Relationships between matrix mineralization, oxidative metabolism, and mitochondrial structure during ATDC5 murine chondroprogenitor cell line differentiation.

The mechanistic relationships between the progression of growth chondrocyte differentiation, matrix mineralization, oxidative metabolism, and mitochondria content and structure were examined in the ATDC5 murine chondroprogenitor cell line. The progression of chondrocyte differentiation was associated with a statistically significant (p ≤ 0.05) ~2-fold increase in oxidative phosphorylation. However, as matrix mineralization progressed, oxidative metabolism decreased. In the absence of mineralization, cartilage extracellular matrix mRNA expression for Col2a1, Aggrecan, and Col10a1 were statistically (p ≤ 0.05) ~2-3-fold greater than observed in mineralizing cultures. In contrast, BSP and Phex that are associated with promoting matrix mineralization showed statistically (p ≤ 0.05) higher ~2-4 expression, while FGF23 phosphate regulatory factor was significantly lower (~50%) in mineralizing cultures. Cultures induced to differentiate under both nonmineralizing and mineralizing media conditions showed statistically greater basal oxidative metabolism and ATP production. Maximal respiration and spare oxidative capacity were significantly elevated (p ≤ 0.05) in differentiated nonmineralizing cultures compared to those that mineralized. Increased oxidative metabolism was associated with both an increase in mitochondria volume per cell and mitochondria fusion, while mineralization diminished mitochondrial volume and appeared to be associated with fission. Undifferentiated and mineralized cells showed increased mitochondrial co-localization with the actin cytoskeletal. Examination of proteins associated with mitochondria fission and apoptosis and mitophagy, respectively, showed levels of immunological expression consistent with the increasing fission and apoptosis in mineralizing cultures. These results suggest that chondrocyte differentiation is associated with intracellular structural reorganization, promoting increased mitochondria content and fusion that enables increased oxidative metabolism. Mineralization, however, does not need energy derived from oxidative metabolism; rather, during mineralization, mitochondria appear to undergo fission and mitophagy. In summary, these studies show that as chondrocytes underwent hypertrophic differentiation, they increased oxidative metabolism, but as mineralization proceeds, metabolism decreased. Mitochondria structure also underwent a structural reorganization that was further supportive of their oxidative capacity as the chondrocytes progressed through their differentiation. Thus, the mitochondria first underwent fusion to support increased oxidative metabolism, then underwent fission during mineralization, facilitating their programed death.

Global transcriptomic analysis of a murine osteocytic cell line subjected to spaceflight.

Bone loss is a major health concern for astronauts during long-term spaceflight and for patients during prolonged bed rest or paralysis. Growing evidence suggests that osteocytes, the most abundant cells in the mineralized bone matrix, play a key role in sensing mechanical forces applied to the skeleton and integrating the orchestrated response into subcellular biochemical signals to modulate bone homeostasis. However, the precise molecular mechanisms underlying both mechanosensation and mechanotransduction in late-osteoblast-to-osteocyte cells under microgravity (µG) have yet to be elucidated. To unravel the mechanisms by which late osteoblasts and osteocytes sense and respond to mechanical unloading, we exposed the osteocytic cell line, Ocy454, to 2, 4, or 6 days of µG on the SpaceX Dragon-6 resupply mission to the International Space Station. Our results showed that µG impairs the differentiation of osteocytes, consistent with prior osteoblast spaceflight experiments, which resulted in the downregulation of key osteocytic genes. Importantly, we demonstrate the modulation of critical glycolysis pathways in osteocytes subjected to microgravity and discovered a set of mechanical sensitive genes that are consistently regulated in multiple cell types exposed to microgravity suggesting a common, yet to be fully elucidated, genome-wide response to microgravity. Ground-based simulated microgravity experiments utilizing the NASA rotating-wall-vessel were unable to adequately replicate the changes in microgravity exposure highlighting the importance of spaceflight missions to understand the unique environmental stress that microgravity presents to diverse cell types. In summary, our findings demonstrate that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

Dosimetry of [212Pb]VMT01, a MC1R-Targeted Alpha Therapeutic Compound, and Effect of Free 208Tl on Tissue Absorbed Doses.

[212Pb]VMT01 is a melanocortin 1 receptor (MC1R) targeted theranostic ligand in clinical development for alpha particle therapy for melanoma. 212Pb has an elementally matched gamma-emitting isotope 203Pb; thus, [203Pb]VMT01 can be used as an imaging surrogate for [212Pb]VMT01. [212Pb]VMT01 human serum stability studies have demonstrated retention of the 212Bi daughter within the chelator following beta emission of parent 212Pb. However, the subsequent alpha emission from the decay of 212Bi into 208Tl results in the generation of free 208Tl. Due to the 10.64-hour half-life of 212Pb, accumulation of free 208Tl in the injectate will occur. The goal of this work is to estimate the human dosimetry for [212Pb]VMT01 and the impact of free 208Tl in the injectate on human tissue absorbed doses. Human [212Pb]VMT01 tissue absorbed doses were estimated from murine [203Pb]VMT01 biodistribution data, and human biodistribution values for 201Tl chloride (a cardiac imaging agent) from published data were used to estimate the dosimetry of free 208Tl. Results indicate that the dose-limiting tissues for [212Pb]VMT01 are the red marrow and the kidneys, with estimated absorbed doses of 1.06 and 8.27 mGyRBE = 5/MBq. The estimated percent increase in absorbed doses from free 208Tl in the injectate is 0.03% and 0.09% to the red marrow and the kidneys, respectively. Absorbed doses from free 208Tl result in a percent increase of no more than 1.2% over [212Pb]VMT01 in any organ or tissue. This latter finding indicates that free 208Tl in the [212Pb]VMT01 injectate will not substantially impact estimated tissue absorbed doses in humans.

Tributyltin protects against ovariectomy-induced trabecular bone loss in C57BL/6J mice with an attenuated effect in high fat fed mice.

Risk factors for poor bone quality include estrogen loss at menopause, a high fat diet and exposures to drugs/chemicals that activate peroxisome proliferator activated receptor gamma (PPARγ). We previously reported that the PPARγ and retinoid X receptor dual ligand, tributyltin (TBT), repressed periosteal bone formation but enhanced trabecular bone formation in vivo. Here, we examined the interaction of diet, ovariectomy (OVX) and TBT exposure on bone structure. C57BL/6J mice underwent either sham surgery or OVX at 10 weeks of age. At 12 weeks of age, they were placed on a low (10% kcal) or high (45% kcal) fat, sucrose-matched diet and treated with vehicle or TBT (1 or 5 mg/kg) for 14 weeks. OVX increased body weight gain in mice on either diet. TBT enhanced body weight gain in intact mice fed a high fat diet, but decreased weight gain in OVX mice. Elemental tin concentrations increased dose-dependently in bone. TBT had marginal effects on cortical and trabecular bone in intact mice fed either diet. OVX caused a reduction in cortical and trabecular bone, regardless of diet. In high fat fed OVX mice, TBT further reduced cortical thickness, bone area and total area. Interestingly, TBT protected against OVX-induced trabecular bone loss in low fat fed mice. The protective effect of TBT was nullified by the high fat. These results show that TBT protects against trabecular bone loss, even in the presence of a strongly resorptive environment, at an even lower level of exposure than we showed repressed homeostatic resorption.

Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms.

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.

Are Narrative Letters of Recommendation for Medical Students Interpreted as Intended by Orthopaedic Surgery Residency Programs?

BACKGROUND: Narrative letters of recommendation are an important component of the residency application process. However, because narrative letters of recommendation are almost always positive, it is unclear whether those reviewing the letters understand the writer's intended strength of support for a given applicant. QUESTIONS/PURPOSES: (1) Is the perception of letter readers for narrative letters of recommendation consistent with the intention of the letter's author? (2) Is there inter-reviewer consistency in selection committee members' perceptions of the narrative letters of recommendation? METHODS: Letter writers who wrote two or more narrative letters of recommendation for applicants to one university-based orthopaedic residency program for the 2014 to 2015 application cycle were sent a survey linked to a specific letter of recommendation they authored to assess the intended meaning regarding the strength of an applicant. A total of 247 unstructured letters of recommendation and accompanying surveys were sent to their authors, and 157 surveys were returned and form the basis of this study (response percentage 64%). The seven core members of the admissions committee (of 22 total reviewers) at a university-based residency program were sent a similar survey regarding their perception of the letter. To answer our research question about whether letter readers' perceptions about a candidate were consistent with the letter writer's intention, we used kappa values to determine agreement for survey questions involving discrete variables and Spearman correlation coefficients (SCCs) to determine agreement for survey questions involving continuous variables. To answer our research question regarding inter-reviewer consistency among the seven faculty members, we compared the letter readers' responses to each survey question using intraclass correlation coefficients (ICCs). RESULTS: There was a negligible to moderate correlation between the intended and perceived strength of the letters (SCC 0.26 to 0.57), with only one of seven letter readers scoring in the moderate correlation category. When stratifying the applicants into thirds, there was only slight agreement (kappa 0.07 to 0.19) between the writers and reviewers. There were similarly low kappa values for agreement about how the writers and readers felt regarding the candidate matching into their program (kappa 0.14 to 0.30). The ICC for each question among the seven faculty reviewers ranged from poor to moderate (ICC 0.42 to 0.52). CONCLUSION: Our results demonstrate that the reader's perception of narrative letters of recommendation did not correlate well with the letter writer's intended meaning and was not consistent between letter readers at a single university-based urban orthopaedic surgery residency program. CLINICAL RELEVANCE: Given the low correlation between the intended strength of the letter writers and the perceived strength of those letters, we believe that other options such as a slider bar or agreed-upon wording as is used in many dean's letters may be helpful.

Bone Mechanical Properties in Healthy and Diseased States.

The mechanical properties of bone are fundamental to the ability of our skeletons to support movement and to provide protection to our vital organs. As such, deterioration in mechanical behavior with aging and/or diseases such as osteoporosis and diabetes can have profound consequences for individuals' quality of life. This article reviews current knowledge of the basic mechanical behavior of bone at length scales ranging from hundreds of nanometers to tens of centimeters. We present the basic tenets of bone mechanics and connect them to some of the arcs of research that have brought the field to recent advances. We also discuss cortical bone, trabecular bone, and whole bones, as well as multiple aspects of material behavior, including elasticity, yield, fracture, fatigue, and damage. We describe the roles of bone quantity (e.g., density, porosity) and bone quality (e.g., cross-linking, protein composition), along with several avenues of future research.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

Differences in Trabecular Microarchitecture and Simplified Boundary Conditions Limit the Accuracy of Quantitative Computed Tomography-Based Finite Element Models of Vertebral Failure.

Vertebral fractures are common in the elderly, but efforts to reduce their incidence have been hampered by incomplete understanding of the failure processes that are involved. This study's goal was to elucidate failure processes in the lumbar vertebra and to assess the accuracy of quantitative computed tomography (QCT)-based finite element (FE) simulations of these processes. Following QCT scanning, spine segments (n = 27) consisting of L1 with adjacent intervertebral disks and neighboring endplates of T12 and L2 were compressed axially in a stepwise manner. A microcomputed tomography scan was performed at each loading step. The resulting time-lapse series of images was analyzed using digital volume correlation (DVC) to quantify deformations throughout the vertebral body. While some diversity among vertebrae was observed on how these deformations progressed, common features were large strains that developed progressively in the superior third and, concomitantly, in the midtransverse plane, in a manner that was associated with spatial variations in microstructural parameters such as connectivity density. Results of FE simulations corresponded qualitatively to the measured failure patterns when boundary conditions were derived from DVC displacements at the endplate. However, quantitative correspondence was often poor, particularly when boundary conditions were simplified to uniform compressive loading. These findings suggest that variations in trabecular microstructure are one cause of the differences in failure patterns among vertebrae and that both the lack of incorporation of these variations into QCT-based FE models and the oversimplification of boundary conditions limit the accuracy of these models in simulating vertebral failure.

Elastic Anisotropy of Trabecular Bone in the Elderly Human Vertebra.

Knowledge of the nature of the elastic symmetry of trabecular bone is fundamental to the study of bone adaptation and failure. Previous studies have classified human vertebral trabecular bone as orthotropic or transversely isotropic but have typically obtained samples from only selected regions of the centrum. In this study, the elastic symmetry of human vertebral trabecular bone was characterized using microfinite element (µFE) analyses performed on 1019 cubic regions of side length equal to 5 mm, obtained via thorough sampling of the centrums of 18 human L1 vertebrae (age = 81.17 ± 7.7 yr; eight males and ten females). An optimization procedure was used to find the closest orthotropic representation of the resulting stiffness tensor for each cube. The orthotropic elastic constants and orientation of the principal elastic axes were then recorded for each cube and were compared to the constants predicted from Cowin's fabric-based constitutive model (Cowin, 1985, "The Relationship Between the Elasticity Tensor and the Fabric Tensor," Mech. Mater., 4(2), pp. 137-147.) and the orientation of the principal axes of the fabric tensor, respectively. Deviations from orthotropy were quantified by the "orthotropic error" (van Rietbergen et al., 1996, "Direct Mechanics Assessment of Elastic Symmetries and Properties of Trabecular Bone Architecture," J. Biomech., 29(12), pp. 1653-1657), and deviations from transverse isotropy were determined by statistical comparison of the secondary and tertiary elastic moduli. The orthotropic error was greater than 50% for nearly half of the cubes, and the secondary and tertiary moduli differed from one another (p < 0.0001). Both the orthotropic error and the difference between secondary and tertiary moduli decreased with increasing bone volume fraction (BV/TV; p ≤ 0.007). Considering only the cubes with an orthotropic error less than 50%, only moderate correlations were observed between the fabric-based and the µFE-computed elastic moduli (R2 ≥ 0.337; p < 0.0001). These results indicate that when using a criterion of 5 mm for a representative volume element (RVE), transverse isotropy or orthotropy cannot be assumed for elderly human vertebral trabecular bone. Particularly at low values of BV/TV, this criterion does not ensure applicability of theories of continuous media. In light of the very sparse and inhomogeneous microstructure found in the specimens analyzed in this study, further work is needed to establish guidelines for selecting a RVE within the aged vertebral centrum.

Pulmonary, aorta, and coronary arteries post-arterial switch in transposition of great arteries: intermediate-term surveillance utilizing conventional echocardiography and cardiac multislice computed tomography.

BACKGROUND: Arterial switch operation (ASO) is the standard surgical choice for D-transposition of great arteries (D-TGA). However, the implications of ASO on pulmonaries, coronaries, and aorta have not been adequately investigated. The current study evaluates arterial morphologic changes post-ASO at intermediate-term surveillance. METHODS: From May 2021 to May 2022, patients with D-TGA who underwent ASO for more than six months were recruited. Preoperative and operative data were collected. Patients were assessed using echocardiography (ECHO) and multislice CT angiography (MSCT) to evaluate pulmonary, coronary, and aortic arterial anatomy. RESULTS: Twenty patients were included with median age of 11 (10-23.25) days at ASO and 14 (7.25-32.75) months on last follow-up. Neo-aortic regurgitation was detected in 12(60%) and neo-pulmonary regurgitation in 3 (15%). Using ECHO, complete evaluation of pulmonary arteries (PAs) was not achieved in 35% and incomplete coronaries assessment in 40% of cases. No stenosis was detected in coronaries using MSCT, although coronary anomalies were found in 9/20 (45%). Dilated Aortic annulus was detected in 16/20 (80%), dilated aortic root in 18/20 (90%), and dilated sinotubular junction in 70%. Right PA stenosis was diagnosed in 10/20 (50%) and left PA(LPA) stenosis in 7/20 (35%). Although Z-score of PAs did not correlate with aortic data, LPA bending angle was positively correlated to neo-aortic root diameter and Z-score (rho = 0.65,p = 0.016; rho = 0.69,p = 0.01), respectively. CONCLUSION: Echocardiography alone is not a conclusive surveillance tool for detecting late post-ASO anatomic changes in D-TGA patients. Cardiac MSCT should be considered for comprehensive evaluation on the intermediate-term follow-up post-ASO to accurately track morphologic abnormalities in the aorta, pulmonary, and coronary arteries.

Antimicrobial resistance patterns among critical priority pathogens in an intensive care unit at a tertiary hospital in Egypt: a descriptive analysis comparing pre- and COVID-19 eras.

Background. The intensified global challenge of antimicrobial resistance, set against the backdrop of the COVID-19 pandemic, is a cause for major concern. Within healthcare settings, intensive care units are recognized as focal points for Gram-negative infections. The study pursued to assess the prevalence and antimicrobial resistance patterns of critical priority pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, comprising Klebsiella pneumoniae and Escherichia coli) during both pre- and COVID-19 periods.Gap Statement. The decision to explore this topic stemmed from the urgent need to understand how the exceptional healthcare crisis of COVID-19 affected AMR patterns.Methods. This was an observational retrospective analysis of 1056 clinical specimens obtained from 950 patients who were admitted to the Medical Intensive Care Unit at Kasr Al-Aini Hospital, Cairo University, Egypt.Results. In the period before COVID-19, 342 pathogenic isolates (135 K. pneumoniae, 83 P. aeruginosa, 76 A. baumannii and 48 E. coli) were obtained from samples collected from 450 patients. Conversely, during the COVID-19 period, 714 isolates (237 K. pneumoniae, 205 A. baumannii, 199 P. aeruginosa and 73 E. coli) were collected from the same number of patients. In the course of the pandemic, there is a slight increase in A. baumannii and P. aeruginosa infections, whereas E. coli and K. pneumoniae exhibit a distinct trend with a noticeable reduction in infection rates during COVID-19. During the COVID-19 period, a noticeable rise in resistance rates was observed for all antibiotics utilized. The results from Fisher's exact test indicated a substantial increase in resistance towards certain antibiotics. Specifically, a significant rise in resistance was observed for E. coli to ciprofloxacin (P = 0.00), gentamicin and P. aeruginosa (P = 0.02), levofloxacin and A. baumannii (P = 0.01), piperacillin-tazobactam and A. baumannii (P = 0.04), and piperacillin-tazobactam and P. aeruginosa (P = 0.01).Conclusion. Our results display how the pandemic impacted bacterial infections and antibiotic resistance, indicating a general increase in resistance rates. These findings are crucial for guiding healthcare practices, emphasizing the need for continued surveillance and potentially checking antibiotic usage schemes.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Effect of specimen-specific anisotropic material properties in quantitative computed tomography-based finite element analysis of the vertebra.

Intra- and inter-specimen variations in trabecular anisotropy are often ignored in quantitative computed tomography (QCT)-based finite element (FE) models of the vertebra. The material properties are typically estimated solely from local variations in bone mineral density (BMD), and a fixed representation of elastic anisotropy ("generic anisotropy") is assumed. This study evaluated the effect of incorporating specimen-specific, trabecular anisotropy on QCT-based FE predictions of vertebral stiffness and deformation patterns. Orthotropic material properties estimated from microcomputed tomography data ("specimen-specific anisotropy"), were assigned to a large, columnar region of the L1 centrum (n = 12), and generic-anisotropic material properties were assigned to the remainder of the vertebral body. Results were compared to FE analyses in which generic-anisotropic properties were used throughout. FE analyses were also performed on only the columnar regions. For the columnar regions, the axial stiffnesses obtained from the two categories of material properties were uncorrelated with each other (p = 0.604), and the distributions of minimum principal strain were distinctly different (p ≤ 0.022). In contrast, for the whole vertebral bodies in both axial and flexural loading, the stiffnesses obtained using the two categories of material properties were highly correlated (R2 > 0.82, p < 0.001) with, and were no different (p > 0.359) from, each other. Only moderate variations in strain distributions were observed between the two categories of material properties. The contrasting results for the columns versus vertebrae indicate a large contribution of the peripheral regions of the vertebral body to the mechanical behavior of this bone. In companion analyses on the effect of the degree of anisotropy (DA), the axial stiffnesses of the trabecular column (p < 0.001) and vertebra (p = 0.007) increased with increasing DA. These findings demonstrate the need for accurate modeling of the peripheral regions of the vertebral body in analyses of the mechanical behavior of the vertebra.

Surgical Mitral Valve Replacement Using a Catheter-Based MelodyTM Valve in a Landing Zone Constructed With a PTFE-Covered Expandable Cheatham-PlatinumTM Stent.

Mitral valve replacement using a Melody valve is a promising solution to the challenge of surgical mitral valve replacement in infants with a hypoplastic annulus. We report the creation of a landing zone in the mitral valve annulus using a Cheatham-Platinum (CP)-covered stent that facilitates Melody valve placement, helps prevent paravalvular leak, minimizes left ventricular outflow tract obstruction, and allows for potential future dilation of the valve.

Retained colistin susceptibility in clinical Acinetobacter baumannii isolates with multiple mutations in pmrCAB and lpxACD operons.

The progressive increase in the resistance rates to first- and second-line antibiotics has forced the reuse of colistin as last-line treatment for Acinetobacter baumannii infections, but the emergence of colistin-resistant strains is not uncommon. This has been long linked to acquired chromosomal mutations in the operons pmrCAB and lpxACD. Hence, such mutations are routinely screened in colistin-resistant strains by most studies. The current study was designed to explore the possible existence of pmrCAB and lpxACD mutations in colistin-susceptible isolates. For this purpose, the whole genome sequences of eighteen multi-/extensively drug resistant A. baumannii were generated by Illumina sequencing and screened for missense mutations of the operons pmrCAB and lpxACD. Most of the isolates belonged to global clones (GCs) including GC1 (n=2), GC2 (n=7), GC7 (n=2), GC9 (n=3), and GC11 (n=1). The minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution assay. Seventeen isolates were fully susceptible to colistin with MICs ranging from (≤0.125 to 0.5 µg/ml). Interestingly, all colistin-susceptible isolates carried missense mutations in pmrCAB and lpxACD operons with reference to A. baumannii ATCC 19606. Overall, 34 mutations were found. Most substitutions were detected in pmrC (n=20) while no mutations were found in pmrA or lpxA. Notably, the mutation pattern of the two operons was almost conserved among the isolates that belonged to the same sequence type (ST) or GC. This was also confirmed by expanding the analysis to include A. baumannii genomes deposited in public databases. Here, we demonstrated the possible existence of missense mutations in pmrCAB and lpxACD operons in colistin-susceptible isolates, shedding light on the importance of interpreting mutations with reference to colistin-susceptible isolates of the same ST/GC to avoid the misleading impact of the ST/GC-related polymorphism. In turn, this may lead to misinterpretation of mutations and, hence, overlooking the real players in colistin resistance that are yet to be identified.

Oxidative metabolism is impaired by phosphate deficiency during fracture healing and is mechanistically related to BMP induced chondrocyte differentiation.

Prior studies of acute phosphate restriction during the endochondral phase of fracture healing showed delayed chondrocyte differentiation was mechanistically linked to decreased bone morphogenetic protein signaling. In the present study, transcriptomic analysis of fracture callus gene expression in three strains of mice was used to identify differentially expressed (FDR = q ≤ 0.05) genes in response to phosphate (Pi) restriction. Ontology and pathway analysis of these genes showed that independent of genetic background, a Pi-deficient diet downregulated (p = 3.16 × 10-23) genes associated with mitochondrial oxidative phosphorylation pathways as well as multiple other pathways of intermediate metabolism. Temporal clustering was used to identify co-regulation of these specific pathways. This analysis showed that specific Ox/Phos, tricarboxylic acid cycle, pyruvate dehydrogenase. Arginine, proline metabolism genes, and prolyl 4-hydroxylase were all co-regulated in response to dietary Pi restriction. The murine C3H10T½ mesenchymal stem cell line was used to assess the functional relationships between BMP2-induced chondrogenic differentiation, oxidative metabolism and extracellular matrix formation. BMP2-induced chondrogenic differentiation of C3H10T½ was carried out in culture media in the absence or presence of ascorbic acid, the necessary co-factor for proly hydroxylation, and in media with normal and 25 % phosphate levels. BMP2 treatment led to decreased proliferation, increased protein accumulation and increased collagen and aggrecan gene expression. Across all conditions, BMP2 increased total oxidative activity and ATP synthesis. Under all conditions, the presence of ascorbate further increased total protein accumulation, proly-hydroxylation and aggrecan gene expression, oxidative capacity and ATP production. Lower phosphate levels only diminished aggrecan gene expression with no other effects of metabolic activity being observed. These data suggest that dietary phosphate restriction controls endochondral growth in vivo indirectly through BMP signaling, which upregulates oxidative activity that is linked to overall protein production and collagen hydroxylation.

Epidemiological features of nosocomial Klebsiella pneumoniae: virulence and resistance determinants.

Aim: The authors aimed to examine antibiotic resistance genes and representative virulence determinants among 100 Klebsiella pneumoniae isolates with an emphasis on capsular serotypes and clonality of some of the isolates. Methods: PCR amplification of (rmpA, rmpA2, iutA, iroN and IncHI1B plasmid) and (NDM, OXA-48, KPC, CTX-M-15, VIM, IMP, SPM) was conducted. Wzi sequencing and multilocus sequence typing (MLST) were performed. Results: K2 was the only detected serotype in the authors' collection. RMPA2 was the most common capsule-associated virulence gene detected. All studied isolates harbored OXA-48-like (100%) and NDM (43%) (n = 43). ST147 was the most common sequence type. Conclusion: This work provides insight into the evolution of the coexistence of virulence and resistance genes in a tertiary healthcare setting in Cairo, Egypt.

Prevalence and molecular characterization of foodborne and human-derived Salmonella strains for resistance to critically important antibiotics.

The primary goals of this cross-sectional study were to screen various food/water, and human samples for the presence of Salmonella species, and to assess the phenotypic and genetic relationship between resistances found in food and human Salmonella isolates to critically important antibiotics. Between November 2019 and May 2021, 501 samples were randomly collected for Salmonella isolation and identification using standard culturing methods, biochemical, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and PCR techniques. Antimicrobial susceptibility testing was performed on confirmed Salmonella species, and PCR was used to investigate the genetic components that confer these resistance traits. Salmonella enterica subspecies enterica was confirmed in 35 (6.99%) of the samples (raw food = 23, ready-to-eat food/drink [REF/D] = 5, human = 7). Seventeen of them were antibiotic-resistant to at least one class, and eight were multidrug-resistant (MDR) isolates (raw food = 7, human = 1). All Salmonella isolates were susceptible to carbapenems, third- and fourth-generation cephalosporins and monobactam antibiotics. Resistance phenotypes to aminoglycosides (48.57%), ß-lactams (20%) and tetracycline (17.14%), as well as associated genes such as aadA, blaTEM , blaZ and tetA, as well as dfrA and sul1, were prevalent in Salmonella isolates. Colistin resistance genotype (mcr1) was detected in three (8.57%) isolates recovered from egg, cattle mince and rabbit meat, and the total incidence was 14.29% when two isolates exhibited resistance phenotypes were considered. Furthermore, four (11.43%) MDR isolates shared the blaTEM and blaZ genes, and one (2.86%) isolate contained three extended spectrum ß-lactams producing genes (ESBL), namely blaCTX , blaTEM and blaZ . The gyrA gene was expressed by one of three foodborne Salmonella isolates (8.57%) with ciprofloxacin resistance phenotypes. To the best of our knowledge, this is the first report from Egypt identifying colistin resistance in Salmonella enterica recovered from cattle minced meat and rabbit meat. Overall, the highest incidence rate of Salmonella enterica was found in cattle-derived products, and it was slightly more prevalent in RTE/D foods than in raw foods. Resistance to critical and clinically important antibiotics, particularly in Salmonella from RTE/D food, suggests that these antibiotics are being abused in the investigated area's veterinary field, and raises the potential of these isolates being transmitted to high-risk humans, which would be a serious problem. Future research using whole-genome sequencing is needed to clarify Salmonella resistance mechanisms to critically important antimicrobial agents or those exhibiting multidrug resistance.

Genetic Configuration of Genomic Resistance Islands in Acinetobacter baumannii Clinical Isolates From Egypt.

In Acinetobacter baumannii (A. baumannii), a wide repertoire of resistance genes is often carried within genomic resistance islands (RIs), particularly in high-risk global clones (GCs). As the first in Egypt, the current study aimed at exploring the diversity and genetic configuration of RIs in the clinical isolates of A. baumannii. For this purpose, draft genomes of 18 isolates were generated by Illumina sequencing. Disk diffusion susceptibility profiling revealed multidrug resistance (MDR) and extensive drug resistance (XDR) phenotypes in 27.7 and 72.2%, respectively. The highest susceptibility was noted for tigecycline (100.0%) followed by colistin (94.4%), for which an MIC50 of 0.25 µg/ml was recorded by the broth microdilution assay. Sequence typing (ST) showed that the majority of the isolates belonged to high-risk global clones (GC1, GC2, and GC9). A novel Oxford sequence type (ST2329) that also formed a novel clonal complex was submitted to the PubMLST database. A novel bla ADC variant (bla ADC-258) was also identified in strain M18 (ST85Pas/1089Oxf). In addition to a wide array of resistance determinants, whole-genome sequencing (WGS) disclosed at least nine configurations of genomic RIs distributed over 16/18 isolates. GC2 isolates accumulated the largest number of RIs (three RIs/isolate) followed by those that belong to GC1 (two RIs/isolate). In addition to Tn6022 (44.4%), the comM gene was interrupted by AbaR4 (5.5%) and three variants of A. baumannii genomic resistance island 1(AbGRI)-type RIs (44.4%), including AbaR4b (16.6%) and two novel configurations of AbGRI1-like RIs (22.2%). Three of which (AbaR4, AbaR4b, and AbGRI1-like-2) carried bla OXA-23 within Tn2006. With less abundance (38.8%), IS26-bound RIs were detected exclusively in GC2 isolates. These included a short version of AbGRI2 (AbGRI2-15) carrying the genes bla TEM-1 and aphA1 and two variants of AbGRI3 RIs carrying up to seven resistance genes [mphE-msrE-armA-sul1-aadA1-catB8-aacA4]. Confined to GC1 (22.2%), sulfonamide resistance was acquired by an ISAba1 bracketed GIsul2 RI. An additional RI (RI-PER-7) was also identified on a plasmid carried by strain M03. Among others, RI-PER-7 carried the resistance genes armA and bla PER-7. Here, we provided a closer view of the diversity and genetic organization of RIs carried by a previously unexplored population of A. baumannii.