Microbial lipopeptides: Properties, mechanics and engineering for novel lipopeptides.

Microorganisms produce active surface agents called lipopeptides (LPs) which are amphiphilic in nature. They are cyclic or linear compounds and are predominantly isolated from Bacillus and Pseudomonas species. LPs show antimicrobial activity towards various plant pathogens and act by inhibiting the growth of these organisms. Several mechanisms are exhibited by LPs, such as cell membrane disruption, biofilm production, induced systematic resistance, improving plant growth, inhibition of spores, etc., making them suitable as biocontrol agents and highly advantageous for industrial utilization. The biosynthesis of lipopeptides involves large multimodular enzymes referred to as non-ribosomal peptide synthases. These enzymes unveil a broad range of engineering approaches through which lipopeptides can be overproduced and new LPs can be generated asserting high efficacy. Such approaches involve several synthetic biology systems and metabolic engineering techniques such as promotor engineering, enhanced precursor availability, condensation domain engineering, and adenylation domain engineering. Finally, this review provides an update of the applications of lipopeptides in various fields.

Novel cancer gene discovery using a forward genetic screen in RCAS-PDGFB-driven gliomas.

BACKGROUND: Malignant gliomas, the most common malignant brain tumors in adults, represent a heterogeneous group of diseases with poor prognosis. Retroviruses can cause permanent genetic alterations that modify genes close to the viral integration site. METHODS: Here we describe the use of a high-throughput pipeline coupled to the commonly used tissue-specific retroviral RCAS-TVA mouse tumor model system. Utilizing next-generation sequencing, we show that retroviral integration sites can be reproducibly detected in malignant stem cell lines generated from RCAS-PDGFB-driven glioma biopsies. RESULTS: A large fraction of common integration sites contained genes that have been dysregulated or misexpressed in glioma. Others overlapped with loci identified in previous glioma-related forward genetic screens, but several novel putative cancer-causing genes were also found. Integrating retroviral tagging and clinical data, Ppfibp1 was highlighted as a frequently tagged novel glioma-causing gene. Retroviral integrations into the locus resulted in Ppfibp1 upregulation, and Ppfibp1-tagged cells generated tumors with shorter latency on orthotopic transplantation. In human gliomas, increased PPFIBP1 expression was significantly linked to poor prognosis and PDGF treatment resistance. CONCLUSIONS: Altogether, the current study has demonstrated a novel approach to tagging glioma genes via forward genetics, validating previous results, and identifying PPFIBP1 as a putative oncogene in gliomagenesis.

An Insight into Efflux-Mediated Arsenic Resistance and Biotransformation Potential of Enterobacter Cloacae RSC3 from Arsenic Polluted Area.

Indiscriminate discharge of heavy metals/metalloids from different sources into the sustainable agro-ecosystem is a major global concern for food security and human health. Arsenic (As), categorized as group one human carcinogen is a quintessential toxic metalloid that alters the microbial compositions and functions, induce physiological and metabolic changes in plants and contaminate surface/ground water. The management of arsenic toxicity, therefore, becomes imminent. Acknowledging the arsenic threat, the study was aimed at identifying arsenic resistant bacteria and evaluating its arsenic removal/detoxification potential. Of the total 118 bacterial isolates recovered from arsenic rich environment, the bacterial strain RSC3 demonstrating highest As tolerance was identified as Enterobacter cloacae by 16S rRNA gene sequence analysis. Enterobacter cloacae tolerated high concentration (6000 ppm) of As and exhibited 0.55 h-1 of specific growth rate as calculated from growth kinetics data. Strain RSC3 also displayed varying level of resistance to other heavy metals and many antibacterial drugs in plate bioassay. The bacterial strain RSC3 possessed gene (arsC) which causes transformation of arsenate to arsenite. The arsenate uptake and efflux of the bacterial cells was revealed by high throughput techniques such as AAS, SEM/TEM and EDX. The simultaneous As reducing ability, and multi metal/multi-antibiotics resistance potentials of E. cloacae provides a promising option in the microbes based remediation of As contaminated environments.

Concrete Crack Restoration Using Bacterially Induced Calcium Metabolism.

Concrete structures are prone to develop cracks and cause devastation. Repair and renovation are not enough to ensure complete eradication of crack development. The entire process is costly and laborious. The microbiologically induced calcium carbonated precipitation can be effective in restoring the cracks. The calcium-based nutrients along with specific bacterial strain have been used in the present investigation. The pellets of calcium as per Fourier transform infrared spectroscopy and energy-dispersive X-ray spectroscopy are deposited in the cracks of the concrete over a period of 7 days of incubation. The presence of bacteria in the calcium precipitates as demonstrated by scanning electron microscope provides adequate strength and adhering quality to the pellets. The effective filling of cracks is confirmed with the help ultrasonic pulse velocity test also. Since, elephantine heritage and high sky buildings have high maintenance costs, the use of present technique will cut down the cost and duration of restoration. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12088-020-00916-0) contains supplementary material, which is available to authorized users.

The polyamine transporter Slc18b1(VPAT) is important for both short and long time memory and for regulation of polyamine content in the brain.

SLC18B1 is a sister gene to the vesicular monoamine and acetylcholine transporters, and the only known polyamine transporter, with unknown physiological role. We reveal that Slc18b1 knock out mice has significantly reduced polyamine content in the brain providing the first evidence that Slc18b1 is functionally required for regulating polyamine levels. We found that this mouse has impaired short and long term memory in novel object recognition, radial arm maze and self-administration paradigms. We also show that Slc18b1 KO mice have altered expression of genes involved in Long Term Potentiation, plasticity, calcium signalling and synaptic functions and that expression of components of GABA and glutamate signalling are changed. We further observe a partial resistance to diazepam, manifested as significantly lowered reduction in locomotion after diazepam treatment. We suggest that removal of Slc18b1 leads to reduction of polyamine contents in neurons, resulting in reduced GABA signalling due to long-term reduction in glutamatergic signalling.

Flower Shaped Gold Nanoparticles: Biogenic Synthesis Strategies and Characterization.

Microbes can serve as mediators for the fabrication of complicated nano-structures, obviating the tedious and time-consuming methods of synthesis. The shape of a nanoparticle has a very prominent role in defining the functionality in prospective arenas. So, the flower shaped nanoparticles are in focus nowadays due to their enhanced electrocatalytic and optical properties as compared to the spherical ones. We present the biosynthesis of flower shaped gold nanoparticles by Bacillus subtilis RSB64 and process parameters optimization using central composite design. The two well-separated scattering spectra showing absorption peaks at 540 nm and 750 nm indicate the presence of anisotropic gold nanoparticles and the results were corroborated by transmission electron microscopy analysis. The presence of gold nanoparticles was further confirmed by energy dispersive X-ray studies. The functional groups responsible for the stability of gold nanoparticles were predicted by Fourier transform infrared spectroscopy. The gold nanoparticles biosynthesis were collective effects of three experimental process parameters viz pH, temperature and precursor concentration. These three parameters were statistically optimized wherein pH 11.0, substrate concentration 1:1 (v/v) and temperature of 50 °C resulted in the synthesis of stable flower shaped gold nanoparticles of 50 nm size. The results indicated the tailored biosynthesis of gold nanoparticles with a flower like morphology by multi process parameter analysis to finalize robust conditions for the synthesis using B. subtilis RSB64. These gold nanoflowers demonstrate increased surface area efficiency/reactivity and could be employed for sustained and controlled delivery of drugs.

Ciprofloxacin Functionalized Biogenic Gold Nanoflowers as Nanoantibiotics Against Pathogenic Bacterial Strains.

PURPOSE: Antibiotics are currently being rendered non-functional by the rising incidence of multi-drug resistance amongst pathogenic bacteria. Research has now been focused on developing solutions to this problem by creating new antibiotics and enhancing the functionality of the existing ones. PATIENTS AND METHODS: In the present study, ciprofloxacin was conjugated to biogenic gold nanoflowers (GNFs) from Bacillus subtilis RSB64 by a robust adsorption method under optimized conditions. The resultant drug-nanoflower conjugate was characterized by UV-visible spectroscopy and Fourier transform infrared spectroscopy (FTIR). Addition of ciprofloxacin to gold nanoflowers changed the extinction spectrum towards longer wavelength. The ciprofloxacin-conjugated gold nanoflowers were tested for the drug release statistically. The prepared nanoflower-drug conjugate was subjected to an in vitro microbiological assay against different Gram-positive and Gram-negative bacterial strains to verify the effect of GNF-ciprofloxacin conjugate on the cell growth inhibitory activity of ciprofloxacin. RESULTS: The GNF-ciprofloxacin conjugates demonstrated enhanced bactericidal activity against Gram-negative bacteria as compared to Gram-positive. The enhancement of the antibacterial activity of the nanoflower-drug conjugate could be attributed to the interaction of the conjugate with phosphate/amine group of the outer membrane of Gram-negative bacterial cell wall making them susceptible to the antibacterial effect of the conjugate. CONCLUSION: This study demonstrates the positive attributes of GNF-ciprofloxacin conjugates as a promising antibacterial therapeutic agent against pathogens.

Mouse Models of Pediatric Supratentorial High-grade Glioma Reveal How Cell-of-Origin Influences Tumor Development and Phenotype.

High-grade glioma (HGG) is a group of primary malignant brain tumors with dismal prognosis. Whereas adult HGG has been studied extensively, childhood HGG, a relatively rare disease, is less well-characterized. Here, we present two novel platelet-derived growth factor (PDGF)-driven mouse models of pediatric supratentorial HGG. Tumors developed from two different cells of origin reminiscent of neural stem cells (NSC) or oligodendrocyte precursor cells (OPC). Cross-species transcriptomics showed that both models are closely related to human pediatric HGG as compared with adult HGG. Furthermore, an NSC-like cell-of-origin enhanced tumor incidence, malignancy, and the ability of mouse glioma cells (GC) to be cultured under stem cell conditions as compared with an OPC-like cell. Functional analyses of cultured GC from these tumors showed that cells of NSC-like origin were more tumorigenic, had a higher rate of self-renewal and proliferation, and were more sensitive to a panel of cancer drugs compared with GC of a more differentiated origin. These two mouse models relevant to human pediatric supratentorial HGG propose an important role of the cell-of-origin for clinicopathologic features of this disease. Cancer Res; 77(3); 802-12. ©2016 AACR.

Multimodal Eph/Ephrin signaling controls several phases of urogenital development.

A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.

mRNA GPR162 changes are associated with decreased food intake in rat, and its human genetic variants with impairments in glucose homeostasis in two Swedish cohorts.

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating intercellular interactions of fundamental physiological importance for survival including regulation of food intake, blood pressure, and hormonal sensing signaling, among other roles. Homeostatic alterations in the physiological status of GPCRs are often associated with underlying causes of disease, and to date, several orphan GPCRs are still uncharacterized. Findings from our previous study demonstrate that the Rhodopsin family protein GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in hypothalamus, amygdala, and ventral tegmental area, regions strictly interconnected and involved in the regulation of energy homeostasis and hedonic feeding. In this study, we provide a further anatomical characterization of GPR162 in mouse brain via in situ hybridization as well as detailed mRNA expression in a panel of rat tissues complementing a specie-specific mapping of the receptor. We also provide an attempt to demonstrate a functional implication of GPR162 in food intake-related behavior via antisense knockdown studies. Furthermore, we performed human genetic studies in which for the first time, variants of the GPR162 gene were associated with impairments in glucose homeostasis.

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

Transport of L-glutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.

Glutamine transporters are important for regulating levels of glutamate and GABA in the brain. To date, six members of the SLC38 family (SNATs) have been characterized and functionally subdivided them into System A (SNAT1, SNAT2 and SNAT4) and System N (SNAT3, SNAT5 and SNAT7). Here we present the first functional characterization of SLC38A8, one of the previous orphan transporters from the family, and we suggest that the encoded protein should be named SNAT8 to adhere with the SNAT nomenclature. We show that SLC38A8 has preference for transporting L-glutamine, L-alanine, L-arginine, L-histidine and L-aspartate using a Na+-dependent transport mechanism and that the functional characteristics of SNAT8 have highest similarity to the known System A transporters. We also provide a comprehensive central nervous system expression profile in mouse brain for the Slc38a8 gene and the SNAT8 protein. We show that Slc38a8 (SNAT8) is expressed in all neurons, both excitatory and inhibitory, in mouse brain using in situ hybridization and immunohistochemistry. Furthermore, proximity ligation assay shows highly similar subcellular expression of SNAT7 and SNAT8. In conclusion, the neuronal SLC38A8 has a broad amino acid transport profile and is the first identified neuronal System A transporter. This suggests a key role of SNAT8 in the glutamine/glutamate (GABA) cycle in the brain.

The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus.

The Rhodopsin family of G protein coupled receptors (GPCRs) includes the phylogenetic α-group consisting of about 100 human members. The α-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempt to study its functional role. We identified the homologue of Gpr153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 and Gpr153 share a common ancestor that split most likely through a duplication event before the divergence of the tetrapods and the teleost lineage. A quantitative real-time PCR study reveals widespread expression of Gpr153 in the central nervous system and all the peripheral tissues investigated. Detailed in situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of Gpr153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and primary functional properties of the Gpr153 gene.

Identification of SLC38A7 (SNAT7) protein as a glutamine transporter expressed in neurons.

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.

Long evolutionary conservation and considerable tissue specificity of several atypical solute carrier transporters.

The superfamily of Solute Carriers (SLCs) has around 384 members in the human genome grouped into at least 48 families. While many of these transporters have been well characterized with established important biological functions, there are few recently identified genes that are not studied regarding tissue distribution or evolutionary origin. Here we study 14 of these recently discovered SLC genes (HIAT1, HIATL1, MFSD1, MFSD5, MFSD6, MFSD9, MFSD10, SLC7A14, SLC7A15, SLC10A6, SLC15A5, SLC16A12, SLC30A10 and SLC21A21) with the purpose to give much better picture over the sequence relationship and tissue expression of the diverse SLC gene family. We used a range of bioinformatic methods to classify each of these genes into the different SLC gene families. We found that 9 of the 14 atypical SLCs are distant members of the Major Facilitator Superfamily (MFS) clan while the others belong to the APC clan, the DMT clan, the CPA_AT clan and the IT clan. We found most of the genes to be highly evolutionary conserved, likely to be present in most bilateral species, except for SLC21A21 that we found only present in mammals. Several of these transporter genes have highly specific tissue expression profile while it is notable that most are expressed in the CNS with the exception of SLC21A21 and SLC15A5. This work provides fundamental information on 14 transporters that previously have not received much attention enabling a more comprehensive view over the SLC superfamily.

The obesity gene, TMEM18, is of ancient origin, found in majority of neuronal cells in all major brain regions and associated with obesity in severely obese children.

BACKGROUND: TMEM18 is a hypothalamic gene that has recently been linked to obesity and BMI in genome wide association studies. However, the functional properties of TMEM18 are obscure. METHODS: The evolutionary history of TMEM18 was inferred using phylogenetic and bioinformatic methods. The gene's expression profile was investigated with real-time PCR in a panel of rat and mouse tissues and with immunohistochemistry in the mouse brain. Also, gene expression changes were analyzed in three feeding-related mouse models: food deprivation, reward and diet-induced increase in body weight. Finally, we genotyped 502 severely obese and 527 healthy Swedish children for two SNPs near TMEM18 (rs6548238 and rs756131). RESULTS: TMEM18 was found to be remarkably conserved and present in species that diverged from the human lineage over 1500 million years ago. The TMEM18 gene was widely expressed and detected in the majority of cells in all major brain regions, but was more abundant in neurons than other cell types. We found no significant changes in the hypothalamic and brainstem expression in the feeding-related mouse models. There was a strong association for two SNPs (rs6548238 and rs756131) of the TMEM18 locus with an increased risk for obesity (p = 0.001 and p = 0.002). CONCLUSION: We conclude that TMEM18 is involved in both adult and childhood obesity. It is one of the most conserved human obesity genes and it is found in the majority of all brain sites, including the hypothalamus and the brain stem, but it is not regulated in these regions in classical energy homeostatic models.

Glutamate, aspartate and nucleotide transporters in the SLC17 family form four main phylogenetic clusters: evolution and tissue expression.

BACKGROUND: The SLC17 family of transporters transports the amino acids: glutamate and aspartate, and, as shown recently, also nucleotides. Vesicular glutamate transporters are found in distinct species, such as C. elegans, but the evolutionary origin of most of the genes in this family has been obscure. RESULTS: Our phylogenetic analysis shows that the SLC17 family consists of four main phylogenetic clades which were all present before the divergence of the insect lineage. One of these clades has not been previously described and it is not found in vertebrates. The clade containing Slc17a9 had the most restricted evolutionary history with only one member in most species. We detected expression of Slc17a1-17a4 only in the peripheral tissues but not in the CNS, while Slc17a5- Slc17a9 are highly expressed in both the CNS and periphery. CONCLUSIONS: The in situ hybridization studies on vesicular nucleotide transporter revealed high expression throughout the cerebral cortex, certain areas in the hippocampus and in specific nuclei of the hypothalamus and thalamus. Some of the regions with high expression, such as the medial habenula and the dentate gyrus of the hippocampus, are important sites for purinergic neurotransmission. Noteworthy, other areas relying on purine-mediated signaling, such as the molecular layer of the dentate gyrus and the periaqueductal gray, lack or have a very low expression of Slc17a9, suggesting that there could be another nucleotide transporter in these regions.