A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm.1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet (‘80%’ sperm) and the 40/80 interface (‘40%’ sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of ‘40%’ and ‘80%’ sperm.Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis . ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65–0.67, lactate/lipid ROC AUC = 0.86–0.87.DGC with two washes minimized the Only 3–4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the ‘40%’ and ‘80%’ sperm.1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility.This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest. Poor sperm quality is a major barrier to conception and is thought to contribute to 30–50% of cases of infertility in heterosexual couples . HoweverMetabolomic studies by Magnetic Resonance Spectroscopy (MRS) have been used in many biological systems to provide insight into functional aspects of cell cultures, tissues and bio-fluids , boar  What sperm preparation methods are suitable to prepare live sperm for 1H MRS analysis?; (b) What is the minimum concentration of sperm required for 1H MRS analysis using a commonly available spectrometer?; and (c) Are there differences in the 1H spectra from populations of sperm recovered from density gradient centrifuge (DGC) that could give important new biological information about their function?While there are a variety of different approaches to conduct MRS, the 1H MRS spectra between the different populations of sperm recovered from DGC (question c) were obtained from men attending the Andrology Laboratory  for diagnostic semen analysis as part of infertility investigations. All samples were produced on-site and men gave their consent for any remaining semen to be used in this study once the routine semen analysis had been completed. Only those samples with sperm concentration and motility above For experiments to determine the best sperm washing method, semen samples were obtained from healthy volunteers following 2–3 days of sexual abstinence. All samples used in questions (a) and (b) were produced by masturbation at home and collected into a sterile plastic container  before being delivered to the laboratory within 1 h. Upon arrival, an assessment of semen volume, sperm concentration and motility was performed on each sample according to World Health Organisation  methods.1H MRS, three techniques of sperm preparation were used and described below. See Fig. To obtain matched samples of (i) unprocessed semen; (ii) sperm-free seminal plasma; and (iii) seminal plasma free populations of sperm, for 1 ml aliquots of each liquefied semen sample (Fraction A) were transferred to a 15 ml centrifuge tube  and centrifuged at 500 g for 15 min in a Sigma 3–16 K . After centrifugation, the (upper) seminal plasma supernatant (Fraction C) was aspirated and transferred to a clean tube, whereas the sperm pellet (Fraction B) was re-suspended in approximately 600 μl phosphate buffered saline (PBS). Aliquots of each fraction were then taken for MRS analysis as described below.1H MRS analysis in order to track the fumarate concentration at each stage. The recovered ‘40%’ sperm (Fraction I) and ‘80%’ sperm (Fraction J) were collected and re-suspended in 600 μl PBS and aliquots taken for 1H MRS analysis as described below.Density gradients were prepared by carefully layering 1.8 ml of a 40% (v/v) isotonic solution of Percoll  in phosphate buffer saline over 1.4 ml of an 80% (v/v) isotonic Percoll in PBS solution as described in . To esti1H MRS analysis (Fractions K and L) and the final pellets were re-suspended with PBS added to yield a final volume of 600 μl (Fractions M and N) before also being analyzed by 1H MRS.To assess the extent of seminal plasma and/or Percoll contamination in the ‘40%’ and ‘80%’ sperm populations recovered from the standard DGC method (Experiment 2 outlined above), a series of further experiments were performed in which an additional wash step was included Fig. . BrieflyFollowing sperm preparation by each of the above methods, sperm concentration and sperm motility was re-measured in each sample according to 2O . Sample tubes were kept at room temperature until they could be scanned. All samples were scanned, in a random order, at 37°C, with up to 10 min equilibration time, using a 9.4 T Bruker Avance III MRS spectrometer , with a 5 mm broadband observe probe. Each spectrum was acquired using a 1H water-gate, excitation sculpting, solvent suppression sequence . Each spectrum was acquired and processed using Bruker Topspin v2.1 software to produce a phase and baseline corrected spectrum. All spectra were referenced to the 1H lactate signal at a frequency offset δ = 1.33 ppm.For each of the fractions recovered from the sperm washing steps illustrated in Fig. Spectra were analyzed using custom Matlab scripts . A noise region with no visible peaks was chosen between 10–11 ppm and all spectra were normalized so that the intensity here had a median value of zero and an interquartile range (IQR) of 1. Spectra from composite fractions  were then fitted to a target spectrum to quantify the proportions of these in samples . A spectral binning method was then used to split the spectrum into 0.04 ppm regions which were integrated to yield a bin area. Estimates for amounts of a component within a spectrum  in washed sperm (Fraction C) in Experiment 1), were generated by dividing the sum of fitted spectrum bin integrals for the target spectrum by the same value for the spectrum being fitted. This is outlined in more detail in the r2 > 0.25 and a significance P < 0.01 were considered to be metabolite peaks from sperm. Bin locations containing a significant correlation were then used to determine the minimum sperm concentration required to yield an observable 1H MRS peak within each bin with a signal to noise ratio (SNR) > 3:1  to determine which metabolite peaks correlated with number of sperm. First, all spectra were normalized as above and binned at 0.04 ppm, generating 193 bins in total after removing the bins associated with the water peak between 4.5 and 5.2 ppm. A Pearson's correlation coefficient was calculated for each bin versus sperm concentration and spectral bins with 1H MRS spectra from ‘40%’ and ‘80%’ sperm populations were examined using the protocol detailed in Experiment 3 . Spectra were processed, normalized and binned at 0.04 ppm as described above for metabolite bin integrals versus sperm concentration. The binned spectral integrals were then normalized to sperm concentration so that differences between ‘40%’ and ‘80%’ sperm spectra could be compared using a two-way ANOVA with a Bonferroni multiple comparison test, where P < 0.05 was regarded as significant. Bin regions identified by the two-way ANOVA as significantly different were then subject to a receiver operator curve (ROC) calculation to examine the specificity and sensitivity of the bin to discriminate sperm from the ‘40%’ or ‘80%’ populations.Differences in P ≤ 0.05) used as the significance threshold. All results are quoted as mean ± SEM unless otherwise stated.Data for progressive sperm motility and sperm concentration in matched samples were compared using Wilcoxon signed rank test using Matlab. Statistical differences between multiple groups were tested in Matlab using a Kruskal–Wallis non-parametric ANOVA test and a multi-comparison Bonferroni post-hoc test with (1H MRS spectrum for (i) unprocessed semen (Fraction A); (ii) the sperm pellet (Fraction B); and (iii) seminal plasma (Fraction C); obtained from a single semen sample processed using the simple centrifugation method of sperm washing. Visually the spectra obtained for sperm and seminal plasma were very similar to the unprocessed semen and fitting the seminal plasma and sperm spectra to the unprocessed semen spectrum estimated the relative proportions of these components as 84 ± 9% and 10 ± 6%, respectively , respectively, compared to its starting value . In addition, the MRS signal from 50% Percoll/PBS solution was also fitted to each sperm spectrum to estimate its remaining content and this showed that Percoll represented the larger component in both sperm spectra; 60 ± 5% of the ‘40%’ sperm ; similarly for ‘40%’ sperm the Percoll content was reduced from 60 ± 2% to 31 ± 1% . Conversely, for ‘40%’ sperm the additional washing step reduced seminal plasma content from 17.5 ± 7.2% (Fraction I) to 4.9 ± 1.1% after washing  an extra centrifugation step was added to the sperm washing protocol. The spectra obtained from ‘40%’ and ‘80%’ sperm (Fractions M and N), along with the supernatant obtained from the additional washing step (Fraction K for ‘40%’ sperm supernatant and Fraction L for ‘80%’ sperm supernatant) are shown in Fig. n = 3) and 1/16 000 for ‘40%’ sperm .Experiment 3 recovered 30 ± 4% of the total sperm , with no difference between the number of recovered sperm in the Experiment 2 and Experiment 3 centrifuge samples, both 15 ± 3%. Importantly the progressive motility was not significantly different between the two centrifugation steps (data not shown). With respect to its original concentration in semen the proportion of seminal plasma in ‘80%’ sperm was estimated to be 1/9100 , processed as outlined above, and bin integrals (0.04 ppm width) correlated against the sperm concentration in the MRS tube. For a range of sperm concentrations from 2.9 to 199 × 106/ml for the ‘80%’ sperm population, 21 correlations were found with a coefficient of determination, r2, greater than 0.25 and significance, P < 0.01  to 199 × 106/ml for a peak assigned as unsaturated aliphatic carbon chain (5.58–5.62 ppm). For the ‘40%’ sperm population (with washed sperm concentrations from 7.0 to 220 × 106/ml), only seven bin correlations were found  was tested by a two-way ANOVA. This showed that there were statistically significant differences between the spectral bins (P < 0.0001), ‘40%’ and ‘80%’ sperm populations (P < 0.0001) as well as a significant interaction term between spectral bins and ‘40%’ and ‘80%’ sperm populations (P < 0.0001). Applying a Bonferroni multivariate comparison to the data identified six bins where metabolite integrals were significantly higher in the ‘40%’ sperm compared to ‘80%’ sperm populations. These were for chemical shift regions associated with lactate , lipid peaks  and choline/GPC , see Table t-test found no significant differences between these populations in terms of sperm concentration or progressive motility.The ability of 1H MRS spectra from live human spermatozoa and that 1H MRS can be used to discriminated between sperm in the ‘40%’ and ‘80%’ populations recovered from ejaculates with normal sperm concentration and motility. However, to obtain suitable spectra of sperm for 1H MRS analysis, three technical challenges had to be overcome.The data presented in this paper are to the best of our knowledge the first to show that it is possible to obtain First it was necessary to be able to efficiently remove sperm from seminal plasma. Since sperm represent a relatively minor component of semen  added by the manufacturer, presumably to provide energy substrates for sperm during the sperm preparation process. Initial experiments with commercially available products proved unsuccessful as the additional media derived components, which were visible on the 1H MRS spectra (data not shown), obscured the sperm derived peaks to an unacceptable degree. Therefore, PBS was chosen because it had been shown to be sufficient to support human sperm viability  is ~3 × 106/ml. Moreover, we conclude that the additional washing step required to prepare sperm did not result in a significant change in the sperm concentration and motility suggesting that this is a reasonable step to take. As such, this may allow the opportunity to use MRS to examine sperm from men with oligozoospermia as well as those with astheno- or teratozoospermia .Third, it was necessary to find a washing method which provided sufficient sperm for MRS scanning and which at the same time did not have a deleterious effect on sperm viability/function. At the start of these experiments, it was not at all clear whether sufficient live sperm could be recovered from a single ejaculate to generate an MRS signal of sufficient intensity. For example, in the experiments by The MRS system used in these experiments was a 9.4 T magnet with a broadband observe MRS probe. The scanning time of each washed sperm sample was 1 h 17 min, therefore potentially making this technology useful in future andrology practice. We envisage that hardware improvements such as use of a higher magnetic field, improved MRS probe detection coil geometry and cryo-cooled coils and amplifiers would improve the sensitivity of detection further and therefore potentially allow detection of MRS signals from lower sperm concentrations. Furthermore, by employing specialized glass MRS tubes, the absolute number of sperm needed for scanning could theoretically be reduced still further and permit the analysis of sperm obtained from oligozoospermic men. Clinical MRI scanning of living patients is restricted in the specific absorption rate of radio frequency radiation (<4 W/kg), and, to a much lesser extent, magnetic field exposure. We do not envisage sperm to be significantly affected by strong magnetic fields or by the radio frequency waves transmitted by the MRS scanning sequence used (estimated as <1.2 W/kg). However, this will need to be specifically investigated in future studies.From the MRS data obtained in this study, we have identified some of the major molecules present in spermatozoa. These include choline/GPC, lipids and acetylcarnitine in one or both sperm populations recovered from DGC. Without specialist techniques, such as multi-dimensional MRS or isotopic labeling strategies, MRS will typically identify molecules of less than 10 000 Daltons . Therefo1H spectrum from ‘40%’ and ‘80%’ sperm populations highlighted significant differences in lactate, lipid, choline and GPC peaks that were greater in the ‘40%’ sperm population compared ‘80%’. Phospholipids are a major component present in the outer cellular structure of sperm and lipid, choline and GPC identified may be associated with this structure  we chose to use DGC as this is commonly used in assisted conception so that our results had the most relevance to clinical practice. Second, we are aware that some of the MRS peaks present in seminal plasma, or the supernatants of the sperm washing steps, could be derived from dead or dying sperm with leaky plasma membranes. For example, analysis of the supernatant recovered from Experiment 3 sperm centrifugation step (Fractions K and L in Fig. 1H MRS can provide information about the molecules present in live human sperm and we propose that this offers the opportunity to study their relative importance and how they relate to sperm function. This may in turn lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility.In conclusion, in this paper we show that Supplementary DataClick here for additional data file.


CCND1 expression and IGH rearrangement. Such markers can, however, be found in other B cell non-Hodgkin lymphomas. Recent studies demonstrate that SOX11 expression is highly specific for MCL and could be used as a marker for measuring MRD. Moreover, evidence shows that SOX11 level could be predictive for overall survival (OS) and progression-free survival (PFS). We have measured MRD level in follow-up samples from 27 patients diagnosed with MCL using the molecular markers: t, IGH rearrangement and SOX11 expression. We compared all markers by their sensitivity, utility and quantitative range. We also examined the predictive value of SOX11 expression for OS and PFS. SOX11 expression was found to have better specificity, quantitative range and utility than the t. The predictive value of SOX11 expression was confirmed. At diagnosis, patients with high SOX11 expression had shorter PFS than patients with low SOX11 expression (p = 0.04*); differences between OS being statistically insignificant. To our best knowledge this is a first study comparing SOX11 with t and IGH rearrangement as markers of MRD level. Moreover, in this study we confirmed that SOX11 is useful in cases when other molecular markers cannot be used.The main cause of death in mantle cell lymphoma (MCL) patients is relapse due to undetermined minimal residual disease (MRD) and therefore monitoring MRD is crucial for making the best treatment decisions. The gold standard method for MRD analysis is the quantitative polymerase chain reaction. The most commonly used molecular markers for measuring MRD in MCL are: t translocation or  Classic MCL with IGH-unmutated B cells and SOX11 expression, which usually follows an aggressive course and can transform to the very aggressive blastoid MCL with activation of certain molecular factors [ factors .Leukemic non-nodal MCL with IGH-mutated B cells, without SOX11 expression, usually having an indolent outcome, but can transform to nodal aggressive MCL with activation of certain molecular factors  [., TP53) .Mantle cell lymphoma (MCL) is a rare and incurable disease , mostly appearing in males  at a median age of 68 years [Relapse due to undetermined minimal residual disease (MRD) is the main cause of death in MCL patients, thereby making monitoring of MRD crucial. Recent studies have shown that consistent monitoring of MRD can improve treatment decisions –7. MRD lCCND1 expression [The gold standard method for measuring minimal residual disease is the quantitative polymerase chain reaction (qPCR). The most widely used molecular markers for measuring MRD in MCL are: t translocation or pression  and IGH pression .CCND1) overexpression can be detected in 90% MCL cases. CCND1 is one of the proteins that regulates cell cycle thus its upregulation drives B cells to proliferation [CCND1 negative subtype of MCL [The t11;14)q13;p32) translocation which causes cyclin D1  negative MCL subtype [SOX11 expression is also more specific for MCL than CCND1 overexpression; being, respectively, found in 90–95% and 90% of MCL cases [SOX11 expression also distinguishes two types of MCL: classic from leukemic non-nodal [SOX11 expression can be used as a measurement of MRD in MCL [SOX11 could potentially appear as a better molecular marker than t. In this study, we compared SOX11 expression, t and IGH rearrangement as MRD molecular markers to determine if SOX11 can be used with at least the same sensitivity and specificity as other molecular markers.Considering these aforementioned limitations, there is a need to find a specific and sensitive molecular marker for monitoring minimal residual disease in MCL. Recent studies have shown that  for MCL  and inde for MCL  and thusCL cases . SOX11 eon-nodal  and can on-nodal , 19. EviD in MCL  with at D in MCL . Therefo6 cells were frozen and stored at − 80 °C. The research protocol was reviewed and approved by the Ethics Committee of Maria Skłodowska-Curie Institute—Oncology Center. Informed consent was obtained from all individual participants included in the study before the collection of biological material.PB and BM from 34 patients treated in the Department of Lymphoproliferative Diseases, Maria Skłodowska-Curie Institute—Oncology Center were collected: 71 samples at diagnosis and 254 samples during treatment. There were at least two samples from every patient at diagnosis and 3–15 samples were collected from 27 patients during treatment. PB samples from healthy donors served as negative controls. White blood cells (WBC) were isolated by density gradient centrifugation. Samples containing 5 × 10SOX11 measurement previously described by Hamborg et al. (2012) [RNA isolation from cell precipitate was performed with the GeneMATRIX Universal Purification Kit  according to the manufacturer’s protocol. RNA concentration and purity were measured using the NanoDrop spectrophotometer (ThermoFisher) and FlashGel system (Lonza). A 500 ng of RNA was reverse transcribed (RT) using the NG dART Reverse Transcription Kit (EURx) according to the manufacturer’s protocol. cDNA quality was estimated by PCR with GAPDH primers: F: 5′-GAAGGTGAAGGTCGGAGTC-3′; R: 5′-GAAGATGGTGATGGGATTTC-3′, using the NG dART RT-PCR kit (EURx) according to the manufacturer’s protocol. cDNA was diluted to a final volume of 35 µl when RT efficiency was low, and to a final 50 µl volume when RT efficiency was high. Diluted cDNA was stored at − 20 °C or +4 °C if used within 24 h. qPCR was performed according to a highly sensitive and specific qPCR assay for DNA isolation was performed using the Sherlock AX isolation kit (A&A Biotechnology) according to the manufacturer’s protocol. For IGH rearrangement, PCR and qPCR reactions were performed according to the BIOMED-2 Concerted Action BMH4-CT98-3936 protocol [Standard curves and follow-up analysis were done according to van der Velden et al. (2007) . PCR andSensitivity of molecular markers was defined by the lowest possible dilution detected by qPCR. The quantitative measurement range was set up as the lowest detected dilution which had a Ct lower than that of the negative controls background by three cycles.MRD level was calculated to allow comparisons between molecular markers to be made. Results from diagnostic samples were set to a 1 (100%) MRD level while MRD level in follow-up samples was calculated as derived proportions.Statistical analysis was performed on Microsoft-Excel using correlation, linear regression and log-rank testing. Graphs were constructed via GraphPad Prism software (GraphPad Prism Inc.).SOX11 expression was measured as a proportion of SOX11 copies/1000 reference genes (RG) copies. Results obtained for patients were very highly variable; from 2 × 10−2SOX11/1000 RG to 353 SOX11/1000 RG. Four patients were diagnosed with blastoid MCL and all four had SOX11 expression higher than 100 SOX11/1000 RG. Eighteen patients with low SOX11 expression in the diagnostic sample were usually diagnosed with less aggressive disease. Three patients with low SOX11 expression in the diagnostic sample diagnosed with aggressive advanced nodal MCL usually had multiple relapses during treatment. Sixteen patients with less than 1 SOX11/1000 RG were excluded from further analysis of MRD.SOX11 expression, 9 patients (26%) by t and 21 patients (61%) using IGH rearrangement. Due to low SOX11 expression and poor sensitivity of the t translocation, in 5 patients (15%) the only molecular marker that could be used was IGH rearrangement. For two patients with a high polyclonal background and low SOX11 expression, the only molecular marker applicable was t. Because of the polyclonal background and poor sensitivity of t in one patient (2%), the only molecular marker that could be used was SOX11 expression.MRD analysis was performed for: 18 patients (53%) by SOX11 expression was very high, at 0.98. Sensitivity for both methods was near 1 × 10−5 but for t the mean quantitative measurement range was 1.2 × 10−4. The difference between MRD level estimated by both markers was not statistically significant, p > 0.98. For further analysis, we estimated a linear regression model using MRD level measured by SOX11 expression and t  and 1;14 and SOX11 expression and the IGH rearrangement the correlation was also high at 0.89. The sensitivity of both molecular markers was near 1 × 10−5 and for IGH rearrangement the mean quantitative range was 5 × 10−5. As in the previous comparison, the difference between MRD level measured by both markers was not statistically significant, p > 0.79. Likewise, as before a linear regression model was estimated using MRD level calculated by SOX11 expression and IGH rearrangement . Mean OS in the SOX11negative/low group of patients was ~ 41 months, and for the SOX11intermediate/high group it was ~ 30 months; however, this difference between groups was statistically insignificant (p = 0.09)  Fig. .Fig. 4PrSOX11 expression, t translocation and IGH rearrangement as the qPCR molecular markers for measuring MRD level in MCL. We have demonstrated that all markers have similar sensitivity but the mean quantitative range for t and for IGH rearrangement was lower than that for SOX11 . For SOX11 expression, the quantitative range was the same as the sensitivity, because there was no background from the negative control in any of the reactions. We have therefore concluded that SOX11 expression is a more specific molecular marker than t and IGH rearrangement. Moreover, there were no statistically significant differences between MRD level obtained using molecular markers: t and SOX11 or IGH rearrangement and SOX11.Our study aimed to compare SOX11 expression and t translocation were comparable, with correlation of 0.98. We thus assumed that these molecular markers could be used interchangeably or that even SOX11 expression can be used as a molecular marker instead of t. In this study, we applied the commonly used PCR primers designed for the major translocation cluster (MTC) which covers about 40% of MCL translocations. Insufficient specificity of t primers used in qPCR reactions limits the use of this marker for monitoring MRD to only 25–40% cases [SOX11 expression to 53% patients, including one patient with CCND1(-) MCL. Our work confirms previous results suggesting that in comparison with the t translocation, SOX11 expression is a more specific molecular marker with similar sensitivity [MRD level measured by 0% cases . Our stusitivity , 21.SOX11 expression was comparable, with a correlation coefficient 0.89. The IGH rearrangement was used as a MRD molecular marker for most patients (61%) compared to the other molecular markers: t translocation and SOX11 expression . These results confirm previous assumptions about the utility of the IGH rearrangement [SOX11 and IGH rearrangement suggests that SOX11 expression could be less sensitive than IGH rearrangement [SOX11 expression was detectable at very low MRD level in the follow-up samples, especially for patients who had high SOX11 expression at diagnosis (> 10 SOX11 copies/1000 RG copies). However, for a few patients SOX11 expression was not detectable in the follow-up samples at very low MRD level while IGH rearrangement was. All such patients had low SOX11 expression at diagnosis (< 10 SOX11 copies/1000 RG copies). Our results confirm previous conclusions, that SOX11 has a lower sensitivity than IGH rearrangement [SOX11 expression at diagnosis. This may be caused by the fact that during treatment, the expression of SOX11 decreases significantly. In patients with low baseline expression of SOX11, the level of expression during treatment decreases to values undetectable by RT-qPCR. However, in patients in whom the initial expression of SOX11 is high, the decrease in the level of SOX11 expression during treatment decreases within the limits of detection. Moreover, the difference in the sensitivity of these two markers may be due to the specification of the genetic material. RNA, which is the material for estimation of SOX11 expression, is less stable thus, it degrades faster than DNA. Therefore, when a small amount of material is analyzed, the detectability of DNA is better than RNA.Our work showed that MRD level measured by IGH rearrangement and ers: t11; translocangement . In our angement , but onlSOX11 expression. We compared PFS and OS in groups of patients with low and high SOX11 expression at diagnosis. Patients with high SOX11 expression (> 10 SOX11 copies/1000 RG copies) had shorter PFS than patients with low SOX11 expression (< 1 SOX11/1000 RG). The difference between mean PFS in both groups was ~ 20 months and was statistically significant (p = 0.04). There was no statistically significant difference between OS for patients with high and low SOX11 expression. Our results confirm the prognostic value of SOX11 expression at diagnosis but only for progression-free survival which contrasts to previous studies in which low SOX11 expression in vitro and in vivo was associated with poor prognosis, shorter OS and PFS [SOX11 in tumor cells could lead to a more aggressive disease course [SOX11 expression may be important for improving therapeutic decisions.Our study also investigated the suggested prognostic value , 19 of S and PFS , 25. Hige course , 19 withe course . TherefoSOX11 expression as a molecular marker for MRD in MCL. Moreover, the quantitative range of MRD level measured by SOX11 was higher than the quantitative range for that obtained with the t translocation and IGH rearrangement. Furthermore, MRD level obtained with the use of SOX11, t and IGH rearrangement was highly correlated. We therefore hope that SOX11 expression will be used as a molecular marker for measuring MRD in MCL with the same frequency of t or IGH rearrangement. However, considering limitations emerging from stability of RNA, for the most efficient estimation of MRD level in MCL patients, SOX11 expression should be used parallelly with IGH rearrangement. Simultaneous usage of both markers allows to confirm results obtained with SOX11 expression. Moreover, this could permit to estimate MRD level in different subtypes of myelomas in rare cases of composite lymphoma. We also confirmed the prognostic value of SOX11 expression, when high SOX11 expression is correlated with poor prognosis. Considering the discordant results on the prognostic value of SOX11 expression, we think that this subject requires further study, including studies on pathways regulated by SOX11, SOX11 methylation profiles and potential isoforms of SOX11.Our results confirm high specificity of


AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes.Salivary amylase in humans is encoded by the copy variable gene AMY1 copy number was significantly correlated with the variation observed in salivary amylase production  and enzyme activity  but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052).AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.Production of salivary amylase is correlated with The online version of this article (doi:10.1186/s40246-017-0097-3) contains supplementary material, which is available to authorized users. AMY1) and an increased level of amylase protein expression , which were included in every experiment in duplicate.PRT PCR reactions were performed using previously described primers PRT_ref12  that ampAMY1 gene copy number, a TATC microsatellite PCR was performed for each sample [For further confirmation of h sample . A singlAMY2A copy number to AMY2B copy number and the ratio of AMY2A copy number to AMY2A pseudogene copy number were measured as previously described [AMY2A (163 bp) to AMY2B (167 bp) and the ratio of AMY2A (197 bp) to AMY2A pseudogene (232 bp).The ratio of escribed . One micAMY2A/2B duplication junction assay is a three-primer assay producing PCR amplicons of 424 bp in all samples and a specific 323 bp only from the duplication junction. The products were visualised on a 2% (w/v) agarose gel, as previously described [The escribed .The concentration of amylase protein antigen present in the saliva was measured using a sandwich amylase ELISA with 1 μg/mL of anti-salivary amylase antibody  and 100 μg/mL of biotinylated detection antibody . Assays were performed using serial dilutions (1:5) of natural human salivary amylase protein of known concentration (200 μg/mL)  to generate a standard curve. Assays were performed in duplicate for each unknown sample, and the standard curve was measured in triplicate.Ultra Amylase Assay Kit  according to manufacturer’s instructions. The saliva samples were added to the substrate solution, vortexed, and the fluorescence of the samples was measured after 10 min incubation at room temperature. Assays were performed using serial dilutions (1:5) of natural salivary amylase protein of known concentration (200 μg/mL)  to generate a standard curve. Assays were performed in duplicate for each unknown sample, and the standard curve was measured in triplicate.The amylase enzyme activity within saliva was measured using the EnzCheck® AMY1. Analysis of repeat measures found that neither of the within-subject factors (time and measurement) was significant for measurement of salivary amylase protein expression or enzyme activity, and therefore, a single time point is suitable for comparing measurements of salivary amylase protein and enzyme activity between individuals.In order to deduce the variation in concentration for protein expression and enzyme activity within each individual, the repeat samples from an initial cohort of 10 individuals were investigated on four separate occasions  according to manufacturer’s instructions. Assays were performed using serial dilutions (1:2) of bovine serum albumin (BSA) protein of known concentration (1.4 mg/mL)  to generate a standard curve. Assays were performed in duplicate for each unknown sample, and the standard curve measured in triplicate.Correlations between groups of copy number data and either protein expression or enzyme activity were assessed using logistic regression in SPSS V22 , and figures were drawn with the software GraphPad Prism . The repeat measures were analysed in SPSS using a general linear model with repeated measures, with the within-subject factors defined as time and measurement and the between-subject factor defined as copy number.


Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20–40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1–20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by ‘cold shocking’ cells at 32 °C for 24–48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or ‘perfect HDR’, as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated . We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing ‘perfect HDR’ compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines. The relative robustness of the CRISPR approach compared to zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs) has made testing accessible on protein coding mutations as well as empirical data generated by genome-wide association studies and other non-coding mutations4. Despite numerous successes, gene editing in iPSCs is challenged by the fact that homology directed repair (HDR), the process by which exogenous donor DNA is used to repair CRISPR-induced double strand breaks, is less efficient in iPSCs than transformed cancer cell lines8.One of the most promising applications of the Clustered Regularly Spaced Palindromic Repeats (CRISPR) technology is its use in creating genetic models of human disease. CRISPR technology can be used on induced pluripotent stem cells (iPSC) isolated from normal individuals to study a disease phenotype, or on IPSCs derived from disease patients to revert putative disease-causing mutations back to wild type9 which, while effective, leaves an undesired insertion of foreign DNA into the genome. Combining positive selection markers with technologies that allow for excision of the selectable marker, such as the Cre/lox system or the footprint-free PiggyBAC transposon, represent notable improvements but extend timelines as clonal selection becomes a two-step process10. Methods that employ single-stranded oligo DNA nucleotide (ssODN) donor molecules avoid the issues that larger, double-stranded DNA molecules present with respect to random integration and unwanted ‘footprints’, but again are subject to the relatively low frequency of successful repair and sequence conversion around the site of the double-stranded break11. To contend with the difficulty of isolating rare clones, Miyaoka and colleagues devised a strategy using droplet digital PCR, pools of clones, and sib selection to enrich for extremely rare clones12. Additional strategies to increase the rate of HDR include timing the delivery of the Cas9 RNP complex to the nuclease by inducing cell cycle synchronization with known chemical inhibitors of cell cycle progression13. Here, while notable increases in HDR, up to 38%, can be achieved with synchronized HEK293 cells, synchronization had minimal effect in human primary fibroblasts or H9 human embryonic stem cells. Specifics of the ssODN structure and composition have also been shown to affect HDR rates. Lin et al. found that oligos with homology arms of at least 60 nucleotides were most effective, but that strand complementarity was not a factor. A more detailed investigation into how the structure of donor oligos affects HDR in HEK293 cells by Richardson et al. used insights gained from in vitro binding studies of Cas9 RNP-dsDNA complex. Using a GFP-reporter assay, they demonstrated that asymmetric donor oligos that are shorter with respect to the PAM site and are complementary to the (+) strand (i.e. non-target strand) were more effective in promoting HDR than symmetric donor oligos. A study optimizing HDR14 in iPSCs by Paquet et al. showed that efficient insertion of an intended mutation could be achieved with oligos that where complementary to the target strand (−) and that the frequency of the intended mutation integration was distance-dependent from the CRISPR cut site. Fidelity of HDR could also be increased by the introduction of silent base changes into the oligo that disrupted the CRISPR recognition sequence15.To overcome low HDR rates, researchers have adopted several strategies such as including antibiotic resistance genes on the CRISPR plasmid and/or donor DNAWe have carried out a systematic evaluation of the gene-editing steps in iPSCs to determine the best combination of delivery, CRISPR modality, and donor oligo design. We then tested the effects of a moderate ‘cold shock’ on the cells’ ability to carry out HDR. Our optimized method can successfully introduce desired genetic alterations into the genome with 10–30% efficiency through our novel combination of lipids designed for large RNA molecule delivery in conjunction with Cas9-encoding mRNA, symmetric donor oligos complementary to the target strand (−), and silent changes to prevent re-editing. Additional exposure of cells to a brief ‘cold shock’ of 32 °C can increase the amount of perfect HDR as much as two- to ten-fold in instances where low efficiency repair is observed at 37 °C.in vitro transcription (IVT)/Cas9 ribronucleoprotein] and delivery methods (e.g. nucleofection or lipids formulated for enhanced delivery of large DNA and RNA molecules) generated the greatest the number of double-stranded breaks at two specific locations within the CAMK2D gene , we tested if exposing the mc-iPS cell line to various 32 °C intervals would have an effect on the efficiency of HDR. The design of the experiment and the resulting percentages of the total alleles having undergone HDR as measured by ddPCR at each temperature are presented in Table 14. To compare the two designs directly and further test the effects of cold shock on HDR, we designed a gene editing experiment whereby we compared the amount of HDR observed with the symmetrical targeted strand oligo donor to that observed with an asymmetrical non-targeted strand oligo donor designed to introduce the same sequence alternations  the amount of total HDR at the locus, i.e. oligo directed repair regardless of whether all or part of the intended changes are present; 2) the percentage HDR that represented ‘perfect HDR’, oligo directed repair with all six intended base changes intact; 3) the percentage of HDR for which the sequence that had putatively undergone re-editing once repaired by virtue of indels being re-introduced into the converted sequence; and 4) the percentage of HDR where partial oligo-directed repair occurred such that sequence is missing the two more distal sequence changes  and with sequence complementarity to the non-targeted strand  are more efficient promoters of HDR than the design we employed for editing the CAMK2D locus i.e., symmetric around the CRISPR cut site and complementary to the targeted strandons Fig. . We dete17 ‘cold shock’ produced an increase in overall indel formation in the absence of any repair oligo but the effects were far less than the overall increase in total HDR. As expected, the percent indel formation drops with the addition of repair oligo as the locus is converted back to ‘wild type’ minus the intended sequence alterations and is commensurate with the amount of total HDR observed under each condition  or 48 hours (Group PL3) produced an increase in HDR; however, the effect was generally minimal given the relativity high rates of HDR to begin with at 37 °C and for the most part did not reach statistical significance Fig. . HoweverThe rate of indel formation for guide TR-CR2 and TR-CR3 in the absence (previously mentioned above) and presence of donor oligo are presented in Supplementary Fig. 8. However, despite the low HDR rates observed at 37 °C, ‘cold shock’ under both sets of conditions, 24 hours (PL2) and 48 hours (PL3), produced for the best sgRNA, CAMK-CR2, a statistically significant 6.9-fold increase in total HDR at both the 24 hour and 48 hour conditions as determined by ddPCR  and diminishes as the base HDR rate increases above 30% or more. In fact, the largest increase in total HDR fold change was in with HEK293 cells where the baseline HDR at 37 °C was in the 1% range. The diminishing of the ‘cold shock’ response at high baseline HDR suggests a theoretical limit to the number of alleles that can be altered, at least with this approach. The exact mechanism by which the ‘cold shock’ increases HDR is currently under investigation. A mechanism similar to how zinc finger nucleases increase indel formation may be plausible17. We observe that with very efficient CRISPRs the effect of the ‘cold shock’ on indel formation is minimal, as observed with CAMK2D guide CAMK-CR1 and TGFBR1 guide TR-CR2. Conversely, when cutting efficiencies and indel formation are lower, as that observed at the CAMK2D locus in HEK293 cells, the increases in cutting efficiency and indel formation are more pronounced. Increased indel formation can clearly contribute to higher HDR rates but does not account for all the increase observed with ‘cold shock’ or explain why ‘perfect HDR’ can be favored under cold conditions  and were maintained on Matrigel (BD Bioscience) coated plates in mTeSR media (Stem Cell Technologies) with 50 units/ml penicillin-streptomycin (Thermo Fisher Scientific) and with daily medium changes22. For passaging, the cells were washed with PBS and treated with Accutase (Thermo Fisher Scientific) at 37 °C°C for 5 min. The cells were re-suspended in mTeSR media, centrifuged at 80 g for 5 min and cell pellets re-plated in mTeSR media supplemented with 10 μM ROCK Inhibitor Y-27632 .Human mc-iPS cellshttp://portals.broadinstitute.org/gpp/public/) and Zhang laboratory CRISPR design tool (http://crispr.mit.edu). The guide sequences were either subcloned into plasmid pX458 (GenScript) or synthesized as IVT sgRNAs (Thermo Fisher Scientific). GeneArt™ Platinum™ Cas9 Nuclease was obtained from Thermo Fisher Scientific and Cas9 mRNA  was obtained from TriLink BioTechnologies. The repair templates  were designed as single-stranded oligodeoxynucleotides (ssODN) with target mutations to the middle of the oligonucleotide with homologous genomic flanking sequence on the both side of mutations14. In some ssODN designs, silent mutations were also introduced within the guide RNA recognition sequences and PAM sites. PCR primers were designed using PRIMER 3 and primers were purchased from Sigma. Sequences of the primers, probes, and oligonucleotide donors used in the study are listed in Supplementary Table CRISPR guide RNAs were designed using the Doench algorithm  per tube. The mixture was incubated at room temperature for 15 min and the entire mixture was then added to the well drop by drop. For homology directed repair experiments, ssODNs were re-suspended in water at 10 µM and various amounts added to the complex before lipid addition. 100 ng of GFP mRNA was also spiked into each mixture to monitor the transfection efficiency. The plates were incubated at 37 °C in a 5% CO2 incubator and the cells were then harvested for genomic DNA extraction 48 h later. Additional transfection methods in iPSC and HEK293 are in supplementary methods.For lipid based transfection of mc-iPSC with IVT sgRNA/ Cas9 mRNA, 1 × 10iPS Cells were harvested by gently aspirating the media from each well and treating the cells with 250 µl of Accutase (Thermo Fisher Scientific) at 37 °C for 10 min. 750 µl of mTeSR media was then added to each well and the cell suspension was transferred to a 1.5 ml Eppendorf tubes and spun at 1000 g for 5 min. Genomic DNA was then extracted using DNeasy Blood & Tissue Kit (QIAGEN) and 100 ng of genomic DNA used for PCR using Q5 polymerse (NEB) and target specific primers  and quantified with a Qubit HS DNA (Invitrogen). The purified PCR products were used as input for the Nextera XT kit (Illumina) using manufacturer’s recommendations. Samples were uniquely indexed with up to 384 unique i5/i7 combinations using Illumina standard indexing kits. Amplification was carried out at 72 °C for 3 min, 98 °C for 1 min, then 12–14 cycles of 98 °C 30 s, 55 °C 30 s, 72 °C 1 min, followed by 5 min final extension at 72 °C, and cooled to 4 °C. Libraries were size selected according to the same Ampure XP bead protocol as described previously, and eluted in 15 µl H2O. Eluted products were run on Tapestation HSD1000 (Agilent) and quantified by qPCR using KAPA Library (Kapa Biosystems) Quantification kit and an ABI Vii7. Individual library concentrations were calculated using the Kapa Library Quantification template. Each library was diluted to 4 nM and pooled. The final pool was denatured and diluted to 12pM following Illumina’s standard protocol using 1% v/v PhiX as a spiked in control. Run parameters were set at 150 bp paired-end, dual indexed 8 bp each using a MiSeq. 300v2 reagent kit (Illumina). Samples were demultiplexed using MiSeq Reporter v2.6 or bcl2fastq v2.17.Sequencing libraries were generated from PCR amplicons as follows: After the PCR step high molecular weight (HMW) template DNA and residual PCR primers were removed in a two-step cleanup process. HMW DNA was first removed by adding 0.6 v/v ratio Ampure XP beads (Beckman Coulter) and transferring the cleared supernatants to a new 96 well plate. Primers were then removed by adding 1.2 v/v Ampure XP beads to the transferred supernatants and placed on a magnet again until clear. The supernatants were then discarded and the beads washed 2 × with 80% ethanol, air dried, and resuspended in 20 µl H16. Quality filtering was performed on paired-end reads using PRINSEQ. Reads with quality score mean below 30 or length shorter than 50 were removed. The remaining reads were then aligned to a reference genome with BWA, followed by realignment using ABRA (Assembly-Based ReAligner) to enhance detection of large indels23. We examined the read coverage for each amplicon, and surveyed the whole amplicon region for insertion and deletion frequencies. To assess CRISPR efficiency, we defined a simple measurement, % indel reads, which represents the percentage of reads that have CRISPR-induced indels among all reads that span the window of sgRNA sequence (about 20 bases without PAM). For a read to be considered an indel read, it must have at least one inserted or deleted base inside this window, whereas a wild-type read must have no indels within the window. To evaluate homology-directed repair (HDR) effectiveness, we examined point mutation frequencies in the window and its flanking regions, and categorized the sequences into four groups: (1) ‘Perfect HDR’: the repair has all intended base changes with no indels between them; (2) Edited HDR: some or all intended base changes occur, but indels are present, likely due to re-editing by CRISPR after the directed repair; (3) Partial HDR: some but not all intended base changes occur, with no indels within the repaired sequence. This could be caused by partial repair or fragmentation of DNA strand during the NGS procedure.A bioinformatics pipeline was developed to analyze the NGS data to determine indel and HDR frequenciesTM Droplet Digital PCR System  was used as suggested by the manufacturer. ddPCR assays for detecting CAMK2D wild-type and mutated sequences were designed using Primer Express and ordered from Life Technologies . ddPCR reactions were assembled using standard protocols: ddPCR Super mix for Probes (no dUTP)  was combined with 160ng of sample genomic DNA, 1 µl of 20 × FAM assay and 1 µl of 20 × VIC assay , 5 units of restriction enzyme BamHI –HF® , and water for a final reaction volume of 20 µl. Reactions were converted into approximately 20,000 one-nanoliter droplets using the QX200 Droplet Generator and transferred to a 96-well plate for thermal cycling per manufacturer recommendation. After thermal cycling, droplets were read on the QX200 Droplet Reader and assigned as positive or negative based on fluorescence amplitude. The primer and probe sequences are listed in Supplementary Table The QX200TM as described. After transfection, group PL1 was kept at 37 °C until harvested 48 hr later while PL2 was transferred to 32 °C for 24 h then returned to 37 °C for the final 24 h. Group PL3 was transferred to 32 °C after transfection and kept at this temperature until harvested 48 h later. The cells were harvested for genomic DNA isolation 48 h post transfection as described and indel formation or HDR were determined by either ddPCR or NGS.One day prior to transfection, mc-IPSCs were seeded in 24 well plates as described in the transfection section and divided into four groups (PL1-PL3). Group PL1 to group PL3 were incubated at 37 °C for 24 h. The cells were then transfected with IVT sgRNA/Cas9 mRNA and ssODN using EditProOne-way ANOVA with Dunnett’s post test was performed using GraphPad Prism 7 (GraphPad Software) to compare the HDR efficiencies among three temperature conditions for each sgRNA and ssODN treatment. Two-tailed Student’s t-test was performed using GraphPad Prism 7 to compare the difference of ‘perfect’ HDR between symmetric and asymmetric donor oligo design, a P-value ≤ 0.05 was used to define statistically significant differences.Supplementary Information


Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells. Bone is capable of regeneration, but the process that leads to scarless restoration of form and function is highly complex and prone to failure . Even to+ memory/effector T cells have been linked to delayed bone healing   was used. RAG1−/− mice lack both mature T and B cells (−/− mice and wild-type (WT) controls  were assessed on various levels: bone biomechanics, by biomechanical torsional testing, morphology by micro computed tomography (microCT) analysis and histological staining, and differential gene expression by microarray, qPCR analysis, and flow cytometry.To assess the global role of T and B cells in bone function, structure, and regeneration process, a mouse model of recombination activating gene 1 homozygous knockout  mice (n = 8)  was investigated at D7 post fracture. Through knockout of genes in the beta- and delta-chain of the T cell receptor, T cells in these animals were unable to express functional receptors that induce cell apoptosis. Bone healing without the influence of T cell was investigated in this group. On the other hand, a second group of immunoglobulin heavy chain, joining region homozygous knockout (JHT−/−) mice (n = 8)  was investigated 7 days post fracture to analyze healing without B cells. These mice harbor a deletion mediated by cre-loxP resulting in the loss of the exons encoding the joining region and the intron enhancer of the immunoglobulin heavy chain locus (IgH). The JHT−/− mice fail to produce functional B-cells as they lack the gene for the assembly of the heavy chain for the production of antibodies.Furthermore, the individual role of each T and B cells in the cartilaginous callus formation (D7) was investigated. A group of T cell receptor β and δ chain homozygous knockout (TCRβδ/−) mice  (n = 8) /−) mice  (n = 8) Adhering to the 3R principles, sample sizes were kept as small as possible—results were confirmed by using different analyzing methods.During the operation, mice were anesthetized with 2.5% isoflurane/oxygen and received pain medication . After opening the skin and the knee joint the patella was dislocated to the lateral side. Through the distal end of the femur a passage for the intramedullary pin was created using a hollow needle . This needle was removed and an intramedullary pin  was inserted into the bone marrow cavity.All experiments were carried out with ethical permission according to the policies and principles established by the Animal Welfare Act, the National Institutes of Health Guide for Care and Use of Laboratory Animals, and the National Animal Welfare Guidelines and were approved by the local legal representative animal rights protection authorities .−/− animals (n = 18 per group). The proximal and distal ends of the femur were embedded into the holding pots using polymethyl methacrylate. Using a laser line, femora were aligned parallel to the loading axis and centered into the holding pots. Bones were brought to the point of failure under torsional loading setup of 0.5°/s and 0.3 N axial preload . Torque of failure (M_max), degree of torsion (angle-at-max), energy at break, and stiffness were evaluated for the intact contralateral bones.To study the initial mechanical properties, torsional testing was carried out on the intact contralateral femora of WT and RAG1n = 56, RAG1−/−n = 39). Femora were scanned with a fixed isotropic voxel size of 10.5 µm . The scan axis equaled the diaphyseal axis of the femora. A diaphyseal volume of interest with a size of 630 slices was analyzed with a global threshold of 190 mg HA/cm3. All analyses were performed on the digitally extracted callus tissue using 3D distance techniques   .−/− mice for paraffin embedding (n = 8 per time point and group). Briefly, bones were harvested with little surrounding muscle tissue and fixated in 4% paraformaldehyde (PFA) at 4°C for 48 h. Afterward, decalcification was done for 3 weeks in a 1:1 solution of 4% PFA and 14% EDTA at 4°C. After dehydration bones were embedded in paraffin and cut into 4 µm sagittal slices.To analyze tissue formation over the course of healing a histological analysis was performed according to the targeted test. Femora were harvested at D3, D7, D14, D21, and D28 of healing from the WT and RAG1−/− mice were also harvested at 7, 14, and 21 days post fracture (n = 3 per group and time point) for plastic embedding. Briefly, femora were fixed with 4% PFA for 48 h, dehydrated, and plastic embedded , and then 7 µm sagittal slices were prepared.Furthermore, femora of WT and RAG1−/−, and TCRβδ−/− groups (n = 3 per group) were collected at 7 days post fracture processed for cryo-sectioning. Briefly, bones were harvested and fixed in 4% PFA for 2 h at 4°C. Afterward, the bones were treated in increasing concentrations of sucrose solution 10%, 20%, and finally 30% for 24 h each at 4°C. Bones were then embedded in SCEM cryo-embedding medium . Cryostat sections of 7 µm were cut sagittal using the Leica CM3050S Cryotom  and Cryofilm .Another set of animals from WT, JHTParaffin- and cryo-sections were used for a general overview of the different tissues that are involved in fracture healing using Movat’s pentachrome staining. The Movat’s pentachrome is a combination of alcian blue solution, Weigert’s iron hematoxylin, brilliant crocein acid fuchsin, phosphotungstic acid, and safran du Gatinais solution. The stain differentiates cartilage formation and hypertrophy (green), matrix mineralization (yellow), connective tissue (light blue), muscle (orange), bone marrow (purple), and was used for histomorphometric analyses , using ain vivo fluorescence dyes were injected i.p., (n = 3). Dyes were dissolved in 1.4% NaHCO3 and injected with respect to the time point of euthanasia: calcein blue  12 days pre-euthanasia, calcein green  7 days pre-euthanasia, and Alizarin red  2 days pre-euthanasia. After sacrifice, femora were dissected and embedded in PMMA as described above. 7 µm thick sections were inspected using fluorescent microscopy and analyzed semiautomated using the provided software .To label the mineral formation near the fracture gap on the endosteal and periosteal regions n = 3) in WT and RAG1−/− animals. Briefly, deparaffinized sections were incubated with hyaluronidase for antigen retrieval, rinsed in phosphate-buffered saline (PBS) and incubated with the primary antibody  overnight at 4°C. Using the avidin/biotin complex method  binding of the secondary antibody  to the primary was visualized by AP substrate . Counterstaining was performed with hematoxylin. As a negative control, sections were processed for each sample in the absence of the suitable primary antibody.Immunohistochemical staining was performed on paraffin sections to visualize collagen type I (ColI) at 7 days post fracture . The approach was used to visualize the following components: 1) B cells using anti-mouse B220 ; 2) T cells using anti-mouse CD3e ; 3) osteocalcin (Ocn) using anti-mouse ; and 4) ColI using anti-mouse ColI . Briefly, sections were brought to room temperature (RT) and washed with PBS. An initial blocking step was performed by applying a solution of PBS containing 5% FCS, 0.1% TWEEN-20, and 10% rat serum for 30 min. For Ocn and ColI staining, sections were blocked with un-labeled Donkey gamma globulin (Jackson). All subsequent washing steps were done using PBS with 5% FCS and 0.1% TWEEN-20. All primary antibodies were incubated for 1 h at RT. Secondary antibodies were used  for detection. DAPI was used to stain nuclei. After staining was completed the slices were mounted with DAKO fluorescent mounting medium . To analyze the staining a LSM 710 confocal microscope  was used, and pictures were taken using the software Zen 2011 . Image analysis was performed using the ImageJ software.Fluorescent IHC was performed on cryo-sections of JHTN = 8 animals were analyzed per group, osteoclast numbers were normalized against the bone surface in the analyzed region of interest.Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine osteoclasts. Counterstaining was done with methyl green. TRAP-positive cells with >3 nuclei and positioned on the bone surface were considered osteoclasts and included in the evaluation. A total of −/− = 4, D7) were used to visualize collagen fibrils in WT and RAG1−/− mice via SHG imaging. Collagen fibrils exhibit endogenous SHG signals arising from their well-known non-centrosymmetric molecular structure  with 100 fs pulse width at 80 MHz and wavelength of 910 nm. The SHG collagen signal was detected in the range of 450–460 nm. Z-Stacks were recorded with 4 µm z-spacing using 25× water immersion objectives with numerical apertures of 0.95. Overview images were created via image stitching and maximal intensity projections of z-stacks.Plastic-embedded samples  were taken. Same region was also collected from intact femora from both groups to assess indigenous differences. Samples were immediately snap frozen in liquid nitrogen and stored at −80°C until further use. Bones were pulverized in liquid nitrogen and transferred to TRIzol  and RNA was isolated according to the manufacturer’s protocol with DNAse I (Invitrogen Life Technologies) digestion included. A total of 150 ng RNA per sample was pooled in a given group (n = 5 per group). The pooled RNA mixture was distributed as 200 ng samples per tube for technical triplicate in the subsequent microarray analysis.For RNA preparation from fracture callus, the soft tissue was removed and the fractured bone samples including 1 mm diaphyseal bone on either side of the fracture . Processing RNA for hybridization, including cRNA synthesis and labeling, was done according to standard protocols described in the instruction manual of Illumina TotalPrep RNA amplification kit . Hybridization of the labeled and fragmented cRNA to the microarray and subsequent staining, washing, and scanning of the arrays was done according to the Illumina Whole-Genome Gene Expression direct Hybridization Assay guide.−/− was calculated using a two-tailed t-test. The p-values were alpha-error adjusted using the Benjamini–Hochberg method. Fold changes (FCs) were calculated from comparison of the mean expressions between the control (WT) and the treatment (RAG1−/−) group. Differentially expressed genes were visualized in a heatmap after applying a t-test analysis to reveal the significant gene regulations for each analyzed time point. Significance FC cutoff of >1 and Benjamini and Hochberg-corrected p-values (p.BH) <0.05 was followed for the guilt-by-association (GBA) analysis and a cutoff of FC > 1 and p.BH < 0.01 for the panel analysis. Genes were then clustered (arranged) according to their expression pattern over the course of healing. Gene lists of the expression clusters were then compared to literature and NCBI-DAVID (an online server for the gene annotation) to characterize the major biological processes, molecular functions, and cellular components for these genes.The arrays were pre-processed and normalized using R , the bea−/−, TCRβδ−/−, and JHT−/− (n = 4) were harvested and instantly frozen in liquid nitrogen and then stored at −80°C until processing. RNA was extracted using a Trizol/chloroform  and the RNAeasy kit . A total of 100 ng RNA per sample have been reverse transcribed using random primer . Primers were tested for the annealing temperature and efficiency prior to use: mCOL1a1 ase: 5′-gttccaggcaatccacgag-3′, mCOL1a1 se: 5′-ggtccacaaggtttccaagg-3′; mOPG ase: 5′-ctgctctgtggtgaggttcg-3′, mOPG se: 5′-agctgctgaagctgtggaaa-3′; mRANKL ase: 5′-cgaaagcaaatgttggcgta-3′, mRANKL se: 5′-gcacacctcaccatcaatgc-3′; mRUNX2 ase: 5′-tgtctgtgccttcttggttcc-3′, and mRUNX2 se: 5′-cgaaatgcctccgctgttat-3′. Cyclophilin A was used as a house keeping gene. PCR cycle: 40 cycles each consisting of 95°C, 30 s, 62°C, 30 s, 72°C, 30 s. Evaluation was done using the 2−ΔCt method.To evaluate the differential expression of collagen I and bone markers  a qRT-PCR was performed. Femora from WT, RAG1−/−, TCRβδ−/−, and JHT−/− (n = 4) and brought into single cell suspension. Cells were washed and resuspended in FACS buffer (PBS/0.5% BSA/0.1% sodium acid). Staining with the fluorescent coupled antibodies was performed for live/dead , CD45 , CD3e , and CD8a . The FACS analysis was done with a BD LSRFortessa (BD Deutschland GmbH). The FlowJo software  was used for data evaluation. Gating strategy: lymphocytes, single cells, live cells, CD45-positive cells, CD3e-positive cells, CD8a-positive cells to determine CD8+ T cell percentage in the respective mouse strains.Splenocytes were harvested from WT, RAG1−/−, JHT−/−, and RAG1−/− mice (n = 4) were pooled and analyzed toward their TNFα and interleukin 10 (IL-10) concentrations. Mouse TNFα ELISA Ready-SET-Go!® and Mouse IL-10 ELISA Ready-SET-Go!® kits  were carried out after manufacturer’s recommendations. Samples were incubated overnight at 4°C, final staining reactions were stopped with 1 M H3PO4 and absorbance was read at 450 nm with 570 nm reference wavelength with Tecan Infinite M200 PRO  and analyzed with i-control 1.9 software (Tecan).Conditioned media of LPS-activated splenocytes of WT, TCRβδ−/−, TCRβδ−/−, and JHT−/− mice (n = 4). Medium change was done twice per week for 14 days. Collagen fibers were stained with Sirius red and analyzed under polarized light.Isolated murine MSCs were cultured in chamber slides with expansion medium for 1 day. Then osteogenic differentiation medium was added which included conditioned medium (1:3 dilution) from LPS (1 µg/ml)-activated splenocytes of WT, RAG1post hoc corrected Mann–Whitney U test was used to test the significance between the two groups in a given time point. p-Values of <0.05 were chosen to indicate an exact two-tailed significance. Data were presented as box plots (median and interquartile range) or bar graphs (mean ± SEM).Tests for statistical significance were performed in SPSS 21.0 . Data from histomorphometry, biomechanics, and microCT analyses were explored for normality by examining skewness and kurtosis. Data were not normally distributed. Therefore, Bonferroni n ≥ 5 animals were included per group. This was the lowest number of samples needed for the present study based on a priori assumption for qualitative statements. For descriptive analyses, samples sizes of n < 5 were used. No experimental animals were excluded from the analysis.In the analyses of statistical significance, −/− mice are in their gross appearance similar to WT. Without mature T and B cells bone developmental processes, however, are altered and this alteration in organogenesis is reflected in fracture healing. Detailed analysis of structural parameters, functional competence, and expression profiling in intact RAG1−/− bone was performed .RAG1−/− and WT, intact bones were investigated using histological and radiological tests. Movat’s pentachrome staining indicated no morphological differences between the femora of both groups. Histomorphological analysis was also performed. However, no significant differences in trabecular number or thickness were found (data not shown). microCT data of unfractured and contralateral bones of WT and RAG1−/− animals showed no differences in total volume, bone volume (BV), or the ratio thereof; however, qualitative bone markers such as tissue mineral density and bone mineral density (BMD) were significantly different  did not differ between RAG1−/− and WT  Microarray data of intact bone between WT and RAG1s Figure ] and typs Figure ] and act−/− was compared to the WT. Differentially expressed genes as described in Figure Guilt-by-association analysis correlates genes regulated in T and B cells and osteogenesis depending on their coexpression and colocalization Figure D. The osMicroarray analysis also showed upregulated B cell-related genes Figure , D.2, su−/− compared to the WT. Another gene was found to be colocalized with these genes: transformation-related protein 53.Interestingly, T cell-related genes Figure , D.3 wer−/− mice: Movat’s pentachrome staining was used to quantify dimensions and distribution of callus tissue during the time course of healing. Histomorphometric methods were used to quantify cartilaginous fractions including zones of hypertrophic chondrocytes as well as ossified regions at the periosteal bone surface area in the fracture callus.To confirm the effects of T and B cells in bone formation processes, bone healing was analyzed in WT and compared to bone healing in RAG1−/−, D7). The earlier callus formation in the RAG1−/− animals lacking mature T and B cells has been reported in detail before .To determine how mature T and B cells influence the material formation of bone, we looked into bone apposition during bone healing. Bone formation was monitored using fluorescent dyes injected 2 days prior to sacrifice at 7, 14, and 21 days after fracture to determine new bone formation in the fracture callus with (WT) and without B and T lymphocytes . While fibers in the WT seem to be aligned along the outer callus border and also surrounding the dark areas that would indicate cartilaginous zones, this was not found in the RAG1s Figure .−/−, ColI was analyzed during bone healing in both animal types by immunohistochemical staining. In the WT animals, a typical outer callus area was positive for ColI, while this ColI-positive area was missing in the RAG1−/− animals  alteration in bone tissues during organogenesis and very pronounced in bone healing, the cells responsible for ColI production were analyzed. ColI is mainly produced by osteoblasts during the bone formation process. Due to the altered ColI pattern in RAG1s Figure . In WT as Figure A,B. In R Figures C,D.The abovementioned data suggested that T and B cells are important for the tissue properties of newly formed bone material and that osteoblasts are located distant to the callus tissue formation front if mature T and B cells are missing. Thus, the interdependency of the immune cells and the bone formation signals were analyzed during fracture healing using expression analysis.−/− showed significantly more osteoclasts (p = 0.037): at this time point occurs typically the maturation from the soft callus to hard callus and the beginning woven bone deposition in WT animals .A detailed examination of osteoclasts, bone cells responsible for bone degradation revealed no significant difference in osteoclasts numbers in the healing cascade up to D14. At D14, RAG1−/− and the WT callus tissue to the gene sets selected in commercial array panels for angiogenesis, cytokines, and growth factors, and bone remodeling and BMP (bone morphogenetic protein) signaling . In RAG1−/− mice, when D0 was taken as control to look into the changes during the bone healing process, the gene list was compared to the panels for an overview of the change in expression. At D3, over 30% of the differentially expressed genes were related to angiogenesis   or B cells (JHT−/−). The histological images showed that in the animals that lacked only T cells an earlier mineralization was detected . CD8+ T cells are present in WT and JHT animals, while they are missing in RAG1−/− and TCRβδ−/− animals  in the bone marrow. Significant difference in expression of collagen I was detected in bone marrow cells of RAG1−/− and TCRβδ−/− mice when compared to wild-type (WT). Bone markers such as osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), and Runt-related transcription factor 2 (RUNX2), however, were not affected  is upregulated only at D7 and D14 in RAG1−/− compared to WT. Dbp is correlated with T cell proliferation and involved in maintaining the BMD, which supports the decrease in bone quality under the lack of mature T cell.Previous studies on fracture healing in lymphopenic RAG1 process . Discrep−/− mice was apparently contradicted by its defective ColI deposition and displacement of Ocn-positive cells in the callus periphery. ColI is the marker ECM molecule of bone, and Ocn is seen as a marker protein for osteoblasts. Therefore, the lack of mature T and B cells has altered two major contributors to bone formation in the RAG1−/− mice. The GBA analysis correlated Spp1, Ctsk, Bglap, Col1a1, and Igf1. Along with the upregulation of Col1a1 at D3 through D7, D14 until D21 those genes suggest the earlier start of mineralization and end of bone formation in the RAG1−/−. The osteoclasts, bone cells important for the bone homeostasis and healing as bone degrading cells, showed no differences in numbers up to day 14 of healing. The changes in collagen I deposition found in our analysis are, therefore, not considered connected to an altered osteoclast function. In addition, our analyses of gene expression in bone marrow cells in mice lacking T cells (TCRβδ−/−) or B cells (JHT−/−) or mature T and B cells (RAG−/−) showed no significant difference for OPG and RANKL, both factors involved in osteoclastogenesis.Faster mineralization in RAG1During endochondral ossification in WT, mineralization starts in the cartilaginous layer with chondrocytes shifting from ColII toward ColX expression. Chondrocytes become hypertrophic and express a number of ECM proteins including osteonectin, fibronectin, and osteopontin –33. At tper se is not a challenge [as illustrated by ectopic bone formation  in the RAG1−/− animals. OPN has been reported to inhibit the mineral crystal growth  . The above are some explanations on how the regulation of T cells impacts matrix mineralization and concurs with our results in animals lacking mature T and B cells. T cells appear to affect cartilage mineralization by slowing the process itself down (when compared to lymphopenic mice), giving matrix organization and mineral deposition adequate time to enable callus structure to concomitant mechanical strains.Immune cells are present during the various phases of bone healing  Figure , 43, a pGD motif . With TGGD motif  this indertrophy , which cl growth  and within (BSP) . BSP is in (BSP) . Lower T−/− animals, we detected lower Ocn expression in the fracture callus. With Ocn binding to hydroxyapatite this could indicate a disturbed bone healing, however, Ocn knockout animals only show a mild bone phenotype and prove that mineralization without Ocn is still possible (−/− animal fracture callus indicates another role for T cells in the bone healing process. T cells seem to be essential for osteoblast maturation. Expression of IL17F and wingless-type MMTV integration site family, member 10B by T cells has been reported to be essential in the osteoblasts maturation process (In the RAG1possible . Ocn, ho process . The clo process  is anoth process  and its  process  might be process . However process . It seem−/− fracture callus could be an indication of another function of immune cells during the bone healing process. Previous studies showed that the lymphatic draining in the injured bone is activated upon fracture and that prolonged lymphonodal activity is associated with successful healing (via blood vessels (in situ for proper trafficking. Further studies should be carried out to elucidate the complete mobilization pathway of these cells during bone repair. This is in accordance with the upregulation of VEGF and the downregulation of Serpinf1 as seen at D7 and D14, which both play a role in osteoblast regulation (The curious finding that Ocn-positive cells line up on the outside of the RAG1 healing , 58. Ocn healing  and oste vessels . However vessels , requiregulation .+ and K+ values (To partake in the healing process, osteoblastic cells would be required to invade the fracture side which directly after the injury would be filled with a hematoma. The hematoma has an unfavorable milieu for cells to survive and being active due to the low oxygen levels, lower pH value, and higher Na+ values –64, howe+ values . Therefo+ values . Among tThis work for the first time presents a direct link between immune cells and matrix formation during regeneration using bone healing as an example. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells.The importance of immune cells during the fracture healing process has already been established. Here, we show that immune cells play an essential part in the regulation of the mineralization in endochondral ossification, which is necessary to generate bone with an elastic quality able to withstand forces. In detail, immune cells are needed to slow down the mineralization process in order to build quality bone, and immune cells are needed to recruit osteogenic precursors and aid their differentiation.Even though callus formation in itself is faster without T and B cells the ensuing bone callus and organ tissue do not have the sufficient quality and thus functionality that is achieved when these immune cells take their active role in the bone organization and formation process.This study was carried out in accordance with the recommendations of the Animal Welfare Act, the National Institutes of Health Guide for Care and Use of Laboratory Animals, and the National Animal Welfare Guidelines and was approved by the local legal representative animal rights protection authorities: Landesamt für Gesundheit und Soziales Berlin. The protocol was approved by the Landesamt für Gesundheit und Soziales Berlin: G 0206/08.TK contributed data acquisition: data evaluation, study design, and manuscript writing; AS contributed data acquisition: histology, data evaluation, and manuscript writing; AP contributed data acquisition: second harmonic imaging and manuscript writing; CB contributed data acquisition: biomechanics, cell culture, and data evaluation; CS contributed data acquisition: histology and manuscript writing; IK contributed data acquisition: histology, data evaluation, and study design; DM contributed data acquisition: data evaluation; SW contributed data acquisition: in cell culture and ELISA, data evaluation, and manuscript writing; HS contributed study design, manuscript writing; H-DV contributed to study design and manuscript writing; KS-B contributed data acquisition: biomechanics study design, data evaluation, and manuscript writing; GD contributed study design and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.