[Allogeneic CAR-T for treatment of relapsed and/or refractory multiple myeloma: four cases report and literatures review].

Objective: To investigate the safety and efficacy of allogeneic CAR-T cells in the treatment of relapsed/refractory multiple myeloma (RRMM) . Methods: CAR-T cells were prepared from peripheral blood lymphocytes of HLA mismatch healthy donors. Median age was 55 (48-60) . Allogeneic cells were derived from 3 HLA haploidentical donors and 1 HLA completely mismatch unrelated donor. Four patients with RRMM were conditioned with FC regimen followed by CAR-T cell transfusion. They were infused into CART-19 (1×10(7)/kg on day 0) and (4.0-6.8) ×10(7)/kg CART-BCMA cells as split-dose infusions (40% on day 1 and 60% on day 2) . The adverse reactions and clinical efficacy were observed during follow-up after infusion, and the amplification and duration of CAR-T cells in vivo were monitored by PCR technique. Results: CAR-T cells were successfully infused in 3 of the 4 RRMM patients according to the study plan, and the infusion in one patient was delayed by 1 day due to high fever and elevated creatinine levels on day 3. The side effects included hematological and non-hematological toxicity, grade 3 hematological toxicity in 2 patients, grade 3 CRS in 1 one, grade 1 CRES in 1 one, prolonged APTT in 3 ones, tumor lysis syndrome in 1 one, mixed chimerism detected STR and clinical GVHD manifestation in 1 one. According to the efficacy criterias of IMWG, 2 patients acquired PR, 1 MR, and 1 SD respectively. Progression-free survival was 4 (3-5) weeks and overall survival was 63 (3-81) weeks. CAR T cells were amplified 2.2 (2-14) times in the patients with a median survival time of 10 (8-36) days. Conclusions: Small sample studies suggested that GVHD may be present in the treatment of RRMM with allogeneic CAR-T cells. There were early clinical transient events after transfusion. Low amplification and short duration of CAR-T cells in vivo may be the main factors affecting the efficacy.

Crystal structure of R-phycoerythrin from Polysiphonia urceolata at 2.8 A resolution.

The structure of R-phycoerythrin (R-PE) from Polysiphonia urceolata was determined at 2.8 A resolution. The crystals belong to space group R3 with unit cell dimensions of a = b = 189.8 A, c = 60.1 A. The subunit composition of R-PE is (alpha 2 beta 2)3 gamma. The three-dimensional structure of R-PE was solved by the multiple isomorphous replacement method. After several cycles of model building and refinement, the crystallographic R-factor of the final model is 18.0% with data from 10.0 to 2.8 A resolution. The four phycoerythrobilin chromophores alpha 84, alpha 140a, beta 84 and beta 155 in an (alpha beta) unit are each covalently bound to a cysteine residue through ring A. The phycourobilin chromophore is bound to cysteine beta 50 by ring A and bound to cysteine beta 61 by ring D. The ring A and ring D of phycourobilin deviate from the conjugate plane formed by ring B and ring C and the four rings form a boat-shaped structure. R-PE contains a 34 kDa gamma subunit that is assumed to lie in the central channel of the molecular disc (alpha 2 beta 2)3. The energy transfer and relationship between cysteine residues and chromophores are discussed.

A proposed interaction model of the insulin molecule with its receptor.

Based on the extensive structural comparisons among the determined structures of the different species and crystal forms of insulin and its derivatives in our laboratory, it was suggested that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle of this amphipathis surface, there was a hydrophobic surface with an area of about 150 A2, while the polar and charged groups distributing around the hydrophobic surface constructed a hydrophilic zone. The hydrophobic surface was usually covered by the extended B-chain C-terminal peptides with great mobility. The angle between the proposed binding interaction surface and the surface of dimerization was about 20 degrees. The results from studies on structures of A1-(L-Trp) insulin and A1-(D-Trp) insulin confirmed the interaction mechanism model we proposed.

Direct-method-aided phasing of MIR diffraction data from proteins.

Direct methods have successfully been used to break the phase ambiguity intrinsic in the single isomorphous replacement (SIR) data of proteins. Based on this, the procedure 'direct-method-aided MIR phasing' (DMIR) has been proposed and applied to the four-derivative multiple isomorphous replacement (MIR) data of a known protein containing 682 amino acid residuals in the asymmetric unit. The data set consists of 14,500 unique reflections at 3 A resolution with F(obs.) greater than 2sigma. Test calculation showed that the phases from conventional MIR phasing could be significantly improved by direct methods leading to obvious improvement in the quality of the resultant Fourier maps.

The effect of surface roughness and contaminant on the dynamic friction of porcelain tile.

Surface roughness affects friction, but it is not clear what surface roughness characteristics are better correlated with friction. The average of the maximum height above the mean line in each cut-off length (Rpm) and the arithmetical average of surface slope (deltaa) had the highest correlation with dynamic friction coefficient in a previous study. The previous study was expanded to two different footwear materials and four different contaminants on a porcelain tile in the current investigation. The results showed that dynamic friction decreased as the interface speed and glycerol content in the contaminant were increased due to the hydrodynamic lubrication effect. Deltaa had the highest correlation with friction for most of the test conditions with neolite. For Four S rubber, friction coefficient appeared to have the highest correlation with the parameters related to the surface void volume at 30% glycerol content, related to the surface slope at 70 and 85% glycerol contents, and related to the peak to valley distance at 99% glycerol content. A good indicator of surface slip resistance probably should consist of the surface parameters representing the surface slope, the surface void volume and the surface peak-to-valley distance with the coefficients determined by the system parameters.

The slip resistance of common footwear materials measured with two slipmeters.

The slip resistance of 16 commonly used footwear materials was measured with the Brungraber Mark II and the English XL on 3 floor surfaces under surface conditions of dry, wet, oily and oily wet. Three samples were used for each material combination and surface condition. The results of a one way ANOVA analysis indicated that the differences among different samples were statistically significant for a large number of material combinations and surface conditions. The results indicated that the ranking of materials based on their slip resistance values depends highly on the slipmeters, floor surfaces and surface conditions. For contaminated surfaces including wet, oily and oily wet surfaces, the slip resistance obtained with the English XL was usually higher than that measured with the Brungraber Mark II. The correlation coefficients between the slip resistance obtained with these two slipmeters calculated for different surface conditions indicated a strong correlation with statistical significance.

Studies on the crystal structure of [L-Met]B0 bovine insulin at 3.0A resolution.

Based on the crystal symmetry of [L-Met]B0 bovine insulin (LMBBI) and the fundamental theory of the molecular packing method, the scheme for the determination of the position and orientation of the molecules by only using one-dimensional rotation and one-dimensional translation was chosen to be used, and therefore the calculation of the rotation function of the molecular replacement method and the refinement of the rotational and translational parameters by using the R-factor search method were simplified greatly. After the preliminary refinement by using the macromolecular rigid body refinement technique, the molecular model was further refined and adjusted by using the energy-minimizing stereochemical-restrained least squares refinement technique assisted by the manual revision on the difference Fourier maps. The L-Met residues on the N-terminus of the B-chain appeared clearly on the final electron density map.

Studies on long-acting insulin: crystal structure of Arg-B31 human insulin at 2.0A resolution.

The crystal structure of Arg-B31 human insulin (ABHI), a long-acting insulin derivative, has been determined at 2.0 A resolution by using X-ray diffraction analysis. The final crystallographic R factor of the structure model after the refinement is 0.189 with the bond length r. m. s deviation of 0.018 A. The refined structure of ABHI showed that the conformation of B-chain C-terminal residues was more stable than that in the native molecule. A striking structural feature of ABHI was an additional ion pair formed between Arg-B31 of molecule I and Glu-B21 of molecule II in a dimer, and three ionic bonds between the neighbouring molecules thereby appeared on the surface of ABHI hexamer. These secondary bonds generated by the insertion of the residue Arg-B31 should make the rate of dissociation of ABHI hexamer slow down when it was injected into the body and the property of protraction should be produced by a 'depot effect'. This ought to be the main structure basis of the prolonged action of ABHI. The results observed here demonstrate that the main idea we used in search for long-acting insulin is reasonable and correct which goes like this: making some additional non-covalent bonds between insulin monomers so as to slow down the dissociation of insulin oligomers and gain the protraction from a 'depot effect', which may be used as a principle in the further research. It also shows an impressive example that the experimental result reported here is in agreement with the theoretical prediction before the structural determination.

The possible mechanism of binding interaction of insulin molecule with its receptor.

Detailed structural comparisons and investigation of DPI, 2 Zn insulin and some other derivatives of insulin were performed by the least-squares superimposition technique and the graphics technique. It is pointed out in this paper that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle, there is a hydrophobic surface with an area of about 150 A2 consisting of many hydrophobic residues; while the polar or charged groups distributing around the hydrophobic surface construct a hydrophilic zone. The hydrophobic surface is usually covered by the extended B-chain C-terminal peptides with great mobility and protected from the solvent molecules. The angle between the amphipathic surface and the surface of dimerization is about 20 degrees. The results from the detailed structural comparison between Al-(L-Trp) insulin and Al-(D-Trp) insulin have provided a very good explanation to their great difference in biological activity, and confirmed our proposed binding interaction model of the insulin molecule with its receptor as well.

The characteristics and motion model of insulin monomer.

The extensive conformational comparisons among the determined structures of the different species and crystal forms of insulin and the varied insulin derivatives were performed by using the least-squares superimposition technique and the graphics technique. The results of the investigation showed that the structure of molecule I in 2Zn insulin was closer to that of the natural monomer; the conformational difference between two molecules of a dimer came out during dimerization and it was further improved and stabilized during the hexamerization and packing of hexamers in crystal; through the hinge peptides, such as A10, B4, B8, B24, B20 and B23, there was a flexible relative motion among the structural segments in the insulin molecule, and the residues at the B-chain C-terminal might have a shift of more than 10A; the mobility for each residue side-chain was very different due to the different surroundings.

Crystal structure determination of desheptapeptide (B24--B30)-insulin.

Desheptapeptide (B24--B30)-insulin (DHPI), an essentially inactive insulin analog, is crystallized in space group P212121 with two molecules in an asymmetric unit. The orientations of the molecules in the crystal cell have been determined by using Patterson search method at 6 A resolution and the positions of the molecules are deduced from translation function calculation and R search at 3 A resolution. After using the rigid body refinement (CORELS) further to refine the orientational and positional parameters as well as the initial energy restrained refinement (EREF) for the model, the crystallographic R value is reduced to 0.384 at 3 A resolution. The initial Fourier map shows that the B-chain N-terminal (B1-B8) and C-terminal (B20-B22) segments, compared with the native 2 zinc insulin, exhibit drastic conformational changes, but the three helices of B- and A-chains and their relative arrangement are essentially kept in DHPI.

The microbial production of amylase inhibitor and its application. I. Isolation and cultivation of Streptomyces nigrifaciens NTU-3314.

In the course of screening amylase inhibitor producing, microorganisms, a strain identified as Streptomyces nigrifaciens NTU-3314 was found to have the highest inhibitor-producing ability among the other isolated strains. This strain was aerobically cultured at 30 degrees C in a 5l jar fermentor with a working volume of 2l. The optimum cultural medium consisted of defatted soybean flake 3.0%, potato starch 4.0%, casein 0.6%, sucrose 0.6%, serine 0.02% and NaCl 0.8% (pH 7.0). With an aeration rate of 1.5 vvm, an agitation speed of 300 rpm and an inoculum of 15% seed (previously grown in seed medium 3), the highest amount of inhibitor was obtained after 24 hours of cultivation. The amylase inhibitor produced had inhibitory effects on both alpha-amylase and glucoamylase, but not on beta-amylase, alpha-glucosidase, beta-glucosidase or dextranase. It was quite stable in 0.1M phosphate buffer (pH 7.0) and nearly 100% of its activity was retained even after boiling at 100 degrees C for 20 min.

Structure of monomeric porcine DesB1-B2 despentapeptide (B26-B30) insulin at 1.65 A resolution.

Insulin has a concentration of 10(-8)-10(-11) M in the blood which ensures that it circulates and exerts its physiological functions in vivo as a monomer. The crystal structure of monomeric porcine desB1-B2 despentapeptide (B26-B30) insulin (DesB1-2 DPI) with M(r) = 4934 Da has been determined at 1.65 A resolution using the molecular replacement method. A structural comparison between DesB1-2 DPI and 2Zn insulin reveals that the conformation of DesB1-2 DPI is more similar to molecule I than molecule II of 2Zn insulin. The remarkable conformational difference between B25-Phe in DesB1-2 DPI and B25-Phe in despentapeptide (B26-B30) insulin (DPI) indicates that the residue B25-Phe possesses great flexibility and mobility.

The measurement of slipperiness--an international scientific symposium.

Occupational slips, trips, and falls (STF) present a tremendous burden on the working people of the world. The precise contribution of slipping to this burden is not completely understood and significant questions exist regarding the definition and measurement of slipperiness. In an attempt to advance slipperiness measurement, a workshop symposium of tribologists, biomechanists, clinicians, engineers, epidemiologists and related scientists was held in order to summarize the state of the science in slipperiness measurement. Organized into issue-focused working groups, participants collaborated on manuscripts addressing conceptual and definitional issues, the contribution of slipperiness to STF injury, and biomechanical, human-centred, and tribological approaches to slipperiness measurement. The conference design, contributions of working groups and outcomes are summarized.

The role of surface roughness in the measurement of slipperiness.

Surface roughness has been shown to have substantial effects on the slip resistance between shoe heels and floor surfaces under various types of walking environments. This paper summarizes comprehensive views of the current understanding on the roles of surface roughness on the shoe and floor surfaces in the measurement of slipperiness and discusses promising directions for future research. Various techniques and instruments for surface roughness measurements and related roughness parameters are reviewed in depth. It is suggested that a stylus-type profilometer and a laser scanning confocal microscope are the preferred instruments for surface roughness measurements in the field and laboratory, respectively. The need for developing enhanced methods for reliably characterizing the slip resistance properties is highlighted. This could be based on the principal understanding of the nature of shoe and floor interface and surface analysis techniques for characterizing both surfaces of shoe and floor. Therefore, surface roughness on both shoe and floor surfaces should be measured and combined to arrive at the final assessment of slipperiness. While controversies around the friction measurement for slipperiness assessment still remain, surface roughness measurement may provide an objective alternative to overcoming the limitations of friction measurements.

Crystal structure of R-phycocyanin and possible energy transfer pathways in the phycobilisome.

The crystal structure of R-phycocyanin from Polysiphonia urceolata (R-PC-PU) at 2.4 A is reported. The R-PC-PU crystal belongs to space group P4(3)2(1)2 with cell parameters a = 135.1 A, c = 210.0 A, and alpha = beta = gamma = 90 degrees. The structure was determined by molecular replacement. The crystallographic R-factor of the refined model is 0.189 (R(free) = 0.239). Comparison of the microenvironment of chromophore beta 155 in R-PC-PU and in C-PC from Fremyolla diphosiphon (C-PC-FD) reveals that their spectral differences may be caused by their different alpha 28 residues. In the R-PC-PU crystal structure, two (alpha beta)(3) trimers assemble face to face to form a hexamer, and two such hexamers assemble in two novel side-to-side arrangements. Possible models for the energy transfer from phycoerythrin to phycocyanin and from phycocyanin to allophycocyanin are proposed based on several phycobiliprotein crystal structures.

Purification and crystal growth of F1-ATPase from pig heart mitochondria.

A method has been evolved toward the aim of getting suitable crystals for high resolution of structural analysis of F1-ATPase by X-ray crystallography. The different conditions for crystal growth of ATPase that were isolated and purified by different methods from pig heart mitochondrial ATP synthase had been compared and screened. A simple method for purification of F1-ATPase was adopted. The F1-ATPase is released with chloroform from submitochondrial particles. Then it was treated with fractional precipitation of (NH4)2SO4 and finally was further purified by employing the sephadex G 200 column. The crystals of F1-ATPase were usually obtained after a few months. They appeared to have uniform morphology of tetrahedron. They diffracted to a resolution of 7A. The diffraction data were collected on the XRD-100 Siemens Area Detector. According to a total of 240 frames, the cell parameters obtained are a = b = 147 A, c = 208 A, alpha = beta = gamma = 90 alpha, the probable space group is P4 or its antipode. The reproducibility of this method for crystallization of F1-ATPase is good.

Purification, crystallization and preliminary crystallographic studies on the N-terminal fragment of human protein disulfide isomerase.

A fragment of human protein disulfide isomerase composed of the thioredoxin-like a and b domains (ab) has been expressed in Escherichia coli as a fusion protein with glutathione-S-transferase and purified after thrombin cleavage. Two forms of ab crystal were obtained with polyethylene glycol as precipitant and different additives at pH 7.5. The space group of form I is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions a = 81.5, c = 259.7 A. The space group of form II is P4(1)22 or P4(3)22, with unit-cell dimensions a = 82.7, c = 86.5 A.