME (10 and 20 mg/kg), PEF (40 and 80 mg/kg) and CF (20 and 40 mg/kg) on the growth of EAC and life span of EAC bearing mice were studied. Hematological profile and biochemical parameters (SALP, SGPT and SGOT) were also estimated. RESULTS: Results of in vivo study showed a significant decrease in viable tumor cell count and a significant increase of life span in the ME and CF treated group compared to untreated one. The life span of ME and CF treated animals was significantly (P<0.05) increased by 43.90% (20 mg ME/kg) and 57.07% (40 mg CF/kg). ME and CF brought back the hematological parameter more or less normal level. ME and CF also restored the altered levels of serum alkaline phosphatase (SALP) and serum glutamate oxaloacetate transaminase (SGOT). CONCLUSIONS: Methanol extract (ME) of C. gigantea root bark and its chloroform soluble fraction (CF) possesses significant antitumor activity.

Tagetes erecta Linn. and its mosquitocidal potency against Culex quinquefasciatus.

OBJECTIVE: To investigate mosquitocidal effects of ethanolic extract of flowers of Tagetes erecta (T. erecta) and its chloroform and petroleum ether soluble fractions against the larvae of Culex quinquefasciatus (Cx. quinquefasciatus). METHODS: The fresh flowers of T. erecta were extracted in cold with ethanol (5.0 L) and after concentration, the ethanol extract was fractionated with chloroform and petroleum ether to afford a brownish syrupy suspension of ethanol extract (50.0 g), petroleum ether soluble fraction (18.6 g) and chloroform soluble fraction (23.8 g). The larvicidal effect of ethanol extract and their solvent fractions were determined by the standard procedure of WHO against different instars of Cx. quinquefasciatus. RESULTS: Among the tested samples the chloroform soluble fractions showed the highest toxicity and consequently, the lowest LC50 values (14.14 µg/mL, 17.06 µg/mL, 36.88 µg/mL and 75.48 µg/mL) for all the instars larvae of Cx. quinquefasciatus. The larvae showed comparative tolerance in the course of increasing age and time. CONCLUSIONS: It can be concluded that the flowers of T. erecta are very effective natural larvicide and could be useful against Cx. quinquefasciatus.

Inhibition of Ehrlich ascites carcinoma by Manilkara zapota L. stem bark in Swiss albino mice.

OBJECTIVE: To evaluate the antitumor activity of Manilkara zapota (M. zapota) L. stem bark against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. METHODS: The in vivo antitumour activity of the ethyl acetate extract of stem bark of M. zapota L. (EASM) was evaluated at 50, 100 and 200 mg/kg bw against EAC using mean survival time. After administration of the extract of M. zapota, viable EAC cell count and body weight in the EAC tumour hosts were observed. The animal was also observed for improvement in the haematological parameters (e.g., heamoglobin content, red and white blood cells count and differential cell count) after EASM treatment. RESULTS: Intraperitoneal administration of EASM reduced viable EAC cells, increased the survival time, and restored altered haematological parameters. Significant efficacy was observed for EASM at 100 mg/kg dose (P<0.05). CONCLUSIONS: It can be concluded that the ethyl acetate extract of stem bark of M. zapota L. possesses significant antitumour activity.