Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Organic Matrix Derived from Host-Microbe Interplay Contributes to Pathological Renal Biomineralization.

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

Contact ratio and adaptations in the maxillary and mandibular dentoalveolar joints in rats and human clinical analogs.

Spatial maps of function-based contact areas and resulting mechanical strains in bones of intact fibrous joints in preclinical small-scale animal models are limited. Functional imaging in situ on intact dentoalveolar fibrous joints (DAJs) in hemimandibles and hemimaxillae harvested from 10 male Sprague-Dawley rats (N = 5 at 12 weeks, N = 5 at 20 weeks) was performed in this study. Physical features including bone volume fraction (BVF), bone pore diameter and pore density, and cementum fraction (CF) of the molars in the maxillary and mandibular joints were evaluated. Biomechanical testing in situ provided estimates of joint stiffness, changes in periodontal ligament spaces (PDL-space) between the molar and bony socket, and thereby localization of contact area in the respective joints. Contact area localization revealed mechanically stressed interradicular and apical regions in the joints. These anatomy-specific contact stresses in maxillary and mandibular joints were correlated with the physical features and resulting strains in interradicular and bony socket compartments. The mandibular joint spaces, in general, were higher than maxillary, and this trend was consistent with age (younger loaded: Mn - 134 ± 55 µm, Mx - 110 ± 47 µm; older loaded: Mn - 122 ± 49 µm, Mx - 105 ± 48 µm). However, a significant decrease (P < 0.05) in mandibular and maxillary joint spaces with age (younger unloaded: Mn - 147 ± 51 µm; Mx - 125 ± 42 µm; older unloaded: Mn - 134 ± 46 µm; Mx - 116 ± 44 µm) was observed. The bone volume fraction (BVF) of mandibular interradicular bone (IR bone) increased significantly with age (P < 0.05) with the percent porosity of coronal mandibular bone lower than its maxillary counterpart. The contact ratio (contact area to total surface area) of maxillary teeth was significantly greater (P < 0.05) than mandibular teeth; both maxillary interradicular and apical contact ratios (IR bone: 41%, 56%; Apical bone: 4%, 12%) increased with age, and were higher than the mandibular (IR bone: 19%, 44%; Apical bone: 1%, 4%) counterpart. Resulting higher but uniform strains in maxillary bone contrasted with lower but higher variance in mandibular strains at a younger age. Anatomy-specific colocalization of physical properties and functional strains in bone provided insights into form-guided adaptive dominance of the maxilla compared to material property-guided adaptive dominance of the mandible. These age-related trends from the preclinical animal model paralleled with age- and tooth position-specific variabilities in mandibular craniofacial bones of adolescent and adult patients following orthodontic treatment.

Standardization of mineral density maps of physiologic and pathologic biominerals in humans using cone-beam CT and micro-CT scanners.

OBJECTIVES: The lack of standardized X-ray imaging remains a challenge for comparative studies on spatial scans acquired from different clinic-specific X-ray scanners. The central objectives of this study are: 1) to delineate mineral density (MD) values, and 2) generate spatial MD maps of various physiologic and pathologic biominerals, and 3) propose a standardization protocol within the safe-operating zone of a CT scanner that underpins normalization of absorbed dose to shape and density of tissues. METHODS: A systematic approach to propose a standardization protocol for CT imaging in vivo included: 1) estimation of pathologic MD ranges by performing a comparative meta-analysis on 2009-2019 data from the PubMed database; 2) calibration of cone-beam CT (CBCT) and micro-CT scanners with phantoms of known mineral densities (0, 250, 500, 750 and 3000 mg/cc) and shapes (cylinders and polyhedrons); 3) scanning craniofacial bones (N = 5) and dental tissues (N = 5), and ectopic minerals from humans (N = 3 each, pulp, salivary gland, kidney and prostrate stones, and penile and vascular plaques); 4) underscoring the effect of shape-factor (surface area-to-volume ratio) on MD of biominerals. RESULTS: Higher MDs of physiologic and pathologic cortical bones (504-1009 mg/cc) compared to trabecular bone (82-212 mg/cc) were observed. An increase in shape-factor increased the CBCT error in MD measurement and revealed that the scanner resolution is dependent on the absorbed dose and shape-factor of detectable features. SIGNIFICANCE: CT scanners should be calibrated with phantoms containing segments of known shape-factors and mineral densities to identify safe-operating zones. The calibrated approach will narrow the gap between length-scale dependent measurements, and will permit spatiotemporal quantitative and reliable detection of pathologies.

Functional Adaptation of LPS-affected Dentoalveolar Fibrous Joints in Rats.

INTRODUCTION: The functional interplay between cementum of the root and alveolar bone of the socket is tuned by a uniquely positioned 70-80 µm wide fibrous and lubricious ligament in a dentoalveolar joint (DAJ). In this study, structural and biomechanical properties of the DAJ, periodontal ligament space (PDL-space also known as the joint space), alveolar bone of the socket, and cementum of the tooth root that govern the biomechanics of a lipopolysaccharide (LPS)-affected DAJ were mapped both in space and time. METHODS: The hemi-maxillae from 20 rats (4 control at 6 weeks of age, 4 control and 4 LPS-affected at 12 weeks of age, 4 control and 4 LPS-affected at 16 weeks of age) were investigated using a hybrid technique; micro-X-ray computed tomography (5 µm resolution) in combination with biomechanical testing in situ. Temporal variations in bone and cementum volume fractions were evaluated. Trends in mineral apposition rates (MAR) in additional six Sprague Dawley rats (3 controls, 3 LPS-affected) were revealed by transforming spatial fluorochrome signals to functional growth rates (linearity factor - RW) of bone, dentin, and cementum using a fast Fourier transform on fluorochrome signals from 100-µm hemi-maxillae sections. RESULTS: An overall change in LPS-affected DAJ biomechanics (a 2.5-4.5X increase in tooth displacement and 2X tooth rotation at 6 weeks, no increase in displacement and a 7X increase in rotation at 12 weeks; 27% increase in bone effective strain at 6 weeks and 11% at 12 weeks relative to control) was associated with structural changes in the coronal regions of the DAJ (15% increase in PDL-space from 0 to 6 weeks but only 5% from 6 to 12 weeks compared to control). A significant increase (p < 0.05) in PDL-space between ligated and age-matched control was observed. The bone fraction of ligated at 12 weeks was significantly lower than its age-matched control, and no significant differences (p > 0.05) between groups were observed at 6 weeks. Cementum in the apical regions grew faster but nonlinearly (11% and 20% increase in cementum fraction (CF) at 6 and 12 weeks) compared to control. Alveolar bone revealed site-specific nonlinear growth with an overall increase in MAR (108.5 µm/week to 126.7 µm/week after LPS treatment) compared to dentin (28.3 µm/week in control vs. 26.1 µm/week in LPS-affected) and cementum (126.5 µm/week in control vs. 119.9 µm/week in LPS-affected). A significant increase in CF (p < 0.05) in ligated specimens was observed at 6 weeks of age. CONCLUSIONS: Anatomy-specific responses of cementum and bone to the mechano-chemo stimuli, and their collective temporal contribution to observed changes in PDL-space were perpetuated by altered tooth movement. Data highlight the "resilience" of DAJ function through the predominance of nonlinear growth response of cementum, changes in PDL-space, and bone architecture. Despite the significant differences in bone and cementum architectures, data provided insights into the reactionary effects of cementum as a built-in compensatory mechanism to reestablish functional competence of the DAJ. The spatial shifts in architectures of alveolar bone and cementum, and consequently ligament space, highlight adaptations farther away from the site of insult, which also is another novel insight from this study. These adaptations when correlated within the context of joint function (biomechanics) illustrate that they are indeed necessary to sustain DAJ function albeit being pathological.

Mineralized Peyronie's plaque has a phenotypic resemblance to bone.

Mineralized Peyronie's plaque (MPP) impairs penile function. The association, colocalization, and dynamic interplay between organic and inorganic constituents can provide insights into biomineralization of Peyronie's plaque. Human MPPs (n = 11) were surgically excised, and the organic and inorganic constituents were spatially mapped using multiple high-resolution imaging techniques. Multiscale image analyses resulted in spatial colocalization of elements within a highly porous material with heterogenous composition, lamellae, and osteocytic lacuna-like features with a morphological resemblance to bone. The lower (520 ±â€¯179 mg/cc) and higher (1024 ±â€¯155 mg/cc) mineral density regions were associated with higher (11%) and lower (7%) porosities in MPP. Energy dispersive X-ray and micro-X-ray fluorescent spectroscopic maps in the higher mineral density regions of MPP revealed higher counts of calcium (Ca) and phosphorus (P), and a Ca/P ratio of 1.48 ±â€¯0.06 similar to bone. More importantly, higher counts of zinc (Zn) were localized at the interface between softer (more organic to inorganic ratio) and harder (less organic to inorganic ratio) tissue regions of MPP and adjacent softer matrix, indicating the involvement of Zn-related proteins and/or pathways in the formation of MPP. In particular, dentin matrix protein-1 (DMP-1) was colocalized in a matrix rich in proteoglycans and collagen that contained osteocytic lacuna-like features. This combined materials science and biochemical with correlative microspectroscopic approach provided insights into the plausible cellular and biochemical pathways that incite mineralization of an existing fibrous Peyronie's plaque. STATEMENT OF SIGNIFICANCE: Aberrant human penile mineralization is known as mineralized Peyronie's plaque (MPP) and often results in a loss of form and function. This study focuses on investigating the spatial association of matrix proteins and elemental composition of MPP by colocalizing calcium, phosphorus, and trace metal zinc with dentin matrix protein 1 (DMP-1), acidic proteoglycans, and fibrillar collagen along with the cellular components using high resolution correlative microspectroscopy techniques. Spatial maps provided insights into cellular and biochemical pathways that incite mineralization of fibrous Peyronie's plaque in humans.

Food hardness can regulate orthodontic tooth movement in mice.

BACKGROUND AND OBJECTIVES: Orthodontic treatment is often accompanied with prescription of softer foods to patients. The question to ask is, is this prescribed load regimen congruent with Wolff's law, and does it provide an adequate mechanical stimulus to maintain the functional health of periodontal complex? This question was answered by studying the effects of mice chewing on soft food (SF) and hard food (HF) while undergoing experimental tooth movement (ETM). METHODS: Three-week-old C57BL/6 mice (n = 18) were fed either hard pellet (HF; n = 9) or soft-chow food (SF; n = 9). ETM was performed on mice at 8 weeks of age, and mice were euthanized at 1 min, 2 weeks, and 4 weeks (8, 10, and 12 weeks old, respectively). A logistic regression model was applied to the experimental data to extrapolate the prolonged effects of ETM on the physical features of the dentoalveolar joint (DAJ). RESULTS: By 12 weeks, mice that chewed on SF expressed wider periodontal ligament space than those that chewed on HF. Mice that chewed on SF demonstrated increased alveolar socket roughness with larger alveoli and decreased bone volume fraction but with significantly lower bone mineral density and reduced overall tooth movement. CONCLUSIONS: These altered physical features when contextualized within the DAJ illustrated that (a) the regions farther away from the "site of insult" also undergo significant adaptation, and (b) these adaptations vary between mesial and distal sides of the periodontal complex and topographically differentiate in the direction of the ETM. These insights underpin the main conclusion, in that there is a need to "regulate chewing loads" as a therapeutic dose following ETM to encourage regeneration of periodontal complex as an effective clinical outcome. The discussed multiscale image analyses also can be used on patient cone beam computed tomography data to identify the effectiveness of orthodontic treatment within the realm of masticatory function.

Functional adaptation of interradicular alveolar bone to reduced chewing loads on dentoalveolar joints in rats.

OBJECTIVES: The effects of reduced chewing loads on load bearing integrity of interradicular bone (IB) within dentoalveolar joints (DAJ) in rats were investigated. METHODS: Four-week-old Sprague Dawley rats (N = 60) were divided into two groups; rats were either fed normal food, which is hard-pellet food (HF) (N = 30), or soft-powdered chow (SF) (N = 30). Biomechanical testing of intact DAJs and mapping of the resulting mechanical strains within IBs from 8- through 24-week-old rats fed HF or SF were performed. Tension- and compression-based mechanical strain profiles were mapped by correlating digital volumes of IBs at no load with the same IBs under load. Heterogeneity within IB was identified by mapping cement lines and TRAP-positive multinucleated cells using histology, and mechanical properties using nanoindentation technique. RESULTS: Significantly decreased interradicular functional space, IB volume fraction, and elastic modulus of IB in the SF group compared with the HF group were observed, and these trends varied with an increase in age. The elastic modulus values illustrated significant heterogeneity within IB from HF or SF groups. Both compression- and tension-based strains were localized at the coronal portion of the IB and the variation in strain profiles complemented the observed material heterogeneity using histology and nanoindentation. SIGNIFICANCE: Interradicular space and IB material-related mechanoadaptations in a DAJ are optimized to meet soft food related chewing demands. Results provided insights into age-specific regulation of chewing loads as a plausible "therapeutic dose" to reverse adaptations within the periodontal complex as an attempt to regain functional competence of a dynamic DAJ.

Structure and elemental composition of Ceftriaxone induced pediatric nephrolithiasis.

Ceftriaxone is a widely used antibiotic because to its broad-spectrum gram-negative coverage, safety, and biological half life (5-9 h) permit dose once-daily administration. It is specifically used in pediatric patients in developing countries. Ceftriaxone forms insoluble sludge/stone when combined with calcium in the urinary system. In this study, Ceftriaxone induced sludge/stones from pediatric patients were collected to identify its microstructure and composition to gather insights into the mechanism of Ceftriaxone induced sludge/stone formation. The results illustrated that Ceftriaxone induced stones formed rapidly following antibiotic administration. Ceftriaxone calcium salt crystals could easily be broken with minimal intervention. However, Ceftriaxone combined with calcium phosphate formed an insoluble stone aggregate.

Structural and chemical heterogeneities of primary hyperoxaluria kidney stones from pediatric patients.

OBJECTIVE: Calcium oxalate stones are the most common type among stone-forming patients and in some cases result from predisposed genetic conditions. In this work, we examined the differences in structure and chemical composition between oxalate stones from patients from three groups: 1) pediatric patients that were genetically predisposed (primary hyperoxaluria) to form stones (PPH); 2) control pediatric patients that did not have such genetic predisposition (PN-PH); 3) adult patients that formed oxalate stones without the genetic predisposition (A-CaOx). A variety of instrumental analyses were conducted to identify physicochemical properties of stones characteristic of predisposed pediatric (PPH), pediatric hyperoxaluria (PN-PH), and adult (A-CaOx) patient populations. METHODS: Genetic variants of 16 stone-forming patients were determined using whole-exome gene sequencing. Components of stones from PPH (n = 6), PN-PH (n = 5), and A-CaOx (n = 5) groups were identified using Fourier transform infrared (FTIR) spectroscopy. Stone morphology and density were evaluated using high resolution X-ray computed tomography (micro-XCT). Stone microstructure and elemental composition were mapped with scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy, respectively. RESULTS: Calcium oxalate bipyramidal crystals were found on stones from all groups. Stones from PPH patients with PH types I and II were composed of calcium oxalate monohydrate (COM) with relatively uniform mineral density (1224 ± 277 mg/cc) and distinct smooth surfaces. By contrast, micro-spherical calcium phosphate particles were found only on PN-PH stones, which also showed a broader range of mineral densities (1266 ± 342 mg/cc). Stones from the PN-PH group also contained phosphorus (P), which was absent in NP-PH stones. A-CaOx stones were of significantly lower mineral density (645 ± 237 mg/cc) than pediatric stones and were more heterogeneous in their elemental composition. CONCLUSION: Unique structural and compositional characteristics were identified in stones from pediatric patients with primary hyperoxaluria. These include the absence of phosphorus, a narrower mineral density distribution, and a uniform elemental composition compared to stones from pediatric patients without the genetic predisposition. Thus, characterization of stones at the macro- and micro-scales in combination with genetic testing of patients can provide insights and accurate diagnosis to develop a treatment plan for effective patient care.

Ectopic biomineralization in kidney stone formers compared to non-stone formers.

BACKGROUND: Kidney stone formers (SFs) are at increased risk of stroke, myocardial infarction, and atherosclerosis of the carotid and coronary arteries. These cardiovascular and urologic pathologies can result from ectopic biomineral deposition. The objectives of this study are: (I) to evaluate risk factors for ectopic biomineralization, and (II) to characterize the overall burden of ectopic minerals in known SFs compared to non-stone formers (NSFs) matched for these risk factors. METHODS: Presence and quantity of biominerals at eight anatomic locations (abdominal aorta, common iliac arteries, pelvic veins, prostate or uterus, mesentery, pancreas, and spleen) were determined in a case control study by retrospective analysis of clinical non-contrast computed tomography scans obtained from 190 SFs and 190 gender- and age-matched NSFs (renal transplant donors). Predictors of biomineralization were determined using negative binomial regression. A subgroup of 140 SFs and 140 NSFs were matched for risk factors for systemic biomineralization, and mineralization was compared between these matched SFs and NSFs using ordinal logistic regression. RESULTS: Hypertension, hyperlipidemia, diabetes mellitus, and smoking were more common amongst SFs. Risk factors for increased systemic biomineralization included history of nephrolithiasis, male gender, older age, and history of hyperlipidemia. When controlling for these comorbidities, SFs had significantly increased biomineralization systemically and at the abdominal aorta, iliac arteries, prostate, mesentery, pancreas, and spleen compared to NSFs. CONCLUSIONS: The current study provides evidence that SFs are at increased risk of biomineralization systemically, independent of common risk factors of atherosclerosis.

Design of drug-like hepsin inhibitors against prostate cancer and kidney stones.

Hepsin, a transmembrane serine protease abundant in renal endothelial cells, is a promising therapeutic target against several cancers, particularly prostate cancer. It is involved in the release and polymerization of uromodulin in the urine, which plays a role in kidney stone formation. In this work, we design new potential hepsin inhibitors for high activity, improved specificity towards hepsin, and promising ADMET properties. The ligands were developed in silico through a novel hierarchical pipeline. This pipeline explicitly accounts for off-target binding to the related serine proteases matriptase and HGFA (human hepatocyte growth factor activator). We completed the pipeline incorporating ADMET properties of the candidate inhibitors into custom multi-objective optimization functions. The ligands designed show excellent prospects for targeting hepsin via the blood stream and the urine and thus enable key experimental studies. The computational pipeline proposed is remarkably cost-efficient and can be easily adapted for designing inhibitors against new drug targets.

High-Throughput Screening: today's biochemical and cell-based approaches.

High-throughput screening (HTS) provides starting chemical matter in the adventure of developing a new drug. In this review, we survey several HTS methods used today for hit identification, organized in two main flavors: biochemical and cell-based assays. Biochemical assays discussed include fluorescence polarization and anisotropy, FRET, TR-FRET, and fluorescence lifetime analysis. Binding-based methods are also surveyed, including NMR, SPR, mass spectrometry, and DSF. On the other hand, cell-based assays discussed include viability, reporter gene, second messenger, and high-throughput microscopy assays. We devote some emphasis to high-content screening, which is becoming very popular. An advisable stage after hit discovery using phenotypic screens is target deconvolution, and we provide an overview of current chemical proteomics, in silico, and chemical genetics tools. Emphasis is made on recent CRISPR/dCas-based screens. Lastly, we illustrate some of the considerations that inform the choice of HTS methods and point to some areas with potential interest for future research.

Fatty acid-binding protein 4 downregulation drives calcification in the development of kidney stone disease.

Nephrolithiasis is a significant source of morbidity, and its incidence has increased significantly over the last decades. This rise has been attributed to concurrent increasing rates of obesity, associated with a 3-time risk of developing NL. To date, the mechanism by which obesity is linked to stone formation has not been elucidated. We aimed to utilize a transcriptomics approach to discover the missing link between these two epidemic diseases. We investigated gene expression profiling of nephrolithiasis patients by two RNA-sequencing approaches: comparison between renal papilla tissue with and without the presence of calcified Randall's plaques (RP), and comparison between the papilla, medulla, and cortex regions from within a single recurrent stone forming kidney. Results were overlaid between differently expressed genes found in the patient cohort and in the severely lithogenic kidney to identify common genes. Overlay of these two RNA-sequencing datasets demonstrated there is impairment of lipid metabolism in renal papilla tissue containing RP linked to downregulation of fatty acid binding protein (FABP) 4. Immunohistochemistry of human kidney specimens and microarray analysis of renal tissue from a nephrolithiasis mouse model confirmed that FABP4 downregulation is associated with renal stone formation. In a FABP4 knockout mouse model, FABP4 deficiency resulted in development of both renal and urinary crystals. Our study revealed that FABP4 plays an important, previously unrecognized role in kidney stone formation, providing a feasible mechanism to explain the link between nephrolithiasis and metabolic syndrome.

Polymicrobial periodontal disease triggers a wide radius of effect and unique virome.

Periodontal disease is a microbially-mediated inflammatory disease of tooth-supporting tissues that leads to bone and tissue loss around teeth. Although bacterially-mediated mechanisms of alveolar bone destruction have been widely studied, the effects of a polymicrobial infection on the periodontal ligament and microbiome/virome have not been well explored. Therefore, the current investigation introduced a new mouse model of periodontal disease to examine the effects of a polymicrobial infection on periodontal ligament (PDL) properties, changes in bone loss, the host immune response, and the microbiome/virome using shotgun sequencing. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum were used as the polymicrobial oral inoculum in BALB/cByJ mice. The polymicrobial infection triggered significant alveolar bone loss, a heightened antibody response, an elevated cytokine immune response, a significant shift in viral diversity and virome composition, and a widening of the PDL space; the latter two findings have not been previously reported in periodontal disease models. Changes in the PDL space were present at sites far away from the site of insult, indicating that the polymicrobial radius of effect extends beyond the bone loss areas and site of initial infection and wider than previously appreciated. Associations were found between bone loss, specific viral and bacterial species, immune genes, and PDL space changes. These findings may have significant implications for the pathogenesis of periodontal disease and biomechanical properties of the periodontium. This new polymicrobial mouse model of periodontal disease in a common mouse strain is useful for evaluating the features of periodontal disease.

Physicochemical and biochemical spatiotemporal maps of a mouse penis.

Spatiotemporal mechanobiology resulting in penile pathologies continues to be investigated using small scale animals models such as mice. However, species-dependent functional biomechanics of a mouse penis, is not known. In this study, spatial mapping of a mechanosensitive transcription factor, scleraxis (Scx), at ages 4, 5, 6 weeks, and 1 year were generated to identify mechanoactive regions within penile tissues. Reconstructed volumes of baculum collected using micro X-ray computed tomography illustrated significantly increased baculum length with decreased porosity, and increased mineral density (p < 0.05) with age. The bony-baculum was held centrally in the Scx positive corpus cavernosum glandis (CCG), indicating mechanoactivity within the struts in a 6 week old mouse. The struts also were stained positive for fibrillar proteins including collagen and elastin, and globular proteins including protein gene product 9.5, and α-smooth muscle actin. The corpus cavernosum penis (CCP) contained significantly (p < 0.05) more collagen than CCG within the same penis, and both regions contained blood vessels with equivalent innervation at any given age. Comparison of volumes of flaccid and erect penile forms revealed functional characteristics of the CCP. Results of this study provided insights into biomechanical function of the CCG; in that, it is a high-pressure chamber that stiffens the penis and is similar to the human corpus cavernosum.

Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins.

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

Fluorescence imaging of nitric oxide in living cells using o-phenylenediamine-rhodamine based polymeric nanosensors.

Direct real-time measurement of nitric oxide (NO) in living cells has proven quite challenging, owing in part to the lack of tools that are selective and sensitive to measure intracellular concentrations of NO. Herein we report the synthesis and characterization of polyvinyl alcohol (PVA) based nanosensors for fluorescence imaging of cytosolic NO using an o-phenylenediamine-rhodamine (OPD-RhB) platform. More specifically, thiol-functionalized PVA incorporating RhB conjugated OPD was disulfide crosslinked to yield NO-responsive nanosensors. The polymeric nanosensors were anionic, averaged 170 nm in hydrodynamic size, and exhibited linear increases in fluorescence intensity (FLI) to micro- and nanomolar concentrations of NO in a sodium nitroprusside (SNP) concentration-dependent manner. In the presence of SNP, the engineered nanosensors demonstrated physical stability at extracellular glutathione (GSH) conditions, while favoring NO detection at cytoplasmic GSH conditions. In addition, the PVA-based nanosensors were non-cytotoxic, cell membrane-permeable and demonstrated hydrogen peroxide-dependent FL increases upon incubation with activated synoviocytes in vitro. Most notably, NO-induced cell FLIs correlated strongly with total nitrite/nitrate content of conventional Griess assays with Pearson correlation coefficients of 0.96. Comprehensively, our results show that OPD-RhB-conjugated PVA nanosensors offer real-time imaging of NO with high sensitivity in living cells that can be employed for direct quantification of NO.