Insights into Oropharyngeal Microbiota, Biofilms and Associated Diseases from Metagenomics and Transcriptomic Approaches

Oral cavity is an ecologically complex environment and hosts a diverse microbial community. Most of these organisms are commensals, however, on occasion, some have the potential to become pathogenic causing damage to the human host. Complex interactions between pathogenic bacteria, the microbiota, and the host can modify pathogen physiology and behavior. Most bacteria in the environment do not exist in free-living state but are found as complex matrix enclosed aggregates known as biofilms. There has been research interest in microbial biofilms because of their importance in industrial and biomedical settings. Bacteria respond to environmental cues to fine-tune the transition from planktonic growth to biofilm by directing gene expression changes favorable for sessile community establishment. Meta-approaches have been used to identify complex microbial associations within human oral cavity leading to important insights. Comparative gene expression analysis using deep sequencing of RNA and metagenomics studies done under varying conditions have been successfully used in understanding and identifying possible triggers of pathogenicity and biofilm formation in oral commensals

Wireless Autonomous Communication and Energy Transfer

The feasibility study of a harmonic RFID chipless sensing tag is presented. The tag sensor is composed by an antenna at f0 = 1:2 GHz, a Schottky diode (HSMS2850), a balanced impedances bridge with a sensing variable element, and a second antenna working at 2f0 = 2:4 GHz. When interrogated with a signal at f0 the tag responds with a signal at 2f0 only when the bridge is unbalanced due to a variation of the sensible impedance value of at least 5. With this approach the system is energetically autonomous, being activated by the received signal and responds only when there is a change in the sensed quantity. No IC for carrier modulation is required to transfer the information

Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation

The spectrum of tumors that arise owing to the overexpression of c-Myc and loss of BLM is very similar. Hence, it was hypothesized that the presence of BLM negatively regulates c-Myc functions. By using multiple isogenic cell lines, we observed that the decrease of endogenous c-Myc levels that occurs in the presence of BLM is reversed when the cells are treated with proteasome inhibitors, indicating that BLM enhances c-Myc turnover. Whereas the N-terminal region of BLM interacts with c-Myc, the rest of the helicase interacts with the c-Myc E3 ligase Fbw7. The two BLM domains act as ‘clamp and/or adaptor’, enhancing the binding of c-Myc to Fbw7. BLM promotes Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase-independent manner. A subset of BLM-regulated genes that are also targets of c-Myc were determined and validated at both RNA and protein levels. To obtain an in vivo validation of the effect of BLM on c-Myc-mediated tumor initiation, isogenic cells from colon cancer cells that either do or do not express BLM had been manipulated to block c-Myc expression in a controlled manner. By using these cell lines, the metastatic potential and rate of initiation of tumors in nude mice were determined. The presence of BLM decreases c-Myc-mediated invasiveness and delays tumor initiation in a mouse xenograft model. Consequently, in tumors that express BLM but not c-Myc, we observed a decreased ratio of proliferation to apoptosis together with a suppressed expression of the angiogenesis marker CD31. Hence, partly owing to its regulation of c-Myc stability, BLM acts as a ‘caretaker tumor suppressor’

Evaluating the fabric performance and antibacterial properties of 3-D piezoelectric spacer fabric

The increasing need of on-demand power for enabling portable low-power devices and sensors has necessitated work in novel energy harvesting materials and devices. In a recent work, we demonstrated the production and suitability of three-dimensional (3-D) spacer all fibre piezoelectric textiles for converting mechanical energy into electrical energy for wearable and technical applications. The current work investigates the textile performance properties of these 3-D piezoelectric fabrics including porosity, air permeability, water vapour transmission and bursting strength. Furthermore, as these textiles are intended for wearable applications, we have assessed their wear abrasion and consequently provide surface resistance measurements which can affect the lifetime and efficiency of charge collection in the piezoelectric textile structures. The results show that the novel smart fabric with a measured porosity of 68% had good air (1855 l/m2/s) and water vapour permeability (1.34 g/m2/day) values, good wear abrasion resistance over 60,000 rotations applied by a load of 12 kPa and bursting strength higher than 2400 kPa. Moreover, the antibacterial activity of 3-D piezoelectric fabrics revealed that owing to the use of Ag/PA66 yarns, the textiles exhibit excellent antibacterial activity against not only Gram-negative bacteria E. coli but they are also capable of killing antibiotic methicillin-resistant bacteria S. aureus

Daughter Cell Separation by Penicillin-Binding Proteins and Peptidoglycan Amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains

Role of Peptidoglycan Amidases in the Development and Morphology of the Division Septum in Escherichia coli▿ †

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan

Structural and functional mapping of ars gene cluster in Deinococcus indicus DR1

Deinococcus indicus DR1 is a novel Gram-negative bacterium, isolated from the Dadri wetlands in Uttar Pradesh, India. In addition to being radiation-resistant, the rod-shaped, red-pigmented organism shows extraordinary resistance to arsenic. The proteins of the corresponding ars gene cluster involved in arsenic extrusion in D. indicus DR1 have not yet been characterized. Additionally, how these proteins regulate each other providing arsenic resistance is still unclear. Here, we present a computational model of the operonic structure and the corresponding characterization of the six proteins of the ars gene cluster in D. indicus DR1. Additionally, we show the expression of the genes in the presence of arsenic using qRT-PCR. The ars gene cluster consists of two transcriptional regulators (ArsR1, ArsR2), two arsenate reductases (ArsC2, ArsC3), one metallophosphatase family protein (MPase), and a transmembrane arsenite efflux pump (ArsB). The transcriptional regulators are trans-acting repressors, and the reductases reduce arsenate (As5+) ions to arsenite (As3+) ions for favourable extrusion. The proteins modelled using RoseTTAFold, and their conformationally stable coordinates obtained after MD simulation indicate their various functional roles with respect to arsenic. Excluding ArsB, all the proteins belong to the α + β class of proteins. ArsB, being a membrane protein, is fully α-helical, with 12 transmembrane helices. The results show the degree of similarity or divergence of the mechanism utilized by these proteins of ars gene cluster in D. indicus DR1 to confer high levels of arsenic tolerance. This structural characterization study of the ars genes will enable new and deeper insights of arsenic tolerance

Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR1

Deinococcus radiodurans exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus strain exhibited cell-morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from D. indicus cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in D. indicus