Disappearance of Imported Cases of Omicron Lineage BA.2.40 in West Kalimantan, Indonesia.

Background: The World Health Organization has declared Omicron as the fifth variant of concern with more than 50 mutations, particularly in the spike protein. Given increased viral infectivity due to mutations, worldwide genomic surveillance and detection of severe acute respiratory syndrome 2 (SARS-CoV-2) is essential. The present study aimed to track Omicron lineage BA.2.40 in West Kalimantan, Indonesia. Methods: In May-August 2022, nasopharyngeal swab samples (n=3,642) were collected from international travelers to West Kalimantan (active surveillance), and patients hospitalized due to SARS-CoV-2 infection (baseline surveillance). The samples were tested for Omicron lineages based on ORF1ab, N, and HV69-70del genes, followed by whole-genome sequencing. The sequences were then identified using two genomic databases, aligned against the reference genome (Wuhan/Hu-1/2019), and then compared with BA.2.40 lineage detected across the world. Phylogenetic analysis between the samples and other SARS-CoV-2 isolates was performed using molecular evolutionary genetics analysis software. Results: Based on the genomic databases, 10 isolates were identified as BA.2.40. All samples tested positive for the ORF1ab and N genes, but negative for the HV69-70del gene, which is a marker to detect the Omicron variant. Phylogenetic analysis showed the isolates were closely related to an isolate from Malaysia, an area dominated by BA.2.40. Conclusion: Omicron lineage BA.2.40 has no HV69-70 deletion in the spike protein, a marker used to screen for the Omicron variant. BA.2.40 showed a high similarity to an isolate from Malaysia and was detected only during certain periods, indicating the effect of internationally imported cases.

Rifampicin adsorption and release study using Santa Barbara amorphous-16 modified Al (SBA-16-Al) for a drug delivery system.

In this study, the surface modification of Santa Barbara Amorphous-16 (SBA-16) with aluminum (SBA-16-Al) was carried out as a rifampicin matrix for the treatment of tuberculosis. Surface modification of SBA-16 was achieved using the direct-synthesis grafting method. Then, the adsorption and release properties of rifampicin from the SBA-16-Al matrix have been studied in batches. In addition, the SBA-16-Al has been characterized using Fourier-Transform Infrared Spectroscopy (FTIR), X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), and Surface Area Analysis (SAA) Brunaur, Emmett and Teller (SAA-BET). The results show that the mesoporous material, the SBA-16-Al has a specific surface area of 843.5 m2 g-1 and 624.3 m2 g-1 for SBA-16, nanometer-sized pore diameters, and an amorphous crystal lattice. The FTIR spectra showed the Al-O bond at 802 cm-1 which indicates the Al group has been successfully added into SBA-16. The adsorption isotherm of rifampicin in SBA-16-Al follows the Freundlich model which illustrates the adsorption is heterogeneous and forms a multilayer. The adsorption of rifampicin is chemisorption which occurs non-spontaneously and is quite stable. The release kinetics of rifampicin in the drug delivery system followed the Higuchi model with k1 0.5472 mg 0.5/hour pH 1.5 and k2 mg 0.5/hour pH 6.5.

Evaluation of safety and effectiveness of remdesivir in treating COVID-19 patients after emergency use authorization study.

Background: This study aimed to determine the real-world safety and effectiveness of remdesivir in hospitalized adult COVID-19 patients with moderate-to-critical disease in Indonesia. Methods: A multicenter, retrospective cohort study was conducted at four COVID-19 referral hospitals in Jakarta. A total of 587 patients were included, of whom 243 received remdesivir within 72 h of admission. The safety endpoints were the proportions of patients with any adverse event (AE), any grade 3 AE, and AE of each system organ class. The effectiveness endpoints were ICU admission >24 h from baseline, live discharge and mortality at day 14, live discharge and mortality at day 28, and virologic conversion. Patients who received remdesivir within 72 h of admission were considered the treatment group, and those who did not were the control group. Multivariate adjustments were performed using a modified Poisson regression. Results: The study found no significant differences in safety endpoints between the two groups. However, the effectiveness endpoints showed that remdesivir was associated with a decreased risk of ICU admission >24 h from baseline (RR 0.71, 95% CI 0.52-0.96), an increased probability of live discharge at day 14 (RR 1.37, 95% CI 1.08-1.74), and an increased probability of live discharge at day 28 (RR 1.28, 95% CI 1.05-1.57). The rate of virologic conversion was not significantly different between the two groups. Conclusion: The study concludes that remdesivir is safe and effective in the treatment of moderate-to-critical COVID-19 in a real-world setting in Indonesia.

Association between cardiovascular diseases and COVID-19 pneumonia outcome in Indonesia: a multi-center cohort study.

Background: COVID-19 is a pandemic affecting 185 countries, including Indonesia. Cardiovascular diseases (CVD) in COVID-19 patients were linked to worse clinical outcomes. However, the association remained inconclusive due to limited data in Indonesia. This study aimed to determine the association between CVD in COVID-19 pneumonia patients with its clinical outcomes. Methods: This retrospective cohort study was conducted in four Indonesian hospitals, enrolling 584 adult COVID-19 pneumonia patients from September 2020 to July 2021. Patients were categorized into two groups: non-CVD and CVD [hypertension, coronary artery disease (CAD), chronic heart failure (CHF), hypertensive heart disease (HHD), arrhythmia, cardiomegaly, left ventricular hypertrophy (LVH), mitral regurgitation (MR), and myocardial injury (MI)]. Clinical outcomes include in-hospital mortality, intensive care unit admission, ventilator use, earlier death, and prolonged hospital stay. Mann-Whitney test was used for analysis. Results: The most common CVD was hypertension (48.1%), followed by MI (10.6%), CAD (9.2%), CHF (6.8%), HHD (3.1%), arrhythmia (1.7%), and others (0.7%). The in-hospital mortality rate was 24%, and patients were hospitalized for a median of 12 days. MI was the only CVD that increased in-hospital mortality (RR 2.105). It was also significantly increased in patients with diabetes mellitus (RR 1.475) and chronic kidney disease (RR 2.079). Meanwhile, prolonged hospital stay was associated with any CVD (RR 1.553), hypertension (RR 1.511), MI (RR 1.969), CHF (RR 1.595), diabetes mellitus (RR 1.359), and cerebrovascular disease (RR 2.203). Conclusion: COVID-19 pneumonia in patients with CVD, specifically MI and hypertension, worsens the COVID-19 clinical outcomes.

Twenty-four hour rhythmicity in mitochondrial network connectivity and mitochondrial respiration; a study in human skeletal muscle biopsies of young lean and older individuals with obesity.

OBJECTIVE: Mitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day-night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h METHODS: Muscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day RESULTS: Young healthy lean individuals displayed a day-night rhythmicity in mitochondrial network morphology, which aligned with the day-night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity CONCLUSIONS: Our data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.

Antidepressant-like Activity of Patchouli Oil var. Tapak Tuan (Pogostemon cablin Benth) via Elevated Dopamine Level: A Study Using Rat Model.

Essential oils are gaining popularity for their use in treating depression, including that extracted from patchouli leaves and stems (Pogostemon cablin). Herein, we used patchouli oil (PO) containing a high amount of patchouli alcohol derived from P. cablin var. Tapak Tuan. The aim of this study was to investigate the antidepressant potential of PO, with a variety of patchouli alcohol concentrations obtained from a separation process using vacuum distillation with different temperature ranges. The initial patchouli oil (iPO) was traditionally distilled by a local farmer and further distilled using a rotary evaporator at temperature ranges of 115−160 °C (POF-1); 120−160 °C (POF-2), and 125−160 °C (POF-3), resulting in products with different patchouli alcohol concentrations. POF-3, with the highest patchouli alcohol content of 60.66% (based on gas chromatography-mass spectrometry), was used for cooling crystallization, resulting in 100% patchouli alcohol crystal (pPA). A tail suspension test (TST) was performed on a rat model to screen the antidepressant potential of iPO and its derivatives. The TST results revealed that POF-3 had the best antidepressant-like effect and was second only to the fluoxetine-based antidepressant, Kalxetin®, where both groups had significant reductions of immobility time post-treatment (p < 0.0001). Other than patchouli alcohol, POF-3 also contained ledol and trans-geraniol, which have been reported for their antidepressant-related activities. Brain dopamine levels increased significantly in the group treated with POF-3 (p < 0.05 as compared with the control group), suggesting its primary anti-depressant mechanism. These findings suggest the potential of vacuum-distilled patchouli oil in reducing depression via dopamine elevation.

Prognostic Scoring System for Mortality of Hospitalized COVID-19 Patients in Resource-Limited Settings: A Multicenter Study from COVID-19 Referral Hospitals.

BACKGROUND: Many studies identified the risk factors and prognostic factors related to in-hospital COVID-19 mortality using sophisticated laboratory tests. Cost and the availability of supporting blood tests may be problematic in resource-limited settings. This multicenter cohort study was conducted to assess the factors associated with mortality of COVID-19 patients aged 18 years and older, based on history taking, physical examination, and simple blood tests to be used in resource-limited settings. METHODS: The study was conducted between July 2020 and January 2021 in five COVID-19 referral hospitals in Indonesia. Among 1048 confirmed cases of COVID-19, 160 (15%) died during hospitalization. RESULTS: Multivariate analysis showed eight predictors of in-hospital mortality, namely increased age, chronic kidney disease, chronic obstructive pulmonary disease, fatigue, dyspnea, altered mental status, neutrophil-lymphocyte ratio (NLR) ≥ 5.8, and severe-critical condition. This scoring system had an Area-under-the-curve (AUC) of 84.7%. With cut-off score of 6, the sensitivity was 76.3% and the specificity was 78.2%. CONCLUSION: The result of this practical prognostic scoring system may be a guide to decision making of physicians and help in the education of family members related to the possible outcome.

Depression and functional disability in stroke patients.

Background: Stroke patients often suffer from depression, a mental disorder that worsens their condition and slows down the recovery process. Depression is the leading cause of functional disability due to inability to cope with daily stressors and to function independently in their activities. The purpose of this study is to analyze the relationship between depression and functional disability levels in post-stroke patients. Design and Methods: This is an analytic observational research with a cross-sectional approach. The population in exam consisted of all 4-12 week post-stroke patient in the hospital (about 139 patients). The study focused on 104 respondents, who were selected using simple random sampling techniques. Results: The results show that 62.5% stroke patients suffered mild depressive episodes after the stroke while 58.7% experienced mild disabilities. Analysis results using the Pearson Product Moment Test obtained P=0,000. This shows there is a relationship between the level of depression and the degree of functional disability in post-stroke patients. Conclusions: It can be concluded that there is a significant relationship between the levels of post-stroke depression and the functional disability. Families are expected to provide a safe, supportive, and comfortable environment to lessen the level of depression.

Pyrophen Isolated from the Endophytic Fungus Aspergillus fumigatus Strain KARSV04 Synergizes the Effect of Doxorubicin in Killing MCF7 but not T47D Cells.

OBJECTIVES: Pyrophen, an amino acid-pyrone derivative isolated from Aspergillus fumigatus strain KARSV04 has been reported to have an anticancer effect on T47D cells by inhibiting the growth of cells and modulating the cell cycle in the S phase. In the present study, the effect of pyrophen in doxorubicin (Dox) chemotherapy in an in vitro model of breast cancers was studied. MATERIALS AND METHODS: The cytotoxicity of pyrophen and Dox separately and in combination were evaluated in T47D and MCF-7 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Modulation of cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: Our findings showed that pyrophen did not significantly potentiate Dox-induced cytotoxicity in T47D cells. Adding Dox-treated T47D cells with pyrophen at a concentration of 9.20 µg/mL induced a slight increase in the S-phase cell population. This compound induced cytotoxicity of MCF-7 cells with IC50 of 70.57 µg/mL. Co-treatment of pyrophen and Dox in MCF-7 cells increased cytotoxicity relative to Dox alone, which was suggested in part to be due to modulation of the cell cycle in the G2/M phase and apoptosis. CONCLUSION: The data suggest different mechanisms of regulation in promoting cell death by two different cell lines in response to administration of pyrophen.

A bioactive compound isolated from Duku ( Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line.

Background: Breast cancer is a major health problem for women globally. Many attempts have been promoted to cure cancer by finding new anticancer medicines from natural resources. Despite the richness of biodiversity discovered, there are some natural resources that remain unexplored. Fruit peels of Duku ( Lansium domesticum Corr.) are rich with compounds that may have the potential to be developed as anticancer drugs. This study aimed to isolate cytotoxic compounds from the fruit peels of L. domesticum and assess their cytotoxic nature against T47D cells. Methods: Powdered peels were macerated with ethyl acetate and the filtrate was evaporated to give EtOAc extract A. Dried extract A was triturated with n-hexane to give n-hexane soluble fraction B and insoluble fraction C. The cytotoxic nature of these three  samples were assessed using MTT assay using T47D cells and doxorubicin as a control. Results: Fraction C that showed the smallest IC50 (25.56 ± 0.64µg/mL) value compared to  extract A and fraction B. Fraction C was further fractionated by vacuum liquid chromatography to give 6 subfractions. Subfraction 2 showed a single compound based on thin layer chromatography, and this compound was identified as Lamesticumin A on the basis of its spectroscopic data. Lamesticumin A demonstrated cytotoxic activity against T47D cell lines with an IC50 value of 15.68 ± 0.30µg/mL. Conclusions: Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.

Tylophorine Abrogates G2/M Arrest Induced by Doxorubicine and Promotes Increased Apoptosis in T47D Breast Cancer Cells

Background: The effects of tylophorine, a natural alkaloid found in Tylophora indica, administered as a single compound or in combination with doxorubicin on cell cycling and apoptosis were assessed in T47D breast cancer cells, selected as a model system for breast cancer. Methods: Cell cycle distribution and apoptosis were examined by flow cytometry. Caspase 3 and 9 expression was determined by immunocytochemistry.Result: We found that tylophorine did not significantly influence the cell cycle distribution of T47D cells. However, the alkaloid did prevent accumulation of cells in the G2/M phase. In addition, tylophorine increased the number of apoptotic cells. Expression of proapoptotic proteins (caspases 3 and 9) was up-regulated upon administration of tyloporine alone or in combination with doxorubicin. Conclusions: Tylophorine alone or in combination with doxorubicin induced apoptosis in T47D breast cancer cells through modulation of the cell cycle and affecting the expression of caspases 3 and 9.

Pyrophen Produced by Endophytic Fungi Aspergillus sp Isolated from Piper crocatum Ruiz and Pav Exhibits Cytotoxic Activity and Induces S Phase Arrest in T47D Breast Cancer Cells.

Ethyl acetate extracts obtained from culture of endophytic fungi Aspergillus sp isolated from Piper crocatum Ruiz and Pav, have been shown to possess cytotoxic activity against T47D breast cancer cells. Investigations were here conducted to determine bioactive compounds responsible for the activity. Bioassay guided fractionation was employed to obtain active compounds. Structure elucidation was performed based on analysis of LC-MS, 1H-NMR, 13C-NMR, COSY, DEPT, HMQC, HMBC data. Cytotoxity assays were conducted in 96 well plates against T47D and Vero cell lines. Bioassay guided isolation and chemical investigation led to the isolation of pyrophen, a 4-methoxy-6-(1'-acetamido-2'-phenylethyl)-2H-pyran-2-one. Further analysis of its activity against T47D and Vero cells showed an ability to inhibit the growth of T47D cells with IC50 values of 9.2 µg/mL but less cytotoxicity to Vero cells with an IC50 of 109 µg/mL. This compound at a concentration of 400 ng/mL induced S-phase arrest in T47D cells.

Endophytic Fungi Isolated from Coleus amboinicus Lour Exhibited Antimicrobial Activity.

PURPOSE: Coleus amboinicus is a medicinal plant traditionally used to treat various diseases such as throat infection, cough and fever, diarrhea, nasal congestion and digestive problems. The plant was explored for endophytic fungi producing antimicrobial agents. METHODS: Screening for endophytic fungi producing antimicrobial agents was conducted using agar plug method and antimicrobial activity of promising ethyl acetate extracts was determined by disc diffusion assay. Thin layer chromatography (TLC) - bioautography was performed to localize the bioactive components within the extract. TLC visualization detection reagents were used to preliminary analyze phytochemical groups of the bioactive compounds. RESULTS: Three endophytic fungi were obtained, two of them showed promising potential. Agar diffusion method showed that endophytic fungi CAL-2 exhibited antimicrobial activity against P. aeruginosa, B. subtilis, S. aureus and S. thypi, whilst CAS-1 inhibited the growth of B. subtilis. TLC bioautography of ethyl acetate extract of CAL-2 revealed at least three bands exhibited antimicrobial activity and at least two bands showed inhibition of B. subtilis growth. Preliminary analysis of the crude extracts suggests that bioactive compounds within CAL-2 extract are terpenoids, phenolics and phenyl propanoid compounds whilst the antimicrobial agents within CAS-1 extract are terpenoids, propylpropanoids, alkaloids or heterocyclic nitrogen compounds. CONCLUSION: These data suggest the potential of endophytic fungi of C. amboinicus as source for antimicrobial agents.

Antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz & Pav

Objective:To investigate antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz&Pav (P. crocatum). Methods:Ethyl acetate extracts were obtained by liquid-liquid partition of fermentation broth of endophytes followed by evaporation. The antimicrobial activities were determined by diffusion techniques followed by thin layer chromatography-bioautography. Cytotoxicity studies were conducted using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Data generated were used to plot a dose-response curve, of which the concentration of extract required to kill 50%of cell population (IC50) was determined. Results: Two endophytes were isolated from leaves and stem of P. crocatum, designated as DS1 and BS1. Ethyl acetate extracts of BS1 was found to inhibit the growth of Bacillus subtilis, Escherichia coli and Staphylococcus aureus at minimum dose of 31.25, 125 and 250 μg, respectively. thin layer chromatography-bioautography of this extract resulted in at least two inhibition zones. BS1 extract also inhibited the growth of WiDr and T47D cell lines with IC50 of 120.38 μg/mL and 37.43 μg/mL, respectively. Conclusions: BS1 extract produced by endophytic fungi of P. crocatum is potential to be developed as source of novel bioactive compounds.

CDC25B overexpression stabilises centrin 2 and promotes the formation of excess centriolar foci.

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 "foci". These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.

Genistein abrogates G2 arrest induced by curcumin in p53 deficient T47D cells.

BACKGROUND: The high cost and low level of cancer survival urge the finding of new drugs having better mechanisms. There is a high trend of patients to be "back to nature" and use natural products as an alternative way to cure cancer. The fact is that some of available anticancer drugs are originated from plants, such as taxane, vincristine, vinblastine, pacitaxel. Curcumin (diferuloylmethane), a dietary pigment present in Curcuma longa rizhome is reported to induce cell cycle arrest in some cell lines. Other study reported that genistein isolated from Glycine max seed inhibited phosphorylation of cdk1, gene involved during G2/M transition and thus could function as G2 checkpoint abrogator. The inhibition of cdk1 phosphorylation is one of alternative strategy which could selectively kill cancer cells and potentially be combined with DNA damaging agent such as curcumin. METHODS: T47D cell line was treated with different concentrations of curcumin and genistein, alone or in combination; added together or with interval time. Flow Cytometry and MTT assay were used to evaluate cell cycle distribution and viability, respectively. The presence of apoptotic cells was determined using acridine orange-ethidium bromide staining. RESULTS: In this study curcumin induced G2 arrest on p53 deficient T47D cells at the concentration of 10 µM. Increasing concentration up to 30 µM increased the number of cell death. Whilst genistein alone at low concentration (≤10 µM) induced cell proliferation, addition of genistein (20 µM) 16 h after curcumin resulted in more cell death (89%), 34% higher than that administered at the same time (56%). The combination treatment resulted in apoptotic cell death. Combining curcumin with high dose of genistein (50 µM) induced necrotic cells. CONCLUSIONS: Genistein increased the death of curcumin treated T47D cells. Appropriate timing of administration and concentration of genistein determine the outcome of treatment and this method could potentially be developed as an alternative strategy for treatment of p53 defective cancer cells.

Phosphorylation of Cdc25B3 Ser169 regulates 14-3-3 binding to Ser151 and Cdc25B activity.

Cdc25B is a major regulator of G(2) phase progression into mitosis and in recovery from a G(2) phase checkpoint arrest. The activity of Cdc25B is regulated by 14-3-3 dimer binding to a high-affinity phosphosite Ser323 and two lower-affinity phosphosites Ser151 and Ser230, which blocks substrate access to the catalytic site and, thereby, activity. Loss of 14-3-3 binding to any of these sites is sufficient to activate the enzyme. Thus, the mechanisms that disrupt 14-3-3 binding are critical regulators of entry into mitosis. Here, we demonstrate that the mechanism regulating the G(2) phase activation of major cdc25B3 isoform is independent of the dephosphorylation of the 14-3-3 binding sites, instead utilizing phosphorylation of Ser169. Mutation of this site to the phosphomimetic Asp reduced 14-3-3 binding to the same extent as Ser151Ala mutation, increased substrate access to the catalytic site and Cdc25B activity but had no effect on Ser151 or Ser230 phosphorylation. Ser169 phosphorylation was observed in G(2)-phase cells. By contrast, Ser249 phosphorylation, which was associated with cyclin B/Cdk1 activation, had no effect on 14-3-3 binding or Cdc25B activity. This indicates that Ser169 phosphorylation can disrupt 14-3-3 binding to Ser151 activating Cdc25B3, providing a mechanism for regulating Cdc25B3 activation without dephosphorylation of the 14-3-3 binding sites.

Mitotic phosphorylation of Cdc25B Ser321 disrupts 14-3-3 binding to the high affinity Ser323 site.

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser(323) being the highest affinity binding and is highly homologous to the Ser(216) 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser(323) increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser(323) phosphorylation is maintained into mitosis, but phosphorylation of Ser(321) disrupts 14-3-3 binding to Ser(323), mimicking the effect of inhibiting Ser(323) phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser(321) appears to have a role in stabilizing 14-3-3 binding to Ser(323), and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser(323). Ser(321) is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser(321) acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser(323) and enhancing the dephosphorylation of Ser(323) to block 14-3-3 binding to this site.

MAPK pathway activation delays G2/M progression by destabilizing Cdc25B.

Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G(2) phase delays entry into mitosis; however, the role of the MAPK pathway during G(2)/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G(2) phase delay independent of known G(2) phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G(2) phase checkpoint arrest. Both the G(2) phase delay and blocked exit from the G(2) checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G(2)/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G(2) phase delay. Thus, we have demonstrated a new function for MEK1 that controls G(2)/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G(2) phase checkpoint arrest.