A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1–Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface

A mobility enabled inpatient monitoring system using a ZigBee medical sensor network

This paper presents a ZigBee In-Patient Monitoring system embedded with a new ZigBee mobility management solution. The system enables ZigBee device mobility in a fixed ZigBee network. The usage, the architecture and the mobility framework are discussed in details in the paper. The evaluation shows that the new algorithm offers a good efficiency, resulting in a low management cost. In addition, the system can save lives by providing a panic button and can be used as a location tracking service. A case study focused on the Princes of Wales Hospital in Hong Kong is presented and findings are given. This investigation reveals that the developed mobile solutions offer promising value-added services for many potential ZigBee applications

Biophysical and nutritional combination treatment for myosteatosis in patients with sarcopenia: a study protocol for single-blinded randomised controlled trial

Introduction Sarcopenia is characterised by age-related loss of skeletal muscle and function and is associated with risks of adverse outcomes. The prevalence of sarcopenia increases due to ageing population and effective interventions is in need. Previous studies showed that β-hydroxy β-methylbutyrate (HMB) supplement and vibration treatment (VT) enhanced muscle quality, while the coapplication of the two interventions had further improved muscle mass and function in sarcopenic mice model. This study aims to investigate the efficacy of this combination treatment in combating sarcopenia in older people. The findings of this study will demonstrate the effect of combination treatment as an alternative for managing sarcopenia. Methods and analysis In this single-blinded randomised controlled trial, subjects will be screened based on the Asian Working Group for Sarcopenia (AWGS) 2019 definition. 200 subjects who are aged 65 or above and identified sarcopenic according to the AWGS algorithm will be recruited. They will be randomised to one of the following four groups: (1) Control+ONS; (2) HMB+ONS; (3) VT+ONS and (4) HMB+VT + ONS, where ONS stands for oral nutritional supplement. ONS will be taken in the form of protein formular once/day; HMB supplements will be 3 g/day; VT (35 Hz, 0.3 g, where g=gravitational acceleration) will be received for 20 mins/day and at least 3 days/week. The primary outcome assessments are muscle strength and function. Subjects will be assessed at baseline, 3-month and 6-month post treatment. Ethics and dissemination This study was approved by Joint CUHK-NTEC (The Chinese University of Hong Kong and New Territories East Cluster) Clinical Research Management Office (Ref: CRE-2022.223-T) and conformed to the Declaration of Helsinki. Trial results will be published in peer-reviewed journals and disseminated at academic conferences

The MapZ-Mediated Methylation of Chemoreceptors Contributes to Pathogenicity of Pseudomonas aeruginosa

The pathogenic bacterium Pseudomonas aeruginosa is notorious for causing acute and chronic infections in humans. The ability to infect host by P. aeruginosa is dependent on a complex cellular signaling network, which includes a large number of chemosensory signaling pathways that rely on the methyl-accepting chemotaxis proteins (MCPs). We previously found that the second messenger c-di-GMP-binding adaptor MapZ modulates the methylation of an amino acid-detecting MCP by directly interacting with a chemotaxis methyltransferase CheR1. The current study further expands our understanding of the role of MapZ in regulating chemosensory pathways by demonstrating that MapZ suppresses the methylation of multiple MCPs in P. aeruginosa PAO1. The MCPs under the control of MapZ include five MCPs (Aer, CtpH, CptM, PctA, and PctB) for detecting oxygen/energy, inorganic phosphate, malate and amino acids, and three MCPs (PA1251, PA1608, and PA2867) for detecting unknown chemoattractant or chemorepellent. Chemotaxis assays showed that overexpression of MapZ hampered the taxis of P. aeruginosa toward chemoattractants and scratch-wounded human cells. Mouse infection experiments demonstrated that a dysfunction in MapZ regulation had a profound negative impact on the dissemination of P. aeruginosa and resulted in attenuated bacterial virulence. Together, the results imply that by controlling the methylation of various MCPs via the adaptor protein MapZ, c-di-GMP exerts a profound influence on chemotactic responses and bacterial pathogenesis

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Functional study of RanBP1 in mitotic progression.

Accurate partitioning of chromosomes in mitosis requires that the states of mitotic spindle assembly and chromosome alignment be monitored and communicated to the cell cycle machinery so that chromosome segregation does not commence until every pair of chromosomes is attached to microtubules (MT) from opposite spindle poles (Bharadwaj and Yu, 2004; Rieder and Malato, 2004). While extensive research has firmly established the importance of the cell cycle machinery in spindle assembly and chromosome segregation, recent studies have revealed that a signaling pathway mediated by RanGTPase plays an equally important role in mitosis (Karsenti and Vernos, 200 I; Zheng, 2004). The regulators of RanGTPase are spatially separated with the nucleotide exchange factor (called RCCI) tethered to the chromatin and the proteins that activate the GTPase, such as RanGAPI and RanBPI, localized in the cytoplasm (Weis, 2002). Consequently, a high RanGTP concentration can be established on the condensed chromosomes in mitosis (Kalab et aI., 2006; Kalab et aI., 2002; Li and Zheng, 2004a; Li and Zheng, 2004b). This spatial distribution of RanGTP is required for mitotic spindle assembly (Karsenti and Vernos, 2001; Zheng, 2004) and it may also regulate spindle checkpoint (Arnaoutov and Dasso, 2003). The chromosome-based RanGTP production appears to determine the spatial distribution of activities that promote microtubule (MT) nucleation and stabilization during spindle assembly (Caudron et aI., 2005). It might also function as a sensor to communicate the progression of spindle assembly to the cell cycle machinery so that anaphase onset is coupled to proper chromosome alignment. Whereas the cyclin B-dependent CDKI appears to directly regulate RanGTP production in mitosis by phosphorylating RCCI (Li and Zheng, 2004a; Li and Zheng, 2004b), there is evidence that RanGTP may in turn influence mitotic progression by regulating the spindle checkpoint and MT-kinetochore association. In Xenopus egg extracts, increasing RanGTP concentration has been shown to cause the dissociation of a number of spindle checkpoint proteins from kinetochores and to silence the spindle checkpoint (Arnaoutov and Dasso, 2003). Since an increased association of RCC1 with mitotic chromosomes coincided with metaphaseanaphase transition in the egg extracts, Arnaoutov and Dasso proposed that elevated RanGTP levels on metaphase chromosomes might silence spindle checkpoint to allow mitosis to progress into anaphase (Arnaoutov and Dasso, 2003). Additional studies have shown that RanGTP is also required to localize both RanGAPI and RanBP2 to kinetochores in mammalian somatic cells where the latter two proteins control the MY-kinetochore attachment (Joseph et aI., 2004; Salina et aI., 2003). More recently, the nuclear export receptor Crml was shown to be a downstream effector of RanGTP in mitosis that regulates MY-kinetochore attachment through RanBP2 and RanGAPI (Arnaoutov et aI., 2005; Arnaoutov and Dasso, 2005). Together, the above studies suggest that RanGTP production and turnover during mitosis could regulate chromosome alignment and metaphase-anaphase transition. However, it remains unclear whether an elevated RanGTP level could lead to checkpoint silencing and metaphaseanaphase transition in somatic cells. In addition, it is unclear how a cell could keep a lower level of RanGTP concentration until all chromosomes have reached proper alignment at the metaphase plate. Moreover, a few misaligned chromosomes are sufficient to trigger spindle checkpoint and mitotic arrest. Therefore, it is difficult to envision how RanGTP concentration is kept low in these situations where the majority of chromosomes have reached alignment at the metaphase plate. Finally, since blocking proper spindle assembly by cithcr nocodazolc or taxol could trigger spindle checkpoint, it is unclear how RanGTP concentration could be kept low under these conditions to allow an active spindle checkpoint. We reasoned that, because RanGTP is produced on each chromosome, the overall state of chromosome alignment might have signi ficant influence on the diffusion of RanGTP and the final RanGTP concentration on chromosomes. Using a computational model, we found that the statc of chromosome alignment has a strong influence on the local concentrations of RanGTP and RanGTP-transport receptor complexes that each chromosome experiences. Aided by this model, we show that elevating the concentration of RanGTP-importin Beta(a nuclear import receptor) by down-regulating RanBP I disrupts both spindle assembly and the association of spindle checkpoint protein Mad2 to unattached kinetochores, which leads to mitotic cell death. Moreover, our studies suggest that the reduced RanGTP concentration during mitotic arrest is controlled by both the degree of chromosome alignment and the interaction of RCC1 with mitotic chromosomes

Regulation of RCC1 and its roles in apoptosis.

This supplement equipment purchase grant is affiliated to the AcRF Teir 2 project ARC07/06. Although a number of signaling pathways have been identified to regulate apoptosis, the mechanism that initiates apoptosis remains incompletely understood. In this project, we aimed to study the possible regulation of the nuclear transport machinery to control the onset of apoptosis. We have found that the nuclear RanGTP level is diminished during early stages of apoptosis, which correlates to the immobilization of RCC1 on the chromosomes. Furthermore, the expression of phospho-mimic histone H2B or caspases activated Mst1 immobilizes RCC1 and causes reduction of nuclear RanGTP levels, which leads to the inactivation of nuclear transport machinery. As a consequence, nuclear localization signal (NLS) containing proteins, including NF-kB-p65, remain bound to importins α and β in the cytoplasm. Knocking down Mst1 allows resumption of nuclear transport and the nuclear entry of NF-kB-p65, which play important roles in rescuing cells from apoptosis. Therefore, we propose that RCC1 reads the histone code created by caspaseactivated Mst1 to initiate apoptosis by reducing the level of RanGTP in the nucleus. The findings in this project suggest that promoting apoptosis is the active disruption of nuclear trafficking before the cells are committed to the execution stages of apoptosis. It is believe that our findings lay the basis for a deeper understanding of how spatial and temporal signals are organized within apoptotic cells.RG150/0

Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. We show here that Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin α and β to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells