Gene Therapy for Skin Aging.

Extrinsic and intrinsic factors contribute to skin aging; nonetheless, they are intertwined. Moreover, intrinsic skin aging mirrors age-related declines in the entire human body's internal organs. There is evidence that skin appearance is an indicator of the general health of somebody. Earlier, it was apparent that the intrinsic factors are unalterable, but the sparkling of skin aging gene therapy on the horizon is changing this narrative. Skin aging gene therapy offers tools for skin rejuvenation and, natural beauty restoration, and therapy for diseases affecting the entire skin. However, skin aging gene therapy is an arduous and sophisticated task relying on precise interim stimulation of telomerase to extend telomeres and wend back the biological clock in the hopes to find the fountain of youth, while preserving cells innate biological features. Finding the hidden fountain of youth will be a remarkable discovery for promoting aesthetics medicine, genecosmetics, and healthy aging. Caloric restriction offers ultimate health benefits and a reproducible way to promote longevity in mammals, while delaying age-related diseases. Moreover, exercise further enhances these health benefits. This article highlights the potential of skin aging gene therapy and foretells the emerging dawn of the genecosmetics era.

Evaluation of skeletal muscle function in male rats with doxorubicin-induced myopathy following various exercise techniques: the significant role of glucose transporter 4.

A common anthracycline antibiotic used to treat cancer patients is doxorubicin (DOX). One of the effects of DOX therapy is skeletal muscle fatigue. Our goal in this research was to study the beneficial effect of exercise on DOX-induced damaged muscle fibers and compare the effect of different exercise strategies (prophylactic, post- toxicity and combined) on DOX toxicity. Five groups were created from 40 male rats: group I, control group; group II, DOX was administered intraperitoneally for 2 weeks over 6 equal injections (each 2.5 mg/kg); group III, rats trained for 3 weeks before DOX; group IV, rats trained for 8 weeks after DOX; and group V, rats were trained for 3 weeks before DOX followed by 8 weeks after. Measures of oxidative damage (H2O2, catalase), inflammation (TNF-α), and glucose transporter 4 (GLUT4) expression on skeletal muscle were assessed. Also, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was estimated. Skeletal performance was evaluated by contraction time (CT), half relaxation time (1/2 RT), and force-frequency relationship by the end of this research. The current study demonstrated a detrimental effect of DOX on skeletal performance as evidenced by a significant increase in CT and 1/2 RT compared to control; in addition, H2O2, TNF-α, and HOMA-IR were significantly increased with a significant decrease in GLUT4 expression and catalase activity. Combined exercise therapy showed a remarkable improvement in skeletal muscle performance, compared to DOX, CT, and 1/2 RT which were significantly decreased; H2O2 and TNF-α were significantly decreased unlike catalase antioxidant activity that significantly increased; in addition, skeletal muscle glucose metabolism was significantly improved as GLUT4 expression significantly increased and HOMA-IR was significantly decreased. Exercise therapy showed significant improvement in all measured parameters relative to DOX. However, combined exercise therapy showed the best improvement relative to both pre-exercise and post-exercise groups.

Advances in Dystrophinopathy Diagnosis and Therapy.

Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the Dystrophin gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and ß-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the Dystrophin gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.

Design and synthesis of 6-arylpyridine-tethered sulfonamides as novel selective inhibitors of carbonic anhydrase IX with promising antitumor features toward the human colorectal cancer.

Hypoxia, a characteristic feature of solid tumors, develops as a result of excessive cell proliferation and rapid tumor growth exceeding the oxygen supply, and can result in angiogenesis activation, increased invasiveness, aggressiveness, and metastasis, leading to improved tumor survival and suppression of anticancer drug therapeutic impact. SLC-0111, a ureido benzenesulfonamide, is a selective human carbonic anhydrase (hCA) IX inhibitor in clinical trials for the treatment of hypoxic malignancies. Herein, we describe the design and synthesis of novel 6-arylpyridines 8a-l and 9a-d as structural analogues of SLC-0111, in the aim of exploring new selective inhibitors for the cancer-associated hCA IX isoform. The para-fluorophenyl tail in SLC-0111 was replaced by the privileged 6-arylpyridine motif. Moreover, both ortho- and meta-sulfonamide regioisomers, as well as an ethylene extended analogous were developed. All 6-arylpyridine-based SLC-0111 analogues were screened in vitro for their inhibitory potential against a panel of hCAs (hCA I, II, IV and IX isoforms) using stopped-flow CO2 hydrase assay. In addition, the anticancer activity was firstly explored against a panel of 57 cancer cell lines at the USA NCI-Developmental Therapeutic Program. Compound 8g emerged as the best anti-proliferative candidate with mean GI% value equals 44. Accordingly, a cell viability assay (MTS) for 8g was applied on colorectal HCT-116 and HT-29 cancer cell lines as well as on the healthy HUVEC cells. Thereafter, Annexin V-FITC apoptosis detection, cell cycle, TUNEL, and qRT-PCR, colony formation, and wound healing assays were applied to gain mechanistic insights and to understand the behavior of colorectal cancer cells upon the treatment of compound 8g. Also, a molecular docking analysis was conducted to provide in silico insights into the reported hCA IX inhibitory activity and selectivity.

LC-MS/MS bioanalytical method for the quantitative analysis of nifedipine, bisoprolol, and captopril in human plasma: Application to pharmacokinetic studies.

In this study, the development and validation of an accurate and highly sensitive LC-MS/MS method were performed for the estimation of nifedipine, bisoprolol and captopril in real human plasma. Liquid-liquid extraction using tert-butyl methyl ether was efficiently applied for extraction of the analytes from plasma samples. The chromatographic separation was carried out using an isocratic elution mode on the X-terra MS C18 column (4.6 × 50 mm, 3.5 µm). The mobile phase consisted of methanol-0.1% formic acid (95:5, v/v) for determination of nifedipine and bisoprolol and acetonitrile-0.1% formic acid (70:30, v/v) for determination of captopril with a flow rate of 0.5 ml/min. Acceptable results regarding the different validation characteristics of the analytes were obtained in accordance with US Food and Drug Administration recommendations for bioanalytical methods. The developed approach was linear over concentration ranges of 0.5-130.0, 50.0-4,500.0 and 0.3-30.0 ng/ml for nifedipine, captopril and bisoprolol, respectively. The method revealed a sufficient lower limit of quantification in the range of 0.3-50.0 ng/ml, as well as high recovery percentages, indicating high bioanalytical applicability. The proposed method was efficiently applied to a pharmacokinetic evaluation of a fixed-dose combination of the analytes in healthy male volunteers.

Gene Therapy for Alzheimer and Parkinson Diseases.

Alzheimer and Parkinson diseases are associated with cholinergic neuron loss and deterioration of bone mineral density. Gene therapy through either gene transfer, CRISPR gene editing, or CRISPR gene modulation holds the potential to cure Alzheimer and Parkinson diseases. The emerging role of weight-bearing exercise in the prevention of, and care for, osteoporosis, obesity, and diabetes has been previously recognized. Moreover, endurance exercise offers a viable alternative to reduce amyloid peptides deposits while increasing bone mineral density in Alzheimer and Parkinson patients. ß-amyloid peptides, α-synuclein, and tau aggregates start building up two decades before the onset of Alzheimer and Parkinson diseases. Therefore, an early intervention program for the detection of these deposits is required to prevent or delay the onset of these diseases. This article spots light on the potential of gene therapy for Alzheimer and Parkinson diseases.

Design and biological evaluation of 3-substituted quinazoline-2,4(1H,3H)-dione derivatives as dual c-Met/VEGFR-2-TK inhibitors.

The dual c-Met/vascular endothelial growth factor receptor 2 (VEGFR-2) TK inhibition is a good strategy to overcome therapeutic resistance to small molecules VEGFR-2 inhibitors. In this study, we designed 3-substituted quinazoline-2,4(1H,3H)-dione derivatives as dual c-Met/VEGFR-2 TK inhibitors. We introduced new synthetic methods for reported derivatives of 3-substituted quinazoline-2,4(1H,3H)-dione 2a-g, in addition to the preparation of some new derivatives namely, 3 and 4a-j. Three compounds namely, 2c, 4b, and 4e showed substantial amount of inhibition for both c-Met and VEGFR-2 TK (IC50 range 0.052-0.084 µM). Both compounds 4b, 4e showed HB with highly conserved residue Asp1222 in the HB region of c-Met TK. For VEGFR-2 TK, compound 4b showed HB with a highly conserved residue Asp1046 in the HB region. Compound 4e showed HB with Glu885 and Asp1046. Moreover, in silico prediction of pharmacokinetic and physicochemical parameters of target compounds was carried out using SwissADME website. The quinazoline-2,4(1H,3H)-dione derivatives are promising antiproliferative candidates that require further optimisation.HighlightsNew 3-substituted quinazoline-2,4(1H,3H)-dione derivatives were synthesised and characterised.Compounds 4b and 4e showed higher cytotoxic activity than cabozantinib against HCT-116 colorectal cell lines.Both compounds 4b and 4e showed less toxicity to WI38 normal cell line compared to HCT 116 colon cancer cell line.Compound 4b was superior to cabozantinib in VEGFR-2 inhibition while compound 2c was equipotent to cabozantinib.Compounds 4b and 4e showed remarkable c-Met inhibitory activity.Compounds 4b and 4e arrested cell cycle and induced significant levels of apoptosis.In silico ADME prediction revealed high oral bioavailability and enhanced water solubility of target compounds as compared to cabozantinib.Target compounds interacted with both c-Met and VEGFR-2 active site in similar way to cabozantinib.

Ligament Preservation in Open Rhinoplasty: Prospective Analysis.

BACKGROUND: In the open rhinoplasty technique, the soft tissue and ligaments are vulnerable to injury. Reconstruction of the ligaments is not performed routinely. The authors aimed to assess the effect of preservation of the nasal ligaments (ie, scroll, septocolumellar, and Pitanguy ligaments) in open-approach rhinoplasty. METHODS: In this prospective cohort study, 32 patients underwent open rhinoplasty with ligament preservation after receiving precise training on five cadavers. RESULTS: All patients had improved aesthetic and functional outcome in the early postoperative period with long-lasting preservation of tip projection and results. No patient needed secondary revision surgery for tip dropping or malrotation. The objective findings and subjective assessments were satisfying for the patients and surgeons. CONCLUSIONS: Refinements of nasal surgery have no limits. This study suggests that nasal ligament reconstruction, including of the scroll, septocolumellar, and Pitanguy ligaments, could maintain nasal tip projection and rotation for a long time. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Duchenne Muscular Dystrophy Gene therapy.

Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that could address the genetic cause of the disease at its root are crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.

A Novel Canine Otoscopy Teaching Model for Veterinary Students.

Otoscopic evaluation using an otoscope is an important tool among the diagnostic modalities for otitis externa and is considered a core component of a canine patient's complete physical examination. Traditionally, otoscopic training in veterinary school involves using live dogs (i.e., laboratory dogs or dogs that are patients of the veterinary teaching hospital). While this approach has its advantages, performing otoscopic examination on live dogs presents several challenges: it requires adequate patient restraint, can cause stress to the dog, and can potentially cause trauma and/or injury to the dog's ear canal when performed by an inexperienced individual. Using an alternative teaching tool for otoscopic evaluation could overcome these challenges and improve veterinary students' learning experience. In this study, we investigated student perceptions of a novel canine teaching model for otoscopic evaluation in first-year veterinary students. The Elnady preservation technique was employed to create a realistic, durable, and flexible model for otoscopic training in a dermatology laboratory session in a first-year veterinary course. Student feedback was assessed on a Likert scale, and overall feedback indicated that students felt that the model was beneficial for skill building and removed many of the stressors incurred with using live animals when training in clinical skills. Most students stated that they would like to have additional similar models incorporated into training and would recommend these models to other students.

Effective ultraviolet C light disinfection of respirators demonstrated in challenges with Geobacillus stearothermophilus spores and SARS-CoV-2 virus.

BACKGROUND: The global COVID-19 pandemic, accompanied by spikes in the number of patients in hospitals, required substantial amounts of respiratory protective devices (respirators), thereby causing shortages. Disinfection of used respirators by applying ultraviolet C (UVC) light may enable safe reuse, reducing shortages. AIM: To determine whether UVC disinfection is applicable to enable repeated safe reuse of respirators. METHODS: The UVC chamber, equipped with low-pressure mercury discharge lamps emitting at 254 nm, was used to determine the sporicidal and virucidal effects. Respirators challenged with spores and viruses were exposed to various UVC energy levels. Deactivation of the biological agents was studied as well as UVC effects on particle filtration properties and respirator fit. FINDINGS: A 5 log10 reduction of G. thermophilus spore viability by a UVC dose of 1.1 J/cm2 was observed. By simulating spores present in the middle of the respirators, a 5 log10 reduction was achieved at a UVC dose of 10 J/cm2. SARS-CoV-2 viruses were inactivated by 4 log10 upon exposure to 19.5 mJ/cm2 UVC. In case UVC must be transmitted through all layers of the respirators to reach the spores and virus, a reduction of >5 log10 was achieved using a UVC dose of 10 J/cm2. Exposure to a six-times higher UVC dose did not significantly affect the integrity of the fit nor aerosol filtering capacity of the respirator. CONCLUSION: UVC was shown to be a mild and effective way of respirator disinfection allowing for reuse of the UVC-treated respirators.

Synthesis, Characterization, Thermal Analysis, DFT, and Cytotoxicity of Palladium Complexes with Nitrogen-Donor Ligands.

Three new palladium complexes ([Pd(DABA)Cl2], [Pd(CPDA)Cl2], and [Pd(HZPY)Cl2]) bearing dinitrogen ligands (DABA: 3,4-diaminobenzoic acid; CPDA: 4-chloro-o-phenylenediamine; HZPY: 2-hydraziniopyridine) were synthesized, characterized, and tested against breast cancer (MCF-7), prostate carcinoma cell line (PC3) and liver carcinoma cell line (HEPG2). [Pd(DABA)Cl2] complex exhibited the highest inhibition percentage, lying between 68-71%. The hydrolysis mechanism of each palladium complex, the key step preceding the binding to the biological target, as well as their photophysical properties were explored by means of DFT and TDDFT computations. Results indicate a faster hydrolysis process for the Pd(DABA)Cl2 complex. The computed activation energies for the first and second hydrolysis processes suggest that all the compounds could reach DNA in their monohydrated form.

Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate.

OBJECTIVE: Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). METHODS: Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 µm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 µm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. RESULTS: Method I showed good linearity over the range 10-100 µg/mL for both dugs. Method II's linear ranges were 0.001-0.1 and 0.02-1 µg/mL for BIS and AML, respectively. CONCLUSION: The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. HIGHLIGHTS: To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol.

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma.

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex® C18 column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x2) over a concentration range of 1-1000, 2.5-800, and 5-500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.

Thermoelastic Processes by a Continuous Heat Source Line in an Infinite Solid via Moore-Gibson-Thompson Thermoelasticity.

Many attempts have been made to investigate the classical heat transfer of Fourier, and a number of improvements have been implemented. In this work, we consider a novel thermoelasticity model based on the Moore-Gibson-Thompson equation in cases where some of these models fail to be positive. This thermomechanical model has been constructed in combination with a hyperbolic partial differential equation for the variation of the displacement field and a parabolic differential equation for the temperature increment. The presented model is applied to investigate the wave propagation in an isotropic and infinite body subjected to a continuous thermal line source. To solve this problem, together with Laplace and Hankel transform methods, the potential function approach has been used. Laplace and Hankel inverse transformations are used to find solutions to different physical fields in the space-time domain. The problem is validated by calculating the numerical calculations of the physical fields for a given material. The numerical and theoretical results of other thermoelastic models have been compared with those described previously.

Multiscale in-vitro analysis of photo-activated riboflavin incorporated in an experimental universal adhesive.

OBJECTIVE: To investigate the effect of blue light photoactivated riboflavin modified universal adhesives on dentin collagen biodegradation resistance, dentin apparent elastic modulus, and resin-dentin bond strength with interfacial morphology. METHODS: Dentin slabs were treated with 0.1% riboflavin-5-phosphate modified (powder added slowly while shaking and then sonicated to enhance the dispersion process) Universal Adhesive Scotch Bond and Zipbond™ along with control (non-modified) and experimental adhesives, photoactivated with blue light for 20s. Hydroxyproline (HYP) release was assessed after 1-week storage. Elastic-modulus testing was evaluated using universal testing machine at 24 h. Resin-dentin interfacial morphology was assessed with scanning electron-microscope, after 6-month storage. 0.1% rhodamine dye was added into each adhesive and analyzed using CLSM. Detection of free amino groups was carried out using ninhydrin and considered directly proportional to optical absorbance. Collagen molecular confirmation was determined using spectropolarimeter to evaluate and assess CD spectra. For molecular docking studies with riboflavin (PDB ID file), the binding pocket was selected with larger SiteScore and DScore using Schrodinger PB software. After curing, Raman shifts in Amide regions were obtained at 8 µm levels. Data were analyzed using Two-way analysis of variance (ANOVA, p ≤ 0.05) and Tukey-Kramer multiple comparison post hoc tests. RESULTS: At baseline, bond strength reduced significantly (p ≤ 0.05) in control specimens. However, at 6 months' storage, UVA Zipbond™ had significantly higher µTBS. Resin was able to diffuse through the porous demineralized dentin creating adequate hybrid layers in both 0.1%RF modified adhesives in CLSM images. In riboflavin groups, hybrid layer and resin tags were more pronounced. The circular dichroism spectrum showed negative peaks for riboflavin adhesive specimens. Best fitted poses adopted by riboflavin compound are docked with MMP-2 and -9 proteases. Amide bands and CH2 peaks followed the trend of being lowest for control UA Scotch bond adhesive specimens and increasing in Amides, proline, and CH2 intensities in 0.1%RF modified adhesive specimens. All 0.1%RF application groups showed statistically significant (p < 0.05) less levels of HYP released compared to controls. Dentin Eappr of riboflavin application was significantly (p < 0.05) more as compared to control groups. CONCLUSION: Blue light photoactivated 0.1% riboflavin modified adhesives improved the biochemical and biomechanical properties of demineralized dentin as well as the long-term resin-dentin interfacial integrity and bond strength of universal adhesive to dentin.

Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany.

AIMS: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. METHODS AND RESULTS: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. CONCLUSIONS: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

Minimally invasive high-intensity focused ultrasound (HIFU) improves dentine remineralization with hydroxyapatite nanorods.

OBJECTIVE: The aim is to investigate the potential significance of combining minimally invasive high-intensity focused ultrasound (HIFU) with hydroxyapatite (HA) nanorods treatment for the remineralization of demineralized coronal dentine-matrix. METHODS: HA having nanorods structure were synthetized using ultrasonication with precipitation method. HA nanorods were characterized by TEM for average-size/shape. Following phosphoric acid demineralization, dentine specimens were treated with HA-nanorods with/without subsequent HIFU exposure for 5 s, 10 s and 20 s then stored in artificial saliva for 1-month. Dentine specimens were characterized using different SEM and Raman spectroscopic techniques. In addition, the biochemical stability and HA-nanorods were examined using ATR-FTIR to observe attachment of nanoparticles. Also, surface nanoindentation properties were evaluated using AFM in tapping-mode. RESULTS: HA-nanorods displayed well-defined, homogenous plate-like nanostructure. TEM revealed intact collagen-fibrils network structure with high density due to obliteration of interfibrillar spaces with clear evidence of remineralization in combined HA/HIFU treatment. With HA-nanorods treatment collagen-network structure was visible, consisting of fibrils interlaced into a compact pattern with evidence of minerals deposition. AFM investigation revealed clear mineral formation with the increase of HIFU exposure time. Bands associated with inorganic phase dominate well in HIFU exposed specimens with PO stretching within dentine mineral identified at 960 cm-1. Characteristic dentine structure for control and HIFU 20 s specimens is reflected as oscillatory mean Amide-I intensity with measurement giving a precise sinusoidal response of polarization angle ß within dentinal tissue. Nanoindentation testing showed a gradual significant increase in elastic-modulus with the increase in HIFU exposure time after 1-month storage. FTIR spectrum of the HIFU exposed dentine displayed bands at 1650 cm-1, 1580 cm-1 and 1510 cm-1 that can be attributed to Amide-I, II and III. SIGNIFICANCE: The synergetic effect of HIFU exposure on remineralization potential of demineralized dentine-matrix following nano-hydroxyapatite treatment was revealed. This synergetic effect is dependent on HIFU exposure time.