Factors associated with treatment response to CD19 CAR-T therapy among a large cohort of B cell acute lymphoblastic leukemia.

CD19-targeted chimeric antigen receptor (CAR) T cell therapy has demonstrated striking responses among B cell acute lymphoblastic leukemia (B-ALL), but analyses of potential factors associated with poor response and relapse are lacking. Here, we summarize the long-term follow-up of 254 B-ALL treated with CD19 CAR-T cells from 5 clinical trials (NCT03173417, NCT02546739, and NCT03671460 retrospectively registered on May 23, 2017, March 1, 2018, and September 7, 2018, respectively, at www.clinicaltrials.gov ; ChiCTR-ONC-17012829, and ChiCTR1800016541 retrospectively registered on September 28, 2017, and June 7, 2018, at www.chictr.org.cn ). Our data showed that TP53 mutation, bone marrow blasts > 20%, prior CAR-T/blinatumomab treatment, and severe cytokine release syndrome (CRS) were associated with a lower complete remission (CR) rate while age, extramedullary disease, complex cytogenetics, history of prior transplant, prior courses of chemotherapy, CAR-T cell dose, and manufacturing source of the cellular product did not affect patients' CR rate. Risk factors related to leukemia-free survival (LFS) and overall survival (OS) were history of prior transplant, complex cytogenetics, TP53 mutation, severe CRS, neurotoxicity, and CAR-T therapy without consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Age and CAR-T cell dose did not influence LFS and OS. Patients with consolidative allo-HSCT after CAR-T therapy had a superior OS and LFS compared to those who did not. This benefit was also observed in both pediatric and adult patients as well as in patients either in high- or low-risk groups. This large study to identify risk factors of CR, LFS, and OS may help to maximize clinical outcomes of CAR-T therapy. Précis TP53 mutation and BM blasts > 20% are two independent factors associated with the CR rate. Patients with high tumor burden as well as those with bone marrow blasts < 5% can benefit from consolidative allo-HSCT post-CAR-T therapy.

Changes in the epidemic of pulmonary tuberculosis in Shanghai from 1992 to 2016.

OBJECTIVE: To estimate whether WHO's End TB Strategy targets can be achieved by analysing the incidence trend of pulmonary tuberculosis in Shanghai during 1992-2016. METHOD: The age-adjusted annual incidence of tuberculosis (TB) was calculated based on data from the national TB registration system. Linear regression was applied to analyse the trend of the epidemic, together with the statistical indicator of annual percent change (APC). RESULTS: The overall age-standardised rate decreased from 34.8/100 000 in 1992 to 21.2/100 000 in 2016, or by 2.15% (t = -13.258, P < 0.05) annually. After rapidly declining between 1999 and 2003 (-5.4% p.a.), the epidemic remained at a stable level with a lower annual declining rate (-1.1% p.a). In 2035, the estimated incidence will be 17.2/100 000 based on the APC in 2004-2016. There were two peaks in average incidence of the total population, 30.9/100 000 in the 20-24 age group and 66.4/100 000 in the 70-74 age group. Overall, the ratio of new to retreated cases continually rose and eventually reached 8.36:1 in 2016. The constituent ratio of smear-positive cases ranged from 35.9% to 47.8% without rising or decreasing trend (P = 0.065). CONCLUSION: The epidemic of TB in Shanghai has steadily declined during last two decades. A new strategy should be developed to rapidly reduce the incidence rate to achieve the WHO Goals in 2035.

Later emergence of acquired drug resistance and its effect on treatment outcome in patients treated with Standard Short-Course Chemotherapy for tuberculosis.

BACKGROUNDS: The failure of current Standard Short-Course Chemotherapy (SCC) in new and previously treated cases with tuberculosis (TB) was mainly due to drug resistance development. But little is known on the characteristics of acquired drug resistant TB during SCC and its correlation with SCC failure. The objective of the study is to explore the traits of acquired drug resistant TB emergence and evaluate their impacts on treatment outcomes. METHODS: A prospective observational study was performed on newly admitted smear positive pulmonary TB (PTB) cases without drug resistance pretreatment treated with SCC under China's National TB Control Program (NTP) condition from 2008 to 2010. Enrolled cases were followed up through sputum smear, culture and drug susceptibility testing (DST) at the end of 1, 2, and 5 months after treatment initiation. The effect factors of early or late emergence of acquired drug resistant TB , such as acquired drug resistance patterns, the number of acquired resistant drugs and previous treatment history were investigated by multivariate logistic regression; and the impact of acquired drug resistant TB emergence on treatment failure were further evaluated. RESULTS: Among 1671 enrolled new and previously treated cases with SCC, 62 (3.7%) acquired different patterns of drug resistant TB at early period within 2 months or later around 3-5 months of treatment. Previously treated cases were more likely to develop acquired multi-drug resistant TB (MDR-TB) (OR, 3.8; 95%CI, 1.4-10.4; P = 0.015). Additionally, acquired MDR-TB cases were more likely to emerge at later period around 3-5 months after treatment starting than that of non-MDR-TB mainly appeared within 2 months (OR, 8.3; 95%CI, 1.7-39.9; P = 0.008). Treatment failure was associated with late acquired drug resistant TB emergence (OR, 25.7; 95%CI, 4.3-153.4; P < 0.001) with the reference of early acquired drug resistant TB emergence. CONCLUSIONS: This study demonstrates that later development of acquired drug resistant TB during SCC is liable to suffer treatment failure and acquired MDR-TB pattern may be one of the possible causes.

Plant hormones and phenolic acids response to UV-B stress in Rhododendron chrysanthum pall.

Our study aims to identify the mechanisms involved in regulating the response of Rhodoendron Chrysanthum Pall. (R. chrysanthum) leaves to UV-B exposure; phosphorylated proteomics and metabolomics for phenolic acids and plant hormones were integrated in this study. The results showed that UV-B stress resulted in the accumulation of salicylic acid and the decrease of auxin, jasmonic acid, abscisic acid, cytokinin and gibberellin in R. chrysanthum. The phosphorylated proteins that changed in plant hormone signal transduction pathway and phenolic acid biosynthesis pathway were screened by comprehensive metabonomics and phosphorylated proteomics. In order to construct the regulatory network of R. chrysanthum leaves under UV-B stress, the relationship between plant hormones and phenolic acid compounds was analyzed. It provides a rationale for elucidating the molecular mechanisms of radiation tolerance in plants.

CED-Net: A more effective DenseNet model with channel enhancement.

In recent years, deep convolutional neural network (CNN) has been applied more and more increasingly used in computer vision, natural language processing and other fields. At the same time, low-power platforms have more and more significant requirements for the size of the network. This paper proposed CED-Net (Channel enhancement DenseNet), a more efficient densely connected network. It combined the bottleneck layer with learned group convolution and channel enhancement module. The bottleneck layer with learned group convolution could effectively increase the network's accuracy without too many extra parameters and computation (FLOPs, Floating Point Operations). The channel enhancement module improved the representation of the network by increasing the interdependency between convolutional feature channels. CED-Net is designed regarding CondenseNet's structure, and our experiments show that the CED-Net is more effective than CondenseNet and other advanced lightweight CNNs. Accuracy on the CIFAR-10 dataset and CIFAR-100 dataset is 0.4 and 1% higher than that on CondenseNet, respectively, but they have almost the same number of parameters and FLOPs. Finally, the ablation experiment proves the effectiveness of the bottleneck layer used in CED-Net.

Design of Longitudinal-Torsional Transducer and Directivity Analysis during Ultrasonic Vibration-Assisted Milling of Honeycomb Aramid Material.

This paper presents a longitudinal-torsional transducer for use during the ultrasonic vibration-assisted milling (UVAM) of honeycomb aramid material. The mechanism of longitudinal-torsional conversion was analyzed to guide the design of a vibration transducer. The transducer features five spiral grooves around the front cover plate, which function under the excitation of a group of longitudinal piezoelectric ceramics. A portion of the longitudinal vibration was successfully converted into torsional vibration. The resonant frequency, longitudinal vibration displacement and torsional amplitude at the top of the disk milling cutter were 24,609 Hz, 19 µm and 9 µm, respectively. In addition, the directivity of the longitudinal-torsional transducer was theoretically analyzed. Compared with conventional milling, UVAM with the longitudinal-torsional could significantly reduce the cutting force (40-50%) and improve the machining stability.

Prognosis and long-term neurodevelopmental outcome in conservatively treated twin-to-twin transfusion syndrome.

BACKGROUND: Amnioreduction remains a treatment option for pregnancies with twin-to-twin transfusion syndrome (TTTS) not meeting criteria for laser surgery or those in which it is not feasible. Amnioreduction is a relatively simple treatment which does not require sophisticated technical equipment. Previous reports of conservative management have indicated that major neurodevelopmental impairment occurs in 14.3-26% of survivors. The purpose of this study was to investigate long-term neurodevelopmental outcome in conservatively treated TTTS. METHODS: During the nine-year study period from January 1996 to December 2004, all pregnancies with TTTS who were admitted to our center were investigated. TTTS was diagnosed by using standard prenatal ultrasound criteria, and staged according to the criteria of Quintero et al. We reviewed gestational age at diagnosis, gestational age at delivery, the stage of TTTS at diagnosis, and diagnosis to delivery interval. Neonatal cranial ultrasound findings were reviewed and the neurodevelopmental outcomes were evaluated. RESULTS: Twenty-one pregnancies with TTTS were included. Thirteen pregnancies (62%) were treated with serial amnioreduction. The mean gestational age at delivery was 28 weeks (22-34 weeks). The perinatal mortality rate was 42.9%. Twenty survivors were followed up until at least 3 years of age. The mean age at follow-up was 6.3 years (3-12 years). Six children (30%) had neurodevelopmental impairment. Four children (20%) had major neurodevelopmental impairment and two children (10%) had minor neurodevelopmental impairment. Children with neurodevelopmental impairment were delivered before 29 weeks of gestation. CONCLUSIONS: Our study showed a high rate of perinatal mortality and a high rate of major neurodevelopmental impairment in conservatively treated TTTS. The long-term outcomes for the survivors with TTTS were good when survivors were delivered after 29 weeks of gestation.

Impact of pro segments on the folding and function of human neutrophil alpha-defensins.

Human neutrophil alpha-defensins (HNPs) are synthesized in vivo as inactive precursor proteins, i.e. preproHNPs. A series of sequential proteolytic events excise the N-terminal inhibitory pro peptide, leading to defensin maturation and storage in azurophilic granules. The anionic pro peptide, required for correct sub-cellular trafficking and sorting of proHNPs, inhibits the antimicrobial activity of cationic defensins, either inter or intra-molecularly, presumably through charge neutralization. To better understand the role of the pro peptide in the folding and functioning of alpha-defensins and/or pro alpha-defensins, we chemically attached the proHNP1 pro peptide or (wt)pro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg(10) (polyR), Ser(10) (polyS), and (cr)pro peptide, a charge-reversing mutant of the pro peptide where Arg/Lys residues were changed to Asp, and Asp/Glu residues to Lys. Comparative in vitro folding suggested that while all artificial pro segments chaperoned defensin folding, with PEG being the most efficient, the pro peptide catalyzed the folding of proHNPs likely through two independent mechanisms: solubilization of and interaction with the C-terminal defensin domain. Further, the N-terminal artificial pro segments dramatically altered the bactericidal activity of HNP1 against both Escherichia coli and Staphylococcus aureus. Surprisingly, (cr)pro peptide and (wt)pro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as alpha-defensin binding, although their binding modes appeared different. Our findings identify a dual chaperone activity of the pro peptide and may shed light on the molecular mechanisms by which pro alpha-defensins fold in vivo.

The proto-oncogene Mer tyrosine kinase is a novel therapeutic target in mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an incurable B cell-derived malignant tumor with a median overall survival of 4-5 years. Mer tyrosine kinase (MerTK) has been reported to be aberrantly expressed in leukemia, melanoma, and gastric cancer, and plays a pivotal role in the process of oncogenesis. This study assessed the role of MerTK in MCL for the first time. METHODS: Immunohistochemistry and western blot were performed to figure out expression of MerTK in MCL. MerTK inhibition by either shRNA or treatment with UNC2250 (a MerTK-selective small molecular inhibitor) was conducted in MCL cell lines. MCL-cell-derived xenograft models were established to evaluate the anti-tumor effects of UNC2250 in vivo. RESULTS: MerTK was ectopically expressed in four of six MCL cell lines. Sixty-five of 132 (48.9%) MCL patients showed positive expression of MerTK. MerTK inhibition by either shRNA or treatment with UNC2250 decreased activation of downstream AKT and p38, inhibited proliferation and invasion in MCL cells, and sensitized MCL cells to treatment with vincristine in vitro and doxorubicin in vitro and in vivo. UNC2250 induced G2/M phase arrest and prompted apoptosis in MCL cells, accompanied by increased expression of Bax, cleaved caspase 3 and poly (ADP-ribose) polymerase, and decreased expression of Bcl-2, Mcl-1 and Bcl-xL. Moreover, UNC2250 delayed disease progression in MCL-cell-derived xenograft models. CONCLUSIONS: Our data prove that ectopic MerTK may be a novel therapeutic target in MCL, and further pre-clinical or even clinical studies of UNC2250 or new MerTK inhibitors are essential and of great significance.

Structure-dependent functional properties of human defensin 5.

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins.

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.

Tuberculosis in an urban area in China: differences between urban migrants and local residents.

BACKGROUND: The increase in urban migrants is one of major challenges for tuberculosis control in China. The different characteristics of tuberculosis cases between urban migrants and local residents in China have not been investigated before. METHODOLOGY/PRINCIPAL FINDINGS: We performed a retrospective study of all pulmonary tuberculosis patients reported in Songjiang district, Shanghai, to determine the demographic, clinical and microbiological characteristics of tuberculosis cases between urban migrants and local residents. We calculated the odds ratios (OR) and performed multivariate logistic regression to identify the characteristics that were independently associated with tuberculosis among urban migrants. A total of 1,348 pulmonary tuberculosis cases were reported during 2006-2008, among whom 440 (32.6%) were local residents and 908 (67.4%) were urban migrants. Urban migrant (38.9/100,000 population) had higher tuberculosis rates than local residents (27.8/100,000 population), and the rates among persons younger than age 35 years were 3 times higher among urban migrants than among local residents. Younger age (adjusted OR per additional year at risk = 0.92, 95% CI: 0.91-0.94, p<0.001), poor treatment outcome (adjusted OR = 4.12, 95% CI: 2.65-5.72, p<0.001), and lower frequency of any comorbidity at diagnosis (adjusted OR = 0.20, 95% CI: 0.13-0.26, p = 0.013) were significantly associated with tuberculosis patients among urban migrants. There were poor treatment outcomes among urban migrants, mainly from transfers to another jurisdiction (19.3% of all tuberculosis patients among urban migrants). CONCLUSIONS/SIGNIFICANCE: A considerable proportion of tuberculosis cases in Songjiang district, China, during 2006-2008 occurred among urban migrants. Our findings highlight the need to develop and implement specific tuberculosis control strategies for urban migrants, such as more exhaustive case finding, improved case management and follow-up, and use of directly observed therapy (DOT).

Total chemical synthesis of human T-cell leukemia virus type 1 protease via native chemical ligation.

Human T-cell leukemia virus 1 (HTLV-1) protease, a member of the aspartic acid protease family, plays critical roles in the pathogenesis of the virus and is an attractive viral target for therapeutic intervention. HTLV-1 protease consists of 125 amino acid residues and functions as a homodimer stabilized in part by a four-stranded beta-sheet comprising the N- and C-termini. Compared with many other viral proteases such as HIV-1 protease, HTLV-1 protease is elongated by an extra 10 amino acid residue "tail" at the C-terminus. The structural and functional role of the extra C-terminal residues in the catalysis of HTLV-1 protease has been a subject of debate for years. Using the native chemical ligation technique pioneered by Kent and coworkers, we chemically synthesized a full-length HTLV protease and a C-terminally truncated form encompassing residues 1-116. Enzyme kinetic analysis using three different peptide substrates indicated that truncation of the C-terminal tail lowered the turnover number of the viral enzyme by a factor of 2 and its catalytic efficiency by roughly 10-fold. Our findings differ from the two extreme views that the C-terminal tail of HTLV-1 protease is either fully dispensable or totally required for enzyme dimerization and/or catalysis.

Development and characterization of monoclonal antibodies against mouse TSLP.

Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that is mainly produced by epithelial cells. It has been shown to play a key role in the development of Th2-type allergic inflammation. Commercial antibodies available against TSLP can only be used in Western blot assay, which further limits investigation of its function. Here we efficiently generated a panel of anti-mouse TSLP monoclonal antibodies in rats with DNA priming-protein boosting strategy. Overall, four MAb strains (4B12, 4E11, 5D7, and 6H10) were obtained and their characterizations were identified. The MAbs can specifically bind to TSLP according to the ELISA and FACS assays. It was found that they recognized distinct epitopes. They are useful in detecting TSLP expression in the tissue by immunoblotting and in the cytoplasm by intracellular staining assay. Thus, these antibodies will be valuable tools for studying TSLP biological functions.

Identification of karyopherin-alpha 2 as an Oct4 associated protein.

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain (containing POU domain and homeodomain) of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopherin-alpha 2 (KPNA-2). KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal (NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

Defining the native disulfide topology in the somatomedin B domain of human vitronectin.

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).

Human defensins: synthesis and structural properties.

Defensins are small, beta-sheet-rich, cationic peptides found in many organisms. All defensins have amphiphilic properties, which are central for antimicrobial activities of the proteins. The human genome encodes many defensin-line molecules, of which 10 have been characterized. Molecules of all known human defensins are stabilized by three intramolecular disulfide bonds arranged in a conserved pattern. To date, studies of human defensins indicate that these proteins are involved in various biological processes associated primarily with defensive and regulatory responses to infections by pathological agents. A comprehensive understanding of the multiple roles played by defensins within the immune system is greatly increased by reviewing the results of detailed structural studies. Emerging structural data, derived by the X-ray crystallography and the NMR spectroscopy in solution combined with functional studies; allow a rational understanding of the different activities of defensins and the mechanisms controlling these activities. Due to their well-established antimicrobial properties, defensins are also being investigated for their potential as therapeutics agents. Recently, increased effort has been focused on studies of the immunoregulatory properties of defensins, associated with their ability to bind and activate the G(i)-protein-coupled seven-transmembrane receptors. Comprehensive studies of defensins require development of simple, efficient, and inexpensive methods to generate these proteins and their derivatives in correctly folded form. This review highlights the current status of the sample generation methods, structural studies, and the structure-function relationships for human defensins.

Total chemical synthesis of human psoriasin by native chemical ligation.

Human psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is richly expressed in keratinocytes of patients suffering from psoriasis. To date, the exact physiological function of psoriasin abundant in many human cell types remains unclear. A recent report by Schröder and colleagues suggests that psoriasin, purified from human stratum corneum extracts, selectively kills Escherichia coli by sequestering Zn(2+) ions essential for bacterial growth, indicative of an important role in innate immune defense against microbial infection. We chemically synthesized the N-terminally acetylated psoriasin of 100 amino acid residues using solid phase peptide synthesis in combination with native chemical ligation. More than 140 mg of highly pure and correctly folded synthetic psoriasin was obtained from a single synthesis on a 0.25 mmol scale. Analysis of synthetic psoriasin by size exclusion chromatography showed that the protein forms a homodimer in solution. Circular dichroism analysis indicated that the alpha-helicity of psoriasin increases by more than 20% in the presence of CaCl(2) or ZnCl(2), suggesting a metal ion binding induced conformational change. Circular dichroism based titration further established that the synthetic protein binds two Ca(2+) and two Zn(2+) ions per dimer, in agreement with the published structural findings. Importantly, the ability of the synthetic protein to kill E. coli and the inhibition of the killing by ZnCl(2) is comparable to that of psoriasin isolated from its natural source. The robust synthetic access to large quantities of human psoriasin should facilitate studies of its biological functions as well as its mode of action.