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Calcium is the key macromineral having a role in skeletal structure and function, muscle contraction, and neurotransmission. Bone remodeling is maintained through a constant balance between calcium resorption and deposition. Calcium deficiency is resolved through calcium supplementation, and among the supplements, water-soluble organic molecules attracted great pharmaceutical interest. Calcium glucoheptonate is a highly water-soluble organic calcium salt having clinical use; however, detailed investigations on its biological effects are limited. We assessed the effects of calcium glucoheptonate on cell viability and proliferation of osteoblast-like MG-63 cells. Calcium uptake and mineralization were evaluated using Alizarin red staining of osteoblast-like MG-63 cells treated with calcium glucoheptonate. Expression of osteogenic markers were monitored by western blotting, immunofluorescence, and qRT-PCR assays. Increased proliferation and calcium uptake were observed in the MG-63 cells treated with calcium glucoheptonate. The treatment also increased the expression of osteopontin and osteogenic genes such as collagen-1, secreted protein acidic and cysteine rich (SPARC), and osteocalcin. Calcium glucoheptonate treatment did not exert any cytotoxicity on colorectal and renal epithelial cells, indicating the safety of the treatment. This is the first report with evidence for its beneficial effect for pharmaceutical use in addressing calcium deficiency conditions.

Understanding the mechanisms of kidney stone formation, development patterns and associated pathological features are gaining importance due to an increase in the prevalence of the disease and diversity in the presentation of the stone composition. Based on the microstructural characteristics of kidney stones, it may be possible to explain the differences in the pathogenesis of pure and mixed types of stones. In this study, the microstructure and distribution of mineral components of kidney stones of different mineralogy (pure and mixed types) were analyzed. The intact stones removed from patients were investigated using synchrotron radiation X-ray computed microtomography (SR-μCT) and the tomography slice images were reconstructed representing the density and structure distribution at various elevation planes. Infrared (IR) spectroscopes, X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to confirm the bulk mineral composition in the thin section stones. Observations revealed differences in the micro-morphology of the kidney stones with similar composition in the internal 3-D structure. Calcium oxalate monohydrate stones showed well-organised layering patterns, while uric acid stones showed lower absorption signals with homogenous inner structure. Distinct mineral phases in the mixed types were identified based on the differential absorption rates. The 3-D quantitative analysis of internal porosity and spatial variation between nine different types of stones were compared. The diversity among the microstructure of similar and different types of stones shows that the stone formation is complex and may be governed by both physiological and micro-environmental factors. These factors may predispose a few towards crystal aggregation and stone growth, while, in others the crystals may not establish stable attachment and/or growth.

Pathological biomineralization in the urinary system leads to urolithiasis. Formation of kidney stones involves a series of events during which they undergo morphological and mineralogical changes. We investigated the mineralization of biogenic struvite (in vitro) and examined the transformation of distinct interior and exterior structure of struvite. In vitro crystallization of struvite was performed in the presence of two bacteria that were originally isolated from the kidney stone patients. Morphological evaluation was carried out using SR-μCT as well as FESEM, XRD and FT-IR. Characteristic internal 3-D morphology and porosity of the stones were studied. For comparison, patient derived struvite stones were used. From the results obtained, we report that the presence of bacteria enhances the crystallization process of struvite in vitro. A series of time-resolved experiments revealed that struvite crystals experienced a significant morphologic evolution from pin pointed structure to X-shaped and tabular morphologies. These X-shaped and unusual tabular habits of struvite resembled biogenic morphologies of struvite. SR-μCT showed similarities between the patient derived and the in vitro derived struvite crystals. In conclusion, these experiments revealed that the bacteria play a major role in the specific morphogenesis of struvite and can able to control the nucleation, modulate crystalline phases, and shape of the growing crystal.

Abstract Terrestrial and aquatic environments contaminated with animal urine may contribute to the transmission of Leptospira, a causative agent of leptospirosis in humans and wild/domesticated animals. Although enormous amounts of work have been done decoding the ecophysiology, the factors governing the cell growth and virulence in Leptospires derived from environmental samples still remain elusive. Here, we show oxidation of a wide array of organic acids including acetoacetate by a new strain of Leptospira interrogans designated as KeTo, isolated from a sewage sample originating from a wildlife enclosure located at Mangalore, India. We further demonstrate the susceptibility of KeTo to ethyl ester of acetoacetate (ethyl acetoacetate, EA). A 4.7 Mbp genome of KeTo shared the highest relatedness to pathogenic L. interrogans RGAT (99.3%), followed by L. kirschneri 3522CT (91.3%) and other related species of Leptospira (80.8‒74.3%), and harbored genes encoding acetoacetyl-CoA synthetase and acetoacetate decarboxylase respectively involved in the acetoacetate utilization and acetone formation. In line with this, strain KeTo oxidized acetoacetate when supplied as a sole carbon. Aqueous EA suppressed biofilm formation (p

To highlight the importance of the spices in the Mediterranean diet, the aim of the paper was to study the essential oil compositions and to clarify the potential differences in the biological activities of the three cardamom species. In the study, we compared the phytochemical profiles and biological activities of essential oils from Elettaria cardamomum, Aframomum corrorima and Amomum subulatum. The oils were analyzed using the GC and GC/MS techniques and were mainly constituted of the oxygenated monoterpenes which represents 71.4%, 63.0%, and 51.0% of all compounds detected in E. cardamomum, A. corrorima and A. subulatum essential oils, respectively, 1,8-cineole was the main common compound between the tree tested volatile oil. The essential oils showed significant antimicrobial activity against Gram-positive and Gram-negative microorganisms tested especially the fungal strains. The Ethiopian cardamom was the most active essential oil with fungal growth inhibition zone ranging from 12.67 to 34.33 mm, MICs values ranging from 0.048 to 0.19 mg/mL, and MBCs values from 0.19 to 1.75 mg/mL. The three tested essential oils and their main component (1,8-cineole) significantly increased the production of elastase and protease production, and motility in P. aeruginosa PAO1 in a dose dependent manner. In fact, at 10 mg/mL concentration, the three essential oils showed more than 50% of inhibition of elastolytic and proteolytic activities in P. aeruginosa PAO1. The same oils inhibited also the violacein production in C. violaceum strain. It was also noticed that at high concentrations, the A. corrorima essential oil significantly inhibited the germination of radish. A thorough knowledge of the biological and safety profiles of essential oils can produce applications of economic importance.

ABSTRACT Background: Formation of struvite stones is associated with urinary tract infection by urease-producing bacteria. Biogenic crystal growth in natural and synthetic materials is regulated by the action of inhibitors, ranging from small ions, molecules to large macromolecules. Materials and Methods: We report the dynamics of in vitro crystallization of struvite in presence of vitamin C in synthetic urine using single diffusion gel growth technique. Sodium metasilicate gel of specific gravity 1.05 and the aqueous solution of ammonium dihydrogen phosphate were used as the medium for growing the struvite crystals. The crystallization process was induced by a urease positive struvite stone associated Pseudomonas aeruginosa to mimic the infection leading to stone formation. The grown crystals were characterized by ATR-FTIR and powder XRD. The surface morphology was analysed through FE-SEM for comparison between treatments. Results: We observed decrease in number, dimension, and growth rate of struvite crystals with the increasing concentrations of vitamin C. Crystals displayed well-defined faces and dendritic morphology of struvite in both control and biogenic systems. Conclusion: The results strongly suggest that, vitamin C can modulate the formation of struvite crystals in the presence of uropathogenic bacteria.

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.

ABSTRACT During microbial surveillance of the Mars 2020 spacecraft assembly facility, two novel bacterial strains, potentially capable of producing lasso peptides, were identified. Characterization using a polyphasic taxonomic approach, whole-genome sequencing and phylogenomic analyses revealed a close genetic relationship among two strains from Mars 2020 cleanroom floors (179-C4-2-HS, 179-J1A1-HS), one strain from the Agave plant (AT2.8), and another strain from wheat-associated soil (V4I25). All four strains exhibited high 16S rRNA gene sequence similarity (>99.2%) and low average nucleotide identity (ANI) with Neobacillus niacini NBRC 15566T, delineating new phylogenetic branches within the genus. Detailed molecular analyses, including gyrB (90.2%), ANI (86.4%), average amino acid identity (87.8%) phylogenies, digital DNA–DNA hybridization (32.6%), and percentage of conserved proteins (77.7%) indicated significant divergence from N. niacini NBRC 15566T. Consequently, these strains have been designated Neobacillus driksii sp. nov., with the type strain 179-C4-2-HST (DSM 115941T = NRRL B-65665T). N. driksii grew at 4°C to 45°C, pH range of 6.0 to 9.5, and 0.5% to 5% NaCl. The major cellular fatty acids are iso-C15:0 and anteiso-C15:0. The dominant polar lipids include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminolipid. Metagenomic analysis within NASA cleanrooms revealed that N. driksii is scarce (17 out of 236 samples). Genes encoding the biosynthesis pathway for lasso peptides were identified in all N. driksii strains and are not commonly found in other Neobacillus species, except in 7 out of 26 recognized species. This study highlights the unique metabolic capabilities of N. driksii, underscoring their potential in antimicrobial research and biotechnology.IMPORTANCEThe microbial surveillance of the Mars 2020 assembly cleanroom led to the isolation of novel N. driksii with potential applications in cleanroom environments, such as hospitals, pharmaceuticals, semiconductors, and aeronautical industries. N. driksii genomes were found to possess genes responsible for producing lasso peptides, which are crucial for antimicrobial defense, communication, and enzyme inhibition. Isolation of N. driksii from cleanrooms, Agave plants, and dryland wheat soils, suggested niche-specific ecology and resilience under various environmentally challenging conditions. The discovery of potent antimicrobial agents from novel N. driksii underscores the importance of genome mining and the isolation of rare microorganisms. Bioactive gene clusters potentially producing nicotianamine-like siderophores were found in N. driksii genomes. These siderophores can be used for bioremediation to remove heavy metals from contaminated environments, promote plant growth by aiding iron uptake in agriculture, and treat iron overload conditions in medical applications.

IntroductionOral 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.MethodsIn 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 discussionWe 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.

Marine ecosystems house a plethora of microorganisms capable of producing therapeutically important metabolites, but remains largely unexplored. The aim of this work was to find a new marine bacterium capable of producing a fibrinolytic enzyme, which is fibrin specific, stable at the physiological pH, exhibiting both anticoagulant and thrombolytic effect in vitro. Once a potential bacterium was found, an effort was made to optimize medium. Based on the screening studies, the isolate C7 was chosen for further studies and was identified as Serratia marcescens subsp. sakuensis. Medium optimisation using Plackett-Burman design and one-factor experiments suggested that nitrogen sources have a profound effect on enzyme production. Fibrinolytic enzyme activity increased to 2.5 fold (487.29 U/mL) in the optimised medium as compared to unoptimised medium (191.65 U/mL). Current study demonstrates that seawater could be an excellent source for novel marine organisms capable of producing fibrinolytic enzymes. Fibrinolytic enzyme produced by Serratia marcescens subsp. sakuensis appears to be a potential candidate for thrombolytic therapy due to its high specific fibrinolytic activity, high ratio of fibrinolytic to fibrinogenolytic activity, activity at the physiological pH, anticoagulant and thrombolytic effects.

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