Outbred Mice with Streptozotocin-Induced Diabetes Show Sex Differences in Glucose Metabolism.

Outbred mice (ICR) with different genotypes and phenotypes have been reported to be more suitable for scientific testing than inbred mice because they are more similar to humans. To investigate whether the sex and genetic background of the mice are important factors in the development of hyperglycemia, we used ICR mice and divided them into male, female, and ovariectomized female (FOVX) groups and treated them with streptozotocin (STZ) for five consecutive days to induce diabetes. Our results show that fasting blood glucose and hemoglobin A1c (HbA1c) levels were significantly higher in diabetes-induced males (M-DM) and ovariectomized diabetes-induced females (FOVX-DM) than in diabetes-induced females (F-DM) at 3 and 6 weeks after STZ treatment. Furthermore, the M-DM group showed the most severe glucose tolerance, followed by the FOVX-DM and F-DM groups, suggesting that ovariectomy affects glucose tolerance in female mice. The size of pancreatic islets in the M-DM and FOVX-DM groups was significantly different from that of the F-DM group. The M-DM and FOVX-DM groups had pancreatic beta-cell dysfunction 6 weeks after STZ treatment. Urocortin 3 and somatostatin inhibited insulin secretion in the M-DM and FOVX-DM groups. Overall, our results suggest that glucose metabolism in mice is dependent on sex and/or genetic background.

One-Pot Synthesis of 1,3,4-Oxadiazines from Acylhydrazides and Allenoates.

The framework of 1,3,4-oxadiazine is crucial for numerous bioactive molecules, but only a limited number of synthetic methods have been reported for its production. In 2015, Wang's group developed a 4-(dimethylamino)pyridine (DMAP)-catalyzed [2 + 4] cycloaddition of allenoates with N-acyldiazenes, which provided an atom-efficient route for 1,3,4-oxadiazines. However, the practicality of this method was limited by the instability of N-acyldiazenes as starting materials. Building upon our ongoing research about the aerobic oxidation of hydrazides and their synthetic applications, we hypothesized that aerobic oxidative cycloadditions using acylhydrazides instead of N-acyldiazenes may provide a more practical synthetic route for 1,3,4-oxadiazines. In this manuscript, we describe a one-pot synthetic protocol for 1,3,4-oxadiazines from acylhydrazides and allenoates. The developed one-pot protocol consists of aerobic oxidations of acylhydrazides into N-acyldiazenes using NaNO2 and HNO3, followed by the DMAP-catalyzed cycloaddition of allenoate with the generated N-acyldiazenes. A variety of 1,3,4-oxadiazines were produced in good to high yields. In addition, the practicality of the developed method was demonstrated by a gram-scale synthesis of 1,3,4-oxadiazine.

Prenylated Rab acceptor RABAC1 inhibits anti-apoptotic protein BCL2A1 and induces apoptosis.

The B cell lymphoma 2 (BCL2) family of proteins constitutes a critical intracellular checkpoint in the intrinsic apoptosis pathway. Among BCL2 members, the anti-apoptotic protein BCL2A1 mediates the resistance to BCL2 inhibitors and may be considered as a target for anti-cancer therapy. Here, we report that prenylated Rab acceptor 1 (RABAC1 or PRA1) inhibits the anti-apoptotic activity of BCL2A1 and induces apoptosis in AGS gastric cancer cells. Protein interaction of BCL2A1 and RABAC1 was verified by an in-vitro glutathione-S-transferase pull-down assay, immunoprecipitation, and confocal microscopy. When apoptosis was induced by cisplatin, the anti-apoptotic activity of BCL2A1 was blocked by RABAC1 expression. RABAC1 caused caspase-3 activation and decreased cell proliferation, clonogenic cell survival, and cell migration and invasion. We suggest RABAC1 as a potential therapeutic target for BCL2A1-related cancer.

A Systemic Immune Inflammation Index and PD-L1 (SP142) Expression as a Potential Combined Biomarker of the Clinical Benefit of Chemo-Immunotherapy in Extensive-Stage Small-Cell Lung Cancer.

Background: No studies have identified combined biomarkers that may be more reasonable for the assessment of current chemo-immunotherapy in patients with extensive stage small-cell lung cancer (ES-SCLC). Methods: This study was conducted to investigate a combined biomarker with prognostic or predictive value in ES-SCLC. We determined the best independent prognostic biomarker among the four complete blood-count-derived inflammatory biomarkers (CBC-IBs). Subsequently, we analyzed the prognostic or predictive value of combining this independent CBC-IB with PD-L1 (SP142) expression. We prospectively assessed the SP142 analyses in tumor samples at diagnosis. Results: All in all, 55 patients with ES-SCLC were classified into four groups according to the systemic immune inflammation index (SII) (low/high) and SP142 (positive/negative). The best survival was observed in the low-SII/ SP142-positive group, whereas the worst survival was observed in the high-SII/SP142-negative group (p = 0.002). The combined SII-SP142 biomarker was better for predicting both survival and disease progression in patients with ES-SCLC. Conclusions: The combined SII-SP142 biomarker can be readily and universally obtained at a low cost in clinical practice, without requiring advanced genomics technology or specialized expertise. Although further studies are needed to confirm that the combined SII-SP142 biomarker is widely applicable, it should help clinicians to identify the best patients for combined chemotherapy with atezolizumab in ES-SCLC.

Long-term cardiovascular outcome in women with preeclampsia in Korea: a large population-based cohort study and meta-analysis.

Recent studies reported the long-term cardiovascular risk of preeclampsia. However, only a few studies have investigated the association between preeclampsia and long-term cardiovascular disease in Asian populations, although there could be racial/ethnic differences in the risk of cardiovascular diseases. Therefore, we aimed to evaluate the long-term effects of preeclampsia on cardiovascular disease in an Asian population. This study included 68,658 parous women in the Health Examinees Study (HEXA) cohort of South Korea and compared the risk of long-term cardiovascular disease, including ischemic heart disease and stroke, according to the history of preeclampsia. We also performed a meta-analysis combining current study data with data from existing literature in the Asian population. Among the study population, 3413 (5.23%) women had a history of preeclampsia, and 767 (1.12%) and 404 (0.59%) women developed ischemic heart disease and stroke for 22 years. Women with a history of preeclampsia were at a higher risk for both ischemic heart disease (adjusted hazard ratio 1.66 [1.19-2.04]) and stroke (adjusted hazard ratio 1.48 [1.02-2.16]) than those without. In the meta-analysis, the pooled hazard ratio of ischemic heart disease and stroke were also increased in women with a history of preeclampsia (ischemic heart disease 1.65 [1.51-1.82]; stroke 1.78 [1.52-2.10]).

Comparison of bolus administration effects of lidocaine on preventing tourniquet-induced hypertension in patients undergoing general anesthesia: a randomized controlled trial.

BACKGROUND: This study assessed the effect of a single bolus administration of lidocaine on the prevention of tourniquet-induced hypertension (TIH) and compared the effect of lidocaine to that of ketamine in patients undergoing general anesthesia. METHODS: This randomized, controlled, double-blind study included 75 patients who underwent lower limb surgery using a tourniquet. The patients were administered lidocaine (1.5 mg/kg, n = 25), ketamine (0.2 mg/kg, n = 25) or placebo (n = 25). The study drugs were administered intravenously 10 min before tourniquet inflation. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were measured before tourniquet inflation, after tourniquet inflation for 60 min at 10 min intervals, and immediately after tourniquet deflation. The incidence of TIH, defined as an increase of 30% or more in SBP or DBP during tourniquet inflation, was also recorded. RESULTS: SBP, DBP, and HR increased significantly over time in the control group compared to those in the lidocaine and ketamine groups for 60 min after tourniquet inflation (P < 0.001, P < 0.001, and P = 0.007, respectively). The incidence of TIH was significantly lower in the lidocaine (n = 4, 16%) and ketamine (n = 3, 12%) group than in the control group (n = 14, 56%) (P = 0.001). CONCLUSION: Single-bolus lidocaine effectively attenuated blood pressure increase due to tourniquet inflation, with an effect comparable to that of bolus ketamine.

Elucidating the characteristics of Mx1 and resistance to influenza A virus subtype H1N1 in the newly developed KWM/Hym mice.

BACKGROUND: Inbred mice have several advantages, including genetic similarity to humans, a well-established gene manipulation system, and strong tolerance to inbreeding. However, inbred mice derived from a limited genetic pool have a small genetic diversity. Thus, the development of new inbred strains from wild mice is needed to overcome this limitation. Hence, in this study, we used a new strain of inbred mice called KWM/Hym. We sequenced the Mx1 gene to elucidate the genetic diversities of KWM/Hym mice and observed the biological alterations of the Mx1 protein upon influenza A infection. RESULTS: The Mx1 gene in KWM/Hym mice had 2, 4, and 38 nucleotide substitutions compared to those in the Mx1 gene in A2G, CAST/EiJ, and Mus spretus mice, respectively. Moreover, the Mx1 protein in KWM/Hym mice had 2 and 25 amino acid substitutions compared to those in the Mx1 protein in CAST/EiJ and M. spretus mice, respectively. To elucidate the function of the Mx1 protein, we inoculated the influenza A virus (A/WSN/1933) in KWM/Hym mice. Nine days after infection, all infected KWM/Hym mice survived without any weight loss. Four days after infection, the lungs of the infected KWM/Hym mice showed mild alveolitis and loss of bronchiolar epithelium; however, the pulmonary viral titers of the infected KWM/Hym mice were significantly lower than that in the infected BALB/c mice (2.17 × plaque-forming units mL-1). CONCLUSIONS: Our results demonstrate that the KWM/Hym mice are resistant to influenza A virus infection. Further, these mice can be used as a model organism to understand the mechanism of influenza A virus susceptibility.

miR-302 Suppresses the Proliferation, Migration, and Invasion of Breast Cancer Cells by Downregulating ATAD2.

Breast cancer is the most common malignant tumor in women. The ATPase family AAA domain-containing protein 2 (ATAD2) contains an ATPase domain and a bromodomain, and is abnormally expressed in various human cancers, including breast cancer. However, the molecular mechanisms underlying the regulation of ATAD2 expression in breast cancer remain unclear. This study aimed to investigate the expression and function of ATAD2 in breast cancer. We found that ATAD2 was highly expressed in human breast cancer tissues and cell lines. ATAD2 depletion via RNA interference inhibited the proliferation, migration, and invasive ability of the SKBR3 and T47D breast cancer cell lines. Furthermore, Western blot analysis and luciferase assay results revealed that ATAD2 is a putative target of miR-302. Transfection with miR-302 mimics markedly reduced cell migration and invasion. These inhibitory effects of miR-302 were restored by ATAD2 overexpression. Moreover, miR-302 overexpression in SKBR3 and T47D cells suppressed tumor growth in the xenograft mouse model. However, ATAD2 overexpression rescued the decreased tumor growth seen after miR-302 overexpression. Our findings indicate that miR-302 plays a prominent role in inhibiting the cancer cell behavior associated with tumor progression by targeting ATAD2, and could thus be a valuable target for breast cancer therapy.

KLK6/PAR1 Axis Promotes Tumor Growth and Metastasis by Regulating Cross-Talk between Tumor Cells and Macrophages.

Kallikrein-related peptidase (KLK)6 is associated with inflammatory diseases and neoplastic progression. KLK6 is aberrantly expressed in several solid tumors and regulates cancer development, metastatic progression, and drug resistance. However, the function of KLK6 in the tumor microenvironment remains unclear. This study aimed to determine the role of KLK6 in the tumor microenvironment. Here, we uncovered the mechanism underlying KLK6-mediated cross-talk between cancer cells and macrophages. Compared with wild-type mice, KLK6-/- mice showed less tumor growth and metastasis in the B16F10 melanoma and Lewis lung carcinoma (LLC) xenograft model. Mechanistically, KLK6 promoted the secretion of tumor necrosis factor-alpha (TNF-α) from macrophages via the activation of protease-activated receptor-1 (PAR1) in an autocrine manner. TNF-α secreted from macrophages induced the release of the C-X-C motif chemokine ligand 1 (CXCL1) from melanoma and lung carcinoma cells in a paracrine manner. The introduction of recombinant KLK6 protein in KLK6-/- mice rescued the production of TNF-α and CXCL1, tumor growth, and metastasis. Inhibition of PAR1 activity suppressed these malignant phenotypes rescued by rKLK6 in vitro and in vivo. Our findings suggest that KLK6 functions as an important molecular link between macrophages and cancer cells during malignant progression, thereby providing opportunities for therapeutic intervention.

The selectivity of austocystin D arises from cell-line-specific drug activation by cytochrome P450 enzymes.

The natural product austocystin D was identified as a potent cytotoxic agent with in vivo antitumor activity and selectivity for cells expressing the multidrug resistance transporter MDR1. We sought to elucidate the mechanism of austocystin D's selective cytotoxic activity. Here we show that the selective cytotoxic action of austocystin D arises from its selective activation by cytochrome P450 (CYP) enzymes in specific cancer cell lines, leading to induction of DNA damage in cells and in vitro. The potency and selectivity of austocystin D is lost upon inhibition of CYP activation and does not require MDR1 expression or activity. Furthermore, the pattern of cytotoxicity of austocystin D was distinct from doxorubicin and etoposide and unlike aflatoxin B(1), a compound that resembles austocystin D and is also activated by CYP enzymes to induce DNA damage. Theses results suggest that austocystin D may be of clinical benefit for targeting or overcoming chemoresistance.

The influence of smoking and alcohol on bone healing: Systematic review and meta-analysis of non-pathological fractures.

BACKGROUND: We aimed to comprehensively evaluate the associations between (i) smoking, (ii) preoperative smoking cessation time, (iii) nicotine replacement therapy (NRT), (iv) vaping, and (v) alcohol consumption and non-pathological fracture healing in adult patients. We also assessed the impacts of preoperative smoking cessation time, NRT, and vaping on wound healing and wound complications after any sort of surgery. METHODS: We searched the MEDLINE, Embase, Cochrane CENTRAL, CINAHL, and AMED electronic databases from their inceptions until August 9th, 2021. Primary outcomes included delayed union rate, nonunion rate, and time to union. A random effects model was used. (Protocol registration: PROSPERO-CRD42019131454). FINDINGS: One hundred and twenty-two studies with 417,767 patients were eligible for the systematic review and 71 of the studies with 39,920 patients were eligible for the meta-analysis. After non-pathological fracture treatment, the nonunion rate was significantly greater in the smoker group than in the non-smoker group (odds ratio [OR], 2·50, 95% confidence interval [1·73-3·61]); additionally, there was no significant difference in the nonunion rate (OR, 0·97 [0·40-2·38]) between the alcohol drinker group and the non-drinker group. The rate of wound infection after surgery was significantly reduced in the smoking cessation group (≥four weeks before surgery) compared to the continuous smoker group (OR, 0·37 [0·16-0·89]). INTERPRETATION: Smoking is associated with higher rates of nonunion and deep surgical site infection after non-pathological fracture treatment. Smoking cessation (≥four weeks before surgery) is associated with a decreased rate of postoperative wound infection. FUNDING: The China Scholarship Council (no. 201809120013).

Collapsin response mediator protein 4 enhances the radiosensitivity of colon cancer cells through calcium­mediated cell signaling.

Radiation therapy is an effective treatment against various types of cancer, but some radiation­resistant cancer cells remain a major therapeutic obstacle; thus, understanding radiation resistance mechanisms is essential for cancer treatment. In this study, we established radiation­resistant colon cancer cell lines and examined the radiation­induced genetic changes associated with radiation resistance. Using RNA­sequencing analysis, collapsin response mediator protein 4 (CRMP4) was identified as the candidate gene associated with radiation sensitivity. When cells were exposed to radiation, intracellular Ca2+ influx, collapse of mitochondrial membrane potential, and cytochrome c release into the cytosol were increased, followed by apoptosis induction. Radiation treatment­ or Ca2+ ionophore A23187­induced apoptosis was significantly inhibited in CRMP4­deficient cells, including radiation­resistant or CRMP4­shRNA cell lines. Furthermore, treatment of CRMP4­deficient cells with low levels (<5 µM) of BAPTA­AM, a Ca2+ chelator, resulted in radiation resistance. Conversely, Ca2+ deficiency induced by a high BAPTA­AM concentration (>10 µM) resulted in higher cell death in the CRMP4­depleted cells compared to CRMP4­expressing control cells. Our results suggest that CRMP4 plays an important role in Ca2+­mediated cell death pathways under radiation exposure and that CRMP4 may be a therapeutical target for colon cancer treatment.

Effectiveness of Non-Pharmacological Interventions for Overweight or Obese Infertile Women: A Systematic Review and Meta-Analysis.

Obesity is a well-known risk factor for infertility, and nonpharmacological treatments are recommended as effective and safe, but evidence is still lacking on whether nonpharmacological interventions improve fertility in overweight or obese women. The aim of this study was to systematically assess the current evidence in the literature and to evaluate the impact of nonpharmacological interventions on improving pregnancy-related outcomes in overweight or obese infertile women. Seven databases were searched for randomized controlled trials (RCTs) of nonpharmacological interventions for infertile women with overweight or obesity through August 16, 2019 with no language restriction. A meta-analysis was conducted of the primary outcomes. A total of 21 RCTs were selected and systematically reviewed. Compared to the control group, nonpharmacological interventions significantly increased the pregnancy rate (relative risk (RR), 1.37; 95% CI, 1.04-1.81; p = 0.03; I² = 58%; nine RCTs) and the natural conception rate (RR, 2.17, 95% CI, 1.41-3.34; p = 0.0004; I² = 19%, five RCTs). However, they had no significant effect on the live birth rate (RR, 1.36, 95% CI, 0.94-1.95; p=0.10, I² = 65%, eight RCTs) and increased the risk of miscarriage (RR: 1.57, 95% CI, 1.05-2.36; p = 0.03; I² = 0%). Therefore, nonpharmacological interventions could have a positive effect on the pregnancy and natural conception rates, whereas it is unclear whether they improve the live birth rate. Further research is needed to demonstrate the integrated effects of nonpharmacological interventions involving psychological outcomes, as well as pregnancy-related outcomes.

Maintenance dose of warfarin beyond time in therapeutic range for preventing vascular events.

BACKGROUND: The quality of anticoagulation is closely associated with efficacy and safety in warfarin users. Although genetic polymorphisms have been related to warfarin dosages and vascular events(VE), genetic evaluations have not been recommended for all warfarin users. The aim is to evaluate the significance of the maintenance dose of warfarin (MDW) on VE, considering the time in therapeutic range (TTR). METHODS: This retrospective study analyzed the data of patients who received warfarin for any reasons. A total of 11,835 patients with warfarin were divided into quartiles by MDW. We assessed TTR using the Rosendaal method and VE. RESULTS: VE occurred in 9.1% of the warfarin users. The mean TTR level was 34.0 ±â€¯25.7%, and the MDW was 3.38 ±â€¯1.06 mg per day. Patients with VE were more likely to have a lower MDW and lower TTR levels. In moderate- or well-controlled TTR status, a lower MDW was significantly related to under-controlled anticoagulation and associated with higher risks of VE. Lower MDW had a higher risk of stroke or arterial/venous thromboembolism (Q1: OR, 1.57; 95% CI 1.25 to 1.96; Q2: OR, 1.40; 95% CI 1.12 to 1.75; Q3: OR, 1.35; 95% CI 1.08 to 1.68). CONCLUSIONS: We suggest that patients with very low MDW might be at risk when using warfarin. Therefore, we propose that patients with a very low MDW might be alternatively considered for novel oral anticoagulants rather than warfarin.

Development of a HA1-specific enzyme-linked immunosorbent assay against pandemic influenza virus A H1N1.

PURPOSE: Enzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein. MATERIALS AND METHODS: rHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice. RESULTS: Our ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model. CONCLUSION: An ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.

Erastin Inhibits Septic Shock and Inflammatory Gene Expression via Suppression of the NF-κB Pathway.

Sepsis is a life-threatening condition that is caused by an abnormal immune response to infection and can lead to tissue damage, organ failure, and death. Erastin is a small molecule capable of initiating ferroptotic cell death in cancer cells. However, the function of erastin in the inflammatory response during sepsis remains unknown. Here, we showed that erastin ameliorates septic shock induced by cecal ligation and puncture or lipopolysaccharides (LPS) in mice, which was associated with a reduced production of inflammatory mediators such as nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. Pretreatment with erastin in bone marrow-derived macrophages (BMDMs) significantly attenuated the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß mRNA in response to LPS treatment. Furthermore, we also showed that erastin suppresses phosphorylation of IκB kinase ß, phosphorylation and degradation of IκBα, and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated BMDMs. Our findings suggest that erastin attenuates the inflammatory response by suppressing the NF-κB signaling pathway, resulting in inhibition of sepsis development. This study provides new insights regarding the potential therapeutic properties of erastin in sepsis.

Evaluation of Mineral Contents of Multi-Vitamin and Minerals Currently Sold in South Korea.

Dietary supplements are popular worldwide and their use has been increasing. The purpose of this study was to evaluate the mineral contents of multi-vitamins and minerals (MVMs) in most commonly sold among dietary supplements. Ninety popular MVM supplements sold in South Korea were surveyed regarding their characteristics and ingredients including minerals listed on the labels through off-line and on-line search. Daily mineral contents of the MVM supplements were compared with Korean Dietary Reference Intakes (DRIs) by target populations. The average price of 90 MVM supplements was $41.3 per bottle, with a price of $0.9 per day and the average number of minerals contained per supplement was 4.7. A total 14 minerals were found in the MVM supplements including calcium, phosphorus, sodium, potassium, and magnesium. Nine minerals (e.g. calcium, magnesium, and iron) were included in more than 30% of the MVM supplements examined. When daily mineral dose of MVMs was compared to DRIs, calcium was the lowest (34.0% of recommended intake [RI]) and chromium was the highest (218.7% of adequate intake [AI]), and zinc, copper, selenium, and chromium were also higher than their RI or AI levels. The daily mineral contents of the 90 MVM supplements were below the tolerable upper-intake level, but some minerals were higher than RI or AI with high variance among products. Therefore, there is a great need to educate the public for the adequate selection and use of MVM supplements based on the contents of MVM supplements and individual's mineral intake derived from the diet.

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.

Physical Activity Frequency and the Risk of Stroke: A Nationwide Cohort Study in Korea.

BACKGROUND: The current guideline recommends moderate- to vigorous-intensity physical activity (PA) at least 40 min/day for 3 to 4 days/week. Although recent evidence has demonstrated that low-dose PA could reduce cardiovascular mortality, the relationship between low-dose PA and the risk of stroke remains uncertain. METHODS AND RESULTS: Using data from a nation-wide sample cohort in Korea, we examined 336 326 individuals who received a general health examination between 2009 and 2010. Level of PA was assessed using a questionnaire for weekly PA frequencies regarding 3 intensity categories: light, moderate, and vigorous. Moderate- to vigorous-intensity PA (MVPA) was classified into 4 frequency categories: none, 1 to 2, 3 to 4, or ≥5 times/week. Cox proportional hazard models were constructed to estimate the risk of stroke. During the average follow-up of 3.6 years, 2213 stroke cases occurred. MVPA was none in 50%, 1 to 2 times/week in 20%, 3 to 4 times/week in 13%, and ≥5 times/week in 18% of the cohort. Individuals with MVPA 1 to 2 times/week had a 16% reduced risk of stroke (adjusted hazard ratio, 0.84; 95% confidence interval, 0.73-0.96) compared with those with no MVPA. The population attributable fraction of no MVPA was 12%, which was the second most important risk factor for a stroke after hypertension. CONCLUSIONS: Even 1 to 2 times a week of MVPA might be beneficial to prevent a first-ever stroke in the general population, although a quantitative validation of the questionnaire is needed. From a public health perspective, we need to encourage inactive people to start exercising with more-achievable goals.

No evidence for a forced-unfolding mechanism during ATP/GroES binding to substrate-bound GroEL: no observable protection of metastable Rubisco intermediate or GroEL-bound Rubisco from tritium exchange.

In tritium-hydrogen exchange experiments, the large GroEL substrate Rubisco was unfolded and exchanged in urea/acid/tritiated water, then diluted into either protic buffer or protic buffer containing GroEL. The respective Rubisco metastable folding intermediate or Rubisco-GroEL binary complex was then separated from residual tritium after varying times of exchange by centrifugation through P-10 or G-25 resin. No significant tritium was recovered in either case, in contrast to an earlier report. Thus, although the earlier-proposed forced unfolding mechanism for the action of GroEL on a bound polypeptide, occurring during ATP/GroES binding, remains an attractive hypothesis, the data here do not provide any indication that it is involved in the folding of Rubisco.