Mycobacterium tuberculosis (Mtb) based on γδ T cells provide a novel approach for TB control. In our previous studies, we found a preponderant complementarity-determining region 3 (CDR3) sequence of the γδ T cell receptor (TCR) in TB patients, and successfully identified a tuberculosis antigen that can effectively activate γδ T cells with a reverse genetic strategy. However, due to the throughput limitation of the method we used, the information we obtained about the γδ TCR repertoire and preponderant CDR3 sequences was limited. In this study, we introduced next generation sequencing (NGS) to study the γδ TCR CDR3 repertoires in TB patients. We found that the CDR3δ tended to be more polyclonal and CDR3γ tended to be longer in TB patients; the γδ T cells expressing CDR3 sequences using a Vγ9-JγP rearrangement expanded significantly during Mtb infection. We also identified new preponderant CDR3 sequences during Mtb infection. This study comprehensively characterized the γδ T cell receptor repertoire changes, and provides useful information for the development of new vaccines and adjuvants against TB.Tuberculosis (TB) is a severe global threat to human health. The immune protection initiated by γδ T cells play an important role in mycobacterial infection. Vaccines for  Mycobacterium tuberculosis (Mtb), the top infectious disease killer worldwide, remains a severe global threat to human health. In 2015, it was estimated that there were 10.4 million new TB cases and 1.4 million deaths due to the disease1. The control of TB lacks new diagnostics, new drugs, and new vaccines; of these, effective vaccines are of vital importance to the prevention of TB. Currently, the only vaccine that is available and widely used against TB is bacille Calmette–Guérin (BCG), which can effectively protect children against active TB diseases, such as TB meningitis, but its efficacy in protecting adults and the aging population against pulmonary tuberculosis is highly variable3. Therefore, safer and more effective vaccines against TB are urgently needed.Tuberculosis (TB) caused by Mtb infection4. Among the unconventional T cells, the γδ T cells are thought to play a critical role in anti-mycobacterial immunity6. The role of γδ T cells in the immune response to Mycobacterium tuberculosis was first reported in 19897. More and more research has demonstrated that the activation of γδ T cells appear rapidly following Mtb infection, and γδ T cells provide protective immunity against Mtb infection with the combined properties of both innate and adaptive immunity10. Vaccines for Mtb based on γδ T cells provide a novel approach to TB control13.To develop efficient TB vaccines, the mechanism of immune protection against TB must be identified. Unconventional T cells, such as lipid-specific CD1-restricted T cells, MAIT cells, and γδ T cells, function at the early stage of 14. However, only a subset of the phosphoantigen-responsive γδ T cells mediate protective tuberculosis immunity15. Based on this research, we established a reverse genetics strategy and successfully identified BCG and Mtb-derived protein antigens, which were not only recognized by γδ TCR, but could also activate γδ T cells and exhibit cytolytic effector function against BCG-infected cells18. In this strategy, we used PCR and Sanger sequencing to find preponderant complementarity-determining region 3 (CDR3) sequences in pulmonary tuberculosis patients. However, due to the limitation of the throughput of this method, we only analyzed 102–103 sequences and obtained limited information about γδ TCR19.Originally, phosphoantigens were reported to activate γδ T cells, and were considered to be the main γδ T cell receptor (TCR)-recognized antigens23. The γδ TCR repertoire in pulmonary tuberculosis and tuberculosis meningitis has been studied with flow cytometry and PCR25; however, with the low throughput of these methods19, the information obtained from these studies is also limited. With the development of next generation sequencing (NGS), the sequencing depth and read length increased tremendously. The introduction of NGS to the reverse genetics strategy of our study will make the analysis of the γδ T cell receptor repertoire more comprehensive and robust.Since the TCR repertoire is a mirror of the human immune response, its characteristics have been widely investigated in infectious and other diseases to study the state of the immune system and the progression of these diseasesMtb infection. Additionally, we found some new preponderant γδ TCR CDR3 sequences in the LTBI group, the TB group, and the LTBI&TB group, which are expected to be more representative than the preponderant CDR3 sequences we obtained in our previous studies and could be used to screen novel Mtb-derived protein ligands of γδ TCR. Taken together, this study comprehensively characterized the TRG and TRD repertoire of TB patients, and provides some information about the preponderant CDR3 sequences of specifically recognized Mtb-derived antigens, which may help to develop new vaccines and adjuvants against TB.In this study, to investigate the profile of the γδ T cells repertoire and to identify more preponderant CDR3 sequences in tuberculosis patients, we analyzed the expressed T cell receptor γ chain (TRG) and T cell receptor δ chain (TRD) repertoire of circulating γδ T cells that were isolated from people with latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (TB). Compared with the healthy controls (HC), we found some changes in the TRG and TRD repertoire of the TB patients: the CDR3δ tended to be more polyclonal; the length of CDR3γ had a trend to be longer; the frequency of TRGV9 and TRGJP usage increased; and the γδ T cells expressing CDR3 sequences using a Vγ9-JγP rearrangement expanded significantly during ®. Data from three samples (two HC subjects and one TB subject) were excluded because their reads were unusually fewer than the others due to an error in library preparation. The general information and clinical characteristics of the subjects are summarized in Table Mtb infection. The differences of total CDR3 reads of TRD between the different groups were minor , 10 HC subjects, 8 LTBI subjects, and 12 TB patients were recruited for the study. The CDR3 of the γδ TCR underwent high-throughput sequencing with the Illumina MiSeq platform by iRepertoireMtb antigens and affects a patient’s immune response to the pathogen. To estimate the repertoire diversity in the different groups, we took the unique CDR3 ratio as the indicator. Unique CDR3 ration was calculated by the number of unique TRG/TRD clones divided by the number of total sequences of each sample. Ideally, the unique CDR3 ration  ranges from 0 to 1, with “0” representing the lowest diversity, while “1” represents the maximal diversity. The results showed that there were no significant differences of TRG diversity between the three groups  that was developed by Krell et al. to analyze the diversity of the TRG and TRD repertoires26, and the results were consistent with the unique CDR3 ratio  developed by Krell et al. to measure the CDR3 length polymorphisms of TRG and TRD. Since the types of the different CDR3 lengths that were observed in each of the TRGV and TRDV families are all more than 8, we did not set the maximum value of the numbers of the different CDR3 lengths contributing to the CSL. The CSL scores of TRG  and TRD  have no significant differences between different groups. The distribution patterns of both unique CDR3γ sequence lengths and unique CDR3δ sequence lengths show an obvious Gaussian-liker distribution28. After nonlinear curve fitting (Gauss function), there was a trend of unique CDR3γ sequence lengths to be longer in the TB patients  developed by Krell et al. to analyze the TRGV-TRGJ and TRDV-TRDJ combinations of the TRG and TRD repertoire in the three groups26. Since the number of reads is different in each sample, we used frequency instead of the reads to calculate the CSC. The results also showed that the CDR3γ sequences using TRGV9-TRGJP rearrangement increased significantly in the TB group . To estimate the junctional diversity of the TRG/TRD repertoire among the different groups, we analyzed the J trimming, V trimming, and N addition of TRG and TRD in the different groups. For total CDR3 sequences, there was no distinct regularity and no significant differences of J trimming, V trimming, and N addition between the three groups. For unique CDR3 sequences, there was no obvious differences in J trimming, V trimming, and N addition of TRD Fig. , and V t30 and some clonotypic γδ T cells exhibited clonal expansion during BCG infection and re-infection31. These studies suggested that clonotypic γδ T cells also clonally expanded during Mtb infection and these γδ T cells carried CDR3 sequences specifically recognized Mtb-derived antigens. These specific CDR3 sequences, which are also known as preponderant CDR3 sequences, may be critical for the recognition of Mtb-derived antigens and instructive for the development of new vaccines. To identify preponderant CDR3 sequences of the γδ TCR in Mtb infection, we analyzed the sequences obtained by NGS. To find preponderant CDR3 sequences in the LTBI, TB, and LTBI&TB groups, we chose unique CDR3 sequences to analyze so as to avoid bias caused by PCR amplification. We first identified the particular sequences in the LTBI, TB, and LTBI&TB groups, and then defined the sequences that occurred in over 50% of the samples as the preponderant CDR3 sequences. We summarize the preponderant CDR3 sequences in Table It has been reported that γδ T cells are present in increased proportions in the peripheral blood of a fraction of tuberculosis patientsMtb infection32. They can expand in response to the stimulation of Mtb antigens in vitro and in vivo34. In our previous work, we demonstrated γδ T cells’ response to Mycobacterium tuberculosis in pulmonary tuberculosis patients using preponderant complementary determinant region 3 sequences16. The γδ TCR repertoire in patients with pulmonary tuberculosis and tuberculosis meningitis has been studied with the flow cytometry and PCR25, but due to the natural limitation of these methods19, the information achieved from these studies is not enough to comprehensively illustrate the γδ TCR repertoire changes during Mtb infection. With the development of NGS, deep sequencing has already been applied in the analysis of TCR repertoire and immunoglobulin repertoire37. Li and colleagues have profiled the β TCR repertoire differences of PBMCs and pleural effusion mononuclear cells (PEMCs) from one of the TB patients using NGS38. To better characterize the changes of γδ TCR repertoire in TB infection, we investigated the γδ TCR repertoire in pulmonary tuberculosis patients by high-throughput sequencing using the Illumina MiSeq platform combined with ARM-PCR. With the combination of the semi-quantitative property of ARM-PCR and the high sequencing depth of NGS, we characterized the profile of the γδ T cells repertoire in TB patients at the level of sequence resolution for the first time.It has been widely reported that γδ T cells, especially Vγ9Vδ2 T cells, play an important role in the protective immune responses to Due to the scarcity of the sample resources, the recruits in our study were limited. We were not able to recruit subjects that were well-matched for age and sex in the beginning. In order to exclude the variation caused by age and sex distribution in the different groups, we analyzed the differences of age or sex among the different groups with analysis of variance and the Chi-square test, respectively. We found that there were no differences in the age and sex distributions among the HC, LITB, and TB groups, except that the age distribution in the LTBI group was different from that in the HC and TB groups with significance , just as the profile of the gene melting spectral pattern of the TCR beta variable gene family in PBMCs from healthy donors28. In our results, there was a trend of CDR3γ to be longer in the TB group, and CDR3γ sequences with a specific number of nucleotides were obviously increased in the TB group. This result suggested that the increased CDR3γ length may be correlated with the Mtb infection, and the γδ T cells expressing CDR3γ sequences with longer lengths may play important roles in the immune response against Mtb infection. The increased length of CDR3γ and the enrichment of CDR3γ sequences with 39, 42, and 45 nucleotides were also demonstrated by the preponderant CDR3 sequences we found in the TB group  of TCR γ chain was important for γδ TCR to recognize the antigens, and the functional γδ TCR repertoire partly depended upon preferentially rearranged Vγ-Jγ gene combinations46. According to our results, the frequency of γδ TCR with the rearranged Vγ9-JγP gene combinations increased obviously in the TB group, which suggested that the Vγ9-JγP gene combinations might be important for γδ T cells to recognize the antigens derived from mycobacteria. Furthermore, the marked γδ T cell expansion in response to mycobacteria stimulation was observed in both adult and newborn subjects, the TCR δ chain of the γδ T cells stimulated with mycobacteria had extensive junctional diversity, which meant the major mechanism of γδ T cell reactivity involves antigen recognition might be mediated by the germline-encoded segments of the TCR52. This might be the reason of the increasing diversity of the TRD CDR3 in TB group, and the increasing diversity of the TCR δ chain caused by the stimulation with Mtb-derived antigens might play important roles in immune response against TB infection. The changes of TCR γ chain and δ chain upon TB infection were also reflected by the preponderant sequences we obtained in TB group. With these preponderant sequences, we can screen more mycobacterial vaccine candidates for adjuvant through the reverse genetic strategy we have stablished in our previous study17, which could induce immune responses against TB infection.Bukowski Mtb infection, and may provide useful information about the γδ T cell-mediated immune response against Mtb. Moreover, we found some preponderant sequences in TB patients, which may also shed light on the development of new vaccines and adjuvants against TB.In summary, we characterized the profile of the γδ TCR repertoire in patients with pulmonary tuberculosis at the level of sequence resolution for the first time with NGS. We found that the more increased the diversity of TRD, the longer CDR3 of TRG in the TB group, which may be caused by the shortened J trimming. We also found that the γδ T cells bearing Vγ9-JγP rearrangement clonally expanded, and the γ chain of the γδ TCR may be more important in antigen recognition than the δ chain. The results promote our understanding of the profile of the γδ TCR repertoire in TB patients. Taking advantage of the analytical power of NGS, this study gave us a comprehensive understanding of the γδ T cells repertoire changes during This work received approval from the Clinical Ethics Committee of the Institute of Pathogen Biology, the Chinese Academy of Medical Sciences, and Beijing Union Medical College. Written informed consent was obtained from all participants. All of the subjects gave written informed consent in accordance with the Declaration of Helsinki.Three study groups  were enlisted to investigate the detailed characteristics of the γδ TCR repertoire. The general information and clinical characteristics of the recruited participants are summarized in Table The participants in the TB group were all in the acute phase; they were newly diagnosed pulmonary tuberculosis patients without prior anti-tuberculosis treatment and with the following clinical parameters: presence of cough/expectoration, a chest x-ray showing infiltration and/or cavities, a minimum of one positive sputum smear, and a positive culture result for acid-fast bacilli, and/or enzyme-linked immunospot assay (ELISpot) detecting IFN-γ release by QuantiFERON-TB Gold kit . This kit measures the cell-mediated response to highly specific TB antigens (ESAT-6/CFP-10/TB-7.7 (p4)) that are associated with M. tuberculosis infection and proven unaffected by the prior BCG vaccination and most non-TB mycobacteria. Participants in the LTBI group were members of the medical staff of the Sixth People’s Hospital of Zhengzhou. They were asymptomatic and had T-cell responses specific to IFN-γ detected by ELISpot. The HC group consisted of healthy volunteer subjects who did not have any changes noted on x-ray and did not have a history of tuberculosis or any other underlying disease. The subjects with viral and other bacterial infections/co-infections, severe hepatic diseases, and renal diseases, especially immunological or autoimmune diseases, were excluded. Diabetes mellitus, medication, pregnancy, and smoking were also included in the exclusion criteria.+ cells were then eluted and immediately resuspended in Trizol reagent . Before sample collection, the efficiency of the magnetic bead-based purification of gamma delta T cells was tested by flow cytometric analysis and the results showed that the purity of isolated γδ T cells was about 97%. During the sample collection and isolation, the isolated γδ T cells from each sample were counted and the ratio of γδ T cells/PBMC in each sample was calculated. The ratio range was from 1.3% to 4.9% and all of them were within the reasonable scope of 0.5–10%.Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient cell separation using Human Lymphocyte Separation tubes  according to the manufacturer’s instructions. Then, the γδ T cells were isolated from the PBMCs with the Anti-TCRγ/δ MicroBead Kit  as follows: the PBMCs were incubated with Anti-TCR γ/δ Hapten-Antibody, followed by fluorescence and magnetic labeling with Anti-Hapten Micro-Beads-FITC. The cell suspension incubated with antibody was then loaded on a column, which was placed in the magnetic field. The magnetically labeled γδ T cells were retained and the unlabeled cells run through. After removal of the column from the magnetic field, the magnetically retained γ/δRT-PCR and sequencing were done by iRepertoire . Specifically, total RNA of the γδ T cells was extracted with Trizol reagent and subjected to RT-PCR using a One-step RT-PCR kit . cDNA was then subjected to Amplicon rescued multiplex PCR (ARM-PCR) using human T cell gamma receptor primers and human T cell delta receptor primers according to the manufacturer’s instructions . Information about the primers can be found in the United States Patent and Trademark Office (US9012148B2). The PCR products were run on 2% agarose gel and the DNA at 212–280 bp was extracted from the gel using a Qiagen Gel Extraction kit. After assessing the DNA samples, 10 sample libraries were pooled and sequenced using the Illumina MiSeq platform .53. iRepertoire® provided filtered DNA sequences. The length of the CDR3 region, information on the V, D, and J gene segment usage, D-D fusion pattern of TRD, and the length of the V trimming, J trimming and N addition, were obtained from these filtered sequences. The total CDR3 sequences referred to all of the CDR3 sequences obtained from the filtered sequences and the unique CDR3 sequences referred to each distinct CDR3 sequence regardless of how many copies appeared.The raw data were analyzed by iRepertoire using IRmap programs with a modified Smith-Waterman algorithm. The CDR3 was identified as the interval between two conserved amino acids for the TCR gamma and delta chain, respectively. The best matches of the germline V gene and J gene and the information of the V trimming and J trimming, and the addition of nontemplated nucleotides (N addition) were searched by determining alignments between the sequencing products and germline sequences in the IMGT/GENE-DB databases as previously describedp < 0.05; very significant, **p < 0.01.The Mann–Whitney test was used to assess differences between any two of the three groups. Analysis was performed with PRISM version 6 (Graph Pad) and Origin Pro. Significant differences among each fraction in all of the figures are indicated by asterisks: significant, *0.01 ≤ Supplementary materials


Optimum production of MgB2, with yields greater than 75%, occurs for autogenous pressure in the range 1.0 MPa to 2.0 MPa, with the reactor at ~500 °C. Autogenous pressure is induced by the decomposition of NaBH4 in the presence of Mg and/or other Mg-based compounds. The morphology, transition temperature and magnetic properties of MgB2 are dependent on the heating regime. Significant improvement in physical properties accrues when the reactor temperature is held at 250 °C for >20 min prior to a hold at 500 °C.High quality, micron-sized interpenetrating grains of MgB These methods include solid state [2 is likely to occur with development of facile methods for bulk synthesis. MgB2 synthesis methods include mixing elemental powders sintered at high temperature (~900 °C) [Since the determination of a superconducting transition temperature  for MgB2id state , liquid id state ,6 and noid state ,8. Whileid state ,10 are d~900 °C) , combust~900 °C) , mechano~900 °C)  at >600 et al. [2 and TiB2 [4 are combined with a solid precursor such as NaBH4 in an autoclave to produce nanoparticles of metal diboride [A comprehensive review by Carenco et al.  describeand TiB2 . In othediboride . In manydiboride . Neverth2 as dense powders without the use of high pressure or the powder-in-sealed-tube (PIST) method [et al. [Jc across a range of physical conditions although synthesis requires high temperature conditions [For many syntheses, the reaction depends on the formation of Mg liquid and/or gas at high temperature or may not afford bulk manufacturing at industrial scale . Synthes) method  is also ) method  for indu [et al.  demonstrnditions  similar nditions .et al. [2 and HfB2, which form at lower temperature (~400 °C–900 °C) utilising inorganic molten salts. The optimum temperature for NbB2 formation using a LiCl/KCl melt is relatively high at 900 °C albeit with the benefit of high purity and nanometre sized crystallites [In general, lower temperature synthesis or solution-based methods are likely to result in products with potential for higher density and commercial production of this technologically important material. Recently, Portehault et al.  demonstrtallites .via the PIST method [2 powders. For example, a synthesis along similar lines to that of Portehault et al. [et al. [2 between 350 °C and 450 °C. In this work [2 and LiBH4 are milled with minor amounts of TiCl3 in a sealed, oxygen-free environment and heated to 400 °C–450 °C for twelve hours. The reaction produces nanometre-sized particles with typical magnetisation and structural characteristics for MgB2 [et al. [2 of high quality with ~85% yield. More recently, Chen et al. [2 using a hybrid physico-chemical vapor deposition (HPCVD) method at constant pressure of 21kPa and ~700 °C.The use of autogenous pressure is implicit in the production of wires T method  and is at et al. , is used [et al.  to form his work , MgH2 anfor MgB2 . In anot [et al.  rapidly n et al.  have dem2 between 420 °C and 500 °C using NaBH4 and Mg metal. High yields of dense, micrometer-scale MgB2 powders are obtained through control of the reaction temperature and heating rate.Autogenous pressure offers the potential to operate at lower temperature compared to conventional solid state synthesis methods with control determined by reaction time and absence of reactive species such as oxygen. In this work, we describe an autogenous reaction without the use of an additional dehydrogenation agent to produce MgB2.2.1.2 is shown in 2. In all cases, MgB2 is the dominant phase. For Runs 1 and 2, the same heating profile is used up to the target temperature as exemplified in i.e., zero ramp) at 50 °C and 250 °C, respectively. For Run 3, the ramp rate is 10 °C/min with a 20 min hold at 50 °C and, subsequently, a 5 h hold at 250 °C. For Run 4, the same temperature increase of 10 °C/min is maintained, but without a hold until 500 °C.A summary of reaction parameters for each run that generated MgB2 as the predominant phase using the same starting materials are shown in Similar parameters for reactions that did not generate MgB4 occur at the top of the reactor and are readily separable from the primary MgB2 product, sometimes with residual Mg, occurring at the bottom of the reactor.In general, the reactor shows distinct layering of products from the base of the vessel, around which the heating element is arranged, to the top region which contains the release valves for gas discharge. This layering reflects a variation in temperature profile with the top of the reactor colder than the centre of the reactor where the thermocouple is situated. In general, comparison of set points, controller values and thermocouple values suggest that the variation in temperature from the centre of the reactor to its extreme is approximately 10%–15% relative. For the experiments listed in in situ thermocouple. In order to evaluate the temperature profile within the reactor—to a rough approximation—“blank” experiments with no reagents or only Mg powder are also undertaken. These experiments suggest that under specific heating rate regimes, the bottom of the reactor may achieve temperatures for short periods of time significantly higher (up to 150 °C) than the value recorded by the thermocouple located in the centre of the reactor. For example, an Mg strip placed on the bottom of the reactor and subject to conditions equivalent to Run 4 results in a sharp increase in pressure to 0.25 MPa with subsequent reduction in pressure similar to Runs 1–4 over a ten hour reaction period. This result and observations of the heated Mg strip after reaction (images not shown), suggest that the bottom of the reactor experiences temperatures > 600 °C for short periods.Results from these experiments imply the formation of gaseous phases at temperatures higher than that nominally specified by the 2.2.2 is formed as determined by X-ray diffraction (XRD) patterns  indicates an MgB2 yield of ~75% on the basis of Mg input.2 is predominant (>96%) with minor amounts of NaH and MgO  is consistent with the presence of Na and/or NaH. XRD of material from the top of the reactor shows that NaH is the predominant phase. 2 grains are clustered within aggregates approximating the size (>100 μm to ~200 μm) of the original Mg grains  used as reactants. Higher magnification images , is widely used in laboratory and industrial practices [2, Mg2FeH6, Mg2NiH4) due to strong interest in industrial applications for hydrogen storage [et al. [Sodium tetrahydroborate, or sodium borohydride  during the reaction(s). The nature of the gas(es) present in the reactor over the temperature range is inferred to include hydrogen, sodium, diborane and magnesium. In addition, the temperature-pressure profiles e.g.,  and 3 co2.5.1.4 reactant to form H2 and/or B2H6 gases, at relatively low temperatures (<250 °C), accelerated decomposition of NaBH4 in the presence of Mg along with formation of intermediate phases such as MgH2 at moderate pressure (<2 MPa), decomposition of MgH2 and NaBH4 to form reactive Mg and Na and possibly Mg(BH4)2 at higher temperatures (200 °C< T <400 °C) and formation of MgB2 and/or residual gas(es) between 400 °C and 500 °C. Formation of NaH along with unreacted or residual NaBH4 occurs between 300 °C and 450 °C on the upper section of the reactor given sufficient production of H−. The overall reaction for this system can be summarised as follows:Over the temperature range 25 °C to 500 °C, a number of key reactions are evident from the composition of the major products formed. The precise mechanism(s) and type(s) of reactions are difficult to detail without additional data collected during the reactions (e.g. composition of gases generated at specific temperatures). However, reactions that reflect the most likely, or dominant, transformations are presented in this section. These key reactions involve decomposition of NaBHHowever, other intermediate reactions are evident from the analysis of all reaction products including those undertaken at lower temperatures see  and 2.4 in the presence of Mg or MgH2 [et al. [2 forms at 400 °C using Mg(s) and H2(g) with up to 4 MPa hydrogen pressure. At lower pressure up to 1 MPa, Mg and MgH2 occur at this temperature [2 from Mg powder via gas-solid reaction at low temperatures is diffusion limited [2(g) [et al. [2 at higher pressure. Data from Run 6, for the reaction held at 300 °C shows that MgH2 is formed and suggests that this phase is an important intermediate in the overall reaction to form coarse grained MgB2. This experiment (Run 6) also implies that H2 is present as a gas above 250 °C and ~0.07 MPa pressure.A significant increase in pressure within the reactor occurs between 250 °C and 500 °C. This pressure increase is consistent with advanced decomposition of NaBH or MgH2 ,27 and,  or MgH2 . Accordi [et al. , MgH2 foperature . While f limited , high qued [2(g) . Figure  [et al.  suggests2 is present as a gas over a range of temperatures in this type of reaction. In addition, Zhu et al. [2 between 250 °C and 600 °C and 1 MPa to 4 MPa pressure. Thus, we propose that the reduction in pressure for Run 3 is predominantly a result of the formation of MgH2. The step-like nature of the pressure drop at constant temperature may be due to competing reactions that include not only the production of MgH2 but also its decomposition in a composite mix of NaBH4 and MgH2 (see below).The reaction parameters for Run 3, for which a five hour hold is maintained at 250 °C, indicate that a gas is consumed at this temperature due to the gradual reduction in reactor pressure . We haveu et al.  and otheu et al.  have shoA rapid increase in temperature that follows the hold at 250 °C is likely to generate localised heating of Mg powder at the bottom of the reactor to more than 500 °C due to a finite delay response time between the thermocouple and temperature controller. Between 600 °C and 650 °C, the vapour pressure for Mg is between 0.13 kPa and 0.41 kPa and thus, may contribute to an increase in pressure as temperature is raised.4)2 decomposes to form MgH2 between 320 °C and 400 °C [4)2 is not detected in the products listed in et al. [2H6 and solid MgH2 at temperatures up to 220 °C. Thus, this reaction may occur as the reactor ramps up in temperature to 250 °C, but direct evidence is not available from the data collected in these experiments.Mgn et al.  suggest 2.5.2.4 is an important component of this reaction. A computational study by Cakir et al. [via lattice defects which, as noted by Cakir et al. [4 as charged defects of Na+ and BH4− ions. Density functional theory calculations show that the formation energies of vacancies corresponding to these ions are the lowest of all possible species and thus, are the primary means for transport of B, H and Na in bulk NaBH4 [4− ion with H− also readily occurs and results in BH3 vacancies which are not easily incorporated into the lattice. The decomposition of BH4− to H− and BH3 occurs at the surface of NaBH4 with H− ions locally forming NaH within the lattice [3 gas which readily polymerizes to form B2H6 and higher forms [Decomposition of NaBHr et al.  providesr et al. , occur plk NaBH4 . Accordi lattice . Thus, fer forms  is promo4 is enhanced in the presence of Mg [2 [2 [4 is 505 °C at standard pressure [4 may commence at temperatures much lower than 400 °C in the presence of Mg while the melting point may increase due to the imposition of autogenous hydrogen pressure [3 from the surface of NaBH4 over a wider temperature range and, consequentially, production of H2.Computational and experimental studies confirm that the decomposition of NaBHce of Mg  or MgH2 of Mg [2  or MgB2 Mg [2 [2  and may Mg [2 [2 . The melpressure  and is apressure ,23 but mpressure . Thus, dpressure . This co4 + MgH2, Kato et al. [2 commences at ~230 °C and is complete at ~330 °C. In addition, the decomposition of NaBH4 in this composite mix begins at ~250 °C and is induced by the migration of Mg to the reaction front [2 is formed at lower temperature in Runs 1–6, the decomposition of both MgH2 and NaBH4—if in intimate mix—occurs at significantly lower temperature than 400 °C. As noted by Kato et al. [In a study of a composite mix of NaBHo et al.  shows thon front . Thus, io et al. , this su2.5.3.2 involves gas phase reaction. Data for experiments at lower temperature  also suggest that gas phase reaction is a mechanism, perhaps of lesser influence, at these temperatures. This implication arises from the temperature-pressure profile during the experiment as well as the presence of MgH2 in Run 6. The precise identity of these gases at specific temperature settings is not available as it is difficult to sample from within the reactor while maintaining pressure. However, we infer from previous studies in similar systems that these gases are likely to be B2H6, H2, Na and possibly Mg [While the generated morphologies and migration of products to the reactor lid, for example, are evidence of vapour phase transfer, another important indicator that gas phase reactions are involved in Runs 1–6 is the reduction of pressure in the reactor while at constant temperature. In all runs, and as shown in sibly Mg ,32,35.4 occurs at low temperature when intimately mixed with a metal chloride and heated above 50 °C [2H6 is optimal at 90 °C while production of H2 increases with higher temperature to ~110 °C [2H6, which may form from the slow decomposition of NaBH4 , generag et al.  who demooduction  and resuoduction ,36. At hoduction ,37. Whil4 (in the presence of Mg or MgH2) and production of H2. At this time, a boron-rich solid may condense in regions that are slightly lower in temperature within the reactor. With increased temperature, the boron-rich solid may also become unstable or susceptible to formation of MgB2 as noted above.This earlier work suggests that at low temperatures, evolution of hydrogen and borohydride gas is possible and is partially responsible for the increase in pressure measured in these reactions up to 250 °C. This increase in pressure is enhanced by holds at 50 °C and 250 °C which ensure continued decomposition of NaBHThe melting point of Na at 0.1 MPa  is 97.7 2.6.2 are micrometre-scale and, in both cases for which the heating rate is held at 250 °C, an intimate mixing and interpenetration of grains is apparent  is generally smaller (~2 μm to 5 μm) and while intermixed, does not show interpenetration of hexagonal-shaped grains at the edges or faces of other individual grains. Images in Images via optical and electron optical methods  and 6 shapparent  and 5. I2 in a nitrogen atmosphere at 750 °C produces nanometre-scale material ranging in size from 40 nm to 80 nm with a BET surface area of 13.9 m2/g and a pore diameter of 0.96 cc/g. The HPCVD method developed by Chen et al. [2 crystals ranging in size from 10 μm to 30 μm. The crystal shapes identified by Chen et al. [et al. [Direct vapour phase production  of MgB2 n et al.  generaten et al.  show a c [et al.  is a dir2 show typical temperature dependence curves for a superconducting transition at T~38K. However, detailed examination of magnetic susceptibility for samples from Runs 3 and 4 shows that subtle differences in the quality of physical properties can be related to the method of formation. For Run 4, where the reactor heating does not include a hold until 500 °C, a higher porosity and lower density of grains  determined from the curves at 20 K for the Run 3 sample is 79 mT while for the Run 4 sample Hc1 is 26 mT . Similar differentials in values for Hc1 occur between these samples for the temperature range 5 K to 30 K. These values suggest that grain connectivity for the sample from Run 3 is significantly better than for Run 4. Jc) determined by the well-known Bean model [Jc are comparable to other measures of MgB2 [2 samples produced by these methods is in preparation.Magnetic hysteresis curves (not shown) determined using Vibrating Sample Magnetometry (VSM) also reflect a difference in grain quality. For example, first critical values  and sodium borohydride powder  supplied by Sigma-Aldrich  are weighed, mixed and placed into a 50 mL Parr reactor within a controlled atmosphere glove box containing Argon (99.99%). Starting materials are pre-heated in an evacuated chamber adjacent to the glove box in order to reduce to the dehydrated state and then transferred to an inert atmosphere. Water and oxygen content in the glove box is normally less than 1 ppm. After weighing, the starting materials are manually mixed in an agate mortar until a consistent texture and greyish colour are achieved. Minor amounts of sample lost during this process are estimated at less than 1% of total volume.The Parr reactor is designed with an internal fixed head and cylinder of Inconel 601 steel with a graphite seal to accommodate a maximum pressure of 20 MPa and a maximum average temperature of 500 °C. Before each reaction, the cylinder is thoroughly cleaned with dilute acid, washed with deionised water and dried in the pre-heating chamber up to 120 °C. The lid is rinsed with water and dried with a hair dryer. In principle, this process removes all products from a prior run and minimises (but does not eliminate) traces of residual water.The reaction mixture is added to the Parr reactor, sealed tightly and removed from the glove box. The reaction chamber is heated according to a standard protocol via thermocouple controller. The change in pressure is monitored during the reaction using a dial pressure gauge and an Ashcroft transducer mounted atop the reaction chamber. The temperature sensor is centred within the reactor and both temperature and pressure are recorded every second. The temperature sensor is not embedded within the precursor materials which, at the start of synthesis, are located at the bottom of the chamber. The typical pressure-temperature profiles shown in via slow pressure equilibration using a gas release valve. Degradation of these samples occurs after periods of more than 24 h exposure in air as noted by Zhu et al. [On cooling the reaction chamber to room temperature, the reactor is opened in the argon-filled glove box u et al. . In gene2 powders are obtained through control of the reaction temperature and heating rate.A consistent heating rate of 10 °C/min is used in all reactions albeit at different temperatures, the heating rate is held constant for varying periods of time. In general, the reactor heating rate is held constant at: (i) ~50 °C; (ii) 250 °C; and (iii) target temperature  for variable periods of time. These specific heating rates and constant temperature periods are identified in 3.2.Reaction products are characterized by optical and electron-optical methods and X-ray diffraction. A Leica multi-focus, stereo optical microscope and a Zeiss Sigma variable pressure Field Emission SEM with Oxford Instruments silicon drift detector (SDD)  are used for microscopy observations and energy dispersive spectroscopy (EDS) elemental analysis. Samples are prepared for SEM/EDS by placing a thin layer of powder onto aluminium stubs with double sided carbon tape. In general, samples are not coated with a conductive coating to avoid analytical interference(s). Elemental analysis is carried out at an accelerating voltage of 20 kV at 8.5 mm working distance. Excessively charging samples are imaged at lower accelerating voltages of 5 kV or 10 kV. If charging is excessive when the sample is uncoated, some samples are re-examined after deposition of a gold coating.i.e., not crushed). Depending on the particle size of the reaction products, X-ray data are collected with a parallel beam configuration, rather than Bragg-Brentano geometry typically used for finely ground powders. Surface area determinations using the BET method and N2 gas are obtained with a Micromeritics Tristar II instrument . Magnetic susceptibility is measured on bulk powders using a Cryogenic Ltd Mini Cryogen-free System  with a 5 T magnet and integrated variable temperature insert which allows for temperature control at the sample between ~300 K and 1.5 K. Hysteresis is measured in the VSM mode at a frequency of 20.4 Hz using a time constant of 300 ms.X-ray diffraction data are collected via a PANalytical X’Pert MPD X-ray diffractometer  using Co Kα radiation. Data are collected for 4° < 2θ < 90° at a step size of 0.02° 2θ on samples as received (4.2 is achievable at moderate temperatures (~420 °C < T < ~500 °C) using pre-mixed starting materials of Mg powder and NaBH4 within an Inconel alloy pressure vessel. This approach differs from earlier in situ or autogenous reactions which use Mg and amorphous boron as starting materials reacting at T > 650 °C. The process described in this work requires the development of autogenous pressure within the reactor initially at relatively low temperature (50 °C < T < 100 °C) through the decomposition of NaBH4 and generation of H2 and/or B2H6. A hold at 50 °C for 20 min enhances gas generation while, with continued heating, a hold at 250 °C for more than 20 min significantly enhances physical properties of the MgB2 product once heated to >420 °C.Formation of MgB2 under these conditions are, as yet unclear, the overall reactions to produce MgB2 involve gas-solid and/or gas-liquid interaction based on known behaviour of related phases observed in reactions at lower temperatures and as minor phases in the final product. The formation of B2H6 and higher order analogues is a key step in these detailed reaction mechanisms. Data from this work suggest that higher order forms of B2H6 may also participate in higher temperature reactions (>250 °C) to form MgB2 and that other phases such as MgH2 are implicit intermediates.While the detailed mechanisms that allow formation of MgB2 forms as dense, euhedral interpenetrating forms with hexagonal habit ranging in size from 40 μm to 80 μm and a bulk Tc of 38.5 °C. This process to produce high quality, micron-sized MgB2 grains offers potential to explore growth conditions that generate high density wires, tapes or ribbons for evaluation of bulk conduction properties.The optimised product from this synthesis method forms at ~500 °C at an initial maximum autogenous pressure ~2.0 MPa after a five hour hold at 250 °C and subsequent heating at 500 °C for ten hours. This MgB


The first crystal structure determination of an organoplutonium(IV) complex revealed an asymmetric sandwich structure for 4 where one COT′′ ring is 1,3‐substituted while the other retains the original 1,4‐substitution pattern. The electronic structure of 4 has been elucidated by a computational study, revealing a probable cause for the unexpected silyl group migration.The blood‐red plutonocene complex Pu ( It was synthesized by Fischer et al. from PuCl3 and BeCp2 in a melt at 70 °C.3 was later improved,IV derivatives.The highly radioactive transuranium element plutonium (Pu) was first produced in 1940 by Glen Seaborg et al. by deuteron bombardment of 2 in 1968,2 , were reported in the early 1970s by Karraker and Streitwieser Jr. et al.2, was prepared by addition of solid [NEt4]2[PuCl6] to a tetrahydrofuran (THF) solution of K2COT and isolated as an air‐sensitive solid giving cherry‐red solutions in toluene.2]  were reported2, An(nBuCOT)2, and An(Me4COT)2 .Following the discovery of iconic uranocene U(COT)2) is still being investigated actively,2 cyclooctatetraenyl) was mentioned.4 with 2 equiv of K2(COT′′′)⋅2 THF, but its characterization was limited to elemental analysis and IR data.4), as well as a detailed structural and theoretical investigation of this new transuranium sandwich complex.Remarkably, while the chemistry of uranocene and thorocene (Th(COT)4 is outlined in Scheme 4 is built from the bottom up. The synthesis starts with 1,4‐bis(trimethylsilyl)cycloocta‐2,5,7‐triene (1), which is readily available according to Cloke et al., by treatment of cycloocta‐1,5‐diene with n‐butyllithium/N,N,N′,N′‐tetramethylethylenediamine followed by reaction of the intermediate Li2(COT′′) with chlorotrimethylsilane.1 with 2 equiv of n‐butyllithium in THF solution affords the intermediate 2, which was treated with 0.45 equiv of anhydrous plutonium(III) chloride, resulting in formation of the green anionic sandwich complex Li[Pu2] (3). The final step, that is, oxidation to the neutral PuIV sandwich, was inspired by our previously discovered method, which employed anhydrous CoCl2 as a mild oxidant to convert the anionic lanthanide(III) sandwich complexes [Ln(COT′′)2]− into the linear neutral triple‐decker sandwich species Ln2(COT′′)3.2 caused an immediate color change to red, indicative of the formation of PuIV species. To date, organometallic complexes of Pu have been reported in the literature for the oxidation states PuIII and PuIV. The organometallic PuIII complexes are generally described as green in color,IV complexes are normally red to brown.n‐pentane. Removal of the n‐pentane left a dark‐red oil which solidified after standing at room temperature for 2 d to give low‐melting, blood‐red crystals of 4 in approximately 85 % isolated yield.The method leading to compound IV sandwich complex 4 was characterized by NMR spectroscopy and single‐crystal X‐ray diffraction. The 1H NMR spectrum of 4 resembled that of the corresponding uranocene complex U2, apart from a very different chemical shift range.3 protons, indicating free rotation of the COT rings at room temperature and potentially rapid chemical exchange. Three multiplets (relative intensity 2 each) at 10.19, 10.08, and 9.48 ppm were assigned to the COT ring protons. In accordance with the X‐ray analysis, which shows that one of the COT rings is substituted at the 1,3‐positions, a very broad resonance observed at 9.78 ppm with a relative intensity is assigned to the proton in the 2‐position of this ring. Additional signals were not resolved. The low number of signals supports a potential chemical exchange. However, radiation safety issues arising from low‐ or high‐temperature NMR experiments could not be performed. Furthermore, the lifetime of the title complex is limited so that all NMR experiments had to be completed within 6 h.The Pu4 confirmed the presence of a neutral PuIV sandwich complex with the two COT′′ rings in a virtually coplanar arrangement 4, however, is the 1,3‐substitution pattern in one of the coordinated COT′′ ligands. All known preparations of f‐element COT′′ sandwich and half‐sandwich complexes start with the precursor 1,4‐bis(trimethylsilyl)cycloocta‐2,5,7‐triene (1). It is generally assumed that the 1,4‐substitution pattern is retained upon formation of various forms of Li2(COT′′), as exemplified by the structurally characterized derivatives Li42(THF)4 (2)2(DME)2.An X‐ray diffraction study of t Figure . The ang2, [Li(THF)4][Ln2], and [Li(DME)3][Ln2], as well as in the triple‐decker sandwich complexes Ln23 .BIG) was employed as a ligand in cerium sandwich complexes. Surprisingly, oxidation of the anionic cerium(III) sandwich [Li(DME)2][Ce2] with AgI resulted in formation of the neutral cerocene Ce2.2(COT′′)3 was found to be the isomers HoHo in which rearrangement of the central COT′′ ligand to the 1,5‐regioisomer had taken place.1,4‐Substitution was also found in all previously reported lanthanide half‐sandwich and sandwich complexes [Ln(μ‐Cl)(THF)]2 (4), with the idea of understanding the cause of the silyl migration. Computations of Pu‐containing molecules are still rather scarce in the literature, mainly because of the number of electrons contained in such systems and the treatment of relativistic/correlation effects. Previous studies4. On the other hand, it was recently demonstrated by the Maron group that DFT can properly account for the geometry, bonding, and reactivity of complexes of transuranic metals such as Pu4, the ground state was assumed to have the same multiplicity as nonsubstituted plutonocene with a ground state of 5Ag.In addition to the X‐ray diffraction analysis, we also carried out a computational study on PuStructural data for the Pu molecule optimized at the ROHF and the MP2 level can be found in Table S9 (Supporting Information) alongside the experimental data. The optimum structure from the MP2 geometry optimization is shown in Figure 3 groups is compensated by a moderate torsion of the rings out of the eclipsed position by about 2.5°. The geometry optimization was carried out using a method described by us in conjunction with other actinide complexes.2 listed in Table S8 (Supporting Information), with a Pu−COT′′ distance of 1.87 Å, C−C bond lengths in the COT′′ moiety of 1.41 Å, and C−H bond lengths of 1.09 Å. As DFT properly reproduces the geometry, silyl migration was considered to find possible explanations for this rather uncommon phenomenon, keeping in mind that this does not occur in the U analogue. Therefore, geometry optimization was carried out at the DFT level on the exclusively 1,4‐substituted plutonocene complex ; that is without the silyl migration on one COT ligand. Interestingly, this complex was also a minimum on the Potential Energy Surface (PES), but slightly less stable than the experimentally determined energy . Therefore, the migration is not due to greater steric effects on the Pu complex relative to those acting on the U complex. A possible explanation for silyl migration is put forward by the molecular orbital diagram of the two complexes. Indeed, as expected from other studies on organoactinide complexes, the highest occupied orbitals are two δ‐orbitals  that are 5f orbitals), which describe the interaction between Pu and the COT′′ ligand . This is a striking difference between U and Pu, which is explained by the molecular orbital diagrams of the two species . Indeed, because of the lower energy of the 5f atomic orbitals for Pu, the δ‐orbitals are higher in energy than the unpaired electrons, which is not the case for the U complex. Therefore, the highest energy of the δ‐orbitals in Pu allows contribution from the antibonding Si−C orbital of the silyl substituents (negative hyperconjugation). This stabilizing effect might be the cause for the silyl migration.Scrutinizing the molecular orbitals, it appears that there is a contribution from the silyl group to the δ‐bond, which is in line with a hyperconjugation effect Figure . Such a IV sandwich complex Pu (4). The synthetic protocol 2] (3) with anhydrous CoCl2) was inspired by our previously discovered method to convert the anionic lanthanide(III) sandwich complexes [Ln(COT′′)2]− into the linear neutral triple‐decker sandwich species Ln2(COT′′)3. Blood‐red crystals of 4 were obtained in 84 % yield after extraction with n‐pentane. Compound 4 is the first organometallic PuIV complex structurally characterized by X‐ray diffraction.4 revealed a sandwich structure with one 1,3‐substituted COT′′ ring while the other ring retains the original 1,4‐substitution pattern. The electronic structure of 4 has been elucidated by computational methods, revealing a plausible cause for this unexpected silyl group migration. These results may contribute to a more profound knowledge of the fascinating properties of transuranium organometallic compounds.In summary, we succeeded in the synthesis of the neutral PuThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


HRV) cause a wide spectrum of disease, ranging from a mild influenza‐like illness (ILI) to severe respiratory infection. Molecular epidemiological data are limited for HRV circulating in the Southern Hemisphere.Human rhinoviruses . Sequencing data of 5′UTR and VP4/VP2 coding regions on RT‐PCR‐positive specimens were analysed.Combined nose and throat swabs or nasopharyngeal aspirates collected from individuals with PCR in 20.9% (116/555) of samples tested. Phylogenetic analysis of 5′UTR and VP4/VP2 on HRV‐positive samples was concordant in the grouping of HRV A and B species but not HRV C species. Eighty per cent (16/20) of sequences that grouped as HRV C in the VP4/VP2 tree clustered as HRV A, alongside some previously described C strains as subspecies C/A. Discordant branching was seen within HRV A group: two sequences clustering as A in the VP4/VP2 tree branched within the C/A subspecies in the 5′UTR tree, and one sequence showed identity to different HRV A strains in the two genes. The prevalence of HRV C and C/A species was greater in paediatric compared to adult patients .Human rhinoviruses were detected by real‐time HRV C is present in the Southern Hemisphere. Sequencing of multiple HRV regions may be necessary to determine exact phylogenetic relationships.Human rhinoviruses are a common cause of respiratory infections, and  This study aimed to identify the species and genotypes of HRV from clinical samples collected in Sydney, Australia, over four consecutive years by analysing the nucleotide homology in the 5′UTR, VP4 and part of the VP2 capsid protein coding regions.22.1Combined nose and throat swabs (NTS) or nasopharyngeal aspirates (NPA) collected from individuals with an influenza‐like illness (ILI) were tested for HRV as described below. The specimens were from three separate sources over four consecutive years: (i) as part of a study assessing the efficacy of face masks in reducing household transmission of respiratory viruses from children to their parents from 2006 to 2008 (n = 268);2.2® in 2007, and sequences generated from the present study.Primers used to amplify all picornaviruses,2.3RNA was prepared directly from combined NTS or NPA using Roche High Pure RNA kits .2.4® 2.0 System  in a final volume of 20 μL with 0.5 μmol L−1 of each primer, 0.2 μmol L−1 of each probe and 4 μL of Roche FastStart hybridization Master Plus probes . The TaqMan real‐time PCR assay conditions consisted of denaturation (95°C for 10 minutes) and amplification .The cDNA was reverse‐transcribed from 10 μL of specimen RNA using 100 units of SuperScript III reverse transcriptase  and 4 μL used for real‐time PCR as described previously.2.5® DNA Polymerase  and its supplied buffer with 4 mmol L−1 of MgCl2, 0.2 mmol L−1 dNTPS, 0.2 μmol L−1 of the forward and reverse primers and 4 μL of cDNA in a volume of 50 μL. PCR cycling conditions with primers EV140 and EV170 had an initial 10‐minute denaturation at 95°C, 40 amplification cycles  and 5‐minute final extension at 72°C. The PCR conditions for primers RHI3A and EVP4R were similar, except that annealing was at 61°C and the cycle number was 50.Human rhinoviruses RT‐PCR‐positive samples were then tested using UTR primers EV140 and EV170 to amplify a 395‐bp product.2.6®, New Jersey, USA). >A homology search was carried out for the 93 sequences generated with NCBI BLAST®. Sequences of non‐ATCC prototype strains and the nearest fully characterized HRV types, but not partial coding sequences (cds), in GenBank® were included in the construction of the phylogenetic tree. Phylogenetic analysis was performed on the 5′UTR and VP4/VP2 regions using the Geneious 6.1.8 software . Maximum‐likelihood (ML) trees with 1000 bootstraps were constructed,PCR amplicons were purified using the QIA PCR purification kit  and sequenced with the respective primer in both directions in an ABI‐3100 Prism Genetic Analyzer using the BigDye Terminator version 3.1 sequencing kit  in 116/555 (20.9%) samples . Age distribution and gender were not recorded in all the subjects that provided specimens. There were 141 children and 125 adults (2 not known) in the study from 2006 to 2008.3.2One hundred and two samples had RNA available for sequencing; 94 samples were amplified using primers EV140 and EV170, and sequencing reactions were successful in 93. Sequencing PCR with primers RHI3A and EVP4R  was successful in 65/93 samples; 19 were negative and nine were insufficient for this PCR. Sixty‐five specimens had sequences of approximately 900 bp, covering part of the 5′UTR, entire VP4 and approximately 232 bp of the VP2 region.3.3®, nearly all the sequences showed >98% similarity to one or more HRV sequences with partial cds available in GenBank®. These were approximately 390–420 bp and did not cover the full sequence from 5′UTR to VP2 of our study samples, and were not included in the construction of the ML tree.Using BLASTAs the data size was large, it was not possible to include each one of the HRV prototype sequences in the tree construction. Figure ® accession numbers are shown in Tables S4 and S5, respectively (supplementary section).Table 3.4Figure Of the 28 samples with 5′UTR alone available, 17, 8 and 3 grouped as HRV A, B and C, respectively. Of the 17 HRV A samples, 12 HRV C samples were incorrectly grouped as HRV A. The 12 samples showed identity to previously reported C strains: CS07‐20 and CS07‐15 with C‐EU840952; MS06‐9 with C‐JF317013; CS07‐23 and CS07‐25 with C‐JQ994498; CS07‐17 with C‐EF‐582387; CS07‐19 with C‐EF077279; CS07‐11 with C‐GQ223227; MS07‐8, CS07‐21 and CS07‐22, respectively, with C17‐JN815244, C‐JF781505 and C‐GU219984 (not shown in figures). Sample CS09‐9 had no close identity.For the 65 samples with both 5′UTR and VP4/VP2 sequences, grouping was similar for most samples that segregated into HRV A and B species as seen in the VP4/VP2 gene, but this was not the case with the HRV C group. All clinical samples that clustered in group A maintained the closest identity to the same reference strains as seen in VP4/VP2, except sample CS09‐2.In comparing the phylogeny between VP4/VP2 and 5′UTR, the grouping of HRV C samples was different. Four of 20 samples in group C with VP4/VP2 analysis also clustered in group C in the 5′UTR along with reference strain C‐EU840728; 16 of the 20 HRV C samples grouped as HRV A, along with reference strains C‐X2‐EF077280, C‐EU840952, C‐DQ875932, C‐JQ994498, C‐EF077279, C‐EF582387 and C‐GQ223227. Four clades that grouped as C species in the VP4/VP2 analysis segregated into the A genogroup in the 5′UTR analysis  in Figs Two samples MS07‐18 (97% homology to A65‐JF781504) and MS08‐7 (closest homology to A71‐JX025555) grouped in a major branch with known strains of HRV Ca subspecies (78%–83% identity to C‐EF077280 and 74% to 1B). In VP4/VP2, the identity for MS07‐18 was 67.2% to EF077280, but maintained around 75% to HRV A viruses like 1B, 55 and 33 reference strains. A VP4/VP2 sequence was not available for MS08‐7.Another sample (CS09‐2) that was closest to A67 (not shown) in VP4/VP2 branched separately and clustered with A89 along with sample MS06‐2. CS09‐2 showed 77.7% relatedness to A89 in the VP4/VP2 region and greater than 95% in the UTR region.Samples with sequences that clustered with more than 99% identity  HRV A, 20 (30.8%) HRV C and one (1.5%) HRV B. Several clades were represented within HRV A and C , C‐EU840952 (five samples), C‐DQ875932 (two samples) and C‐JQ994498 (two samples) and four samples to the following reference strains not shown in the figure: C26‐JX193796, C‐JF317015 and C‐JF317015. Three samples  did not show close identity to any characterized HRV C strains in GenBank3.6The grouping of samples as C and CA in the 5′UTR/VP4/VP2 analysis was very similar to that seen in the 5′UTR analysis. The discordant branching pattern seen with samples MS06‐3 and HRV A12 (EF173415), MS07‐18 and MS08‐7 and CS09‐2 (indicated with ●) in the 5′UTR ML tree was not seen; it was in agreement with that seen in the VP4/VP2 ML tree.3.7In the mask study group where samples were collected from 2006 to 2008, sequences were available for 40 samples collected from 12 adults, 26 children aged 10 years or less and two of unknown age. Sequences of clinical samples collected in 2007 (CS07) were from patients presenting to ED and/or hospitalized. Sequences from clinical samples collected in 2009 (CS09) were from individuals not presenting to hospital but in the community suffering from symptoms of ILI. The distribution of the different HRV subtypes is shown in Table 4®. Retrospective analyses show that HRV C has been circulating for nearly 20 years and now considered a third genetically distinct HRV subtype.® sequences of known HRV A and B strains  to which the clinical samples showed greatest homology. The few field isolates compared were closely identified with the reference types as minor variants.Since 2007, new global HRV variants that are not closely related to any of the existing 101 HRV serotypes have been classified into a novel lineage, HRV C.In our study, the sequences grouped into three phylogenetically distinct species: A (52.6%), C/CA (37.6%) and B (9.6%). However, there was discordance between proposed phylogeny groups when sequences from the 5′UTR and VP4/VP2 coding regions were analysed. Sixteen of the 20 samples that clustered as HRV C in the VP4/VP2 ML tree segregated as A in the 5′UTR analysis, as did some of the early, well‐characterized HRV C strains from New York (EU840952 and DQ875932),There is no designated region within the HRV genome that is known to give consistent genotyping results for HRV strains. It is also difficult to compare the findings when different groups have used different genes. The 5′UTR, VP4/VP2 and VP1 coding regions have been previously used to characterize genotypes. Kiang et al.In this study, the 5′UTR primers and the primers chosen to amplify the entire VP4 and partial VP2 region produced amplicons with overlapping sequences that resulted in an approximately 900‐bp continuous sequence. We used a single set of primers for each of the two PCR assays but did not use cloning, which has been used in other studies.Real‐time diagnostic PCR primers target the 5′UTR, a region with high interstrain homology, because of the ease of using a single set of primers that detect the majority of HRV strains. However, HRV strains with low copy numbers may remain undetected. In this study, the sensitivity of the 5′UTR sequencing primers and the second set of primers for the amplification of the full length of the VP4 and part of VP2 in comparison with the real‐time assay was 92.1% and 69.56%, respectively.P = .032, Fisher's exact test). All clinical samples were collected from patients with respiratory illness who were sick enough to warrant testing or in many cases hospitalization, but our data are insufficient to attribute clinical severity to any of the HRV species.It is unclear whether the severity of infections caused by HRV C species is greater compared to HRV A and HRV B. The mask study had 2.5 times more children than adults who were positive for HRV, but the study selected children with a febrile respiratory illness, with well adult contacts at baseline; children who had specimens collected all presented to a paediatric ED. The majority of these HRV sequences were either C or the C/A variant (16/27). CS07 samples were from patients either presenting to ED or hospitalized and had an equal proportion of HRV C or C/A strains among adults and children. CS09 samples were from adults with an ILI who did not present to hospital during the A(H1N1)pdm09 influenza pandemic; HRV C/Ca infection was seen in only 13%. Overall, there was a greater representation of HRV C/Ca species in the paediatric compared to the adult group (47.9% vs 25.6%, It is possible that HRV C or the C/A variants may cause exacerbation of respiratory infections in infants that require presentation to ED compared to HRV A or B, and this may contribute to the higher percentage of HRV C or C/A infection in the paediatric population. One of the early reports of HRV C‐QPM variant severity was from Australian samples collected in 2003 from children with lower respiratory infection.In conclusion, the present study shows that sequencing of one region alone is insufficient for determining the lineage of the HRV variants. The presence of many diverse strains has become apparent, and it is likely that more will emerge. Genotypic assignment and identification of HRV types will facilitate monitoring of emerging novel variants, and investigations into type‐associated differences in disease epidemiology, transmission and outcomes.per se, but as part of a study assessing the use of face mask in controlling respiratory virus transmission in households following approval by the local institutional review board or for laboratory diagnosis of patients with an influenza‐like illness. The present study does not involve the reporting of patient data, and no patient intervention occurred with the obtained results.Samples used in this study were not collected for this study VMR participated in the study design, analysed the results and drafted the manuscript. FZ and LD performed nucleic acid testing. CRM was the chief investigator of the study on the use of face masks to reduce household transmission of respiratory viruses. JK had input in the preparation and editing of the manuscript. DED participated in the design of the study and was involved in manuscript editing. All authors have read and accepted the manuscript. Click here for additional data file.


II(tame)2]Cl2⋅MeOH ethane) as a function of temperature reveals a smooth crossover between a high temperature high‐spin octahedral d6 state and a low temperature low‐spin ground state without change of the symmetry of the crystal structure. The temperature at which the high and low spin states are present in equal proportions is T1/2=140 K. Single crystal, variable‐temperature optical spectroscopy of [FeII(tame)2]Cl2⋅MeOH is consistent with this change in electronic ground state. These experimental results confirm the spin activity predicted for [FeII(tame)2]2+ during its de novo artificial evolution design as a spin‐crossover complex , offering the first experimental validation of a functional transition‐metal complex predicted by such in silico molecular design methods. Additional quantum chemical calculations offer, together with the crystal structure analysis, insight into the role of spin‐passive structural components. A thermodynamic analysis based on an Ising‐like mean field model (Slichter–Drickammer approximation) provides estimates of the enthalpy, entropy and cooperativity of the crossover between the high and low spin states.Single crystal structural analysis of [Fe In contrast, even simple correlations based on geometry parameters may sometimes accurately predict the relative spin state stability of a spin‐active complex,II(tame)2]Cl2⋅MeOH [Scheme II(tame)2]2+ emerged as the most promising candidate predicted by the above‐mentioned de novo design study.Experimental validation will help determine to what extent molecular computational design including the local spin‐active complex alone may be useful in development of SCO materials. In this report we present the case of [FeH Scheme  . A selection of these are shown in Scheme II(tacn)2]2+ and [FeII(trans‐diammac)]2+ (Scheme There are relatively few ligand systems that have been shown to form hexaamine complexes with FeII(tame)2]2+ complex in a pure form. Both oxygen and water were problematic. If oxygen is present, immediate oxidation of the mixture to give an insoluble precipitate of ferric oxide results, probably coupled with ligand dehydrogenation. Secondly, unless anhydrous MeOH is employed, addition of the tame free base leads to formation of metal hydroxides. Thirdly, the desired product [FeII(tame)2]Cl2⋅MeOH must be crystallised by slow addition of chloride to a solution of ferrous triflate and tame free ligand followed by slow evaporation of the MeOH solution. If chloride is present in a stoichiometric amount during FeII/tame complexation then the reaction does not yield a crystalline product. For these reasons, high yields of pure compound were not pursued and the crystalline product was separated from other material manually under a microscope. In spite of its instability in solution and susceptibility to oxidation, in the solid state the crystals are robust (for more than a year) if protected from oxygen and free from solvent. Furthermore, crystals of [FeII(tame)2]Cl2⋅MeOH can be manipulated in air for periods of hours without any noticeable degradation.This presented a challenge in isolating the [FeII(tame)2]Cl2⋅MeOH was determined at a number of temperatures from 10 K to 333 K. In all cases a trigonal structure was defined (space group R3‾m), which is isomorphous with [NiII(tame)2]Cl2.3‾m (Dd3) symmetry. The methyl and quaternary C‐atoms lie on a threefold axis . An initial refinement found the methylene and amine groups of the ligand on a crystallographic mirror plane, as reported for the NiII analogue.2N‐CH2 bond length (1.433(5) Å) indicated that the N‐atom was actually displaced from the mirror plane and disordered either side as are the methylene protons. Indeed, DFT calculations (see Supporting Information) confirm that the [FeII(tame)2]2+ cation gains stability by twisting each tripodal “cap” about its C3 axis in either direction, thus removing the mirror plane symmetry elements. The disorder is fixed by symmetry and there seems to be no ordering process correlated with spin crossover at least at the level of the average structure. So the structure amounts to a disordered mixture of S6‐symmetric complex cations related by a C2 symmetry operation . The crystal structure is built from layers of cationic spin‐active Fe complexes and Cl− counter‐ions, the layers are separated by layers of disordered MeOH molecules; all the layers are orthogonal to the (threefold) c‐axis . This reflects an anisotropy of the packing forces with stronger Coulomb interactions within the anion–cation layers and weaker between the layers.Spin inversion (LS→HS) is associated with elongation of Fe−N bonds by ≈0.15 Å. This deformation mainly affects the c, increase in unit cell volume, and increase in the apparent Fe−N bond length are all correlated. The two extreme Fe−N bond lengths at 333 K (2.189(3) Å) and 10 K (2.035(3) Å) are consistent with high spinII structure. In the intermediate range (60–250 K) a sigmoidal variation in the apparent Fe−N bond length as a function of temperature is seen, which reflects a weighted average of the relative proportions of HS and LS FeII in the crystal (Supporting Information Eqn S2).The elongation of 3C axis is qualitatively consistent with that predicted by previous molecular‐level modeling of [FeII(tame)2]2+.II(tame)2]2+ cation with and without the chloride counter ions (see Supporting Information). The results suggest that the [FeII(tame)2]2+ complex engages in a network of NH⋅⋅⋅Cl interactions that are stronger in the more compact LS coordination. These interactions thus also limit the LS→HS transition‐induced elongation compared to that of FeIIN6 complexes of other ligands than tame.The LS→HS spin‐transition‐induced elongation along the c axis with the polarisation ∥ c (π spectrum) and ⊥ c (σ spectrum) and unpolarised propagating parallel to the c axis (α spectrum). The relationship σ=α≠π implies that the transitions are electric dipole allowed in this direction and consistent with threefold‐symmetry of the complex cation (see Supporting Information for more details).The polarised single crystal visible spectrum at 11 K is shown in Figure 1A1g→1T1g, 1T2g transitions (Oh term symbols) decrease with increasing temperature. Figure 1A1g→1T1g transition in π‐polarisation for several cooling and heating cycles.The temperature dependent behaviour of the spectra 2]Cl2⋅MeOH. Since no superstructure has been observed, the title compound may serve as a candidate for observation of short‐range correlations, associated diffuse scattering, and other non‐linear phenomena near T1/2. Importantly, the central cation [FeII(tame)2]2+ was automatically designed from scratch by assembling small and unconstrained structural fragments (in silico de novo molecular design) to give FeIIN6 complexes. These complexes were optimized  to express spin bistability.Single crystal structural analysis and variable‐temperature optical spectroscopy have confirmed the spin‐crossover properties of [FeSyntheses, instrumental and computation details are in the Supporting Information.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


A triple‐nuclear, same‐breath imaging examination of the lungs with 1H, 3He, and 129Xe can therefore provide exclusive functional information from the gas images. In addition, the 1H images provide complementary co‐registered structural information in the same physiological time frame. The goal of this study was to design an RF system for triple nuclear lung MRI at 1.5T, consisting of a dual‐tuned transceiver coil for 3He and 129Xe, RF switches and a nested 1H receiver array.The hyperpolarized gases 3He and 129Xe was designed and constructed to work in unison with a nested 1H receiver array.A dual‐tuned transmit‐receive dual‐Helmholtz RF coil for 1 maps and volumetric ventilation imaging using a three‐dimensional, balanced steady‐state free precession pulse sequence performed with both hyperpolarized 3He and 129Xe indicate good stand‐alone performance of the coil for the respective nucleus.Triple‐nuclear imaging  and apparent diffusion coefficient mapping of the human lungs was performed in the same breath‐hold using the integrated RF system. B3He and 129Xe) and a nested 1H array has been demonstrated with a custom RF system. Magn Reson Med 75:1841–1848, 2016. © © The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.Triple‐nuclear same‐breath lung imaging with a dual‐tuned coil ( In a recent study 1H lung SNR in same‐breath imaging can be improved with a nested 1H receive array, which is compatible with operation with either a 3He or a 129Xe T‐R coil.Therefore, same‐breath, multinuclear lung imaging with 3He and 129Xe at 1.5T. For 1H imaging, we incorporated the 1H array developed in our previous study designed to nest within either 3He or 129Xe T‐R RF coils 3He and 129Xe ventilation images and 1H anatomical images. With the same system ADC measurement of mixtures of 3He and 129Xe were performed in the same breath at a particular lung inflation state.The motivation of this study was the design and construction of an integrated radiofrequency (RF) coil and T‐R switching system for triple nuclear lung imaging in the same breath. To achieve this, we developed a new dual‐tuned flexible T‐R RF coil to operate in quadrature for both 3He‐129Xe) flexible quadrature T‐R coil was constructed in‐house. The conducting elements were made from self‐adhesive copper tape  of 66‐μm thickness and 6‐mm width, which was fixed on a substrate of 0.5‐mm‐thick polytetrafluoroethylene  as shown in Figure 1H frequency to enable 1H imaging with this coil in situ and the other trap to dual‐tune the coil to the 129Xe and 3He Larmor frequencies. The trap design was based on the formalism established in our earlier study for multituned resonators 3He (48.62 MHz) and 129Xe (17.65 MHz) at 1.5T. The 1H trap was tuned with a 47‐pF capacitor and a seven‐turn wire wound inductor with a diameter of 6 mm. The trap for dual‐tuning the coil was tuned with a 56‐pF capacitor and a nine‐turn wire wound inductor with a diameter of 6 mm. Wire wound inductors were constructed from 21 AWG insulated copper wire. RF measurements were performed with an Agilent 5061B Network Analyzer . For the RF measurement, the dual‐tuned coil was wrapped longitudinally around the thorax of the subject, as shown in Figure 1H chest receiver array from our earlier study 1H lung imaging.A dual‐tuned 3He‐29Xe flex3He and 129Xe was performed with approval from the National Research Ethics Committee. The imaging was performed on a healthy male volunteer . Lung MRI was performed on a GE whole body 1.5T Signa HDx system with 3He and 129Xe gas polarized with spin exchange optical pumping 3He and 129Xe gas was delivered in separate Tedlar bags and was mixed at the mouth piece at the time of inhalation, as illustrated in Figure 3He had polarization of 25% (≈100% of He is 3He). 129Xe had a polarization of 40%–50% (87% of Xe is 129Xe).All in vivo imaging with 3He‐129Xe) from the appropriate T‐R switch on the scanner to the dual‐tuned coil and to route the received RF signal (3He‐129Xe) from the dual‐tuned coil back to the appropriate T‐R switch on the scanner, a 2‐kW rated coaxial antenna RF switch  was used. The RF power required for the desired flip angle for the 3He and 129Xe sequences was calculated based on a standard calibration procedure, whereby the rate of depletion of polarization was calculated from the decay of signal resulting from a set of hard RF pulse‐acquires of equal amplitude. The period to prescribe calibration values on the spectrometer between the end of imaging a particular nucleus and initiation of the sequence for imaging the next nucleus was less than 4 s. The time required to operate the RF switch manually between acquisitions was 3 s.To route the transmit RF signal , high‐resolution, three‐dimensional (3D) imaging data sets were acquired with a 3D balanced steady state sequence Flip angle maps of the dual‐tuned coil at the 1H trap) generated three resonant modes at 17.65 MHz (129Xe Larmor frequency), 48.62 MHz (3He Larmor frequency), and 79.2 MHz. The isolation between the two ports of the Helmholtz was less than −15 dB. The quality (Q) factor of the dual‐tuned coil at the 129Xe Larmor frequency (17.65 MHz) was 61 in the unloaded condition and 17 in the loaded condition. The Q factor of the dual‐tuned coil at the 3He Larmor frequency (48.62 MHz) was 32 in the unloaded condition and 7 in the loaded condition. Thus, the ratio of Q factor unloaded to loaded condition was 3.5 at the 129Xe Larmor frequency (17.65 MHz) and 4.5 at the 3He Larmor frequency (48.62 MHz). Under the loaded condition, the dual‐tuned coil was matched to less than −20 dB at both ports at both the 129Xe (17.65 MHz) and 3He (48.62 MHz) Larmor frequencies, as shown in Figure 1H array was less than −15 dB, as shown in Figure 3He and Figure 129Xe. The standard deviation of the flip angle map was calculated to be 0.7° (mean = 8°) for 3He and 0.3° (mean = 9°) for 129Xe.The two traps on the coil at 47.81 MHz and 63.86 MHz  images are shown in Figure 3He and 129Xe are shown in Figure 4a and Figure 4b, respectively.Volumetric ventilation images from the 3D balanced steady‐state free precession sequence for the coil in operation as a stand‐alone transceiver for 1 field homogeneity of the dual Helmholtz design is inherently inferior to that of a birdcage design 1 transmit homogeneity  is comparable to 3He and 129Xe lung imaging.The construction of the flexible dual‐tuned coil is in the form of a bib, which enables a close fit to the subject's thorax irrespective of body type. The design was optimized to the typical subject size mentioned earlier. As the shape/form deviates from the optimal design with other body types, the distributed inductance and T‐R efficiency of the dual‐tuned coil changes accordingly. The B3He‐1H lung imaging 1H). Both the dual‐tuned RF coil and the nested 1H array from the earlier study Because the RF switches are currently manually operated, and the spectrometer has an inherent delay time for precalibration for each nucleus, the method is not currently compatible with repetition time resonant frequency interleaved imaging, as demonstrated in our earlier study with same‐breath 3He is 0.88 cm2 · s−12 · s−1 for 3He in the trachea (slightly lower than 3He free diffusion). The free diffusion of 129Xe is 0.14 cm2 · s−12 · s−1 for 129Xe in the trachea. The higher ADC value for 129Xe in the trachea, we presume is due to its mixture with the highly diffusive 3He . These findings are currently being investigated in future work studying the physiology of gas mixing in the lung with the two gases.The free diffusion (in air) of 1H body T‐R coil, a 3He birdcage T‐R coil, and a nested 129Xe T‐R vest coil 3He‐129Xe coil minimizes the number of individually tuned coils, and the nested 1H array 1H SNR by closer proximity to the lung. Applications of this triple nuclear RF system for lung MRI are manifold and allow the different physical and physiological properties of the two gases to be explored in the same time course with added provision of high‐quality and coregistered 1H structural images.In contrast to our previous triple nuclear same‐breath lung imaging experiments demonstrated at 3T on a Philips system using the 1H with hyperpolarized gases 3He and 129Xe at 1.5T using a custom integrated RF system. This system incorporates a new design of dual‐tuned RF coil for 3He and 129Xe and RF switches, together with a nested receiver array for 1H imaging. With this system, we have demonstrated high‐quality, same‐breath 1H with 3He and 129Xe ventilation imaging and the capability for ADC mapping of 3He and 129Xe in the same lung‐inflation state. In addition, the image quality on all three nuclei is comparable with those acquired with separate RF coils for the given nucleus.In conclusion, we have demonstrated a system for triple nuclear same‐breath lung imaging of