Burn Wound Healing Abilities of a Uronic Acid Containing Exopolysaccharide Produced by the Marine Bacterium Halomonas malpeensis YU-PRIM-29 T.

Bacterial exopolysaccharides (EPS) are an emerging class of biopolymers with extensive applications in different fields due to their versatile physico-chemical and biological properties. The role of EPS in healing of different wound types is gaining interest in the tissue engineering sector. Burn is one of the devitalizing injuries that causes greater physical harm and can be fatal. Appropriate treatment modalities have to be followed for faster healing outcomes and to minimize the risk. In this study, a bacterial EPS (EPS-H29) from the marine bacterium Halomonas malpeensis YU-PRIM-29 T was used to treat the burn wound in vivo. The biochemical and structural characterizations of EPS-H29 were carried out using standard methods. In addition, FE-SEM, conformational, rheological, and HP-GPC analyses were carried out. In vitro biocompatibility of EPS-H29 was studied in human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). Scratch assay was used to study the wound healing in vitro. For in vivo evaluation, burn wound (second-degree) was created on Wistar albino rats and treated with EPS-H29 along with appropriate control groups. The total sugar and protein contents of EPS-H29 were 72.0 ± 1.4% and 4.0 ± 0.5%, respectively, with a molecular weight of 5.2 × 105 Da. The lyophilized samples exhibited porous surface features, and in solution, it showed triple helical conformation and shear thickening behavior. In vitro cell-based assays showed biocompatibility of EPS-H29 up to 200 µg/mL concentration. At a concentration up to 50 µg/mL, EPS-H29 promoted cell proliferation. Significant increase in the HDF cell migration was evident with EPS-H29 (15 µg/mL) treatment in vitro and induced significantly higher (p ≤ 0.0001) closure of the scratch area (90.3 ± 1.1%), compared to the control (84.3 ± 1.3%) at 24 h. Enhanced expression of Ki-67 was associated with the cell proliferative activities of EPS-H29. The animals treated with EPS-H29 showed improved healing of burn wounds with significantly higher wound contraction rate (80.6 ± 9.4%) compared to the positive control (54.6 ± 8.0%) and untreated group (49.2 ± 3.7%) with histopathological evidence of epidermal tissue formation at 15 days of treatment. These results demonstrate the biocompatibility and burn wound healing capability of EPS-H29 and its potential as an effective topical agent for the burn wound care.

Biotechnological interventions for the production of forskolin, an active compound from the medicinal plant, Coleus forskohlii.

Coleus forskohlii, an Indian-origin medicinal plant is the sole natural source of the labdane terpenoid forskolin (C22H34O7), with growing demand. Forskolin emerged as an industrially important bioactive compound, with many therapeutic applications in human health. It has established potential effects in the treatment of various diseases and conditions such as glaucoma, asthma, obesity, allergies, skin conditions and cardiovascular diseases. Moreover, clinical trials against different types of cancers are progressing. The mechanism of action of forskolin mainly involves activating adenylyl cyclase and elevating cAMP, thereby regulating different cellular processes. For the extraction of forskolin, tuberous roots of C. forskohlii are used as they contain the highest concentration of this metabolite. Approximately 2500 tonnes of the plant are cultivated annually to produce a yield of 2000-2200 kg ha-1 of dry tubers. The forskolin content of the root is distributed in the range of 0.01-1%, which cannot meet the increasing commercial demands from industries such as pharmaceuticals, cosmetics, dietary supplements, food and beverages. Hence, various aspects of micropropagation with different culture methods that employ precursors or elicitors to improve the forskolin content have been explored. Different extraction and analytical methods are also introduced to examine the yield and purity of forskolin. This review discusses the significance, clinical importance, mechanism of action and different approaches used for mass production including tissue culture for the lead compound forskolin to meet market needs.

High Incidence of Antibiotic Resistance in the Uropathogenic Bacteria Associated with Different Urological Diseases and Metabolic Complications: A Single Center Cross-Sectional Study.

Urological diseases affect all age groups and are associated with different urinary complications. Presence of pathogenic bacteria complicates the urological diseases such as chronic kidney disease (CKD), kidney stone disease (KSD), emphysematous pyelonephritis (EPN), and urological cancers (UCs) coinciding with urinary metabolic complications. The One Health concept for preventing the spread of antibiotic resistant opportunistic pathogens necessitates detailed investigation on the virulence and the antibiotic sensitivity patterns of the pathogens from the urinary tract infections (UTIs). This cross-sectional study was aimed to profile the pathogenic bacteria associated with different urological diseases that included urine samples from the patients from a tertiary care hospital. The study included 258 patients representing CKD (15.1%), KSD (28.7%), EPN (15.5%), UC (12.0%), and UTI patients without any urological diseases (28.7%) with overall 70.5% patients showing positive urine culture. Furthermore, other than UTI in patients without any urological diseases (100%), higher culture positive cases were seen in KSD (64.9%), followed by CKD (61.5%), EPN (52.5%), and UC (48.4%). Escherichia coli was the most predominant bacteria in UTI (35.1%) and EPN (66.7%). In KSD, Pseudomonas aeruginosa (41.7%), Staphylococcus aureus (18.8%), and Proteus mirabilis (14.6%) were more common. S. aureus (86.7%) was the most isolated bacteria from the UC cases. Overall rate of multidrug resistance (MDR) was 77.8%. All (100%) E. coli, K. pneumoniae, P. mirabilis, and S. aureus strains were MDR. Among the strains, strong biofilm formation was observed in 73.6%, and 66.7% strains were urease positive. Biofilm was positively correlated with MDR and urease activity. The abundance and distribution of bacteria differed among the urological diseases suggesting their association with the urine metabolite profile. Colonization of MDR pathogens in patients with urological diseases is a serious concern requiring steps to control the emergence of drug resistance and their further spread into the ecosystem.

Differential expression of urease genes and ureolytic activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa isolates in different nutritional conditions.

Uropathogens have adaptation strategies to survive in the host urinary tract by efficiently utilizing and tolerating the urinary metabolites. Many uropathogens harbour the enzyme urease for the breakdown of urea and the enzymatic breakdown of urea increases the pH and facilitate the struvite crystallization. In this study, the differential urease activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa strains was investigated under different nutritional conditions. The experiments included measurement of growth, pH, urease activity, NH4-N generation and urease gene (ureC) expression among the bacterial strains under different conditions. Further, the implications of urea breakdown on the struvite crystallization in vitro and biofilm formation were also assessed. The study included urease positive isolates and for comparison urease negative isolates were included. Compared to the urease negative strains the urease positive strains formed higher biofilms and motility. The urease positive P. aeruginosa showed significantly higher (p < 0.01) pH and urease activity (A557-A630) compared to E. coli under experimental conditions. Further, supplementation of glucose to the growth media significantly increased the urease activity in P. aeruginosa and in contrast, it was significantly lower in E. coli. The expression profile of urease gene (ureC) was significantly higher (p < 0.001) in P. aeruginosa compared to E. coli and was consistent with the biochemical results of the urease activity under the nutritional conditions. The differential urease activity under two nutritional conditions influenced the biogenic struvite crystallization. It correlated with the urease activity showing higher crystallization rate in P. aeruginosa compared to E. coli. The results highlight the differential urease activity in two common uropathogens under different nutritional conditions that may have significant role on the regulation of virulence, pathogenicity and in the kidney stone disease.

Dysbiosis of the oral microbiota composition is associated with oral squamous cell carcinoma and the impact of radiotherapy: a pilot study.

Radiotherapy can potentially influence the diversity and composition of the oral microbiome. We performed a study comparing the composition of oral microbiota in patients with oral squamous cell carcinoma (OSCC) before radiotherapy (n = 6), at three months (n = 6), and six months (n = 6) post-radiotherapy, and controls (n = 6). We profiled the oral microbiome by 16S rRNA gene sequencing using Illumina MiSeq. Alpha diversity (Chao1 index) showed significant differences in species richness between healthy controls and OSCC patients (P = 0.014). Conversely, no noteworthy distinctions were observed in the Chao1 index when comparing the pre-and post-radiation periods at both three and six months. The beta diversity of the oral microbiota differed significantly between the controls and OSCC patients (P = 0.014). However, no significant differences were observed in beta diversity between pre- and post-radiation at three months, whereas a significant difference was observed at six months (P = 0.038). Linear Discriminant Analysis Effect Size (LEfSe) demonstrated lower abundance of Corynebacterium, Actinomyces, Veillonella, and Haemophilus, and higher abundance of Selenomonas and Mycoplasma in OSCC patients than in healthy controls. The oral microbiome composition varied among healthy controls, patients with OSCC, and post-radiation therapy patients with OSCC. The observed recovery in the numerical dominance of specific beneficial oral taxa and the reduction in pathogenic bacteria after radiation therapy highlights the need for further investigations into their clinical implications.

Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker.

Introduction: Oral Squamous Cell Carcinoma (OSCC), a common malignancy of the head and neck region, is frequently diagnosed at advanced stages, necessitating the development of efficient diagnostic methods. Profiling autoantibodies generated against tumor-associated antigens have lately demonstrated a promising role in diagnosis, predicting disease course, and response to therapeutics and relapse. Methods: In the current study, we, for the first time, aimed to identify and evaluate the diagnostic value of autoantibodies in serum samples of patients with OSCC using autoantibody profiling by an immunome protein array. The utility of anti-NUBP2 antibody and tissue positivity in OSCC was further evaluated. Results and discussion: We identified a total of 53 autoantibodies with significant differential levels between OSCC and control groups, including 25 that were increased in OSCC and 28 that were decreased. These included autoantibodies against Thymidine kinase 1 (TK1), nucleotide-binding protein 2 (NUBP2), and protein pyrroline-5-carboxylate reductase 1 (PYCR1), among others. Immunohistochemical validation indicated positive staining of NUBP2 in a large majority of cases (72%). Further, analysis of OSCC data available in TCGA revealed higher NUBP2 expression correlated with better disease-free patient survival. In conclusion, the differential serum autoantibodies identified in the current study, including those for NUBP2, could be used as potential biomarkers for early diagnosis or as screening biomarkers for OSCC pending investigation in a larger cohort.

Emerging cell cycle related non-coding RNA biomarkers from saliva and blood for oral squamous cell carcinoma.

The unnotified or undifferentiable early stages of oral squamous cell carcinoma (OSCC) progression are the prime reasons for late-stage detection and poor survival outcomes of oral cancer. This review summarizes the prior research and recent advancements on the influence of dysregulated non-coding RNA (ncRNA) on cell cycle and their employability as diagnostic and prognostic biomarkers of oral cancer. The literature search was performed using the following keywords: 'serum/saliva non-coding RNAs' and 'serum/saliva non-coding RNAs and cell cycle', 'serum/saliva dysregulated ncRNAs and cell cycle', 'Cdk/CKI and ncRNAs', 'tissue ncRNAs' concerning 'oral cancer''. The compiled data focuses mainly on the diagnostic and prognostic significance of MicroRNAs (miRNAs), Circular RNAs (circRNAs), and Long noncoding RNAs (lncRNAs) on oral cancer and all other cancers as well as subject-relevant articles published in languages other than English are beyond the scope of this review and excluded from the study. Moreover, articles focusing on DNA, protein, and metabolite markers are eliminated from the study. While there exist various potential biomolecules such as DNA, RNA, proteins, metabolites, and specific antigens representing predictive biomarkers in body fluids for oral cancer, this review completely focuses on non-coding RNAs restricted to saliva and blood, picking out a few of the reliable ones amongst the recent investigations based on the sophisticated techniques, cohort, and sensitivity as well as specificity, i.e., salivary miR-1307-5p, miR-3928, hsa_circ_0001874 and ENST00000412740, NR_131012, ENST00000588803, NR_038323, miR-21 in circulation. Thus, further studies are required to clinically confirm the usage of these non-invasive biomarkers in oral cancer.

Description and host-range determination of phage PseuPha1, a new species of Pakpunavirus infecting multidrug-resistant clinical strains of Pseudomonas aeruginosa.

A new phage PseuPha1, infecting multiple multi-drug resistant strains of Pseudomonas aeruginosa with strong anti-biofilm activities, was isolated from wastewater in India. PseuPha1 showed optimal multiplicity of infection at 10-3, maintained the infectivity at wide ranges of pH (6-9) and temperature (4-37 °C), and exhibited 50 minutes latent period and a burst size of 200 when tested against P. aeruginosa PAO1. PseuPha1 shared 86.1-89.5% pairwise intergenomic similarity with Pakpunavirus species (n = 11) listed by the International Committee on Taxonomy of Viruses and established distinct phyletic lineages during phylogenetic analyses of phage proteins. While genomic data validated the taxonomic novelty and lytic attributes of PseuPha1, BOX-PCR profiling asserted the genetic heterogeneity of susceptible clinical P. aeruginosa. Our data supported the affiliation of PseuPha1 as a new Pakpunavirus species and provided the first line of evidence for its virulence and infectivity that can be harnessed in wound therapeutics.

Development of a bioink using exopolysaccharide from Rhizobium sp. PRIM17.

Biopolymers play a significant role in tissue engineering, including in the formulation of bioinks that require careful selection of the biopolymers having properties ideal for printability and supporting biological entities such as cells. Alginate is one of the most widely explored natural biopolymers for tissue engineering applications due to its biocompatibility, cross-linking ability, hydrophilic nature, and easy incorporation with other polymers. Here, a succinoglycan-like exopolysaccharide (EPS-R17) produced by a bacterial strain Rhizobium sp. PRIM17 was incorporated with alginate for the development of a bioink. The physicochemical characterization of EPS-R17 was performed before formulating the bioink with alginate. The bioink formulation was prepared by mixing different concentrations of EPS with an alginate solution at room temperature under sterile atmosphere. The prepared bioink was characterized for rheological properties, biocompatibility, and a bioplotting experiment was also conducted to mimick the extrusion bioprinting. The EPS-R17 was composed of glucose, galactose, and rhamnose with a molecular weight of 69.98 kDa. It was thermally stable up to 260 °C and showed characteristic FT-IR peaks (1723.3 cm-1) for succinyl groups. The EPS-R17 showed biocompatibility with keratinocytes (HaCaT), and fibroblasts (HDF) in vitro. The rheological properties of EPS-R17-alginate bioink at different combinations showed shear thinning behavior at 25 and 37 °C. Amplitude sweep measurements showed the gel-like nature of the polymer combinations in the solution system superior to alginate or EPS-R17 alone. The combination of 1 % EPS-R17 and 1.5 % alginate showed good compressive strength and swelling behavior. Extrusion bioprinting mimicked using a bioplotting experiment showed the sustained cell viability in the polymer matrix of EPS-R17-alginate bioink. The results indicate that the EPS-R17 can be used in combination with alginate for bioinks for bioprinting applications for providing physical properties and favorable bioactivities.

Genomic potential for exopolysaccharide production and differential polysaccharide degradation in closely related Alteromonas sp. PRIM-21 and Alteromonas fortis 1T.

Members of the genus Alteromonas are widely distributed in diverse marine environments and are often associated with marine organisms. Their ability to produce exopolysaccharides (EPS) and depolymerize sulfated algal polysaccharides has provided industrial importance to some species. Here, we describe the draft genome of an algae-associated strain namely, Alteromonas sp. PRIM-21 isolated from the southwest coast of India to understand the EPS biosynthetic pathways as well as polysaccharide depolymerization system in comparison to the closely related strain Alteromonas fortis 1T that shares 99.8% 16S rRNA gene sequence similarity. Whole-genome shotgun sequencing of Alteromonas sp. PRIM-21 yielded 50 contigs with a total length of 4,638,422 bp having 43.86% GC content. The resultant genome shared 95.9% OrthoANI value with A. fortis 1 T, and contained 4125 predicted protein-coding genes, 71 tRNA and 10 rRNA genes. Genes involved in Wzx/Wzy-, ABC transporter- and synthase-dependent pathways for EPS production and secretion were common in both Alteromonas sp. PRIM-21 and A. fortis 1T. However, the distribution of carbohydrate-active enzymes (CAZymes) was heterogeneous. The strain PRIM-21 harbored polysaccharide lyases for the degradation of alginate, ulvan, arabinogalactan and chondroitin. This was further validated from the culture-based assays using seven different polysaccharides. The depolymerizing ability of the bacteria may be useful in deriving nutrients from the biopolymers produced in the algal host while the EPS biosynthesis may provide additional advantages for life in the stressful marine environment. The results also highlight the genetic heterogeneity in terms of polysaccharide utilization among the closely related Alteromonas strains.

Urease-negative uropathogen Kalamiella piersonii YU22 metabolizes urea by urea carboxylase and allophanate hydrolase enzyme system.

Urea is one of the major components of the human urine and its breakdown by the uropathogens occurs mainly through the activity of the enzyme urease. However, a few reports suggest the presence of an alternate enzyme system for urea breakdown namely urea carboxylase (UC) and allophanate hydrolase (AH). We have previously reported the UC and AH system in the genome of a urease-negative uropathogen Kalamiella piersonii YU22 of the novel genus Kalamiella (reclassified recently as Pantoea).To validate the UC and AH activity in the presence of urea, we investigated the growth and urea utilization patterns of this bacterium. Growth kinetics, variations in media pH, NH4-N generation and UC and AH gene expressions were probed using urea-containing media. YU22 was able to grow in M9 media containing urea and increase the pH of the media due to the urea breakdown. Further, significantly higher concentrations of extracellular NH4-N (p < 0.001) was also detected in the cultures along with over-expression of UC and AH genes. The bacterium formed biofilm, and displayed swimming and swarming motilities in presence of urea. Additional glucose supply to urea boosted the colonization but ameliorated the media alkalization and ammonification through suppression of gene expressions encoding UC and AH. These results show that the urease-negative strain YU22 can utilize the UC and AH system for urea metabolism. We propose to further investigate the UC and AH system in other urease-negative uropathogens and its implications for pathogenicity and urinary tract colonization.

Competitive interaction of thymol with cviR inhibits quorum sensing and associated biofilm formation in Chromobacterium violaceum.

Biofilm formation associated with quorum sensing (QS) is a community behaviour displayed by many gram-negative pathogenic bacteria that provide survival advantages in hostile conditions. The inhibitors of QS interrupt bacterial communication and coordinated cell signalling for community aggregation in the biofilm. Thymol, a natural monoterpenoid, was tested against QS in Chromobacterium violaceum. As the first step, the interaction of thymol with cviR protein was investigated using in silico approach followed by validation using detailed in vitro experiments. The QS and biofilm studies were performed using the wild type of strain C. violaceum ATCC 12,472 and a mini-Tn5 mutant CV026. The MIC of thymol was established by the broth micro-dilution method, and IC50 value for violacein inhibition was quantified spectrophotometrically by extracting the violacein from the treated cells. Inhibitory effect of thymol on the biofilm was quantified by the crystal violet staining method, and scanning electron microscopy (SEM) was employed for biofilm visualization. The expression of biofilm associated genes (hmsH, hmsR, pilB, and pilT) was evaluated by qRT-PCR analysis. The in silico molecular interactions of thymol with cviR exhibited a G-score of - 5.847 kcal/mol, binding with TYR-80 and SER-155 by Pi-Pi stacking and H-bond, respectively. The MIC of thymol was 160 µg/mL, and the IC50 for violacein inhibition was estimated to be 28 µg/mL. The thymol treatment significantly reduced the biofilm viability and biomass by > 80% along with disruption of the well-organized biofilm architecture. QS inhibitory activity of thymol resulted in the reduction of exopolysaccharide production, swarming motility, and downregulation of biofilm-associated hmsH, hmsR, pilB, and pilT genes. This data establishes the QS inhibitory role of thymol in the biofilm formation in C. violaceum.

Extraction and characterization of an exopolysaccharide from a marine bacterium / Extracción y caracterización de un exopolisacárido de una bacteria marina

The marine bacterial exopolysaccharides (EPS) have transfigured the biotech sector with their myriad applications and prospects. This work was carried out to characterize and analyze the functional and biochemical properties of an EPS (EPS-DR3A) produced by a marine bacterium, Pseudoalteromonas sp. YU16-DR3A. The bacterium was cultured in Zobell marine broth for the production of EPS. The extracted EPS designated as EPS-DR3A was composed of 69% carbohydrates and 7.6% proteins with a molecular weight of 20 kDa. FT-IR spectra showed the presence of different functional groups. The monosaccharide analysis performed using GC–MS showed the presence of fucose, erythrotetrose, ribose, and glucose as monomers. EPS-DR3A showed excellent emulsifying activity against the tested hydrocarbons and food oils with stable emulsions. Rheological analysis of EPS-DR3A revealed the pseudoplastic behavior. The EPS-DR3A displayed good thermal stability with a degradation temperature of 249 °C and a melting point at 322 °C. Further, it had the ability to scavenge DPPH and nitric oxide free radicals with good total antioxidant activity. The in vitro biocompatibility study of EPS-DR3A showed high degree of biocompatibility with human dermal fibroblast cells at the tested concentrations. Taken together, the findings such as thermostability, emulsifying activity, pseudoplasticity, antioxidant activity, and biocompatibility of EPS-DR3A make this biomolecule an important candidate for a wide range of biomedical applications.(AU)

Self-assembled polyelectrolyte complexes of chitosan and fucoidan for sustained growth factor release from PRP enhance proliferation and collagen deposition in diabetic mice.

Diabetic wound management is a serious health care challenge due to higher rates of relapse, expensive treatment approaches, and poor healing outcomes. Among cell-based therapies, use of platelet-rich plasma (PRP) has been shown to be effective for diabetic wounds, but its poor shelf-life limits its clinical use. Here, we demonstrate a simple but effective polymer system to increase the shelf-life of PRP by developing a polyelectrolyte complex with dropwise addition of chitosan solution containing PRP by simple mixing at room temperature. Thus, prepared chitosan-fucoidan (CF) carrier complex encapsulated more than 95% of the loaded PRP. The resulting CF/PRP colloids were spherical in shape and ensured extended PRP release up to 72 h at 37 °C. Routine characterization (FT-IR, XRD, SEM) showed the material properties. The biological assays showed that CF complexes were biocompatible while CF/PRP enhanced the proliferation of fibroblasts and keratinocytes via higher Ki67 expression and fibroblast migration. Further investigations using a diabetic mouse model demonstrated significantly higher wound contraction and histopathological observations showed increased fibroblast migration, and collagen and cytokeratin deposition in treatment groups. The results are suggestive of the efficacy of CF/PRP as a cost-effective topical formulation for the sustained delivery of growth factors in treating chronic diabetic wounds.

Carvone - a quorum sensing inhibitor blocks biofilm formation in Chromobacterium violaceum.

Carvone is a natural monoterpenoid and in this study it was tested for its role in attenuating quorum sensing (QS) controlled biofilm formation in Chromobacterium violaceum. It showed significant QS inhibition in terms of reduction in violacein at a concentration range of 60 to 70 µg/mL against C. violaceum ATCC 12472. At the same concentration, carvone also inhibited biofilm formation by more than 80%. The biofilm morphology of C. violaceum is unique with a well organised pattern of cell arrangement in a tight matrix. The same was evident in Scanning electron microscopy, however, carvone treatment not only showed reduction in biofilm density but also disruption of biofilm matrix. Interruption of biofilm formation was attributed to reduction in the exopolysaccharide production and swarming motility. Molecular investigations (RT-PCR) showed that the important genes involved in biofilm regulation such as pilS, pilR, pilB and pilT were downregulated significantly in the treatment groups.

Extraction and characterization of an exopolysaccharide from a marine bacterium.

The marine bacterial exopolysaccharides (EPS) have transfigured the biotech sector with their myriad applications and prospects. This work was carried out to characterize and analyze the functional and biochemical properties of an EPS (EPS-DR3A) produced by a marine bacterium, Pseudoalteromonas sp. YU16-DR3A. The bacterium was cultured in Zobell marine broth for the production of EPS. The extracted EPS designated as EPS-DR3A was composed of 69% carbohydrates and 7.6% proteins with a molecular weight of 20 kDa. FT-IR spectra showed the presence of different functional groups. The monosaccharide analysis performed using GC-MS showed the presence of fucose, erythrotetrose, ribose, and glucose as monomers. EPS-DR3A showed excellent emulsifying activity against the tested hydrocarbons and food oils with stable emulsions. Rheological analysis of EPS-DR3A revealed the pseudoplastic behavior. The EPS-DR3A displayed good thermal stability with a degradation temperature of 249 °C and a melting point at 322 °C. Further, it had the ability to scavenge DPPH and nitric oxide free radicals with good total antioxidant activity. The in vitro biocompatibility study of EPS-DR3A showed high degree of biocompatibility with human dermal fibroblast cells at the tested concentrations. Taken together, the findings such as thermostability, emulsifying activity, pseudoplasticity, antioxidant activity, and biocompatibility of EPS-DR3A make this biomolecule an important candidate for a wide range of biomedical applications.

Characterization of an exopolysaccharide produced by Enterobacter sp. YU16-RN5 and its potential to alleviate cadmium induced cytotoxicity in vitro.

Natural biopolymers have gained remarkable attention for bioremediation particularly in heavy metal removal and oil degradation due to their non-toxic nature and lack of secondary pollution. The exopolysaccharides (EPS) produced by the bacteria have become an important class of biopolymers that are employed in bioremediation. The bacteria isolated from the rhizospheric soil have higher metal tolerance and their EPS are effective in biosorption of heavy metals. Here, we report the characterization of an EPS (EPS-RN5) isolated from the root nodule-associated bacteria, Enterobacter cancerogenus strain YU16-RN5 and its heavy metal biosorption abilities. The bacteria isolated from the West coast of India was cultured in yeast extract mannitol (YEM) medium for EPS extraction and to study the production kinetics on a temporal scale. The biochemical composition, rheological properties and thermostability of EPS-RN5 was characterized by standard methods. The biosorption potential of EPS-RN5 against the selected heavy metals was analyzed by employing the inductively coupled plasma atomic emission spectroscopy (ICP-AES) technique. Further, cell culture experiments were used to test the role of EPS-RN5 in reducing the cytotoxicity exerted by the heavy metals in vitro using a human embryonic kidney cell line (HEK 293T). The bacteria showed good growth in YEM media and the maximum EPS yield was 1800 mg/L at 96 h. The molecular weight of EPS-RN5 was 0.7 × 106 Da and it contained 61.5% total sugars and 14.5% proteins. The monosaccharide composition of the EPS included glucose, sorbose and galactose in the ratio 0.25:0.07:1.0. The EPS-RN5 showed high thermal stability with a degradation temperature of 273 °C. Rheological analysis revealed the non-Newtonian behavior, with pseudoplastic characteristics. The EPS-RN5 efficiently absorbed cadmium and other heavy metals such as mercury, strontium, copper, arsenic, and uranium. In vitro studies revealed significant protective effect against the cadmium-induced cytotoxicity in HEK 293T cells. These results indicate the potential applications of EPS-RN5.

Serum and salivary immunoglobulin G4 levels in children with autism spectrum disorder from south India: a case-control study.

BACKGROUND: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with wide spectrum of symptoms and few effective therapies. Evidence is suggestive of an association between immune system dysfunction and autism spectrum disorders (ASD) among children with ASD. Immunoglobulins (Ig) are found to be increased in the circulation of individuals with autism. The prospective study was aimed to estimate and correlate the levels of IgG4 in blood and saliva of children with autism. METHODOLOGY: Blood and unstimulated saliva were collected from 172 children (55 ASD, 57 healthy control, and 60 suspected parasitic infection) aged 0-18 years. Routine blood investigations were done. Serum and salivary IgG4 levels were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Data were subjected to statistical analysis. RESULTS: ELISA tests showed that the IgG4 levels in serum and saliva were significantly increased (P<0.05) in children with ASD as compared to normal control children. Both serum and saliva IgG4 levels showed a significant positive correlation (P<0.05). CONCLUSION: IgG4 can be used as a potential biomarker for the early detection of ASD. Further, saliva can be a diagnostic, noninvasive assessment tool for health monitoring of children with autism. Lay summary: The collection of saliva is easy and painless compared to other sample collection methods. The present study shows that, among children with autism, brain-reactive antibody, immunoglobulin G4 (gG4), is increased both in blood and saliva, and there is a significant correlation between the two levels. Therefore, the study recommends IgG4 as a potential biomarker for the early detection of autism, and saliva can be helpful in diagnosis and health monitoring of children with ASD.

Potential synergistic activity of quercetin with antibiotics against multidrug-resistant clinical strains of Pseudomonas aeruginosa.

Development of drug resistance in opportunistic pathogens is one of the major healthcare challenges associated with infection management. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. However, selection of the drugs should offer safety and synergistic interaction against most of the strains. Here, the efficacy of antibiotics in combination with quercetin, a natural flavonoid capable of targeting quorum sensing was tested against biofilm-forming Pseudomonas aeruginosa strains previously isolated from catheter associated urinary tract infection. Based on the antibiotic susceptibility pattern, synergistic effect of quercetin with selected antibiotics (levofloxacin, ceftriaxone, gentamycin, tobramycin and amikacin) was tested at the fractional concentrations of MIC by the checkerboard method and the fractional inhibitory concentration index (FICi) was calculated to estimate the synergistic effect. Effect of the synergistic combinations were further tested using time-kill assay, and against biofilm formation and biofilm cell viability. Cytotoxicity assays were performed using Human Embryonic Kidney 293T cells (HEK-293T) using the effective drug combinations with respective controls. The biofilm formation and biofilm cell viability were drastically affected with quercetin and selected antibiotics combinations with ≥80% inhibition. In vitro infection studies showed that all the strains could exert significant cell killing (68 to 85%) and the drug combinations decreased the infection rate significantly by reducing the cell killing effect of P. aeruginosa (p<0.05). The synergistic effect of quercetin is attributed to its quorum sensing inhibitory properties. These findings indicate that quercetin along with existing antibiotics can potentiate the treatment against P. aeruginosa infection and may reduce the selection pressure due to antibiotic overuse.

First Report of Pathogenic Bacterium Kalamiella piersonii Isolated from Urine of a Kidney Stone Patient: Draft Genome and Evidence for Role in Struvite Crystallization.

Uropathogenic bacteria are widely distributed in the environment and urinary tract infection is implicated in kidney stone disease. Here, we report on a urease negative bacterium Kalamiella piersonii (strain YU22) isolated from the urine of a struvite stone (MgNH4PO4·6H2O) patient. The closest species, K. piersonii IIIF1SW-P2T was reported from International Space Station samples. However, there are no earlier reports on its human association. Using whole genome and experimental analysis, its involvement in urinary tract colonization and struvite crystallization was explored. The strain YU22 showed many virulence factors that are needed for host cell invasion and colonization including cell adhesion factors, swimming and swarming motilities, biofilm and siderophore among others. In vitro infection studies in HEK-293T cells demonstrated the host cell attachment and killing. It was able to utilize amino acids as sole carbon source and showed growth in synthetic and healthy urine establishing metabolic adaptation to urinary tract. Increased pH and availability of ammonium ions from amino acid breakdown promoted struvite crystallization. The results from this study support the involvement of urease negative uropathogen in the struvite lithogenesis. Further studies on other isolates of K. peirsonii are warranted to assess its health risks.