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Background: Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples., Methods: A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample., Findings: In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel β-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo., Interpretation: This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections., Funding: Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU., Competing Interests: Declaration of interests AO and RC have participated in educational programs organised by MSD, Pfizer and Shionogi and conducted research studies financed by MSD and Shionogi. JPH has participated in educational programs organised by MSD, Pfizer, Menarini and Angelini and in advisory boards organised by Advanz Pharma, Tillots, and GILEAD., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aim: To monitor prospectively the occurrence of colorectal anastomotic leakage (CAL) in patients with colon cancer undergoing resectional surgery, characterizing the microbiota in both faeces and mucosal biopsies of anastomosis. In a second stage, we investigated the ability to predict CAL using machine learning models based on clinical data and microbiota composition., Method: A total of 111 patients were included, from whom a faecal sample was obtained, as well as biopsy samples from proximal and distal sites in the healthy margins of the tumour piece. The microorganisms present in the samples were investigated using microbial culture and 16S rDNA massive sequencing. Collagenase and protease production was determined, as well as the presence of genes responsible for expressing enzymes with these activities. Machine learning analyses were developed using clinical and microbiological data., Results: The incidence of CAL was 9.0%, and CAL was associated with collagenase/protease-producing Enterococcus. Significant differences were found in the microbiota composition of proximal and distal biopsy samples, but not in faecal samples, among patients who developed CAL. Clinical predictors of CAL were 5-day C-reactive protein and heart disease, whereas 3-day C-reactive protein and diabetes were negative predictors., Conclusion: Biopsy samples from surgical margins, rather than faecal samples, are the most appropriate samples for exploring the contribution of the intestinal microbiota to CAL. Enterococci are only enriched in the anastomosis after surgery, and their collagenases and proteases are involved in the degradation of the anastomotic scar., (© 2023 The Authors. Colorectal Disease published by John Wiley & Sons Ltd on behalf of Association of Coloproctology of Great Britain and Ireland.)

Objective: To evaluate the activity of cefiderocol against sequential P. aeruginosa isolates from chronically-infected cystic fibrosis patients as well as to investigate the potential mechanisms involved in resistance through whole genome sequencing., Methods: Three sequential P. aeruginosa isolates from each of 50 chronically-colonized cystic fibrosis patients were studied. MICs for novel and classical antipseudomonal agents were determined by broth microdilution and whole genome sequences (n = 150) were obtained to investigate the presence of mutations within a set of chromosomal genes involved in P. aeruginosa antibiotic resistance (n = 40) and iron uptake (n = 120)., Results: Cefiderocol showed the lowest MIC 50/90 values and its susceptibility rate was comparable to other novel antipseudomonal agents. Clinical resistance was documented in 9 isolates from 6 patients. Resistance genes associated with a statistically significant increase in cefiderocol MICs included ampC, pmrAB, galU, fusA1 and those coding the penicillin-binding proteins PBP2 and PBP3. Likewise, mutations within several genes participating in different iron-uptake systems were found to be significantly associated with resistance, including genes participating in the pyochelin and pyoverdin biosynthesis and several tonB-dependent receptors. Mutator and small colony variants isolates were also associated with increased cefiderocol MICs., Discussion: Cefiderocol resistance is modulated by a complex mutational resistome, potentially conferring cross-resistance to novel beta-lactam beta-lactamase combinations, as well as an extended list of mutated iron-uptake genes. Monitoring the acquisition of mutations in all these genes will be helpful to guide treatments and mitigate the emergence and spread of resistance to this novel antibiotic., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Streams and rivers act as landscape-scale bioreactors processing large quantities of terrestrial particulate organic matter (POM). This function is linked to their flow regime, which governs residence times, shapes organic matter reactivity and controls the amount of carbon (C) exported to the atmosphere and coastal oceans. Climate change impacts flow regimes by increasing both flash floods and droughts. Here, we used a modelling approach to explore the consequences of lateral hydrological contraction, that is, the reduction of the wet portion of the streambed, for POM decomposition and transport at the river network scale. Our model integrates seasonal leaf litter input as generator of POM, transient storage of POM on wet and dry streambed portions with associated decomposition and ensuing changes in reactivity, and transport dynamics through a dendritic river network. Simulations showed that the amount of POM exported from the river network and its average reactivity increased with lateral hydrological contraction, due to the combination of (1) low processing of POM while stored on dry streambeds, and (2) large shunting during flashy events. The sensitivity analysis further supported that high lateral hydrological contraction leads to higher export of higher reactivity POM, regardless of transport coefficient values, average reactivity of fresh leaf litter and differences between POM reactivity under wet and dry conditions. Our study incorporates storage in dry streambed areas into the pulse-shunt concept (Raymond and others in Ecology 97(1):5-16, 2016. 10.1890/14-1684.1), providing a mechanistic framework and testable predictions about leaf litter storage, transport and decomposition in fluvial networks., Supplementary Information: The online version contains supplementary material available at 10.1007/s10021-022-00802-4., (© The Author(s) 2022.)

Ticks are blood feeder able to transmit a wide diversity of microbes including pathogens. Therefore, it is of our interest to detect the diversity of microorganisms residing within ticks using massive sequencing of 16S rDNA. In this study, 200 adult ticks were collected from healthy camels in two localities from Hail province (Saudi Arabia). The analysis showed high microbial diversity dominated by the two domains (Archaea and Bacteria) associated with Hyalomma dromedarii from both regions. Proteobacteria (61.3%) and Firmicutes (31.2%) dominated the ticks from the Al Khotha region. While, the microbiome of ticks from the Al Gayed region was dominated by Proteobacteria (81.2%) and Firmicutes (9.2%). Twenty-three families were identified in the DNA-pool from the Al Gayed region, and was dominated by Pseudomonadaceae (45.37%), and Marinobacteraceae (14.39%) families. Francisellaceae (46%), Staphylococcaceae (24.26%) dominated the microbiome of the ticks collected from Al Gayed region. Thus, the genera Pseudomonas, Francisella, Proteus, Marinobacter, Glutamicibacter, Pedobacter, and Staphylococcus are largely distributed in the two identified microbiomes. This study concluded that ticks collected from the studied localities contained a wide range of microbial communities. These data have a great veterinary and medical importance in near future., (© 2020 Biomedical Informatics.)

Purpose: The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals., Methodology: In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR., Results: A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3')-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %)., Conclusion: To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

A highly topical set of perovskite oxides are high-sensitivity piezoelectric ones, among which Pb(Zr,Ti)O 3 at the morphotropic phase boundary (MPB) between ferroelectric rhombohedral and tetragonal polymorphic phases is reckoned a case study. Piezoelectric ceramics are used in a wide range of mature, electromechanical transduction technologies like piezoelectric sensors, actuators and ultrasound generation, to name only a few examples, and more recently for demonstrating novel applications like magnetoelectric composites. In this case, piezoelectric perovskites are combined with magnetostrictive materials to provide magnetoelectricity as a product property of the piezoelectricity and piezomagnetism of the component phases. Interfaces play a key issue, for they control the mechanical coupling between the piezoresponsive phases. We present here main results of our investigation on the suitability of the high sensitivity MPB piezoelectric perovskite BiScO 3 -PbTiO 3 in combination with ferrimagnetic spinel oxides for magnetoelectric composites. Emphasis has been put on the processing at low temperature to control reactions and interdiffusion between the two oxides. The role of the grain size effects is extensively addressed.

Mg and Mg-HAP composites containing 5, 10 and 15 wt% of hydroxyapatite have been produced following a powder metallurgy route that consists of mixing raw powders and consolidation by extrusion. The microstructure, texture, mechanical behavior and resistance to corrosion under a PBS solution have been studied. Addition of HAP increases the microhardness of the composites, however the yield strength under compression slightly decreases. Texture analyses reveal a fiber texture for pure Mg that is weakened increasing the HAP fraction. This texture promotes twinning and softening of Mg and Mg-5HAP during the initial deformation stages. Mg-10HAP and Mg-15HAP present a strain-hardening dependence showing no softening. The volume fraction of HAP particles weakens the texture and favors the activation of secondary slip systems. Corrosion experiments in PBS solution have shown that Mg-5HAP exhibits the best resistance to corrosion. Texture and porosity appear to be the main material features controlling the corrosion rates of Mg-HAP composites under the present conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Proportional Hazards models have been widely used to analyze survival data. In many cases survival data do not verify the assumption of proportional hazards. An alternative to the PH models with more relaxed conditions are Accelerated Failure Time models. These models are fairly commonly used in the field of manufacturing, but they are more and more frequent for modeling clinical trial data. They focus on the direct effect of the explanatory variables on the survival function allowing an easier interpretation of the effect of the corresponding covariates on the survival time. Optimal experimental designs are computed in this framework for Type I and random arrival. The results are applied to clinical models used to prevent tuberculosis in Ugandan adults infected with HIV., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RIETE is an ongoing registry of consecutive patients with acute venous thromboembolism (VTE). First we learned that in RIETE the prevalence of positive tests was 32%. One in every 2 patients younger than 50 years tested positive, with no differences between idiopathic or secondary, first event or recurrent VTE. In contrast, one in every 4 patients older than 50 years tested positive. Then, we found that the younger age of women with factor V Leiden or prothrombin G20210A may be attributed to the higher proportion of women who had the VTE during pregnancy or contraceptive use. At variance with this, 60% of men had idiopathic VTE, and only those with factor V Leiden were younger than those who tested negative. We need more patients, more data and a longer follow-up. In a near future we might be able to learn more about the outcome of VTE patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization.

Objective: To identify infection-causing Enterococcus species in Cuban hospitals and determine their susceptibility to antimicrobial drugs, as well as their resistance mechanisms., Methods: A total of 687 Enterococcus isolates from 30 Cuban hospitals in nine provinces of the country were studied over the period 2000-2009. The species were identified using both the conventional method and the automatic API(®) system. The minimum inhibitory concentration was determined for 13 antimicrobial drugs following the standards recommended by the Clinical Laboratory and Standards Institute. The polymerase chain reaction technique was used to characterize the genes that were resistant to aminoglycosides, erythromycin, tetracycline, and glucopeptides. The presence of beta-lactamase was determined by the chromogenic cephalosporin test., Results: The most prevalent species were Enterococcus faecalis (82.9%) and E. faecium (12.2%). Resistance to glucopeptides (1.0%) was mediated by the vanA and vanB genes. The strains resistant to ampicillin (6%) did not produce beta-lactamases. A high percentage of resistance to aminoglycosides was observed. Gentamicin (31.0%) and streptomycin and amikacin (29.1%) were mediated by the aac(6')Ie-aph(2")Ia, aph(3')-IIIa, ant(6)Ia, and ant(3")(9) genes. A correlation was found between resistance to tetracycline (56.0%) and presence of the tet(M) (75.1%) and tet(L) genes (7.0%), while resistance to erythromycin (34.1%) was due to the erm(B) gene (70.9%)., Conclusions: Resistance to vancomycin is infrequent in Cuba, as opposed to a high level of resistance to aminoglycosides, which may be indicative of treatment failures. The microbiology laboratory is a cornerstone of Enterococcus infection surveillance, along with ongoing monitoring of the susceptibility of these infections to antimicrobial drugs at a time when resistance of this microorganism is on the rise.

All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sodA gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sodA gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphysial plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.

The purposes of these experiments were to study the biosynthetic and postbiosynthetic relationships between proteoglycans in noncalcified growth cartilage and calcified cartilage in metaphysis from the costochondral junctions of immature rabbits. Based on in vivo experiments in which 35 S-sodium sulfate was injected into rabbits, it is shown that proteoglycans from the hypertrophic region becomes part of the calcified cartilage matrix which is to be incorporated into the metaphysis. The proteoglycan aggregates in the growth apparatus undergo partial disaggregation and degradation. There is approximately a 25% decrease in aggregation from regions of the rib distal to the metaphyseal-growth plate junction (69%) to the region proximal to it (50%). In contrast, in their final state in calcified cartilage, the proteoglycans are more completely disaggregated and the proteoglycans subunits are smaller, as adjudged from gel chromatography. Control experiments indicate that although some artifactual disaggregation is produced by the extraction process, it is not of the same magnitude as that seen in the actual isolation experiments nor are the subunits reduced in size.

Recent immunofluorescent and histochemical data did not detect changes in the concentration of proteoglycans between noncalcified and calcified cartilage in fetal bovine growth plate or metaphyseal bone. These findings were constant, regardless of prior fixation before demineralization with disodium ethylenediaminetetraacetate (EDTA) or prior demineralization before fixation. Previous experience has shown that EDTA can extract proteoglycans from calcified cartilage. With this in mind, we determined the amount of proteoglycan extracted from calcified cartilage in metaphyseal bone and uncalcified growth plate cartilages during decalcification of unfixed fresh tissues with EDTA. To this end, fresh growth plate cartilages and metaphyses were decalcified at 5 degrees C for 48 hours in a buffered solution of EDTA to which several protease inhibitors were added. Under these conditions 20-25% of the total proteoglycan (measured as uronic acid and hexosamine) was extracted from mineralized cartilage but only about 1% from the uncalcified (growth plate) cartilages. Thus, histochemical and immunohistochemical studies appear to be insensitive measures of proteoglycan concentrations in histological sections of mineralized tissue and may not give quantitative information.

Soluble proteoglycans (SPG) were extracted from bovine (BCC) and human (HCC) costal cartilages by the dissociative method using 4 M guanidinium chloride (GuHCl). Proteoglycans which are resistant to extraction (RPG) were obtained following collagenase digestion or hydroxylamine treatment of the cartilage residues. Similarly, SPG were extracted from bovine metaphyseal and cortical bone using EDTA. The RPG were extracted from the bones using hydroxylamine. Density gradient fractionation under dissociative conditions of cartilage SPG and RPG followed by chromatography on Sepharose 2B revealed that A1D1 RPG are smaller than the SPG. SPG reacted with either collagenase or hydroxylamine are also smaller than the parent SPG. A1D1 fractions obtained from BCC-SPG and RPG or from mixtures of SPG and acid-soluble collagen are free of hydroxyproline. Hydroxyproline is not completely separated from HCC-RPG. Density gradient fractionation of bone proteoglycans and Sepharose chromatography of the A1 and A1D1 fractions showed that those obtained from metaphysis are larger than those from cortical bone. This was attributed to the presence of calcified cartilage in metaphyseal bone. The A1D1 fractions of the metaphyseal proteoglycans seemed to undergo self-association since this fraction is larger than the A1 fraction from which it is derived. Cortical bone proteoglycans do not behave similarly. Density gradient purification under dissociative conditions failed to separate hydroxyproline from the proteoglycans obtained from bone. It is hypothesized that in bone proteoglycans and collagen might be linked.

Slices of fresh ovine and bovine epiphyseal cartilages swell following extraction in 0.05 M disodium ethylenediaminetetraacetate (EDTA) in Tris buffer, pH 5.8 and 7.4, at 4 degrees and 37 degrees. The swelling is strikingly visible to the unaided eye and is most pronounced in the growth plate region of the epiphysis. Other chelators--ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), and citrate buffer--also induce swelling. Swelling is associated with increased degradation of proteoglycans (PG) especially at pH 5.8, however, collagen seems to be unaffected. These effects are prevented by the addition of certain divalent cations (Ca, Mg, Zn) to the extraction media. At higher concentrations, the monovalent cation sodium also prevents swelling. It is concluded that divalent cations are required to maintain structure and function of cartilage. Freezing and thawing the cartilage did not prevent swelling or degradation, which suggests that these phenomena are not dependent on living chondrocytes. Although PG degradation and loss is markedly increased at 37 degrees as compared with 4 degrees, swelling is unaffected. It is concluded therefore that the degradative effects are enzymatic but the swelling is physicochemical. Other cartilages (nasal, manubrium) also swell and show histochemical evidence of PG degradation. These effects are minimal compared with the effects induced in the growth plate. It is inferred that growth plate contains more proteases than other cartilages and has properties that make it more susceptible to swelling. Swelling of the growth plate occurs even when the metaphysis is attached to it albeit to a lesser extent than when it is freed of underlying bone. A hypothesis is offered which attempts to link these phenomena with chondrocyte and matrical imbibition of water (swelling) in the zone of hypertrophy of the growth plate.

On the basis of an examination of autoradiograms of knee-joints fixed so as to remove chondroitin sulfate or inorganic sulfate, or to minimize the loss of both, it is suggested that the cartilage is permeable to inorganic sulfate in vivo and in vitro. In vivo and in vitro, almost as rapidly as it enters the cartilage, inorganic sulfate is utilized by the cells in the synthesis of chondroitin sulfate. The net result is a continuing low concentration of inorganic sulfate in the cartilage.

Tibiae and humeri were removed from suckling rats at intervals of time after intraperitoneal injection of C(14)-L-phenylalanine, C(14)-L-leucine, S(35)-sulfate, or Ca(45) Cl(2). Autoradiograms of sections of the bones were prepared. Ca(45) was removed from sections treated with dilute acetic acid; neither the concentration of S(35) nor that of C(14) was thereby markedly decreased. The S(35) was removed from the demineralized sections on incubation in a solution of testicular hyaluronidase; the C(14) was not. These results are interpreted as indicating that most of the S(35) was present in the bones as chondroitin sulfate and that most of the C(14) in the bones was present as protein. In the epiphyses, the C(14) was initially concentrated in the proliferaing and hypertrophic chondrocytes, as was the S(35). Secretion of S(35)- and C(14)-labeled materials into the matrix followed. Thereafter, however, although the S(35)-labeled material (chondroitin sulfate) persisted in the matrix, albeit at a diminished concentration, and was incorporated into metaphyseal bone, the C(14)-labeled material (protein) was almost completely removed from the matrix. When rats were given repeated doses of 17-beta-estradiol benzoate so as to inhibit resorption of their metaphyses, repeated doses of S(35)-sulfate were discerned as strata of S(35) in their metaphyses. This was not the case if the rats received repeated doses of C(14)-L-phenylalanine or C(14)-L-leucine. On the basis of the results in these experiments it is suggested that although a portion of the chondroitin sulfate produced by the chondrocytes of the epiphyseal plate is retained and becomes part of the cores of metaphyseal spicules of bone, the protein of the proteinpolysaccharide is somehow removed before calcification of the cartilage ensues.