Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Hydrogen stable isotope probing of lipids demonstrates slow rates of microbial growth in soil.

The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen-stable isotope probing of lipids with 2H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that growth rates in soil are quite slow and correspond to average generation times of 14 to 45 d but are also highly variable at the compound-specific level (4 to 402 d), suggesting differential growth rates among community subsets. We observe that low-biomass microbial communities exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid-stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities.

The number of arthroscopies performed by trainees does not deduce the level of their arthroscopic proficiency.

PURPOSE: It is reasonable to question whether the case volume is a suitable proxy for the manual competence of an arthroscopic surgeon. The aim of this study was to evaluate the correlation between the number of arthroscopies previously performed and the arthroscopic skills acquired using a standardized simulator test. METHODS: A total of 97 resident and early orthopaedic surgeons who participated in arthroscopic simulator training courses were divided into five groups based on their self-reported number of arthroscopic surgeries: (1) none, (2) < 10, (3) 10 to 19, (4) 20 to 39 and (5) 40 to 100. Arthroscopic manual skills were evaluated with a simulator by means of the diagnostic arthroscopy skill score (DASS) before and after training. Seventy-five points out of 100 must be achieved to pass the test. RESULTS: In the pretest, only three trainees in group 5 passed the arthroscopic skill test, and all other participants failed. Group 5 (57 ± 17 points; n = 17) scored significantly higher than the other groups (group 1: 30 ± 14, n = 20; group 2: 35 ± 14, n = 24; group 3: 35 ± 18, n = 23; and group 4: 33 ± 17, n = 13). After a two-day simulator training, trainees showed a significant increase in performance. In group 5, participants scored 81 ± 17 points, which was significantly higher than the other groups (group 1: 75 ± 16; group 2: 75 ± 14; group 3: 69 ± 15; and group 4: 73 ± 13). While self-reported arthroscopic procedures were n.s. associated with higher log odds of passing the test (p = 0.423), the points scored in the pretest were found to be a good predictor of whether a trainee would pass the test (p < 0.05). A positive correlation was observed between the points scored in the pretest and the posttest (p < 0.05, r = 0.59, r2 = 0.34). CONCLUSIONS: The number of previously performed arthroscopies is not a reliable indicator of the skills level of orthopaedic residents. A reasonable alternative in the future would be to verify arthroscopic proficiency on the simulator by means of a score as a pass-fail examination. LEVEL OF EVIDENCE: III.

Significant increase in quantity and quality of knee arthroplasty related research in KSSTA over the past 15 years.

PURPOSE: This study aimed to evaluate both publication and authorship characteristics in Knee Surgery, Sports Traumatology, Arthroscopy journal (KSSTA) regarding knee arthroplasty over the past 15 years. METHODS: PubMed was searched for articles published in KSSTA between January 1, 2006, and December 31st, 2020, utilising the search term 'knee arthroplasty'. 1288 articles met the inclusion criteria. The articles were evaluated using the following criteria: type of article, type of study, main topic and special topic, use of patient-reported outcome scores, number of references and citations, level of evidence (LOE), number of authors, gender of the first author and continent of origin. Three time intervals were compared: 2006-2010, 2011-2015 and 2016-2020. RESULTS: Between 2016 and 2020, publications peaked at 670 articles (52%) compared with 465 (36%) published between 2011 and 2016 and 153 articles (12%) between 2006 and 2010. While percentage of reviews (2006-2010: 0% vs. 2011-2015: 5% vs. 2016-2020: 5%) and meta-analyses (1% vs. 6% vs. 5%) increased, fewer case reports were published (13% vs. 3% vs. 1%) (p < 0.001). Interest in navigation and computer-assisted surgery decreased, whereas interest in perioperative management, robotic and individualized surgery increased over time (p < 0.001). There was an increasing number of references [26 (2-73) vs. 30 (2-158) vs. 31 (1-143), p < 0.001] while number of citations decreased [30 (0-188) vs. 22 (0-264) vs. 6 (0-106), p < 0.001]. LOE showed no significant changes (p = 0.439). The number of authors increased between each time interval (p < 0.001), while the percentage of female authors was comparable between first and last interval (p = 0.252). Europe published significantly fewer articles over time (56% vs. 47% vs. 52%), whereas the number of articles from Asia increased (35% vs. 45% vs. 37%, p = 0.005). CONCLUSION: Increasing interest in the field of knee arthroplasty-related surgery arose within the last 15 years in KSSTA. The investigated topics showed a significant trend towards the latest techniques at each time interval. With rising number of authors, the part of female first authors also increased-but not significantly. Furthermore, publishing characteristics showed an increasing number of publications from Asia and a slightly decreasing number in Europe. LEVEL OF EVIDENCE: IV.

Exploring the Potential of Electrospray-Orbitrap for Stable Isotope Analysis Using Nitrate as a Model.

Widely used isotope ratio mass spectrometers have limited capabilities to measure metabolites, drugs, or small polyatomic ions without the loss of structural isotopic information. A new approach has recently been introduced that uses electrospray ionization Orbitrap to measure multidimensional isotope signatures of intact polar compounds. Using nitrate as a model compound, this study aims to establish performance metrics for comparisons with conventional IRMS at the natural abundance level. We present a framework on how to convert isotopolog intensities to δ values that are commonly used in the isotope geochemistry community. The quantification of seven nitrate isotopologs provides multiple pathways for obtaining the primary N and O δ values including non-mass-dependent O isotope variations, as well as opportunities to explore nonrandom isotopic distributions (i.e., clumping effects) within molecular nitrate. Using automation and the adaptation of measurement principles that are specific to isotope ratio analysis, nitrate δ15NAIR, δ18OVSMOW, and δ17OVSMOW were measured with a long-term precision of 0.4‰ or better for isotopic reference materials and purified nitrate from environmental samples. In addition, we demonstrate promising results for unpurified environmental samples in liquid form. With these new developments, this study connects the two largely disparate mass spectrometry fields of bioanalytical MS and isotope ratio MS, thus providing a route to measure new isotopic signatures in diverse organic and inorganic solutes.

Determination of the δ2 H values of high molecular weight lipids by high-temperature gas chromatography coupled to isotope ratio mass spectrometry.

RATIONALE: The hydrogen isotopic composition of lipids (δ2 Hlipid ) is widely used in food science and as a proxy for past hydrological conditions. Determining the δ2 H values of large, well-preserved triacylglycerides and other microbial lipids, such as glycerol dialkyl glycerol tetraether (GDGT) lipids, is thus of widespread interest but has so far not been possible due to their low volatility which prohibits analysis by traditional gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS). METHODS: We determined the δ2 H values of large, polar molecules and applied high-temperature gas chromatography (HTGC) methods on a modified GC/P/IRMS system. The system used a high-temperature 7-m GC column, and a glass Y-splitter for low thermal mass. Methods were validated using authentic standards of large, functionalised molecules (triacylglycerides, TGs), purified standards of GDGTs. The results were compared with δ2 H values determined by high-temperature elemental analyser/pyrolysis/isotope ratio mass spectrometry (HTEA/P/IRMS), and subsequently applied to the analysis of GDGTs in a sample from a methane seep and a Welsh peat. RESULTS: The δ2 H values of TGs agreed within error between HTGC/P/IRMS and HTEA/IRMS, with HTGC/P/IRMS showing larger errors. Archaeal lipid GDGTs with up to three cyclisations could be analysed: the δ2 H values were not significantly different between methods with standard deviations of 5 to 6 ‰. When environmental samples were analysed, the δ2 H values of isoGDGTs were 50 ‰ more negative than those of terrestrial brGDGTs. CONCLUSIONS: Our results indicate that the HTGC/P/IRMS method developed here is appropriate to determine the δ2 H values of TGs, GDGTs with up to two cyclisations, and potentially other high molecular weight compounds. The methodology will widen the current analytical window for biomarker and food light stable isotope analyses. Moreover, our initial measurements suggest that bacterial and archaeal GDGT δ2 H values can record environmental and ecological conditions.

Enzyme-Specific Coupling of Oxygen and Nitrogen Isotope Fractionation of the Nap and Nar Nitrate Reductases.

Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR is catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases and fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O), which is reflected in residual environmental nitrate pools. Data on the relationship between the pattern in oxygen vs nitrogen isotope fractionation (18ε/15ε) suggests that systematic differences exist between marine and terrestrial ecosystems that are not fully understood. We examined the 18ε/15ε of nitrate-reducing microorganisms that encode Nar, Nap, or both enzymes, as well as gene deletion mutants of Nar and Nap to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of 18ε/15ε fractionation ratios of all examined nitrate reductases forms two distinct peaks centered around an 18ε/15ε proportionality of 0.55 (Nap) and 0.91 (Nar), with the notable exception of the Bacillus Nar reductases, which cluster isotopically with the Nap reductases. Our findings may explain differences in 18ε/15ε fractionation between marine and terrestrial systems and challenge current knowledge about Nar 18ε/15ε signatures.

Isolated gracilis tendon harvesting is not associated with loss of strength and maintains good functional outcome.

PURPOSE: The gracilis tendon is a commonly used autologous graft. Most information on knee function and outcomes after its harvest is related to both semitendinosus- and gracilis tendon harvest. Therefore this study analyzed the effect of isolated gracilis tendon harvest from healthy, uninjured knees on thigh muscle strength and patient reported outcome measures (PROMs). METHODS: Stabilization of the acromioclavicular joint because of chronic instability was performed with autologous gracilis tendon in 12 patients. After a mean of 44 ± 25 months after surgery, isokinetic peak-torque measurements of specific functions of the gracilis muscle were performed: knee flexion in a sitting position (flexion angles 0-90°) and in prone position (flexion angles > 70°), internal tibial rotation and hip adduction. The contralateral limb was control. Knee specific PROMs were collected including IKDC-2000 subjective evaluation form, Lysholm score, the Marx Activity Rating Scale and SF-36 health survey. RESULTS: No significant side-to-side differences were found regarding torque measurements. Excellent results were shown regarding the PROMs, which even in terms of IKDC-2000 (97 vs. 82 points, p = 0.001) exceeded significantly the age- and gender matched reference-data. CONCLUSION: Isolated gracilis tendon harvesting was not associated with loss of strength in knee flexion, internal tibial rotation and thigh adduction. Additionally, good functional outcome as well as excellent knee-specific subjective outcome was found. LEVEL OF EVIDENCE: III.

Management of traumatic meniscus tears: the 2019 ESSKA meniscus consensus.

PURPOSE: The importance of meniscus integrity in the prevention of early osteoarthritis is well known, and preservation is accepted as the primary goal. The purpose of the ESSKA (European Society for Sports Traumatology, Knee Surgery and Arthroscopy) European consensus on traumatic meniscus tears was to provide recommendations for the treatment of meniscus tears based on both scientific evidence and the clinical experience of knee experts. METHODS: Three groups of surgeons and scientists elaborated and ratified the so-called formal consensus process to define the recommendations for the management of traumatic meniscus tears. A traumatic meniscus tear was defined as a tear with an acute onset of symptoms caused by a sufficient trauma. The expert groups included a steering group of eight European surgeons and scientists, a rating group of another nineteen European surgeons, and a peer review group. The steering group prepared twenty-seven question and answer sets based on the scientific literature. The quality of the answers received grades of A (a high level of scientific support), B (scientific presumption), C (a low level of scientific support) or D (expert opinion). These question and answer sets were then submitted to and evaluated by the rating group. All answers were scored from 1 (= totally inappropriate) to 9 (= totally appropriate) points. Thereafter, the comments of the members of the rating group were incorporated by the steering group and the consensus was submitted to the rating group a second time. Once a general consensus was reached between the steering and rating groups, the finalized question and answer sets were submitted for final review by the peer review group composed of representatives of the ESSKA-affiliated national societies. Eighteen representatives replied. RESULTS: The review of the literature revealed a rather low scientific quality of studies examining the treatment of traumatic meniscus tears. Of the 27 questions, only one question received a grade of A (a high level of scientific support), and another received a grade of B (scientific presumption). The remaining questions received grades of C and D. The mean rating of all questions by the rating group was 8.2 (95% confidence interval 8.1-8.4). A general agreement that MRI should be performed on a systematic basis was not achieved. However, MRI was recommended when arthroscopy would be considered to identify concomitant pathologies. In this case, the indication for MRI should be determined by a musculoskeletal specialist. Based on our data, stable left in situ lateral meniscus tears appear to show a better prognosis than medial tears. When repair is required, surgery should be performed as early as possible. Evidence that biological enhancement such as needling or the application of platelet-rich plasma would improve healing was not identified. Preservation of the meniscus should be considered as the first line of treatment because of an inferior clinical and radiological long-term outcome after partial meniscectomy compared to meniscus repair. DISCUSSION: The consensus was generated to present the best possible recommendations for the treatment of traumatic meniscus tears and provides some groundwork for a clinical decision-making process regarding the treatment of meniscus tears. Preservation of the meniscus should be the first line of treatment when possible, because the clinical and radiological long-term outcomes are worse after partial meniscectomy than after meniscus preservation. The consensus clearly states that numerous meniscus tears that were considered irreparable should be repaired, e.g., older tears, tears in obese patients, long tears, etc. LEVEL OF EVIDENCE: II.

Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum.

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

No correlation between rotation of femoral components in the transverse plane and clinical outcome after total knee arthroplasty.

PURPOSE: It was hypothesized that malrotation of femoral component in total knee arthroplasty (TKA) will significantly impact clinical outcome. METHODS: Eighty-eight consecutive patients with primary osteoarthritis of the knee were prospectively evaluated. They received a cemented posterior stabilized TKA (NexGen, Zimmer/Biomet® Inc., Warsaw, IN, USA). The femoral component was placed in 3° of external rotation referenced to the posterior condylar line. Postoperatively, a CT scan was performed to evaluate rotation of femoral component in the transverse plane. SF-36, KSS, and WOMAC, as well as their range of motion was assessed prior to surgery, after 6 and 24 months. Data are presented as mean and standard deviation (SD), as well as range if applicable. Correlation analysis was performed between the placement of the femoral component in the transverse plane and the clinical outcome. RESULTS: Femoral component placement was on average 0.1° [SD 2.5°, range - 6.5° to + 6.5°] referenced to the surgical transepicondylar line showing a wide range between the two landmarks after surgery. After 6 months, WOMAC category 'physical function' correlated significantly with femoral component rotation (r = - 0.28, p = 0.007). After 24 months, WOMAC categories 'physical function' and 'pain' correlated significantly with femoral rotation (r = - 0.41, p < 0.001; and r = - 0.33, p = 0.001). No significant correlations were found between femoral component rotation and range of motion (r = 0.04), WOMAC category "stiffness", KSS, as well as SF-36 questionnaires. These reported formally significant correlations were without any clinical relevance. DISCUSSION: The study showed that there is a significant patients specific femoral component placement in the transverse plane. Internal or external malrotation of the femoral component does not correlate automatically with poor knee function. The lack of correlation between femoral component position and clinical as well as functional outcome underlines complexity and significant individuality of each patient. The surgeon should be aware of the finding and attention should be paid during surgery when significant divergency is seen between the two landmarks. Soft tissue balancing might be very crucial in these specific patients, which needs to be studied in depth in the future. LEVEL OF EVIDENCE: II.

A whole leg radiograph is not necessary for postoperative determination of the mechanical leg axis after total knee arthroplasty.

BACKGROUND: Anteroposterior (AP) whole leg radiographs (WLR) in the standing position for assessment of the mechanical leg axis are generally performed preoperatively for the planning of total knee replacement (TKR) and postoperatively to assess the leg axis. The objective of the present study was to investigate whether, if preoperative WLR are available, postoperative AP standard knee radiographs in the standing position are sufficient for calculating the mechanical leg axis. METHODS: In the present prospective study, the mechanical and the anatomical leg axes were determined on the basis of WLR from 104 patients prior to implantation of a TKR and the difference was calculated. Twelve weeks postoperatively, standing long AP radiographs and WLR were prepared. In addition, the mechanical axis was calculated by adding the preoperative difference between the anatomical and mechanical axis to the anatomical axis from the postoperative AP radiographs. Accuracy, bias and level of agreement for calculated relative to measured mechanical alignment were determined. RESULTS: Mean accuracy of calculated mechanical alignment was 0.5° ± 0.4°, and mean bias was 0.0° ± 0.6° (p = 1.00). Bland-Altman analysis revealed a 95% upper and lower level of agreement of - 1.3° and 1.3°, respectively. CONCLUSION: A preoperative WLR and a postoperative long AP knee standard radiograph are sufficient to determine the mechanical leg axis after TKR. If these are available, it is possible to do without WLR after TKR, particularly since they involve higher radiation exposure, are time-consuming, and are also prone to errors in the first postoperative weeks. LEVEL OF EVIDENCE: II diagnostic study.

Alterations in structure of the muscle-tendon unit and gait pattern after percutaneous repair of Achilles tendon rupture with the Dresden instrument.

BACKGROUND: Functional deficits after Achilles tendon (AT) ruptures are observed. The relationship between musculotendinous structural alterations and functional outcome is not clear. METHODS: Kinematic analyses (level walking, stair climbing), patient-reported outcome measures (PROMs), calf atrophy (maximum calf circumference (MCC)), and AT length were evaluated in patients after percutaneous AT repair with the Dresden instrument (n=20min. FOLLOW-UP: 24 months). RESULTS: Patients achieved good results in PROMs. However, MCC decreased significantly and AT length increased significantly postoperatively. Side-to-side MCC differences over 2cm resulted in significantly lower PROMs. AT lengthening correlated with increased dorsiflexion and decreased plantarflexion. CONCLUSION: Calf atrophy and AT lengthening after minimally invasive AT repair resulted in inferior ankle kinematics and PROMs.

Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain ("anteiso") fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward "best practices" for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite.IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods.

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

Towards measuring growth rates of pathogens during infections by D2 O-labeling lipidomics.

RATIONALE: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. METHODS: Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. RESULTS: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. CONCLUSIONS: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.

The influence of applied internal and external rotation on the pivot shift phenomenon.

PURPOSE: The pivot shift test is performed in different techniques and the rotation of the tibia seems to have a significant impact on the amount of the pivot shift phenomenon. It has been hypothesised that external rotation will increase the phenomenon due to less tension at the iliotibial band in knee extension. METHODS: Twenty-four patients with unilateral anterior cruciate ligament insufficiency were included prospectively. The pivot shift test was performed bilaterally in internal and external tibial rotation under general anaesthesia. Knee motion was captured using a femoral and a tibial inertial sensor. The difference between positive and negative peak values in Euclidean norm of acceleration was calculated to evaluate the amount of the pivot shift phenomenon. RESULTS: The pivot shift phenomenon was significantly increased in patients with ACL insufficiency when the test was performed in external [mean 5.2 ms- 2 (95% CI 4.3-6.0)] compared to internal tibial rotation [mean 4.4 ms- 2 (95% CI 3.5-5.4)] (p = 0.002). In healthy, contralateral knees did not show any difference between external [mean 4.0 ms- 2 (95% CI 3.3-4.7)] and internal tibial rotation [mean 4.0 ms- 2 (95% CI 3.4-4.6)] (ns). CONCLUSIONS: The pivot shift phenomenon was increased with external rotation in ACL-insufficient knees, and therefore, one should perform the pivot shift test, rather, in external rotation to easily evoke the, sometimes difficult to detect, pivot shift phenomenon. LEVEL OF EVIDENCE: I (diagnostic study).

Heavy water and (15) N labelling with NanoSIMS analysis reveals growth rate-dependent metabolic heterogeneity in chemostats.

To measure single-cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and (15) N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single-cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single-cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % (2) H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.