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We present a tutorial to carry out umbrella-sampling free-energy simulations with a combined quantum mechanical and molecular mechanical (QM/MM) potential, which may also be used in a computational or biophysical chemistry curriculum for first-year graduate and undergraduate students. In this article, we choose the Type II S N 2 Menshutkin reaction between ammonia and chloromethane to construct the potential of mean force (PMF) for the reaction in aqueous solution. In this exercise, we wish to accomplish three tasks: (1) an understanding of the concept of PMF and the umbrella-sampling free-energy simulation method, (2) the use of a combined QM/MM potential in molecular dynamics simulation of chemical reactions, and (3) an understanding of solvent effects and intermolecular interactions on chemical reactions through comparison with gas-phase results. Analysis of the simulation results allows students to appreciate the difference between the transition state along a PMF from statistical simulations versus the optimized saddle point in the gas phase, the shift of transition state with respect to solvation, and the significance of electronic polarization. Through this tutorial, students will gain the necessary skills to carry out free energy simulations of chemical reactions in solution and in enzymes both in a classroom and in the laboratory.

Objective: To explore the clinical significance of a series of cytokines and peripheral blood immunocyte subsets before and after immunosuppressive therapy in patients with immune thrombocytopenia (ITP)., Methods: The percentages of immunocyte subsets in the peripheral blood of 20 patients with ITP and 20 healthy controls were detected by flow cytometry, including CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+), CD19(+). ELISA was applied to detect the level of serum TNFα, IL-2, IL-6, IL-4, IL-10, IL-11, IL-17, IL-27, transforming growth factor β (TGFβ), thrombopoietin (TPO) of 20 patients with ITP and 20 healthy controls., Results: The percentage of CD3(+) T lymphocyte, CD4(+) T lymphocyte and the ratio of CD4(+)/CD8(+) T lymphocyte in patients with ITP were lower than those in healthy controls[(62.66 ± 6.58)% vs (69.93 ± 4.81)%, (29.46 ± 5.02)% vs (39.08 ± 3.50)%, 0.97 ± 0.35 vs 1.56 ± 0.26, all P<0.05]. After immunosuppressive therapy, the percentage of CD3(+) T lymphocyte, CD4(+) T lymphocyte and the ratio of CD4(+)/CD8(+) T lymphocyte [(71.49 ± 5.16)%, (39.25 ± 3.21)% and 1.56 ± 0.28] recovered to the same levels in healthy controls. The percentage of CD8(+) T lymphocyte and CD19(+) B lymphocyte in patients with ITP were higher than those in the healthy controls [(30.28 ± 4.63)% vs (25.90 ± 3.06)%, (18.92 ± 4.27)% vs (13.17 ± 3.64)%, all P<0.05]. After treatment of immunosuppressive therapy, the percentage of CD8(+) T lymphocyte and CD19(+) B lymphocyte [(25.16 ± 3.45)% and (11.98 ± 3.68)%] recovered to the similar levels in healthy controls. The serum levels of IL-4, IL-6, IL-11, IL-17 and TPO in patients with ITP were significantly higher than those in healthy controls. While TGFβ level was significantly decreased. There was no significant difference of IL-27 between ITP patients and healthy controls. After the treatment of immunosuppressive therapy, IL-4, IL-6, IL-11, IL-17, TPO and TGFβ were down-regulated while IL-27 was up-regulated. There was no significant difference of IFNγ, TNFα, IL-2 and IL-10 among ITP patients before or after immunosuppressive therapy and healthy controls., Conclusions: The present study suggests that the aberrant immunocyte subsets and cytokines are involved in the pathogenesis of ITP. Hyper-function of Th2 and Th17, dysfunction of Treg cells, up-regulation of IL-27, IL-11, TPO and other factors may contribute to the pathogenesis of ITP.

Objective: To evaluate the semen quality of the donors., Methods: The semen parameters of the primary samples of 512 donors were examined following the World Health Organization (WHO) guide and computer-aided sperm analysis (CASA, product by Harmilton Thorne) system., Results: Of the 246 (246/512, 48%) donors with potential fertility, only 146 (146/512, 28.5%) came up to the semen standard set by the Chinese Ministry of Health, while 266 (266/512, 52%) were below the WHO reference values of semen parameters., Conclusion: Repeat semen analyses may increase the success rate in screening semen donors. The semen quality of the donors suggests that it is necessary to pay more attention to male reproductive health.

Objective: To observe sperm motion parameters in pre-freeze and post-thaw semen samples using computer-assisted sperm analysis (CASA) system., Methods: Semen analyses of 238 samples before freezing and after thawing were separately performed by Hamilton-Thorne Sperm Analyzer., Results: Sperm motility in post-thaw samples was significantly decreased. There was significant correlation and difference between pre-freeze and post-thaw samples in sperm motion parameters, including average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), straightness (STR) and linearity (LIN), except heat cross frequency (BCF). The percentage of sperm movement velocity parameters (VAP, VSL and VCL) and moving pattern parameters (ALH) significantly decreased, while that of LIN and STR significantly increased in post-thaw samples., Conclusion: CASA system is of clinically applied value and is a useful tool for evaluating sperm motion parameters in pre-freeze and post-thaw semen samples.