ANXA9 facilitates S100A4 and promotes breast cancer progression through modulating STAT3 pathway.

Breast cancer has the highest global incidence and mortality rates among all cancer types. Abnormal expression of the Annexin family has been observed in different malignant tumors, including upregulated ANXA9 in breast cancer. We found highly expressed ANXA9 in metastatic breast cancer tissues, which is correlated with breast cancer progression. In vitro, the functional experiments indicated ANXA9 influenced breast cancer proliferation, motility, invasion, and apoptosis; in vivo, downregulation of ANXA9 suppressed breast cancer xenograft tumor growth and lung metastasis. Mechanically, on one side, we found that ANXA9 could mediate S100A4 and therefore regulate AKT/mTOR/STAT3 pathway to participate p53/Bcl-2 apoptosis; on the other side, we found ANXA9 transferred S100A4 from cells into the tumor microenvironment and mediated the excretion of cytokines IL-6, IL-8, CCL2, and CCL5 to participate angiogenesis via self- phosphorylation at site Ser2 and site Thr69. Our findings demonstrate significant involvement of ANXA9 in promoting breast cancer progression, thereby suggesting that therapeutic intervention via targeting ANXA9 may be effective in treating metastatic breast cancer.

[Advances in the study of the actin nucleation factor Spire].

There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

Otolith microchemistry reveals diverse habitat uses and migratory patterns of two Coilia species (Engraulidae) in the Min River Estuary, southern China.

A recent study based on gonad histology revealed that the existence of the spawning grounds for Gray's grenadier anchovy (Coilia grayii) and Osbeck's grenadier anchovy (C. mystus) in the Min River Estuary, the largest in Fujian Province, southern China. Further confirming their natal sources and migratory patterns is essential to understand their life histories. We used otolith microchemistry to assess the origins and habitat uses of 23 C. grayii and 22 C. mystus, collected the Min River Estuary and the adjacent waters. The results showed that C. grayii spawned in both freshwater (n| = 14) and brackish water (n = 9), and C. mystus spawned mainly in brackish water (n| = 20) with minor in freshwater (n| = 1) and marine water (n| = 1). The migratory patterns of C. grayii (four types) and C. mystus (five types) were diverse, mainly exhibiting anadromous and semi-anadromous behaviors. The first migratory behavior of C. grayii and C. mystus occurred within the age of the first year. The findings have significant implications for fishery stock management of the Min River Estuary and its adjacent waters.

Formamide Electrosynthesis from Methanol and Ammonia in Water over Pr-Doped MnO2.

A rare earth element doping strategy is reported to boost the activity and enhance the stability of MnO2 for selective formamide production through electrocatalytic oxidation coupling (EOC) of methanol and ammonia. MnO2 doped with 1% Pr was selected as the best candidate with an optimized formamide yield of 211.32 µmol cm-2 h-1, a Faradaic efficiency of 22.63%, and a stability of more than 50 h. The easier formation of Mn6+ species and the lower dissolution rate of Mn species over Pr-doped MnO2 revealed by in situ Raman spectra were responsible for the boosted formamide production and enhanced stability. In addition, a two-electrode flow electrolyzer was developed to integrate EOC with C2H2 semihydrogenation for simultaneously producing value-added products in both the anode and cathode.

α1-Antitrypsin Binds to the Glucocorticoid Receptor with Anti-Inflammatory and Antimycobacterial Significance in Macrophages.

α1-Antitrypsin (AAT), a serine protease inhibitor, is the third most abundant protein in plasma. Although the best-known function of AAT is irreversible inhibition of elastase, AAT is an acute-phase reactant and is increasingly recognized to have a panoply of other functions, including as an anti-inflammatory mediator and a host-protective molecule against various pathogens. Although a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Because AAT has anti-inflammatory properties, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in human macrophages. We report the finding that AAT binds to GR using several approaches, including coimmunoprecipitation, mass spectrometry, and microscale thermophoresis. We also performed in silico molecular modeling and found that binding between AAT and GR has a plausible stereochemical basis. The significance of this interaction in macrophages is evinced by AAT inhibition of LPS-induced NF-κB activation and IL-8 production as well as AAT induction of angiopoietin-like 4 protein, which are, in part, dependent on GR. Furthermore, this AAT-GR interaction contributes to a host-protective role against mycobacteria in macrophages. In summary, this study identifies a new mechanism for the gene regulation, anti-inflammatory, and host-defense properties of AAT.

Littoral Water in Hong Kong as a Potential Transient Habitat for Juveniles of a Temperate Deepwater Gnomefish, Scombrops boops (Acropomatiformes: Scombropidae).

A total of 40 juveniles belonging to a temperate deepwater gnomefish species, Scombrops boops, were sampled from littoral habitats (2-5 m depth) of eastern Hong Kong waters in April and May 2017 and March 2019. The presence of gnomefish juveniles in subtropical southern China is reported for the first time at a record low latitude of 22°11'-22°21'N. The specimens were identified based on the COI gene sequence. The genetic composition between Japan and Hong Kong gnomefish populations were compared by sequencing the mitochondrial Cytb gene, which showed no genetic differentiation. The juveniles ranged from 3.5-10.1 cm (n = 40) in total length, with 35 individuals caught from Sargassum beds and five from rocky reefs. Our findings highlighted that the littoral habitats in Hong Kong waters, in particular the seasonal Sargassum beds, are important for small juveniles of S. boops.

Complete mitochondrial genome and the phylogenetic position of a new species, Johnius taiwanensis (Perciformes: Sciaenidae) from Chinese waters.

In this study, the complete mitogenome of a new species, Johnius taiwanensis (Chao et al. 2019) was obtained. Its mitogenome is 18,451 bp in length, consisting of 37 genes with the typical gene order and direction of transcription in vertebrates. Gene rearrangement was found in J. taiwanensis. The overall nucleotide composition is: 24.2% A; 18.0% C; 21.1% G, and 36.7% T. Sizes of the 22 tRNA genes range from 66 to 75 bp. Two start codons (ATG and GTG) and three stop codons (TAG, AGA and TAA/TA/T) were detected in 13 protein-coding genes. In the Bayesian tree based on the complete mitogenomes of 26 species (including J. taiwanensis) from the family Sciaenidae, all nodes were strongly supported. The result shows that J. taiwanensis was placed as sister to the Trewavas croaker J. trewavasae of the same genus. The mechanism of gene rearrangement in the genus Johnius merits further investigation.

Differential Responses by Human Macrophages to Infection With Mycobacterium tuberculosis and Non-tuberculous Mycobacteria.

Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) are formidable causes of lung diseases throughout the world. While MTB is considered to be more virulent than NTM, host factors also play a key role in disease development. To elucidate whether there are differential immune responses to various mycobacteria, THP-1 macrophages were temporally infected with MTB H37Rv or with four different NTM species. We found that cells infected with MTB had greater bacterial burden and p65 nuclear factor-kappa B (NF-κB) activation than cells infected with NTM. There was also differential expression of mRNA for interleukin-1-ß (IL-1ß), IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) with no distinct pattern of mRNA expression among the different mycobacteria. In contrast, at the protein level, some generalizations can be made of the cytokines and chemokines expressed. Compared to uninfected cells, the rapid-growing Mycobacterium smegmatis but not Mycobacterium abscessus induced significantly greater pro-inflammatory cytokines and IL-10, whereas both NTM individually induced greater levels of chemokines. Compared to uninfected control cells, the two slow-growing NTM and MTB differentially induced cytokine expression with Mycobacterium avium inducing more pro-inflammatory cytokines and IL-10, whereas M. avium, Mycobacterium intracellulare, and MTB inducing greater but similar levels of chemokines. MTB-infected THP-1 cells also demonstrated lower level of phagosome-lysosome fusion and apoptosis than NTM-infected cells while there were differences in these macrophage functions among the NTM species. Interestingly, M. intracellulare, M. avium, and MTB have similar levels of autophagosome formation, but the levels displayed by all three were lower than for M. smegmatis and M. abscessus. This study demonstrates the differences in bacterial burden and macrophage effector functions among several clinically relevant mycobacterial species. Such disparities may, in part, account for differences in clinical outcomes among patients infected with various species of NTM as has been seen for different strains of MTB.

Separation and Recycling of Concentrated Heavy Metal Wastewater by Tube Membrane Distillation Integrated with Crystallization.

Tube membrane distillation (MD) integrated with a crystallization method is used in this study for the concurrent productions of pure water and salt crystals from concentrated single and mixed system solutions. The effects of concentrated Zn2+ and Ni2+ on performance in terms of membrane flux, permeate conductivity, crystal recovery rates, and crystal grades are investigated. Preferred crystallization and co-crystallization determinations were performed for mixed solutions. The results revealed that membrane fluxes remained at 2.61 kg·m-2·h-1 and showed a sharp decline until the saturation increased to 1.38. Water yield conductivity was below 10 µs·cm-1. High concentrated zinc and nickel did not have a particular effect on the rejection of the membrane process. For the mixed solutions, membrane flux showed a sharp decrease due to the high saturation, while the conductivity of permeate remained below 10 µs·cm-1 during the whole process. Co-crystallization has been proven to be a better method due to the existence of the SO42- common-ion effect. Membrane fouling studies have suggested that the membrane has excellent resistance to fouling from highly concentrated solutions. The MD integrated with crystallization proves to be a promising technology for treating highly concentrated heavy metal solutions.

[Advances in polydopamine surface modification for capillary electrochromatography].

Capillary electrophoresis (CE) has a wide range of applications in analytical fields due to its advantages of low sample consumption, short separation time, and high separation efficiency. The cathodic electroosmotic flow (EOF) and single electrophoretic separation mechanism are not optimal for many CE applications. Hence, the use of an unmodified fused-silica capillary leads to insufficient separation performance that cannot meet the requirements for various complex sample systems, especially neutral and chiral compounds. Therefore, it is necessary to introduce various capillary modification strategies in CE so that its potential for practical application can be expanded. Mussel-inspired polydopamine (PDA) and PDA-derived coating materials have fascinating advantages such as simple surface coating procedures, strong surface adhesiveness, good chemical stability, latent reactivity with many functionalized molecules, and good biocompatibility. Thus, they have been widely utilized in different research fields, including catalysis, sensing, water treatment, sample pretreatment, biomedicine, chromatographic separation, and CE. The preparation of PDA coatings is simple as it involves physical adsorption, and the obtained surface adhesive coatings possess good stability similar to covalently bonded coatings. Therefore, PDA and PDA-derived coatings are well suited for the modification of fused-silica capillaries. More importantly, the PDA coating can be utilized as an intermediate reaction platform for diverse subsequent surface modification because of its strong surface adhesive property and strong latent reactivity with many functionalized molecules (such as polymers, proteins, and nanomaterials). Consequently, various chromatographic retention mechanisms can be introduced on the inner wall of the capillary, thereby contributing to the fabrication of multi-functional PDA-based stationary phases for CEC. Owing to these outstanding advantages, researchers are paying increasing attention to the great application potential of PDA and PDA-derived coatings in CEC. In this paper, recent advances in the methods for preparing PDA coatings, especially the recently developed fabrication strategies and various applications of PDA-modified silica capillary in open tubular-capillary electrochromatography (OT-CEC) and capillary electrochromatography monolithic columns, are summarized and discussed. Furthermore, the application prospects of PDA-based coating materials in CEC are prospected in this review. Although PDA and PDA-derived coating materials are seeing widespread utilization in field of CEC, researchers have still not reached a definite conclusion regarding the PDA formation and coating mechanisms, and further investigation is needed in this direction. The PDA coatings formed using existing methods are generally thin. In the early stage, many studies adopted the strategy of repeated coating to improve the coating effect of PDA in capillaries, but this method was found to be time-consuming and less efficient. In order to improve the preparation efficiency of PDA-modified CEC columns, many researchers have focused on fast deposition induced by a strong oxidant to obtain PDA-coated columns. However, the controllability of the PDA coating obtained by this method is poor. Thus, it is necessary to further explore new preparation strategies for PDA-coated CEC capillaries with better reproducibility and stronger operability. On the other hand, although a strategy for directly synthesizing functional PDA-coated CEC columns in the organic phase has been proposed, its application potential in CEC remains to be further explored. In addition, the PDA coating itself has poor porosity and a small specific surface area, which may be significantly improved by modifying the coating on the porous monolithic column surface. However, there has been limited research on the use of PDA coatings in monolithic columns, and their application potential remains to be expanded. With in-depth research into the formation mechanism and preparation methodologies of PDA coatings, PDA, which is a highly malleable biomimetic material, will play a more important role in advances in the fields of CE and CEC.

Johnius taiwanensis, a new species of Sciaenidae from the Taiwan Strait, with a key to Johnius species from Chinese waters.

A new sciaenid fish, Johnius taiwanensis, is described from the southeast coast of mainland China from Zhejiang to Guangdong, Hong Kong, and west coast of Taiwan. Johnius taiwanensis sp. nov. can be distinguished from other Johnius species by having a grayish dorsal half of body divided by a clear line from a whitish ventral half, and a black spot at the dorsal half of pectoral-fin axil, appearing as a distinct dot at the most dorsal point of the pectoral-fin base. First dorsal fin black tipped, other fins pale to dusky but never darkly pigmented. The species lacks distinctly enlarged teeth on upper and lower jaws. Body scales ctenoid, moderately large, with five or six rows between first dorsal-fin origin and lateral line. It is one of the most abundant sciaenids found in the shallow coastal waters (20 m) of southeast mainland China and the west coast of Taiwan. It has often been misidentified as J. macrorhynus in the region. Phylogenetic analysis from all 27 sciaenid species found in Chinese waters based on the complete COI and 16S rRNA gene sequences confirmed that the genus Johnius is monophyletic and J. taiwanensis is placed as a sister species of J. trewavasae. Acoustic analysis has shown that J. taiwanensis produces a unique sound among fishes in Taiwan coastal waters.

Alpha-1-Antitrypsin Enhances Primary Human Macrophage Immunity Against Non-tuberculous Mycobacteria.

Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare-induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.

Nicotine Impairs Macrophage Control of Mycobacterium tuberculosis.

Pure nicotine impairs macrophage killing of Mycobacterium tuberculosis (MTB), but it is not known whether the nicotine component in cigarette smoke (CS) plays a role. Moreover, the mechanisms by which nicotine impairs macrophage immunity against MTB have not been explored. To neutralize the effects of nicotine in CS extract, we used a competitive inhibitor to the nicotinic acetylcholine receptor (nAChR)-mecamylamine-as well as macrophages derived from mice with genetic disruption of specific subunits of nAChR. We also determined whether nicotine impaired macrophage autophagy and whether nicotine-exposed T regulatory cells (Tregs) could subvert macrophage anti-MTB immunity. Mecamylamine reduced the CS extract increase in MTB burden by 43%. CS extract increase in MTB was also significantly attenuated in macrophages from mice with genetic disruption of either the α7, ß2, or ß4 subunit of nAChR. Nicotine inhibited autophagosome formation in MTB-infected THP-1 cells and primary murine alveolar macrophages, as well as increased the intracellular MTB burden. Nicotine increased migration of THP-1 cells, consistent with the increased number of macrophages found in the lungs of smokers. Nicotine induced Tregs to produce transforming growth factor-ß. Naive mouse macrophages co-cultured with nicotine-exposed Tregs had significantly greater numbers of viable MTB recovered with increased IL-10 production and urea production, but no difference in secreted nitric oxide as compared with macrophages cocultured with unexposed Tregs. We conclude that nicotine in CS plays an important role in subverting macrophage control of MTB infection.

[Modified Bianchi orchiopexy for median or low cryptorchidism].

OBJECTIVE: To investigate the effect of modified Bianchi (single incision in the midline of the scrotum) orchiopexy (MBO) versus that of traditional surgery in the treatment of median or low cryptorchidism. METHODS: Eighty-two children with median or low cryptorchidism were treated from February 2013 to February 2014, 46 (53 testes) by MBO and the other 36 by the traditional method of inguinal incision (control, 40 testes). Comparisons were made in the operation time and postoperative complications between the two surgical strategies. RESULTS: The mean operation time was significantly shorter in the MBO group than in the control (ï¼»25±6ï¼½ vs ï¼»35±4ï¼½ min, P<0.05). No testicular atrophy, hernias or hydrocele was found in either group during the 1－2 years of follow-up. Testis retraction was observed in 3 cases in the MBO group as compared with 2 in the control (P>0.05). The incision scar was obvious in all the controls, with 1 case of postoperative inguinal hematoma, but almost invisible in all the MBO cases. CONCLUSIONS: Modified Bianchi orchiopexy is superior to traditional surgery in the treatment of median or low cryptorchidism for its advantages of short operation time, few complications, and satisfactory appearance of the healed incision.

Complete mitochondrial genome and the phylogenetic position of the Pawak croaker Pennahia pawak (Perciformes: Sciaenidae).

In this study, the complete mitogenome of the Pawak croaker Pennahia pawak was first determined. This mitogenome is 16,408 bp in length, and consists of 37 genes with the typical gene order and direction of transcription in vertebrates. The overall nucleotide composition is: 27.7% A, 29.5% C, 15.9% G, and 26.9% T. Sizes of the 22 tRNA genes range from 66 to 75 bp. One start codons (ATG) and two stop codons (AGA and TAA/TA/T) were detected in 13 protein-coding genes. In the Bayesian tree based on the complete mitogenomes of 17 species (including P. pawak) from the family Sciaenidae, all nodes were strongly supported. The phylogenetic results suggested that P. pawak has the closest relationship to the silver croaker P. argentata, a species from the same genus.

Curcumin enhances human macrophage control of Mycobacterium tuberculosis infection.

BACKGROUND AND OBJECTIVE: With the worldwide emergence of highly drug-resistant tuberculosis (TB), novel agents that have direct antimycobacterial effects or that enhance host immunity are urgently needed. Curcumin is a polyphenol responsible for the bright yellow-orange colour of turmeric, a spice derived from the root of the perennial herb Curcuma longa. Curcumin is a potent inducer of apoptosis-an effector mechanism used by macrophages to kill intracellular Mycobacterium tuberculosis (MTB). METHODS: An in vitro human macrophage infection model was used to determine the effects of curcumin on MTB survival. RESULTS: We found that curcumin enhanced the clearance of MTB in differentiated THP-1 human monocytes and in primary human alveolar macrophages. We also found that curcumin was an inducer of caspase-3-dependent apoptosis and autophagy. Curcumin mediated these anti-MTB cellular functions, in part, via inhibition of nuclear factor-kappa B (NFκB) activation. CONCLUSION: Curcumin protects against MTB infection in human macrophages. The host-protective role of curcumin against MTB in macrophages needs confirmation in an animal model; if validated, the immunomodulatory anti-TB effects of curcumin would be less prone to drug resistance development.

Caspase-3-independent apoptotic pathways contribute to interleukin-32γ-mediated control of Mycobacterium tuberculosis infection in THP-1 cells.

BACKGROUND: Macrophages are the primary effector cells responsible for killing Mycobacterium tuberculosis (MTB) through various mechanisms, including apoptosis. However, MTB can evade host immunity to create a favorable environment for intracellular replication. MTB-infected human macrophages produce interleukin-32 (IL-32). IL-32 is a pro-inflammatory cytokine and has several isoforms. We previously found that IL-32γ reduced the burden of MTB in human macrophages, in part, through the induction of caspase-3-dependent apoptosis. However, based on our previous studies, we hypothesized that caspase-3-independent death pathways may also mediate IL-32 control of MTB infection. Herein, we assessed the potential roles of cathepsin-mediated apoptosis, caspase-1-mediated pyroptosis, and apoptosis-inducing factor (AIF) in mediating IL-32γ control of MTB infection in THP-1 cells. RESULTS: Differentiated human THP-1 macrophages were infected with MTB H37Rv alone or in the presence of specific inhibitors to caspase-1, cathepsin B/D, or cathepsin L for up to four days, after which TUNEL-positive cells were quantified; in addition, MTB was quantified by culture as well as by the percentage of THP-1 cells that were infected with green fluorescent protein (GFP)-labeled MTB as determined by microscopy. AIF expression was inhibited using siRNA technology. Inhibition of cathepsin B/D, cathepsin L, or caspase-1 activity significantly abrogated the IL-32γ-mediated reduction in the number of intracellular MTB and of the percentage of GFP-MTB-infected macrophages. Furthermore, inhibition of caspase-1, cathepsin B/D, or cathepsin L in the absence of exogenous IL-32γ resulted in a trend toward an increased proportion of MTB-infected THP-1 cells. Inhibition of AIF activity in the absence of exogenous IL-32γ also increased intracellular burden of MTB. However, since IL-32γ did not induce AIF and because the relative increases in MTB with inhibition of AIF were similar in the presence or absence of IL-32γ, our results indicate that AIF does not mediate the host-protective effect of IL-32γ against MTB. CONCLUSIONS: The anti-MTB effects of IL-32γ are mediated through classical caspase-3-dependent apoptosis as well as caspase-3-independent apoptosis.

Human IL-32 expression protects mice against a hypervirulent strain of Mycobacterium tuberculosis.

Silencing of interleukin-32 (IL-32) in a differentiated human promonocytic cell line impairs killing of Mycobacterium tuberculosis (MTB) but the role of IL-32 in vivo against MTB remains unknown. To study the effects of IL-32 in vivo, a transgenic mouse was generated in which the human IL-32γ gene is expressed using the surfactant protein C promoter (SPC-IL-32γTg). Wild-type and SPC-IL-32γTg mice were infected with a low-dose aerosol of a hypervirulent strain of MTB (W-Beijing HN878). At 30 and 60 d after infection, the transgenic mice had 66% and 85% fewer MTB in the lungs and 49% and 68% fewer MTB in the spleens, respectively; the transgenic mice also exhibited greater survival. Increased numbers of host-protective innate and adaptive immune cells were present in SPC-IL-32γTg mice, including tumor necrosis factor-alpha (TNFα) positive lung macrophages and dendritic cells, and IFN-gamma (IFNγ) and TNFα positive CD4(+) and CD8(+) T cells in the lungs and mediastinal lymph nodes. Alveolar macrophages from transgenic mice infected with MTB ex vivo had reduced bacterial burden and increased colocalization of green fluorescent protein-labeled MTB with lysosomes. Furthermore, mouse macrophages made to express IL-32γ but not the splice variant IL-32ß were better able to limit MTB growth than macrophages capable of producing both. The lungs of patients with tuberculosis showed increased IL-32 expression, particularly in macrophages of granulomas and airway epithelial cells but also B cells and T cells. We conclude that IL-32γ enhances host immunity to MTB.

[Optimal expression and activity detection of recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies in P.pastoris].

AIM: To express the recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) using yeast cells secreting type carrier pPIC9K, and screen for optimal conditions of engineered yeast cells expressing cRFB4FC and cRFB4CH3. METHODS: The genes of cRFB4FC and cRFB4CH3 were cloned into P. pastoris eukaryotic expression vector pPIC9K to construct the recombinant plasmids pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3, then identified by DNA sequencing. The recombinant plasmid pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were transfected into P. pastoris SMD1163. The recombinant yeast cell line chosen by G418 resistance was identified by PCR. After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blotting, and biologic activity was identified by ELISA. Finally, orthogonal test was conducted to optimize the expression conditions of the recombinant yeast cell lines so as to increase the expression level of the antibodies. RESULTS: The secretory type yeast carriers pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were successfully constructed and chosen by G418 as well as identified by PCR to obtain the highly copied and integrated recombinant yeast cell line. The cRFB4FC and cRFB4CH3 proteins were expressed by yeast cells containing pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 induced by methanol. The relative molecular mass (M(r);) of cRFB4FC and cRFB4CH3 were about 326 000 and 295 000 Da. They could be identified by goat anti-human IgG(Fc) monoclonal antibody with Western blotting on SDS-PAGE, and higher biologic activity was confirmed by ELISA. Under the optimized expression conditions, the mean expression levels of cRFB4FC and cRFB4CH3 in recombinant yeast increased by 196.4% and 151.7%. CONCLUSION: The recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) proteins can be highly expressed in the P.pastoris SMD1163 expression system, and possess immunological activity.