Predictive Ppk calculations for biologics and vaccines using a Bayesian approach - a tutorial.

In pharmaceutical manufacturing, especially biologics and vaccines manufacturing, emphasis on speedy process development can lead to inadequate process development, which often results in less robust commercial manufacturing process after launch. Process performance index (Ppk) is a statistical measurement of the ability of a process to produce output within specification limits over a period of time. In biopharmaceutical manufacturing, progression in process development is based on Critical Quality Attributes meeting their specification limits, lacking insight into the process robustness. Ppk is typically estimated after 15-30 commercial batches at which point it may be too late/too complex to make process adjustments to enhance robustness. The use of Bayesian statistics, prior knowledge, and input from Subject matter experts (SMEs) offers an opportunity to make predictions on process capability during the development cycle. Developing a standard methodology to assess long term process capability at various stages of development provides several benefits: provides opportunity for early insight into process vulnerabilities thereby enabling resolution pre-licensure; identifies area of the process to prioritize and focus on during process development/process characterization (PC) using a data-driven approach; and ultimately results in higher process robustness/process knowledge at launch. We propose a Bayesian-based method to predict the performance of a manufacturing process at full manufacturing scale during the development and commercialization phase, before commercial data exists. Under Bayesian framework, limited development data for the process of interest at hand, data from similar products, general SME knowledge, and literature can be carefully formulated into informative priors. The implementation of the proposed approach is presented through two examples. To allow for continuous improvement during process development, we recommend to embed this approach of using predictive Ppk at pre-defined commercialization stage-gates, for example, at completion of process development, prior to and completion of PC, prior to technology transfer runs (Engineering/Process Performance Qualification, PPQ), and prior to commercial specification setting.

Interaction of VvDELLA2 and VvCEB1 Mediates Expression of Expansion-Related Gene during GA-Induced Enlargement of Grape Fruit.

Exogenous gibberellin treatment can promote early growth of grape fruit, but the underlying regulatory mechanisms are not well understood. Here, we show that VvDELLA2 directly regulates the activity of the VvCEB1 transcription factor, a key regulator in the control of cell expansion in grape fruit. Our results show that VvCEB1 binds directly to the promoters of cell expansion-related genes in grape fruit and acts as a transcriptional activator, while VvDELLA2 blocks VvCEB1 function by binding to its activating structural domain. The exogenous gibberellin treatment relieved this inhibition by promoting the degradation of VvDELLA2 protein, thus, allowing VvCEB1 to transcriptionally activate the expression of cell expansion-related genes. In conclusion, we conclude that exogenous GA3 treatment regulates early fruit expansion by affecting the VvDELLA-VvCEB1 interaction in grape fruit development.

PCDD/Fs and PBDD/Fs in sediments from the river encompassing Guiyu, a typical e-waste recycling zone of China.

Severe pollution of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and their brominated analogues (PBDD/Fs) was frequently reported for the waters located near unregulated e-waste recycling areas. However, the migrations of these high-level dioxins via waterways and their potential threats to the lower reaches were seldom investigated. In this study, we analyzed PCDD/Fs and PBDD/Fs in 27 surficial sediments collected from the Lian River encompassing the Guiyu, China e-waste recycling zone, and investigated their distributions, sources, migration behaviors and risks. Both PCDD/Fs and PBDD/Fs in these sediments exhibited a spatial trend of Guiyu > Guiyu downriver > Guiyu upriver, illustrating that the Guiyu e-waste recycling activities were the uppermost dioxin contributors in this watershed. Sediments from different Guiyu villages demonstrated big gaps in PCDD/F concentrations and congener compositions, and the reason was attributed to the diverse e-waste recycling activities practiced in these villages. Sediments near the e-waste open-burning areas demonstrated extremely high PCDD/F concentrations and unique PCDD/F profiles featured by low-chlorinated PCDFs (tetra- to hexa-), which is quite different from the OCDD-dominant PCDD/F profile found in most of the Lian River sediments. The geographical distributions of PCDD/F concentrations and profiles illustrate that the substantial amount of PCDD/Fs in Guiyu sediments were mainly retained in local and vicinal water bodies. The principal component analysis (PCA) results further confirm that the high-level PCDD/Fs in Guiyu sediments exhibited quite limited translocations downstream and therefore exerted little influences on the lower reaches. Pentachlorophenol use in history, ceramic industry and vehicle exhaust were diagnosed as the major PCDD/F sources for most sediments of the Lian River. Total toxicity equivalent quantities (TEQs) of 70% of the Lian River sediments surpassed the high-risk limit specified for mammalian life by the U.S.EPA (25 pg TEQ g-1), and most of these sediments were from Guiyu and its near downstream, which merit continuous attention and necessary remediation measures.

Characterization of Five Meloidogyne incognita Effectors Associated with PsoRPM3.

Root-knot nematodes (RKNs) are devastating parasites that invade thousands of plants. In this study, five RKN effectors, which might interact with Prunussogdiana resistance protein PsoRPM3, were screened and identified. In situ hybridisation results showed that MiCal, MiGST_N_4, MiEFh and MiACPS are expressed in the subventral oesophageal glands (SvG), and MiTSPc hybridization signals are found in the dorsal esophageal gland (DG) of Meloidogyne incognita in the pre-J2. RT-qPCR data indicated that the expression of MiCal, MiGST_N_4, MiEFh, and MiACPS genes are highly expressed in M. incognita of pra-J2 and J3/J4 stages. The expression of MiTSPc increased significantly in the female stage of M. incognita. Moreover, all effectors found in this study localize in the cytoplasm and nucleus when transiently expressed in plant cells. In addition, MiGST_N_4, MiEFh, MiACPS and MiTSPc can elicit the ROS burst and strong hypersensitive response (HR), as well as significant ion leakage. Our data suggest that MiGST_N_4, MiEFh, MiACPS and MiTSPc effectors may be involved in triggering the immune response of the host plant.

Overexpression of PsoRPM3, an NBS-LRR gene isolated from myrobalan plum, confers resistance to Meloidogyne incognita in tobacco.

KEY MESSAGES: We reported an NBS-LRR gene, PsoRPM3, is highly expressed following RKN infection, initiating an HR response that promotes plant resistance. Meloidogyne spp. are root-knot nematodes (RKNs) that cause substantial economic losses worldwide. Screening for resistant tree resources and identifying plant resistance genes is currently the most effective way to prevent RKN infestations. Here, we cloned a novel TIR-NB-LRR-type resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz.) and demonstrated that its protein product localized to the nucleus. In response to Meloidogyne incognita infection, PsoRPM3 gene expression levels were significantly higher in resistant myrobalan plum plants compared to susceptible plants. We investigated this difference, discovering that the - 309 to - 19 bp region of the susceptible PsoRPM3 promoter was highly methylated. Indeed, heterologous expression of PsoRPM3 significantly enhanced the resistance of susceptible tobacco plants to M. incognita. Moreover, transient expression of PsoRPM3 induced a hypersensitive response in tobacco, whereas RNAi-mediated silencing of PsoRPM3 in transgenic tobacco reduced this hypersensitive response. Several hypersensitive response marker genes were considerably up-regulated in resistant myrobalan plum plants when compared with susceptible counterparts inoculated with M. incognita. PsoPR1a (a SA marker gene), PsoPR2 (a JA marker gene), and PsoACS6 (an ET signaling marker gene) were all more highly expressed in resistant than in susceptible plants. Together, these results support a model in which PsoRPM3 is highly expressed following RKN infection, initiating an HR response that promotes plant resistance through activated salicylic acid, jasmonic acid, and ethylene signaling pathways.

Themis controls thymocyte selection through regulation of T cell antigen receptor-mediated signaling.

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

The VvSUPERMAN-like Gene Is Differentially Expressed between Bicarpellate and Tricarpellate Florets of Vitis vinifera L. Cv. 'Xiangfei' and Its Heterologous Expression Reduces Carpel Number in Tomato.

Multicarpellate fruits are larger and produce more seeds than mono- or bicarpellate fruits, enhancing the reproductive capacity of the plant. To identify the phenotypic and molecular differences among florets of different carpel types, we studied carpel formation and fusion in the grapevine (Vitis vinifera) cultivar 'Xiangfei', which produces a high proportion of multicarpellate fruit. We also determined the function of VvSUPERMAN-like (VvSUP-like) and explored its relationship with VvWUS (VvWUSCHEL) and VvAG1 (VvAGAMOUS), which is related to the formation of carpel primordia. We showed that carpel formation and fusion were largely consistent between bicarpellate and tricarpellate ovaries, which both involve congenital fusion; rather, the differences between these ovary types arose from variation in carpel primordia number and location. Transgenic tomato (Solanum lycopersicum) plants expressing VvSUP-like produced significantly fewer carpels and other floral organs than the wild type. Moreover, transcriptome sequencing results indicate that VvSUP-like was more highly expressed in bicarpellate than in tricarpellate 'Xiangfei' florets. Luciferase reporter assays indicated that VvSUP-like inhibits the expression of VvAG1 and VvWUS by directly binding to their promoters, and VvWUS promotes VvAG1 expression by directly binding to its promoter. VvSUP-like inhibits the feedback signaling between VvWUS and VvAG1. Together, these results suggest that VvSUP-like negatively regulates the number of carpels that develop by inhibiting VvAG1 and VvWUS expression.

Atmospheric bulk deposition of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in the vicinity of MSWI in Shanghai, China.

Atmospheric bulk deposition samples were gathered month by month throughout a year at two sites in vicinity of a MSWI in Shanghai, to carry out an investigation on the atmospheric bulk deposition fluxes and seasonal variations of polychlorinated dibenzo-p-dioxinsand dibenzofurans (PCDD/Fs). The atmospheric bulk deposition fluxes of PCDD/Fs ranged from 23.5 to 560 pg m-2·d-1 (1.01-23.9 pg WHO-TEQ·m-2·d-1), with an average value of 136 pg m-2·d-1 (5.08 pg WHO-TEQ·m-2·d-1) in the Vicinity of the MSWI in Shanghai. The measured concentrations were well compared with those from urban or industrial sites in other regions in China and abroad. The seasonal trend of atmospheric bulk deposition fluxes of PCDD/Fs throughout a year exhibited as high levels in summer, moderate levels in winter, and low levels in spring and autumn. The principal component analysis (PCA) indicated not only the MSWI, but also vehicle emission was the indispensable source of PCDD/Fs in the vicinity of the MSWI, especially for the urban areas. The positive matrix factorization (PMF) apportioned 5 source categories: MSWI, diesel vehicles, atmosphere background, industrial combustion and un-leaded gas vehicles, accounting for 43.3%, 38.1%, 6.89%, 6.19% and 5.50% in average, respectively of PCDD/Fs in atmospheric bulk deposition in the vicinity of the MSWI in Shanghai, China.

Dendritic cell-MHC class II and Itk regulate functional development of regulatory innate memory CD4+ T cells in bone marrow transplantation.

MHC class II (MHCII)-influenced CD4(+) T cell differentiation and function play critical roles in regulating the development of autoimmunity. The lack of hematopoietic MHCII causes autoimmune disease that leads to severe wasting in syngeneic recipients. Using murine models of bone marrow transplantation (BMT), we find that MHCII(-/-)→wild-type BMT developed disease, with defective development of innate memory phenotype (IMP, CD44(hi)/CD62L(lo)) CD4(+) T cells. Whereas conventional regulatory T cells are unable to suppress pathogenesis, IMP CD4(+) T cells, which include conventional regulatory T cells, can suppress pathogenesis in MHCII(-/-)→wild-type chimeras. The functional development of IMP CD4(+) T cells requires hematopoietic but not thymic MHCII. B cells and hematopoietic CD80/86 regulate the population size, whereas MHCII expression by dendritic cells is sufficient for IMP CD4(+) T cell functional development and prevention of pathogenesis. Furthermore, the absence of Tec kinase IL-2-inducible T cell kinase in MHCII(-/-) donors leads to preferential development of IMP CD4(+) T cells and partially prevents pathogenesis. We conclude that dendritic cells-MHCII and IL-2-inducible T cell kinase regulate the functional development of IMP CD4(+) T cells, which suppresses the development of autoimmune disorder in syngeneic BMTs.

Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli.

Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

Halogenated aromatic pollutants in routine animal-derived food of south China: Occurrence, sources, and dietary intake risks.

Halogenated aromatic pollutants (HAPs) including polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs), polybrominated dibenzo-p-dioxins/furans (PBDD/Fs), and polybrominated diphenyl ethers (PBDEs) exhibit diverse toxicities and bio-accumulation in animals, thereby imposing risks on human via animal-derived food (ADF) consumption. Here we examined these HAPs in routine ADFs from South China and observed that PBDEs and PCBs showed statistically higher concentrations than PCDD/Fs and PBDD/Fs. PCDD/Fs and PCBs in these ADFs were mainly from the polluted feed and habitat of animals, except PCDD/Fs in egg, which additionally underwent selective biotransformation/progeny transfer after the maternal intake of PCDD/F-polluted stuff. PBDEs and PBDD/Fs were mostly derived from the extensive use of deca-BDE and their polluted environments. Significant interspecific differences were mainly observed for DL-PCBs and partly for PBDD/Fs and PBDEs, which might be caused by their distinct transferability/biodegradability in animals and the different living habit and habitat of animals. The dietary intake doses (DIDs) of these HAPs via ADF consumption were all highest for toddlers, then teenagers and adults. Milk, egg, and fish contributed most to the DIDs and risks for toddlers and teenagers, which results of several cities exceeded the recommended thresholds and illustrated noteworthy risks. Pork, fish, and egg were the top three risk contributors for adults, which carcinogenic and non-carcinogenic risks were both acceptable. Notably, PBDD/Fs showed the lowest concentrations but highest contributions to the total risks of these HAPs, thereby meriting continuous attention.

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFU mL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

[Effect of early intervention of liver-smoothing and blood-activating decoction combined with acupuncture on patients with post-stroke depression].

To study the effect of early intervention of liver-soothing and Blood-activating decoction combined with acupuncture in improving neurological functions, depressive symptom and life quality of patients with post-stroke depression, and compare with fluoxetine hydrochloride. Specifically, 63 patients with post-stroke depression were randomly divided into the traditional Chinese medicine (TCM) acupuncture group (31 cases) and the western medicine group (32 cases). On the basis of the conventional treatment of the primary disease, the TCM acupuncture group was treated with liver-soothing and blood-activating decoction and acupuncture, while the western medicine group was treated with fluoxetine hydrochloride for four weeks. In the follow-up visit six months later, scores of HAMD, NIHSS and SS-QOL were observed. The scores of HAMD and NIHSS of both groups were significantly decreased (P < 0.01), while the scores of SS-QOL increased significantly, with a notable difference compared with that before the treatment (P < 0.01). Specifically, the TCM acupuncture group's was superior to the western medicine group (P < 0.05). The study suggests that the early intervention of liver-soothing and blood-activating decoction combined with acupuncture on patients with post-stroke depression has the effect in relieving depression symptom and improving neurological functions, thereby improving their quality of life and prognosis.

Polybrominated dibenzo-p-dioxins/furans and their chlorinated analogues in sediments from a historical hotspot for both brominated flame retardants and organochlorine pesticides.

Polybrominated dibenzo-p-dioxin/furans (PBDD/Fs) and polychlorinated dibenzo-p-dioxin/furans (PCDD/Fs) in the environment are closely related to their precursors, brominated flame retardants (BFRs) and organochlorine pesticides (OCPs). However, their change trends following the regulation of BFRs and OCPs remain incompletely characterized. Here, we examined PBDD/Fs and PCDD/Fs in sediments from a historical hotspot for both BFRs and OCPs, namely the Pearl River Delta (PRD), China. PBDD/Fs showed ubiquity in these samples but significantly lower concentrations than PCDD/Fs. Spatially, the occurrence of PBDD/Fs was positively correlated with local development levels and sediments from highly urbanized/industrialized areas showed higher and increasing PBDD/F concentrations. Polybrominated diphenyl ether (PBDE)-related products/industries were the greatest PBDD/F contributors to the PRD, followed by bromo-phenol/benzene-related products/industries. PCDD/Fs in PRD sediments showed significant positive correlations with local grain planting area, yield, and pesticide consumption. The historical use of pentachlorophenol (PCP)/PCP-Na and biomass open-burning were the leading PCDD/F sources of the PRD agricultural/rural areas, where the concentrations and toxic equivalent quantities (TEQs) of PCDD/Fs in sediments changed very little over the past decade. Anthropogenic thermal processes involved in metallurgy, waste incineration, and vehicles were the greatest PCDD/F contributors in the PRD urban/industrial areas, where the PCDD/F concentrations in sediments almost doubled over the last decade. This finding indicates the increasing PCDD/F contributions of industrial and municipal activities in the PRD, despite the implementation of strict emission standards. Over sixty percent of the samples showed TEQs that surpassed the low-risk threshold specified for mammalian life by the U.S. EPA (2.5 pg TEQ g-1) and warrant continuous attention.

Atmospheric PCDD/F concentrations in 38 cities of China monitored with pine needles, a passive biosampler.

Many studies have shown that pine needles are a good biomonitor for atmospheric concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). However, this biomonitor has not been tested in large areas, and the quantitative relationship between concentrations in pine needles and air has not been established. In the present study, we collected pine needles from 38 cities in China. The concentrations of 2,3,7,8-PCDD/Fs were measured by gas chromatography combined with high-resolution mass spectrometry. The total PCDD/F concentrations ranged from 5.4 to 330 pg/g dry weight of pine needles. The pine needles from cities located in central China and three major city agglomerations contained high concentrations of PCDD/Fs, while those from cities in the west and on the coast contained relatively low concentrations. Primary emission and diffusion processes were the main controls of the PCDD/F concentrations in pine needles. Homologue profiles of PCDD/Fs in pine needles were characterized by PCDFs/PCDDs >1 in the industrialized cities and <1 in unindustrialized cities. The congener patterns were dominated by 1,2,3,4,6,7,8-HpCDF and OCDD, indicating that combustion was the major source of the PCDD/Fs. By comparing the PCDD/F concentrations in the pine needles and those in air, we established a preliminary linear relationship between them. Finally, the concentrations in air of PCDD/Fs in the 38 cities of China could be predicted from this relationship.

APANet: Auto-Path Aggregation for Future Instance Segmentation Prediction.

Despite the remarkable progress achieved in conventional instance segmentation, the problem of predicting instance segmentation results for unobserved future frames remains challenging due to the unobservability of future data. Existing methods mainly address this challenge by forecasting features of future frames. However, these methods always treat features of multiple levels (e.g., coarse-to-fine pyramid features) independently and do not exploit them collaboratively, which results in inaccurate prediction for future frames; and moreover, such a weakness can partially hinder self-adaption of a future segmentation prediction model for different input samples. To solve this problem, we propose an adaptive aggregation approach called Auto-Path Aggregation Network (APANet), where the spatio-temporal contextual information obtained in the features of each individual level is selectively aggregated using the developed "auto-path". The "auto-path" connects each pair of features extracted at different pyramid levels for task-specific hierarchical contextual information aggregation, which enables selective and adaptive aggregation of pyramid features in accordance with different videos/frames. Our APANet can be further optimized jointly with the Mask R-CNN head as a feature decoder and a Feature Pyramid Network (FPN) feature encoder, forming a joint learning system for future instance segmentation prediction. We experimentally show that the proposed method can achieve state-of-the-art performance on three video-based instance segmentation benchmarks for future instance segmentation prediction.

Polybrominated dibenzo-p-dioxins/furans (PBDD/Fs) in soil around municipal solid waste incinerator: A comparison with polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs).

Polybrominated dibenzo-p-dioxins/furans (PBDD/Fs) and polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) share similar toxicities and thermal origins, e.g., municipal solid waste incinerator (MSWI). Recently, PBDD/Fs from MSWI attracted rising concern because their important precursors, i.e., brominated flame retardants (BFRs), were frequently found in various wastes for landfill or MSWI feedstock. So far, however, little is known about PBDD/Fs and their associated risks in the vicinal environments of MSWI. Here we analyzed PBDD/Fs and PCDD/Fs in 29 soil samples collected around a multiyear large-scale MSWI, and compared their spatial distributions, sources and risks. PBDD/Fs demonstrated comparable concentrations and toxic equivalent quantities (TEQs) to PCDD/Fs in these samples. Spatially, both the concentrations of PBDD/Fs and PCDD/Fs decreased outwards from the MSWI, and exhibited significant linear correlations with the distances from the MSWI in the southeast downwind soil, suggesting the influence of the MSWI on its vicinal soil environment. However, the existence of other dioxin sources concealed its influence beyond 6 km. PBDD/Fs in the soils were characterized by highly-brominated PBDFs, especially Octa-BDF, and their sources were diagnosed as the MSWI and diesel exhaust; PCDD/Fs, however, were dominated by highly-chlorinated PCDDs, particularly Octa-CDD, and were contributed individually or jointly by the MSWI, automobile exhaust and pentachlorophenol (PCP)/Na-PCP. The non-carcinogenic risks of dioxins in all the soil samples were acceptable, but their carcinogenic risks in 17% of the samples were unacceptable. These samples were all located close to the MSWI and highways, therefore, the land use of these two high-risk zones should be cautiously planed.

Astragalus polysaccharide (APS) exerts protective effect against acute ischemic stroke (AIS) through enhancing M2 micoglia polarization by regulating adenosine triphosphate (ATP)/ purinergic receptor (P2X7R) axis.

Clinically, the effective treatment for patients with acute ischemic stroke (AIS) is very limited. Therefore, this paper aims to investigate the mechanism how astragalus polysaccharide (APS) exerts protective effect against AIS and provide a new method for the treatment of AIS. Cell surface antigen flow cytometry and immunofluorescence were used to identify M1 and M2 microglia. Western blot was used to evaluate the expression of associated protein. Oxygen-glucose deprivation (OGD) was used to simulate the effect of AIS on rat microglia. The middle cerebral artery occlusion (MCAO) model was established to simulate the effect of AIS in vivo. Evans blue dye (EBD) was used to evaluate the permeability of blood-brain barrier (BBB). Western blot and cell surface antigen flow cytometry results showed that APS promoted the M2 polarization of rat microglia by inhibiting the expression of purinergic receptor (P2X7R). APS reversed the effect of OGD on the polarization of rat microglia M1/ M2 by regulating P2X7R. APS reversed the effect of MCAO on the polarization of rat microglia M1/ M2 in vivo. Furthermore, APS inhibited the expression of P2X7R by promoting the degradation of adenosine triphosphate (ATP) in the cerebral cortex of MCAO rats. In addition, APS contributed to maintain the integrity of BBB. Summarily, APS can reduce brain injury by promoting the degradation of ATP in microglia and inhibiting the expression of P2X7R after AIS.

Enhanced development of CD4+ gammadelta T cells in the absence of Itk results in elevated IgE production.

The Tec kinase Itk is critical for the development of alphabeta T cells as well as differentiation of CD4(+) T cells into Th2 cells. Itk null mice have defects in the production of Th2 cytokines; however, they paradoxically have significant elevations in serum IgE. Here we show that Itk null mice have increased numbers of gammadelta T cells in the thymus and spleen. This includes elevated numbers of CD4(+) gammadelta T cell, the majority of which carry the Vgamma1.1 and Vdelta6.2/3 gammadelta T-cell receptor with a distinct phenotype. The development of these CD4(+) gammadelta T cells is T cell intrinsic, independent of either major histocompatibility complex class I or class II, and is favored during development in the absence of Itk. Itk null CD4(+) gammadelta T cells secrete significant amounts of Th2 cytokines and can induce the secretion of IgE by wild-type B cells. Our data indicate that Itk plays important role in regulating gammadelta T-cell development and function. In addition, our data indicate that the elevated IgE observed in Itk-deficient mice is due in part to the enhanced development of CD4(+) gammadelta T cells in the absence of Itk.

Fifteen-year trend in the use of male reproductive surgery: analysis of the healthcare cost and utilization project data.

OBJECTIVE: • To determine trends in male reproductive surgeries. MATERIALS AND METHODS: • A database analysis was performed using the Healthcare Cost and Utilization Project databases from 1988, 1992 and 1998-2002. SAS statistical software was used to estimate the total numbers of reproductive surgeries and their variances for each year. RESULTS: • The number of male reproductive surgeries has declined during the period of study, both in the inpatient and outpatient arenas. The number of procedures with a diagnosis of infertility has also declined. CONCLUSION: • The number of male reproductive surgeries has declined from 1988 to 2002.