Epinephrine Stimulates Mycobacterium tuberculosis Growth and Biofilm Formation.

The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this study, we screened several human catecholamine stress hormones (epinephrine, norepinephrine, and dopamine) for their effects on Mycobacterium growth. Our results showed that epinephrine significantly stimulated the growth of M. tuberculosis in the serum-based medium as well as macrophages. In silico analysis and molecular docking suggested that the extra-cytoplasmic domain of the MprB might be the putative adrenergic sensor. Furthermore, we showed that epinephrine significantly enhances M. tuberculosis biofilm formation, which has distinct texture composition, antibiotic resistance, and stress tolerance. Together, our data revealed the effect and mechanism of epinephrine on the growth and biofilm formation of M. tuberculosis, which contributes to the understanding of the environmental perception and antibiotic resistance of M. tuberculosis and provides important clues for the understanding of bacterial pathogenesis and the development of novel antibacterial therapeutics.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Strategies to package recombinant Adeno-Associated Virus expressing the N-terminal gasdermin domain for tumor treatment.

Pyroptosis induced by the N-terminal gasdermin domain (GSDMNT) holds great potential for anti-tumor therapy. However, due to the extreme cytoxicity of GSDMNT, it is challenging to efficiently produce and deliver GSDMNT into tumor cells. Here, we report the development of two strategies to package recombinant adeno-associated virus (rAAV) expressing GSDMNT: 1) drive the expression of GSDMNT by a mammal specific promoter and package the virus in Sf9 insect cells to avoid its expression; 2) co-infect rAAV-Cre to revert and express the double-floxed inverted GSDMNT. We demonstrate that these rAAVs can induce pyroptosis and prolong survival in preclinical cancer models. The oncolytic-viruses induce pyroptosis and evoke a robust immune-response. In a glioblastoma model, rAAVs temporarily open the blood-brain barrier and recruit tumor infiltrating lymphocytes into the brain. The oncolytic effect is further improved in combination with anti-PD-L1. Together, our strategies efficiently produce and deliver GSDMNT into tumor cells and successfully induce pyroptosis, which can be exploited for anti-tumor therapy.

Screening of Compounds for Anti-tuberculosis Activity, and in vitro and in vivo Evaluation of Potential Candidates.

Tuberculosis (TB) is a debilitating infectious disease responsible for more than one million deaths per year. The emergence of drug-resistant TB poses an urgent need for the development of new anti-TB drugs. In this study, we screened a library of over 4,000 small molecules and found that orbifloxacin and the peptide AK15 possess significant bactericidal activity against Mycobacterium tuberculosis (Mtb) in vitro. Orbifloxacin also showed an effective ability on the clearance of intracellular Mtb and protect mice from a strong inflammatory response but not AK15. Moreover, we identified 17 nucleotide mutations responsible for orbifloxacin resistance by whole-genome sequencing. A critical point mutation (D94G) of the DNA gyrase (gyrA) gene was found to be the key role of resistance to orbifloxacin. The computational docking revealed that GyrA D94G point mutation can disrupt the orbifloxacin-protein gyrase interactions mediated by magnesium ion bridge. Overall, this study indicated the potential ability of orbifloxacin as an anti-tuberculosis drug, which can be used either alone or in combination with first-line antibiotics to achieve more effective therapy on TB.

Rv3722c Promotes Mycobacterium tuberculosis Survival in Macrophages by Interacting With TRAF3.

Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-ß expression and decrease of IL-1ß, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.

Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing.

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

Author Correction: In vitro biological screening of a critically endangered medicinal plant, Atropa acuminata Royle Ex Lindl of north western Himalaya.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo pharmacological investigation of Monotheca buxifolia and Bosea amherstiana using animal models.

Experimental based evidence suggests that most of the medicinal plants possess wide-ranging pharmacological and biological activities that may possibly use in treatment of inflammation-related diseases. The current study was aimed to explore the acute toxicity, analgesic, sedative and antipyretic activities of Monotheca buxifolia and Bosea amherstiana in mices. In vivo experimental models were used in this study. Acute toxicity was evaluated for 24 h' interval at concentration of 500, 1000, 1500 and 2000 mg/kg. The analgesic activity was estimated by acetic acid induced writhing test. White wood apparatus enclosed in stainless steel was used for sedative experiment and antipyretic activity was evaluated in brewer's yeast induced hyperthermic at 50, 100 and 150 mg/kg i.p. Both plants were found safe at all tested doses. Monotheca buxifolia and Bosea amherstiana dose-dependently reduced abdominal constrictions in mice. Both plants exhibited significant (P < 0.0001) sedative effects in dose of 50, 150 and 150 mg/kg. Both plants markedly (P < 0.0001) reduced yeast induced hyperthermia. The inhibitions were dose-dependent and remained significant up to five hours of administration. These investigational results have linked a pharmacological indication for the traditional claim of the drugs to be used as an anti-inflammatory, analgesics and antipyretic agents.

Antimicrobial potentials of medicinal plant's extract and their derived silver nanoparticles: A focus on honey bee pathogen.

Infectious (or Communicable) diseases are not only the past but also the present problem in developing as well as developed countries. It is caused by various pathogenic microbes like fungi, bacteria, parasites and virus etc. The medicinal plants and nano-silver have been used against the pathogenic microbes. Herbal medicines are generally used for healthcare because they have low price and wealthy source of antimicrobial properties. Like medicinal plants, silver nanoparticles also have emergent applications in biomedical fields due to their immanent therapeutic performance. Here, we also explore the various plant parts such as bark, stem, leaf, fruit and seed against Gram negative and Gram-positive bacteria, using different solvents for extraction i.e. methanol, ethyl acetate, chloroform, acetone, n. hexane, butanol, petroleum ether and benzene. Since ancient to date most of the countries have been used herbal medicines, but in Asia, some medicinal plants are commonly used in rural and backward areas as a treatment for infectious diseases. In this review, we provide simple information about medicinal plants and Silver nanoparticles with their potentialities such as antiviral, bactericidal and fungicidal. Additionally, the present review to highlights the versatile applications of medicinal plants against honey bee pathogen such as fungi (Ascosphaera apis), mites (Varroa spp. and Tropilaelaps sp.), bacteria (Melissococcus plutonius Paenibacillus larvae), and microsporidia (Nosema apis and Nosema ceranae). In conclusion, promising nonchemical (plant extracts) are innocuous to adult bees. So, we strongly believed that this effort was made to evaluate the status of medicinal plants researches globally.

Nano-biotechnology: a new approach to treat and prevent malaria.

Malaria, the exterminator of ~1.5 to 2.7 million human lives yearly, is a notorious disease known throughout the world. The eradication of this disease is difficult and a challenge to scientists. Vector elimination and effective chemotherapy for the patients are key tactics to be used in the fight against malaria. However, drug resistance and environmental and social concerns are the main hurdles in this fight against malaria. Overcoming these limitations is the major challenge for the 21st-century malarial researchers. Adapting the principles of nano-biotechnology to both vector control and patient therapy is the only solution to the problem. Several compounds such as lipids, proteins, nucleic acid and metallic nanoparticles (NPs) have been successfully used for the control of this lethal malaria disease. Other useful natural reagents such as microbes and their products, carbohydrates, vitamins, plant extracts and biodegradable polymers, are also used to control this disease. Among these particles, the plant-based particles such as leaf, root, stem, latex, and seed give the best antagonistic response against malaria. In the present review, we describe certain efforts related to the control, prevention and treatment of malaria. We hope that this review will open new doors for malarial research.

In vitro biological screening of a critically endangered medicinal plant, Atropa acuminata Royle Ex Lindl of north western Himalaya.

Atropa acuminata Royle Ex Lindl (Atropa acuminata) under tremendous threat of extinction in its natural habitat. However, the antimicrobial, antileishmanial and anticancer effects of the plant's extracts have not been reported yet. In the current study, an attempt has been made to evaluate the pharmacological potential of this plant's extracts against microbes, Leishmania and cancer. The roots, stems and leaves of Atropa acuminata were ground; then, seven different solvents were used alone and in different ratios to prepare crude extracts, which were screened for pharmacological effects. The aqueous, methanolic and ethanolic extracts of all parts carried a broad spectrum of anti-bacterial activities, while no significant activity was observed with combined solvents. Three types of cytotoxicity assays were performed, i.e., haemolytic, brine shrimp and protein kinase assays. The aqueous extract of all the parts showed significant haemolytic activity while n-hexane extracts of roots showed significant activity against brine shrimp. The acetone extracts strongly inhibited protein kinase while the methanolic extracts exhibited significant cytotoxic activity of roots and stem. The anti-leishmanial assays revealed that the methanolic extract of leaves and roots showed significant activity. These findings suggest that this plant could be a potential source of natural product based drugs.

Honey: Single food stuff comprises many drugs.

Honey is a natural food item produced by honey bees. Ancient civilizations considered honey as a God gifted prestigious product. Therefore, a huge literature is available regarding honey importance in almost all religions. Physically, honey is a viscous and jelly material having no specific color. Chemically, honey is a complex blend of many organic and inorganic compounds such as sugars, proteins, organic acids, pigments, minerals, and many other elements. Honey use as a therapeutic agent is as old as human civilization itself. Prior to the appearance of present day drugs, honey was conventionally used for treating many diseases. At this instant, the modern research has proven the medicinal importance of honey. It has broad spectrum anti-biotic, anti-viral and anti-fungal activities. Honey prevents and kills microbes through different mechanism such as elevated pH and enzyme activities. Till now, no synthetic compound that works as anti-bacterial, anti-viral and anti-fungal drugs has been reported in honey yet it works against bacteria, viruses and fungi while no anti-protozoal activity has been reported. Potent anti-oxidant, anti-inflammatory and anti-cancerous activities of honey have been reported. Honey is not only significant as anti-inflammatory drug that relieve inflammation but also protect liver by degenerative effects of synthetic anti-inflammatory drugs. This article reviews physico-chemical properties, traditional use of honey as medicine and mechanism of action of honey in the light of modern scientific medicinal knowledge.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Potassium and zinc increase tolerance to salt stress in wheat (Triticum aestivum L.).

Potassium and zinc are essential elements in plant growth and metabolism and plays a vital role in salt stress tolerance. To investigate the physiological mechanism of salt stress tolerance, a pot experiment was conducted. Potassium and zinc significantly minimize the oxidative stress and increase root, shoot and spike length in wheat varieties. Fresh and dry biomass were significantly increased by potassium followed by zinc as compared to control C. The photosynthetic pigment and osmolyte regulator (proline, total phenolic, and total carbohydrate) were significantly enhanced by potassium and zinc. Salt stress increases MDA content in wheat varieties while potassium and zinc counteract the adverse effect of salinity and significantly increased membrane stability index. Salt stress decreases the activities of antioxidant enzymes (superoxide dismutase, catalase and ascorbate peroxidase) while the exogenous application of potassium and zinc significantly enhanced the activities of these enzymes. A significant positive correlation was found of spike length with proline (R2 = 0.966 ∗∗∗), phenolic (R2 = 0.741∗) and chlorophyll (R2 = 0.853∗∗). The MDA content showed significant negative correlation (R2 = 0.983∗∗∗) with MSI. It is concluded that potassium and zinc reduced toxic effect of salinity while its combine application showed synergetic effect and significantly enhanced salt tolerance.

Hyperbilirubinemia in Neonates: Types, Causes, Clinical Examinations, Preventive Measures and Treatments: A Narrative Review Article.

BACKGROUND: Hyperbilirubinemia, or jaundice, is a life threatening disorder in newborns. It is a multifactorial disorder with many symptoms. Generally, the physiological jaundice is the most prevalent type however in some regions pathological jaundice is also common. This review article focuses on a brief introduction to jaundice, its types and causes, measuring the bilirubin level, clinical approaches towards hyperbilirubinemia, different precautionary measures for the parents of babies suffering from hyperbilirubinemia and different remedial therapeutic measures for its treatment. METHODS: The main databases including Scopus, Pubmed, MEDLINE, Google scholar and Science Direct were researched to obtain the original papers related to the newborns' hyperbilirubinemia. The main terms used to literature search were "newborns' hyperbilirubinemia", "newborns' jaundice", "Physiological Jaundice" and "Patholigical Jaundice". The timeframe included the obtained articles was from 1952 to 2015. RESULTS: Neonatal jaundice due to breast milk feeding is also sometimes observed. Hemolytic jaundice occurs because of the incompatibility of blood groups with ABO and Rh factors, when the fetus and mother blood groups are not compatible and the fetus blood crosses the barrier of the umbilical cord before birth causing fetus blood hemolysis owing to severe immune response. CONCLUSION: Jaundice is easily diagnosable however require quick and on the spot treatment. If not treated properly, it leads to many complications. Currently the treatment options for jaundice include photo therapy, chemotherapy, and vaccinations.