Ye-Jin Lee

Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB.

BACKGROUNDnThe exact role of neutrophils in the pathogenesis of TB is poorly understood. Recent evidence suggests that neutrophils are not simply scavenging phagocytes in Mycobacterium tuberculosis (Mtb) infection.nnnMETHODSnThree different types of clinical specimens from patients with active pulmonary TB who underwent lung surgery were examined: sputum, BAL fluid, and cavity contents. Differential cell separation and quantification were performed for intracellular and extracellular bacteria, and bacterial length was measured using microscopy.nnnRESULTSnNeutrophils were more abundant than macrophages in sputum (86.6% +/- 2.2% vs 8.4% +/- 1.3%) and in BAL fluid (78.8% +/- 5.8% vs 11.8% +/- 4.1%). Inside the cavity, lymphocytes (41.3% +/- 11.2%) were the most abundant cell type, followed by neutrophils (38.8% +/- 9.4%) and macrophages (19.5% +/- 7.5%). More intracellular bacilli were found in neutrophils than macrophages in sputum (67.6% +/- 5.6% vs 25.2% +/- 6.5%), in BAL fluid (65.1% +/- 14.4% vs 28.3% +/- 11.6%), and in cavities (61.8% +/- 13.3% vs 23.9% +/- 9.3%). The lengths of Mtb were shortest in cavities (1.9+/- 0.1 microm), followed by in sputum (2.9 +/- 0.1 microm) and in BAL fluid (3.6 +/- 0.2 microm).nnnCONCLUSIONSnOur results show that neutrophils are the predominant cell types infected with Mtb in patients with TB and that these intracellular bacteria appear to replicate rapidly. These results are consistent with a role for neutrophils in providing a permissive site for a final burst of active replication of the bacilli prior to transmission.

Generation of donor natural killer cells from CD34 + progenitor cells and subsequent infusion after HLA-mismatched allogeneic hematopoietic cell transplantation: a feasibility study

Post transplant infusion of donor-type natural killer (NK) cells has been shown to have an anti-leukemia-enhancing effect without evoking GVHD in murine hematopoietic cell transplantation (HCT) models. Here, we tested 14 patients (age, 23–65 years), 12 with acute leukemia and 2 with myelodysplastic syndrome, who underwent HLA-mismatched HCT and subsequently received donor NK cell infusions. Cell donors (age, 16–51 years), comprising seven siblings, five offspring, and two mothers of the patients, underwent growth factor-mobilized leukapheresis for 3–5 days. Cells collected on the first 2–4 days were used for HCT, whereas those collected on the last day were CD34 selected by magnetic-activated cell sorting (median, 2.22 × 106 cells/kg; range, 0.29–5.66). Donor NK cells were generated from the CD34+ cells by ex vivo cell culture over a 6-week period (median, 9.28 × 106 cells/kg; range, 0.33–24.50; CD122/CD56+ 64%; CD3+ 1.0%; and viability 88%). There were no signs of acute toxicity in patients infused with these cells 6–7 weeks post transplant. Overall, one and five patients developed acute and chronic GVHD during post transplant period, respectively. These results showed that clinical-grade donor NK cell production from CD34+ cells is feasible.

Association of Antigen-Stimulated Release of Tumor Necrosis Factor-Alpha in Whole Blood with Response to Chemotherapy in Patients with Pulmonary Multidrug-Resistant Tuberculosis

Background: We have previously reported that TNF-α levels correlate to total mycobacterial burden in tuberculosis (TB) patients. Objective: To characterize the dynamics of cytokine responses in TB patients during chemotherapy to identify potential surrogate markers for effective treatment. Methods: Following induction by culture filtrate proteins in whole blood, production patterns of TNF-α, IL-10, IFN-γ and IL-12 were measured in 23 non-multidrug-resistant (MDR)-TB and 16 MDR-TB patients and in 31 healthy controls. Rates of mycobacterial clearance from the sputum were then measured and compared. Results: Prior to the initiation of chemotherapy, TNF-α and IL-10 levels were significantly higher in TB patients than in healthy controls while IFN-γ and IL-12 levels were similar. During chemotherapy, the levels of all 4 cytokines increased. We evaluated these responses separately in patients that did and did not clear their sputum culture at 2 and 6 months. At 2 months, decreases in both IFN-γ and IL-12 correlated strongly with a successful early response, while after 6 months of therapy, when half (7/14) of MDR-TB patients were still sputum culture positive, downregulation of TNF-α was uniquely correlated with sputum conversion between the groups. Conclusion: Our findings suggest the possibility that the regulation of TNF-α production in whole blood may be a more specific indicator of sputum conversion at 6 months than IFN-γ, IL-12 or IL-10 in MDR-TB patients.

Recent advances in mass spectrometry-based proteomics of gastric cancer.

The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-and-coming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.

Anti-mycobacterial Effects of the Extract of Humulus japonicus

Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of HealthAbstract The present study aimed to evaluate the in vitro antimycobacterial effects of hop plant, Humulus japonicus.Methanol extract of H. japonicus (MeOH extract) showed strong direct bactericidal effects against Mycobacteriumtuberculosis in vitro. Furthermore, the MeOH extract significantly inhibited M. tuberculosis growth in human macrophages.When five fractions obtained from MeOH extract were examined using the same methods, the hexane and ethyl acetatefractions showed bactericidal effects against M. tuberculosis in vitro, whereas the butanol and water fractions inhibited M.tuberculosis growth in macrophages. Because H. japonicus extract exhibited antimycobacterial activity against both free M.tuberculosis in culture medium and intracellular M. tuberculosis in human macrophages, this plant might be a goodcandidate for development of a new anti-tuberculosis drug.Keywords: Humulus japonicus, tuberculosis, bactericidal effect, macrophageThe present study aimed to evaluate the in vitro antimycobacterial effects of hop plant, Humulus japonicus. Methanol extract of H. japonicus (MeOH extract) showed strong direct bactericidal effects against Mycobacterium tuberculosis in vitro. Furthermore, the MeOH extract significantly inhibited M. tuberculosis growth in human macrophages. When five fractions obtained from MeOH extract were examined using the same methods, the hexane and ethyl acetate fractions showed bactericidal effects against M. tuberculosis in vitro, whereas the butanol and water fractions inhibited M. tuberculosis growth in macrophages. Because H. japonicus extract exhibited antimycobacterial activity against both free M. tuberculosis in culture medium and intracellular M. tuberculosis in human macrophages, this plant might be a good candidate for development of a new anti-tuberculosis drug.

Sensitivity of three widely used questionnaires for measuring psychological distress among patients with type 2 diabetes mellitus

BackgroundAlthough a range of generic and diabetes-specific instruments are available to assess emotional distress, no studies have evaluated sensitivity in relation to sample size requirement. The present study sets out to compare the sensitivity among the Diabetes Health Profile Psychological Distress scale (DHP-PD), Problem Areas in Diabetes (PAID) and Kessler-10 Psychological Distress scale (K10). We hypothesized that the diabetes-specific measures (DHP-PD and PAID) would require smaller sample sizes than the generic measure (K10), yet remain specific.Research designA total of 208 patients with type 2 diabetes mellitus (mean age 45.2 (12.4) years; 63.1xa0% males, 45.8xa0% Chinese, 11.3xa0% Malay and 26.6xa0% Indian), recruited from a Singaporexa0tertiary hospital diabetes clinic, completed the English DHP-PD, PAID and K10. Clinical information derived from medical records and HbA1c was recorded. Effect sizes (ES), ratio of ES and sample size requirement relative to the most sensitive questionnaire were computed.ResultsA comparison of patients with good versus poor glycaemic control (HbA1c ≥7.0) revealed that using K10 will require 4 times the sample size of a study using the PAID in order to detect the same level of psychological distress. The DHP-PD and PAID had similar sensitivity when comparing between patients with good versus poor glycaemic control.ConclusionsAs hypothesized, sample size requirement is largest for K10 and remarkably similar for PAID and DHP-PD. This information is useful for designing clinical trials and studies.

Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function.

Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. Here, we employ a single-molecule fluorescence approach to characterize nucleosome remodeling by yeast CHD1 (Chd1p). We show that Chd1p translocates nucleosomes in steps of multiple base pairs per ATP. ATP binding to Chd1p induces a transient unwrapping of the exit-side DNA, and facilitates nucleosome translocation. ATP hydrolysis induces nucleosome translocation, which is followed by the rewrapping upon the release of the hydrolyzed nucleotide. Multiple Chd1ps binding to a single nucleosome sequentially moves a histone octamer with a preference to the center of DNA fragments, suggesting a new mechanism for regularly spaced nucleosome generation by Chd1p. Our results reveal the unique mechanism by which Chd1p remodels nucleosomes. One Sentence Summary Yeast CHD1 uses one ATP to unwrap and translocate a nucleosome by multiple base pairs.

ANKRD9 is associated with tumor suppression as a substrate receptor subunit of ubiquitin ligase

BACKGROUNDnHuman ANKRD9 (ankyrin repeat domain 9) expression is altered in some cancers.nnnMETHODSnWe tested genetic association of ANKRD9 with gastric cancer susceptibility and examined functional association of ANKRD9 with altered proliferation of MKN45 gastric cancer cells. We then identified ANKRD9-binding partners in HEK 293 embryonic kidney cells using quantitative proteomics, western blotting and complex reconstitution assays. We finally demonstrated ANKRD9s role of recognizing substrates for ubiquitination using in vitro ubiquitylation assay.nnnRESULTSnANKRD9 is associated with cancer susceptibility in a comparison of single-nucleotide polymorphisms between 1092 gastric cancer patients and 1206 healthy controls. ANKRD9 depletion accelerates tumor progression by increasing cellular proliferation, piling up, and anchorage-independent growth of MKN45 cells. We discovered that ANKRD9 is a ubiquitin ligase substrate receptor subunit and has an anti-proliferative activity. ANKRD9 associates with CUL5 (not CUL2), ELOB, ELOC, and presumably RNF7 subunits, which together assemble into a cullin-RING superfamily E3 ligase complex. ANKRD9 belongs to the ASB family of proteins, which are characterized by the presence of ankyrin repeats and a SOCS box. In addition to its interactions with the other E3 ligase subunits, ANKRD9 interacts with two isoforms of inosine monophosphate dehydrogenase (IMPDH). These IMPDH isoforms are cognate substrates of the ANKRD9-containing E3 enzyme, which ubiquitinates them for proteasomal degradation. Their ubiquitination and turnover require the presence of ANKRD9.nnnCONCLUSIONnANKRD9, a previously unidentified E3 substrate receptor subunit, functions in tumor suppression by recognizing the oncoprotein IMPDH isoforms for E3 ubiquitination and proteasomal degradation.

PDMS Sylgard 527 Based Freely Suspended Ultrathin Membranes Exhibiting Mechanistic Characteristics of Vascular Basement Membranes

In the past, significant effort has been made to develop ultrathin membranes exhibiting physiologically relevant mechanical properties, such as thickness and elasticity of native basement membranes. However, most of these fabricated membranes have a relatively high elastic modulus, ∼MPa-GPa, relevant only to retinal and epithelial basement membranes. Vascular basement membranes exhibiting relatively low elastic modulus, ∼kPa, on the contrary, have seldom been mimicked. Membranes demonstrating high compliance, with moduli ranging in ∼kPa along with sub-microscale thicknesses have rarely been reported, and would be ideal to mimic vascular basement membranes in vitro. To address this, we fabricate ultrathin membranes demonstrating the mechanistic features exhibited by their vascular biological counterparts. Salient features of the fabricated ultrathin membranes include free suspension, physiologically relevant thickness ∼sub-micrometers, relatively low modulus ∼kPa, and sufficiently large culture area ∼20 mm2. To fabricate such ultrathin membranes, undiluted PDMS Sylgard 527 was utilized as opposed to the conventional diluted polymer-solvent mixture approach. In addition, the necessity to have a sacrificial layer for releasing membranes from the underlying substrates was also eliminated in our approach. The novelty of our work lies in achieving the distinct combination of membranes having thickness in sub-micrometers and the associated elasticity in kilopascal using undiluted polymer, which past approaches with dilution have not been able to accomplish. The ultrathin membranes with average thickness of 972 nm (thick) and 570 nm (thin) were estimated to have an elastic modulus of 45 and 214 kPa, respectively. Contact angle measurements revealed the ultrathin membranes exhibited hybrophobic characteristics in unpeeled state and transformed to hydrophilic behavior when freely suspended. Human umbilical vein endothelial cells were cultured on the polymeric ultrathin membranes, and the temporal cell response to change in local compliance of the membranes was studied by evaluating the cell spread area, density, percentage area coverage, and spread rate. After 24 h, single cells, pairs, and group of three to four cells were noticed on highly compliant thick membranes, having average thickness of 972 nm and modulus of 45 kPa. On the contrary, the cell monolayer was noted on the glass slide acting as a control. For the thin membranes featuring average thickness of 570 nm and modulus of 214 kPa, the cells tend to exhibit response similar to that on control with initiation of monolayer formation. Our results indicate, the local compliance, in turn, the membrane thickness governs the cell behavior and this can have vital implications during disease initiation and progression, wound healing, and cancer cell metastasis.