Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp.

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

Design and Synthesis of a Cell-Permeable,Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

Background

Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.

Methodology/Principal Findings

In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.

Conclusion/Significance

We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.     

Association of ischemic stroke onset time with presenting severity,acute progression,and long-term outcome: A cohort study

BackgroundPreclinical data suggest circadian variation in ischemic stroke progression, with more active cell death and infarct growth in rodent models with inactive phase (daytime) than active phase (nighttime) stroke onset. We aimed to examine the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in human ischemic stroke.Methods and findingsIn a Korean nationwide multicenter observational cohort study from May 2011 to July 2020, we assessed circadian effects on initial stroke severity (National Institutes of Health Stroke Scale [NIHSS] score at admission), END, and favorable functional outcome (3-month modified Rankin Scale [mRS] score 0 to 2 versus 3 to 6). We included 17,461 consecutive patients with witnessed ischemic stroke within 6 hours of onset. Stroke onset time was divided into 2 groups (day-onset [06:00 to 18:00] versus night-onset [18:00 to 06:00]) and into 6 groups by 4-hour intervals. We used mixed-effects ordered or logistic regression models while accounting for clustering by hospitals. Mean age was 66.9 (SD 13.4) years, and 6,900 (39.5%) were women. END occurred in 2,219 (12.7%) patients. After adjusting for covariates including age, sex, previous stroke, prestroke mRS score, admission NIHSS score, hypertension, diabetes, hyperlipidemia, smoking, atrial fibrillation, prestroke antiplatelet use, prestroke statin use, revascularization, season of stroke onset, and time from onset to hospital arrival, night-onset stroke was more prone to END (adjusted incidence 14.4% versus 12.8%, p = 0.006) and had a lower likelihood of favorable outcome (adjusted odds ratio, 0.88 [95% CI, 0.79 to 0.98]; p = 0.03) compared with day-onset stroke. When stroke onset times were grouped by 4-hour intervals, a monotonic gradient in presenting NIHSS score was noted, rising from a nadir in 06:00 to 10:00 to a peak in 02:00 to 06:00. The 18:00 to 22:00 and 22:00 to 02:00 onset stroke patients were more likely to experience END than the 06:00 to 10:00 onset stroke patients. At 3 months, there was a monotonic gradient in the rate of favorable functional outcome, falling from a peak at 06:00 to 10:00 to a nadir at 22:00 to 02:00. Study limitations include the lack of information on sleep disorders and patient work/activity schedules.ConclusionsNight-onset strokes, compared with day-onset strokes, are associated with higher presenting neurologic severity, more frequent END, and worse 3-month functional outcome. These findings suggest that circadian time of onset is an important additional variable for inclusion in epidemiologic natural history studies and in treatment trials of neuroprotective and reperfusion agents for acute ischemic stroke.

Wi-Sun Ryu and colleagues investigate the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in ischemic stroke.     

Changes in the Pulmonary Function Test after Radioactive Iodine Treatment in Patients with Pulmonary Metastases of Differentiated Thyroid Cancer

ObjectivePulmonary function test (PFT) is a useful tool for an objective assessment of respiratory function. Impaired pulmonary function is critical for the survival and quality of life in patients with pulmonary metastases of solid cancers including thyroid cancer. This study aimed to evaluate clinical factors associated with severely impaired pulmonary function by serial assessment with PFT in patients with pulmonary metastasis of differentiated thyroid cancer (DTC) who received radioactive iodine treatment (RAIT).PatientsThis retrospective study enrolled 31 patients who underwent serial PFTs before and after RAIT for pulmonary metastasis of DTC. We evaluated the risk factors for severe impairment of pulmonary function.ResultsThe median age of the patients was 44.1 years and 18 of them were female patients. Severe impairment of pulmonary function was observed in five patients (16%) after a median of three RAITs (cumulative I-131 activity = 20.4 GBq). These patients were older and more frequently had mild impairment of baseline pulmonary function, respiratory symptoms, or progressive disease compared with patients with stable pulmonary function. Neither cumulative dose nor number of RAIT was associated with decreased pulmonary function. Coexisting pulmonary diseases, presence of respiratory symptoms, and metastatic disease progression were significantly associated with severe decrease in forced vital capacity during follow-up (p =.047, p =.011, and p =.021, respectively).ConclusionsPulmonary function was severely impaired during follow-up in some patients with pulmonary metastasis of DTC after a high-dose RAITs. Neither the number of RAIT nor the cumulative I-131 activity was associated with decreased pulmonary function. Serial PFT might be considered for some high-risk patients during follow-up.     

Clinical Correlates of Mass Effect in Autosomal Dominant Polycystic Kidney Disease

Mass effect from polycystic kidney and liver enlargement can result in significant clinical complications and symptoms in autosomal dominant polycystic kidney disease (ADPKD). In this single-center study, we examined the correlation of height-adjusted total liver volume (htTLV) and total kidney volume (htTKV) by CT imaging with hepatic complications (n = 461) and abdominal symptoms (n = 253) in patients with ADPKD. “Mass-effect” complications were assessed by review of medical records and abdominal symptoms, by a standardized research questionnaire. Overall, 91.8% of patients had 4 or more liver cysts on CT scans. Polycystic liver disease (PLD) was classified as none or mild (htTLV < 1,600 mL/m); moderate (1,600 ≤ htTLV <3,200 mL/m); and severe (htTLV ≥ 3,200 mL/m). The prevalence of moderate and severe PLD in our patient cohort was 11.7% (n = 54/461) and 4.8% (n = 22/461), respectively, with a female predominance in both the moderate (61.1%) and severe (95.5%) PLD groups. Pressure-related complications such as leg edema (20.4%), ascites (16.6%), and hernia (3.6%) were common, and patients with moderate to severe PLD exhibited a 6-fold increased risk (compared to no or mild PLD) for these complications in multivariate analysis. Similarly, abdominal symptoms including back pain (58.8%), flank pain (53.1%), abdominal fullness (46.5%), and dyspnea/chest-discomfort (44.3%) were very common, and patients with moderate to severe PLD exhibited a 5-fold increased risk for these symptoms. Moderate to severe PLD is a common and clinically important problem in ~16% of patients with ADPKD who may benefit from referral to specialized centers for further management.